Journal of Chromatography A, 1430 (2016) 1678

Contents lists available at ScienceDirect

Journal of Chromatography A
journal homepage: www.elsevier.com/locate/chroma

Review article

Recent advances and trends in the liquid-chromatographymass

spectrometry analysis of avonoids
Andr de Villiers , Pieter Venter, Harald Pasch
Department of Chemistry and Polymer Science, Stellenbosch University, Private Bag X1, Matieland 7602, South Africa

a r t i c l e i n f o a b s t r a c t

Article history: Flavonoids have elicited signicant attention as a result of their importance in plants, their inuence
Received 14 September 2015 on the properties of natural-product derived commodities and especially as a consequence of their pur-
Accepted 25 November 2015 ported health benets. Research in all of these elds relies heavily on accurate analytical data, and in
Available online 28 November 2015
this LCMS has come to play an inuential role by allowing relatively fast tentative identication and
accurate quantication of low levels of avonoids in a variety of matrices. The eld has undergone rapid
Keywords:
expansion in the last decade due to important developments in both HPLC and MS instrumentation,
Comprehensive two-dimensional LC
which nowadays allow much faster and more accurate analysis of avonoids. This contribution aims to
(LC LC)
Flavonoids
provide an overview of these developments and their application in avonoid analysis since 2009. The
Liquid-chromatographymass discussion is focussed rst on methodologies which provide improved LC separation of avonoids in
spectrometry (LCMS) terms of speed and/or resolution, including ultra high pressure LC (UHPLC), monolithic and supercially
MS/MS porous phases, high temperature LC (HTLC) and comprehensive two-dimensional LC (LC LC). The fun-
Supercially porous phases damental background relevant to each of these will be briey outlined, as well as the implications and
Ultra high pressure liquid chromatography promise of their hyphenation to MS. Secondly, the possibilities and limitations of a range of the latest
(UHPLC) MS instruments available in combination with advanced LC analysis will be discussed, including ion trap,
triple quadrupole, time-of-ight, Orbitrap, ion mobility and various hybrid instruments. Examples from
the latest literature will be used to illustrate the performance gains achievable in avonoid analysis by
the hyphenation of advanced LC separation and high-end MS instrumentation.
2015 Elsevier B.V. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
2. LCMS analysis of avonoids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
2.1. General aspects of LC separation of avonoids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
2.2. Detectors used in the LC analysis of avonoids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
2.3. General aspects of LCMS(n) analysis of avonoids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
2.3.1. Flavonoid glycosides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
2.3.2. Flavonoid aglycones . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
3. Advances in HPLC separation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
3.1. One-dimensional HPLC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24
3.1.1. Ultra high pressure liquid chromatography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25
3.1.2. Monolithic columns . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26
3.1.3. Supercially porous stationary phases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
3.1.4. High temperature liquid chromatography (HTLC) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34
3.2. Comprehensive multi-dimensional LC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35
Fundamental aspects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35
4. Advances in LCMS analysis of avonoids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40

Corresponding author.
E-mail address: ajdevill@sun.ac.za (A. de Villiers).

http://dx.doi.org/10.1016/j.chroma.2015.11.077
0021-9673/ 2015 Elsevier B.V. All rights reserved.
 A. de Villiers et al. / J. Chromatogr. A 1430 (2016) 1678 17

4.1. Ionisation modes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41

4.2. Mass analysers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42
4.2.1. Nominal mass instruments: ion trap (IT) and triple quadrupole (QqQ) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43
4.2.2. Time-of-ight (TOF) instruments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50
4.2.3. Fourier transform ion cyclotron resonance (FT-ICR) MS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59
4.2.4. Orbitrap instruments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59
4.2.5. Ion mobility spectrometry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63
4.2.6. Overview of mass spectrometers used in avonoid analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64
5. Conclusions and perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65
Symbols and abbreviations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67

1. Introduction mechanisms, including protection of DNA and low density lipo-

proteins, inhibition of enzymes and of platelet aggregation. The
Flavonoids are secondary plant metabolites dened by a antioxidant activity and metal chelating properties of avonoids
diphenylpropane (C6 C3 C6 ) skeleton. Classication of avonoids are considered important properties in determining their contri-
is based on the oxidation state and degree of unsaturation of bution to human health [22]. More recent epidemiological studies
the central heterocyclic ring (or the lack thereof in the case of however report more ambiguous ndings regarding the benecial
chalcones), according to which the major families of avones, properties ubiquitously ascribed to avonoids [23,24], especially
isoavones, avanones, avanonols, avanols, avonols, antho- since knowledge on the metabolism of these compounds is still
cyanidins and chalcones can be distinguished (Fig. 1). Oligomeric limited [24,25]. Much current avonoid research is therefore
and polymeric avan-3-ols collectively comprise the proantho- focussed on the metabolism of avonoids in vivo [26,27].
cyanidin family [1], while additional bi-, tri-, tetra-, penta- and The second major development that has propelled avonoid
hexa-avonoids originate mainly, although not exclusively, from research involves improvements in the analytical techniques used
the oxidative coupling of constituents of the other avonoid classes for their characterisation. In early research, scientists relied on
[2]. Within each of these chemical classes, structural diversity techniques such as thin layer chromatography (TLC) and UV/vis
stems from different substitution patterns of primarily the A- and spectrophotometry, which lack sufcient specicity and sensi-
B-aromatic rings, as well as extensive conjugation including glyco- tivity. As analytical techniques applicable to avonoids have
sides (both C- and O-glycosylated avonoids occur in nature) and developed, progressively more information about the numbers of
acylated glycosides. The number of identied avonoids was esti- compounds and their structural properties has become available,
mated to exceed 4000 in 1994 [3]; by 2005 the number was more thereby supporting more detailed research in the eld. Inuential
than 7000 [4] and has certainly grown signicantly in the interim developments in this regard include the application of gas chro-
due to extensive research focussed on these compounds. matography (GC, although mainly used for sugar determination, the
Flavonoids are biosynthesized via condensation of requirement of derivatisation to render avonoids volatile means
phenylalanine-derived hydroxycinnamic acids and malonoyl- that the technique is rarely used in avonoid analysis nowadays)
moieties originating from phenylpropanoid metabolism and the and 1 H nuclear magnetic resonance (NMR) spectroscopy in the
shikimate and arogenate pathways, respectively, which ultimately late 1960s, followed by 13 C NMR in the 1970s and subsequent
form the B- and A-rings of avonoids [6]. Only plants are capa- advanced two-dimensional NMR techniques from this middle of
ble of avonoid synthesis, and therefore the consumption of this decade. High performance liquid chromatography (HPLC) was
plant-derived products provides the most important source of rst applied for avonoid analysis in the late 1970s [2830]
avonoids in the human diet. Flavonoids have been shown to full and liquid chromatographymass spectrometry (LCMS) from the
several important roles in plants, including attraction of insects, 1980s. Since the last decade of the 20th century the application
protection against abiotic and biotic stress factors such as UV of capillary electrophoresis (CE, including various electrophoretic
radiation, harmful insects and animals as well as regulation of separation modes) has also found application in avonoid analy-
physiological and metabolic processes [3,7,8]. sis [3133], while countercurrent separation techniques such as
Initially thought to be metabolic waste products and of interest countercurrent chromatography (CCC) and centrifugal partition
only for taxonomic reasons, interest in avonoids has grown almost chromatography (CPC) have more recently found application pri-
exponentially since the late 1960s, supported by two key develop- marily as purication methods for natural products [3336].
ments. In the rst instance, recognition of the biological activities From a structural elucidation perspective, NMR is indispens-
of these compounds prompted substantive research (interestingly, able in avonoid research as the technique allows the complete
one of the rst studies to provoke such interest reported Vita- assignment of avonoid structures, including the aglycone identity,
min P in oranges [9], although the name was later revoked when stereochemistry, nature and position of sugars and acylated sugars,
the compound was identied as the avonol rutin). Especially at etc. [37]. NMR is however hampered by cost and throughput limita-
the end of the 20th century, a large body of evidence pointed to tions and is not suited to all applications, especially because of the
several benecial physiological effects associated with avonoid- requirement of sufcient concentrations of relatively pure analytes.
rich diets [1013]. Recognition of the contribution of phenolics The hyphenation of HPLC with NMR has been demonstrated to be
to the French Paradox [14], where the French population was a powerful method for avonoid characterisation, circumventing
found to exhibit low incidences of cardiovascular mortality despite the need for exhaustive isolation of compounds prior to their iden-
a high fat diet a phenomenon explained in part by the relatively tication [3840]. However, this technology is, due instrumental
high consumption of red wine by the French proved inuential complexity and cost, not in widespread use.
in stimulating further research in avonoids and their contri- It is fair to say that the hyphenation of LC with mass spectrom-
bution to the human diet. Subsequent studies have highlighted etry (MS), especially once successfully attained using atmospheric
several potentially important roles of avonoids in vitro, including pressure ionisation sources, has revolutionised the analysis of com-
their protective effects against cardiovascular diseases and some plex avonoid fractions; LCMS has become an obligatory tool
forms of cancer [1521], amongst others, through a variety of in avonoid characterisation as a complementary technique to
18 A. de Villiers et al. / J. Chromatogr. A 1430 (2016) 1678
Fig. 1. Structures of the main classes of avonoids, with numbering of rings and carbons indicated. Note the difference in the numbering scheme for chalcones [5]. Substituents
on the backbones are mostly hydroxyl and methoxy groups, while additional mono- to oligosaccharides with varying degrees of acylation are also common.
NMR. Whereas NMR provides average compositional information
on complex mixtures, MS addresses the composition of single
molecules. Developments in commercial mass analysers over the
last decade and a half now routinely allow determination of molec-
ular formulae and molecular fragmentation using collision induced
dissociation (CID) on single or multi-stage MS systems, and are
largely responsible for the fact that LCMS has assumed its current
position of eminence in the eld of avonoid analysis. The eld
of LCMS has experienced exceptional growth during this period
due to the commercial availability of a range of high resolution and
hyphenated MS detectors based on atmospheric pressure ionisa-
tion (API) ionisation sources [41,42].
The combination of chromatographic resolution provided by
HPLC and structural information provided by MSn enables rou-
tine separation and tentative identication of complex mixtures
of avonoids spanning several orders of magnitude in concentra-
tion, something that was not possible 25 years ago. Furthermore,
important advances in LC technology have been realised in the
last decade. Signicant developments include the use of ultra high
pressures and sub-2 m stationary phases (ultra high pressure liq-
uid chromatography, UHPLC), alternative stationary phases such as
monoliths and supercially porous phases, high temperature HPLC
[43,44] and multidimensional HPLC [4549].
As a consequence of the intensive research activity involving
avonoids, a wealth of information may be obtained regarding
important aspects such as their analysis, biochemistry, health prop-
erties and recently identied compounds. Especially a number of
comprehensive book series serve as excellent reference works that
provide detailed overviews of the eld and are updated periodi-
cally [3,4,5052]. More dedicated information on the analysis of
avonoids may be found in books such as [53,54], while landmark
reports provide comprehensive overviews of particular aspects
such as the analytical chemistry of avonoids in food [55,56],
extraction and analysis of phenolics [57,58], the role of MS in the
structural analysis of avonoids [59,60], also in biological samples
[27] and wine chemistry [61], separation and detection methods
for avonoids [62] and advanced separation methods for selected
avonoids [33]. In light of the spectacular developments in the
eld during the last 510 years, however, these seminal reference-
works are somewhat outdated. Several more recent review papers
have addressed particular areas of the HPLC or LCMS analy-
sis of avonoids, such as developments in the HPLC separation
of phenolic compounds [63,64], analysis of food polyphenols by
ultra high pressure LC (UHPLC) combined with MS [65], chromato-
graphic analysis of avonoids and metabolites [66], developments
in the analysis of natural product [67] and food phenolics [68]
and metabolites [6973]. None of these works comprehensively
address the fundamental and instrumental aspects pertinent to
the current state-of-the-art in avonoid analysis by LCMS; in this
work we will endeavour to ll this gap.
The emphasis of the current report will be on the most impor-
tant advances in the HPLC separation and MS instrumentation
and their application in the LCMS analysis of avonoids in a
variety of matrices. Considering the very large number of liter-
ature reports appearing annually in this eld, we will limit the
scope of this review to the last 7 years (since 2009). We will fur-
ther limit the scope to UHPLC separation (dened here as HPLC
separations performed on columns packed with sub-2 m phases
and/or operated at pressures above 400 bar), the use of elevated
temperatures (50 C), alternative stationary phase morphologies
(including monoliths, supercially porous phases), comprehensive
two-dimensional LC separations (LC LC) and multi-stage and/or
high resolution MS.1 The fundamental background of advanced
HPLC methods and MS(n) instrumentation and their application
to avonoid analysis will be addressed briey, and the benets of
these developments will be highlighted on the hand of important
literature reports. We will attempt to highlight the most important
contemporary trends in the LCMS analysis of avonoids based on
these reports.
Note that sample preparation and extraction of avonoids,
although essential in all analytical workows for avonoids and
especially so in the case of LCMS analysis to reduce matrix
effects, will not be addressed in detail in the current work.
This aspect has been covered in detail by several reviews
[16,33,5659,62,65,66,74,75] and developments in the eld are less
dramatic than those involving the analysis step.
Finally, a caveat should be added: because of the very large
number of papers falling within the scope of this review, a com-
prehensive list of all relevant reports is not possible in this format;
neither will we attempt to discuss each report in detail. Rather,
tables will be used to summarise the information, and the discus-
sion will focus on important contributions and trends deduced from
these works.
2. LCMS analysis of avonoids
2.1. General aspects of LC separation of avonoids
Contemporary HPLC separation of avonoids is almost exclu-
sively performed using reversed phase liquid chromatography
1
This of course implies that a large number of excellent research papers published
in the last 7 years will not be included due to the criteria dened for the scope of
the current work. This was however important to limit the number of papers to
manageable numbers.
 A. de Villiers et al. / J. Chromatogr. A 1430 (2016) 1678
(RP-LC), with the notable exceptions being normal phase liq-
uid chromatography (NP-LC) for oligomeric proanthocyanins [76],
and the recent increasing application of hydrophilic interaction
chromatography (HILIC). Other modes of separation which have
been applied to avonoid separation include mixed-mode ion-
exchange-reversed phase [7779] separation of anthocyanins, size
exclusion chromatography (SEC) analysis of avonol glycosides
[80,81] and theaavins and proanthocyanidins [81]. Because of the
relatively sparse use of the latter modes, however, they will not
be discussed in detail in the current work (except when used in
LC LC), which will focus primarily on RP-LC in accordance with
the scope and preponderance of this mode in avonoid literature.
RP-LC has demonstrated its suitability for the separation of
avonoids based on the nature of the aglycone (including the
oxidation state, substitution patterns and stereochemistry), the
nature and degree of glycosylation as well as the nature and
degree of acylation. By far the majority of RP-LC separations are
performed using C18 octadecyl-silica (ODS) phases, although C8
[82,83], C12 [84], phenyl or phenyl-hexyl [76,8590], pentauo-
rophenyl (PFP) [9095] and polar embedded RP phases [88,9698]
as well as polymeric RP-LC phases [99] have also found appli-
cation in avonoid analysis [100102]. Mobile phases typically
comprise aqueous/organic phases containing methanol, acetoni-
trile and less commonly tetrahydrofuran [103], isopropanol [101]
or ethanol [99] and acidic modiers such as acetic acid, formic acid,
ammonium acetate or triuoroacetic acid (TFA) [101] (phosphoric,
citric or perchloric acids have also been used in combination with
UV detection, although these are not suited to hyphenation with
MS). For anthocyanins, highly acidic mobile phases (>410% formic
acid, 0.10.6% TFA) [104107] are used to ensure prevalence of
the avylium cationic species in solution and therefore improve
chromatographic efciency [33,100,101,105].
Retention of avonoids in RP-LC is based on hydrophobicity;
some selectivity differences may be observed based on the nature of
the stationary phase, endcapping, carbon loading, polar embedded
groups as well as the nature of the silica phase (e.g. organo-silanes)
[108,109]. However, the general elution order of avonoids on
RP-LC columns is mostly consistent in the following sequence for
avonoid aglycones: avanols < avanones < avonols < avones
[4,59,110,111] (anthocyanins elute close to avanols using generic
(i.e. not highly acidic) mobile phases [111,112]). For each of these
classes, retention decreases with the number of hydroxyl groups
on the avonoid backbone, and increases with the number of
methoxy substituents. Glycosylation decreases RP-LC retention,
whereas acylation of glycosyl groups increases retention relative
to the corresponding glycosylated derivatives. Common sugars
encountered in glycosylated avonoids include glucose, galac-
tose, rhamnose, xylose and arabinose, with mannose, fructose
and glucoronic and galactoronic acids being rarer. The general
elution order of glycosylated avonoids in RP-LC is -galactosides < -
glucosides < -arabinosides < -xylosides < -rhamnosides [101]. Di-
and higher oligomeric saccharides are common in some prod-
ucts, with the most common disaccharides being rutinoside
(rhamnosyl-(16)-glucose) and neo-hesperidose (rhamnosyl-(-
(12)-glucose) [59]. The most common aliphatic and aromatic
acids involved in acylation include (in rough order of their RP-LC
elution sequence) malic, acetic, malonic, succinic, gallic, proto-
catechuic, hydroxybenzoic, vanillic, caffeic, syringic, p-coumaric,
ferulic and synaptic acids [101]. For more detailed informa-
tion regarding the relative elution orders of different classes of
avonoids in RP-LC, the reader is referred to [101].
As a rule, NP-LC is not suited to avonoid analysis due to the lim-
ited solubility of these compounds in typical NP-LC mobile phases,
the exception being apolar avonoids such as polymethoxylated
avones [113] and acetylated avonoids [114]. One notable appli-
cation of NP-LC is however the separation of proanthocyanins
19
according to molecular weight (MW), rst reported in 1979 [76]
(and based on previous ndings on silica gel TLC [115]) and sub-
sequently adapted to HPLC format and rened by several groups
[116,117]. This type of separation is typically performed in adsorp-
tive mode on bare silica stationary phases and mobile phases
containing dichloromethane/methanol/acidied water [116,117],
hexane/acetone or hexane/methanol/ethyl acetate [118]. Proan-
thocyanins elute in order of their degree of polymerisation (DP)
under these conditions, with isomeric compounds of the same DP
co-eluting.
A more recent trend is the application of HILIC for avonoid anal-
ysis. HILIC can be considered an aqueous variant of NP-LC, where
retention is primarily governed by partitioning of polar compounds
into an aqueous-rich layer immobilised on the surface of polar
bonded phases [119,120]. The rst application of HILIC to avonoid
analysis, and one of the rst applications of this mode of separa-
tion [121] (although the term and a description of its separation
mechanism was to follow only much later), was reported by Lea
[76], who used a pellicular polyamide phase and a mobile phase
comprising methanol, acetic acid and water for the separation of
epicatechin and phloridzin. Similar retention of proanthocyanins
is observed in HILIC compared to NP-LC [122,123]. However, the
more polar mobile phases used in HILIC, typically comprising binary
mixtures of acetonitrile and acidied water, makes the technique
more suitable for avonoid analysis than NP-LC and improves ion-
isation efciency for these compounds compared to RP-LC due to
higher organic content of the mobile phase [124]. Indeed, the appli-
cation of HILIC for the analysis of avones and avonols [125127],
dihydrochalcones and avanones [127], anthocyanins [128] and
phlorotannins [129] has been demonstrated recently. HILIC sta-
tionary phases used in avonoid analysis include silica [130], diol
[122,131], polyethylene glycol (PEG) [131], cyclodextrin (CD) [130],
ZIC-HILIC [132] and amide [128,130] phases. One of the major driv-
ing forces behind the interest in HILIC for avonoid analysis is the
alternative separation mechanism to RP-LC offered by this separa-
tion mode [133], which typically results in classtype separations
of avonoids according to the degree of glycosylation and/or acy-
lation. As a consequence, HILIC has found increasing application in
the LC LC separation of avonoids in combination with RP-LC (see
Table 2).
2.2. Detectors used in the LC analysis of avonoids
A wide range of detectors can and have been used in combi-
nation with HPLC separation to detect and/or identify avonoids.
These include electrochemical detection (ED) [134,135], uores-
cence (FL) [62,136], UVvis, diode array [101,137], NMR [3840]
and of course MS detectors [59,60,62,110,138]. Of these, by far the
most common nowadays are diode array and MS detectors, which
will be discussed briey in this and the next section, respectively.
The conjugated aromatic character of avonoids proved to be a
signicant benet especially in early avonoid research: absorption
at relatively long wavelengths increases the selectivity of qual-
itative and quantitative spectrophotometric methods, while the
characteristic spectra of different classes of avonoids allow dis-
tinction between them. These characteristics are equally useful
when UVvis detection is performed in combination with HPLC
separation.
Flavonoids display two UVvis absorption maxima: an absorp-
tion band in the region of 240285 nm (Band II) ascribed to
the A-ring, and a second band at 300550 nm due to the B-ring
(Band I). Of these, the latter is more useful because it provides
more selective information, since all avonoids absorb in the
region of 240285 nm. Flavanols exhibit only Band II absorption
(269279 nm) due to a lack of conjugation between A- and B-rings;
the same applies to avanones, dihydroavonols and isoavones.
20 A. de Villiers et al. / J. Chromatogr. A 1430 (2016) 1678

Fig. 2. Examples of UVvis spectra of different classes of avonoids: (A) anthocyanins, (B) avanols, (C) avanones, (D) avones, (E) avonols and (F) isoavones.
Source: Reproduced with permission from [56].

Flavonols and avones show Band I absorption between 300 and
380 nm, whereas anthocyanidins are easily distinguished by their
Band I absorption between 460 and 550 nm in the visible range.
Typical examples of the UVvis absorbance spectra of the principal
classes of avonoids are presented in Fig. 2.
As a rule, increasing hydroxylation of A- and B-rings leads to
bathochromic shifts in the wavelength of maximum absorption,
max , of Bands II and I; this effect is somewhat offset by methylation
of the hydroxyl groups. Glycosylation does not have a signicant
effect on the absorbance spectra of most avonoids, and the nature
of the sugar moiety has no effect. Exceptions are glycosylation at C-
3, which causes a hypsochromic shift of 1520 nm in absorption
Band I for avonols and avones, C-glycosylation which results
in a bathochromic shift of Band II, and the absence of a shoul-
der at 440 nm for anthocyanidin-3,5-di-O-glycosides compared
to anthocyanidin-3-O-diglycoside [101,139]. Acylation involving
aromatic acids leads to increased absorbance in the UV region
close to Band II (260290 nm), whereas for hydroxycinnamic acids
increased absorbance is observed in the region 305325 nm. In the
interpretation of UV spectra obtained using on-line diode array
detection, it is important to consider the effect of the solvent (both
the nature of mobile phase constituents and their composition at
the point of elution) when comparing with reference spectra, since
this can have a signicant effect on avonoid spectra. For more
detailed information on the UVvis absorption characteristics of
avonoids, the reader is referred a range of excellent dedicated
texts available [101,110,140].
While the structural analysis of avonoids based on their UVvis
spectra has in recent years largely been surpassed by more pow-
erful tools such as MS and NMR, useful information can easily be
obtained based on UVvis spectral properties, the most impor-
tant being the chemical class of avonoids. For this reason UVvis
 A. de Villiers et al. / J. Chromatogr. A 1430 (2016) 1678
detection remains a very useful technique in combination with
HPLC and MS for avonoid analysis. Because UVvis detection is
non-destructive, the detector can be connected before other detec-
tors such as MS and NMR, is compatible with high mobile phase
ow rates and provides extremely reliable quantication (provided
that co-elution of compounds with similar absorbance properties
can be avoided) [137]. The sensitivity of UV detection depends on
the avonoid target and instrument used, but is generally in the
region of 0.0210 ng injected mass [94,95,103,141,142], which is
sufcient for most natural product and food applications. Further-
more, UVvis spectra can be used to distinguish between different
classes of avonoids as outlined above. While this may sound trivial,
this information in many cases may prove pivotal. For example, sev-
eral glycosylated avonoids exhibit identical MS behaviour due to
identical molecular formulae and fragmentation, yet belong to dif-
ferent avonoid families [143]. As an example, even accurate mass
MS cannot distinguish between the delphinidin and cyanidin gly-
cosides (anthocyanins) and quercetin and kaempferol glycosides
(avonols), respectively, unless high-collision energy CID exper-
iments are performed. However, UVvis spectra of anthocyanins
and avonols are clearly distinct (Fig. 2) and therefore allow facile
assignment of the chemical class without additional experiments.
Furthermore, UV absorbance at 500 nm can be used for selec-
tive detection and accurate quantication of anthocyanins, even
in cases where co-elution occurs with avonols. In light of the fact
that UV detectors are relatively cheap and commonly available, it
is no surprise that on-line hyphenation of HPLC separation with UV
and MS detection in sequence has become the norm in avonoid
analysis, especially in proling methods where the aim is to obtain
maximum information [144].
Post-column addition of UV shift reagents has been used to
study avonoid substitution patterns, and post-column or pre-
column derivatisation (for example uorescent labelling to exploit
the benets of this mode of detection) has also been used in
avonoid analysis, although these are less common nowadays
[145,146].
Fluorescence detection is used much less often in the LC analysis
of avonoids, a consequence of the fact that only few avonoid
classes exhibit uorescent behaviour: isoavones, methoxylated
avones and avonoids with an OH substituent at carbon 3 (Fig. 1)
[62]. Signicantly, this includes avanols such as (epi)catechin and
the procyanidins, which makes uorescence detection extremely
useful for the selective and sensitive analysis of these compounds,
and the technique has continued to nd application in research
focussed on these classes [147150].
NMR clearly remains the most important spectroscopic tech-
nique for avonoid analysis, although the technique is still
relatively rarely used on-line in combination with HPLC separation
due to the challenges associated with this hyphenation. In LC-NMR,
two approaches are possible: direct coupling using a ow probe
for the NMR, or stop-ow NMR measurements. In the former case,
deuterated solvents are used in combination with pulse sequences
aimed at suppressing background signals. Stop-ow NMR involves
the use of a valve triggered from an upstream detector to park the
peak of interest in the ow cell, and provides much higher sensitiv-
ity as a consequence. The power of LC-NMR for natural products has
been demonstrated admirably by Wolfender and co-workers; since
the topic falls outside the scope of this work, readers are referred
to several important overviews of the eld for further information
[3840].
2.3. General aspects of LCMS(n) analysis of avonoids
In this section, a brief overview of the background infor-
mation relevant to the MS detection and MS/MS structural
elucidation of avonoids will be presented, with the emphasis
21
on general ionisation and fragmentation processes. The contem-
porary importance of LCMS in avonoid analysis is reected
by the large number of excellent review articles reported in
the last decade on this topic [59,60,110], and the reader is
referred to these for more in-depth information on avonoid
identication by LCMS. Furthermore, much more detailed infor-
mation may be obtained regarding specic classes of avonoids
such as dihydroavonols [151], isoavones [152], avonoid-
[153] and avone-di-C-glycosides [154], avonoid aglycones
[155,156], avonoid-O-glycosides [157,158] and avonoid glyco-
sides [108,159,160] in dedicated research reports.
The following discussion will be limited to the currently most
relevant LCMS ionisation sources, the API sources electrospray
ionisation (ESI), atmospheric pressure chemical ionisation (APCI)
and atmospheric pressure photoionisation (APPI). Fortunately, the
general ionisation and fragmentation behaviour of avonoids is
largely independent of the ionisation source and instrument used
[60,62,161,162], although relative ion intensities may vary some-
what [60,153,163] and some differences have been noted between
CID-MS/MS and in-source CID spectra [164] and between MS2 and
pseudo MS3 spectra on IT and Q-TOF instruments [162]. Never-
theless, the following brief overview can be considered generally
applicable to most instruments.
For all API sources, the composition of the mobile phase plays
a critical role in ionisation, since analytes should either be ionised
in solution (ESI) or in the gas phase after mobile phase evaporation
(APCI and APPI). In fact, divergent conclusions have been drawn
regarding the best mobile phase composition for avonoid analysis.
For negative mode ESI ionisation, optimal mobile phases reported
in literature include 0.1% formic acid [108], 10 mM ammonium
acetate (pH 4) [165] or 0.5% acetic acid [156], whereas for posi-
tive mode, 0.20.75% formic acid [108,165] is preferred; although
triuoroacetic acid has been used, this additive has also been
shown to reduce ESI-MS sensitivity due to its ion-pairing prop-
erties [108,165]. For anthocyanins, highly acidic mobile phases
containing 510% formic acid are commonly used, and since
the anthocyanidins occur as avylium cations in such mobile
phases, positive ionisation is almost exclusively used [138]. The
two most common organic modiers used in the RP-LC anal-
ysis of avonoids, methanol and acetonitrile, have little direct
effect on the ionisation of molecules, although nebulisation ef-
ciency will be affected by the % organic modier in the mobile
phase (generally low percentages of acetonitrile are detrimental)
[108].
Negative ionisation has been shown to be more sensitive than
positive ionisation for all ionisation sources [165], with ESI being
the most sensitive [62]. This is not so much due to ionisation ef-
ciency, but is rather a consequence of increased chemical noise in
positive ionisation mode [108,165]. Positive ionisation generally
results in more extensive fragmentation than negative ionisation
[59,108,165], and remains popular due to the amount of struc-
tural information that may be obtained using this mode [59,62,110],
especially for the analysis of avonoid-C-glycosides [153].
In rst-order MS spectra of avonoids, with low cone voltages,
protonated [M+H]+ and de-protonated [MH] ions are most com-
mon, with little fragmentation present. Depending on the mobile
phase composition, however, adducts such as [MH+AcONa] ,
[M+HSO4 ] , [M+AcO] and [MH+AcONa+MeOH] may be
detected, in addition to molecular complexes such as [2MH] ,
[2M+H]+ and [2M+NaH]+ [110,153]. Complexation with metals
and chelating ligands (added post-column in the case of LCMS)
has also been used in the MS/MS study of avonoids [37,60].
For most of the fragmentation pathways outlined below, tan-
dem mass spectrometers operated under conditions of relatively
high collision energies (1047 eV) are used to generate second-
order mass spectra. However, single stage mass spectrometers may
22 A. de Villiers et al. / J. Chromatogr. A 1430 (2016) 1678
be used to obtain fragmentation information by adaptation of the
cone voltage to attain in-source CID [110,166].
2.3.1. Flavonoid glycosides
The nomenclature proposed by Domon and Costello [167] has
found widespread acceptance for the fragmentation of glycosylated
avonoids, and is illustrated for avonoid O- and C-glycosides in
Fig. 3A and B, respectively. (Note that the ionisation mode is spec-
ied by the superscript + or in this nomenclature [62]). When
the charge is located on the aglycone fragment, ions are labelled
k,l X , Y and Z , where the subscript j denotes the number of
j j j containing sugars of different molar masses.
the interglycosydic bond cleaved (the aglyconeglycosidic bond is
numbered 0) and superscripts k and l indicate cleavages within
the carbohydrate rings. When more than one glycosylation pos-
itions are involved, an additional superscript m is used to denote
the position of aglycone substitution, e.g. ions k,l Xm j , Ym j . For
charged carbohydrate fragments, ions are designated k,l A i , B i
and C i , where the subscript i (1) represents the number of
the glycosidic bond cleaved, counting from the non-reducing end
numbered 1.
Flavonoids commonly occur in nature as glycosidic species,
with two types distinguished based on the nature of the
avonoidsaccharide linkage. Flavonoid-O-glycosides are more
common (and exclusively occur in the case of anthocyanins), sugars
are attached to one or more hydroxyl groups through an acid-
labile C O bond; hydroxyl groups at C-3 and C-7 are common
glycosylation sites (C-3 and C-5 for anthocyanidins). Flavonoid-
C-glycosides contain acid-resistant C C bonds linking the sugar
directly to the avonoid nucleus (Fig. 3B). C-glycosidic bonds
occur virtually exclusively at carbons C-6 and C-8 of the aglycone
[108].
The labile nature of the O-glycosidic bond ensures that the
bond is cleaved under low collision energy conditions, lead-
ing to the loss of neutral sugar residues (loss of 162, 146 and
132 amu for hexosides, deoxyhexosides and pentosides, respec-
tively) and the corresponding Y 0 aglycone ion in the case of
monoglycosides [59,60,110]. The mechanism for this fragmenta-
tion has been shown to entail rearrangement involving hydrogen
migration [169]. In some cases the corresponding saccharide ions,
B 1 , are observed at m/z 163 or 147 for hexoses and pentoses,
respectively [59,110]. In negative ionisation mode, a radical agly-
cone ion, [Y0 H] , resulting from the homolytic cleavage of
the glycosidic bond, may also be observed [59,170]. Flavonoid-
O-diglycosides, where a disaccharide is attached to the aglycone,
show Y 1 and Y 0 fragments, with higher collision energies
favouring the formation of the Y 0 aglycone ion. An interesting
exception is observed for avonol- and avanone-O-rutinosides
(rhamnosyl-(16)-glucose) and avanone-7-O-neohesperidoses
(rhamnosyl-(12)-glucose), where an Y+ * ion is observed. This
ion results from the loss of the inner glucose residue ([M+H162]+ )
through hydrogen migration from the C-5 hydroxyl of the agly-
cone to the terminal rhamnose, followed by a rearrangement
reaction [59,110,171]. The ratio of the Y 1 and Y 0 fragments
in positive ionisation mode can be used to assign the class of
avonoid-O-disaccharide, whereas negative or positive ionisation
second order spectra can be used to distinguish between iso-
meric avonoid-O-neohesperidosides and -O-rutinosides, which
differ in terms of their interglycosidic linkage (1 2 and 1 6,
respectively) [157,158,168]. This approach can be extended to
di- to penta-glycosylated avonoids to allow characterisation of
the interglycosidic linkage and to differentiate positional isomers
based on negative ionisation MS/MS spectra [172]. Especially the
relative abundance of Y 1 , Z 1 , Y 0 and Y7 0 ions are determina-
tive in this regard. The stereochemical assignment of the terminal
monosaccharide units in avonoid-O-glycosides using ESI and
FAB-MS/MS following acetylation has been reported [173],
although this approach has found limited application since.
Flavonoid-di-O-glycosides, where two monosaccharides are
linked to different aglycone hydroxyls, show Y3 0 , Y7 0 fragments
due to the cleavage of one of the glycosidic bonds (for anthocyanins
these will be Y3 0 , Y5 0 ). In some cases Y3 0 Y7 0 fragments corre-
sponding to cleavage of both sugar linkages are also observed [110].
The susceptibility of the sugaraglycone bond to cleavage varies
according to position, with fragmentation preferentially occurring
at the C-3 position compared to the C-7 position [174]. This allows
assignment of glycan substitution for avonoid-di-O-glycosides
The relative stability of the C-glycosidic bond means that high
collision energies are required for fragmentation, which mainly
involves cross-ring cleavages of the saccharide residue and the
loss of water molecules. Typical fragmentation patterns are pre-
sented in Fig. 3B, with the main fragment ions corresponding to
0,2 X , 0,1 X , and 0,4 X , often coinciding with the loss of water
0 0 0
or CH2 O fragments [175,176]. The 0,3 X 0 ion is only present at
noticeable levels for avonoid-C-8-glycosides [110]. 0,3 X 0 , 0,2 X 0 ,
and 0,1 X 0 fragment ions correspond to losses of 90, 120 and
150 amu, respectively, for avonoid-hexosides. The corresponding
fragments for avonoid-pentosides result in losses of 60, 90 and
120 amu [59,110].
C-6 and C-8 positional isomers can be distinguished based on
more extensive fragmentation of 0,2 X 0 fragments for the former;
the 0,3 X+ 0 and [M+H4H2 O]+ ions have been proposed as being
diagnostic for the differentiation of C-8 and C-6 isomers in posi-
tive ESI-MS/MS [153]. Waridel et al. [162] compared Q-TOF and IT
instruments in positive and negative mode APCI, and found loss of
water to be characteristic for the C-6 glycosides, whereas MS/MS
of the 0,2 X+ ion in positive mode allowed distinction if the iso-
mers based on the losses of small molecules (and the presence
of a 1,3 A+ ion observed in Q-TOF spectra only). A more complete
set of guidelines has been reported in both positive and negative
ionisation [177]. Similar to avonoid-di-O-glycosides, avonoid-
di-C-glycosides containing sugar residues of different mass can
be assigned based on the more extensive fragmentation of the
C-6-glycoside moiety [59]. More recently, an approach to iden-
tify avone-di-C-glycosides based on negative ionisation MS/MS
spectra has been reported [154] (refer to Section 4.2.1 for further
details).
Flavonoid-O,C-diglycosides can contain the O-glycoside moiety
linked either to a hydroxyl group of the aglycone or to a hydroxyl
group of the C-bound glycoside. Fragmentation of these compounds
entails both cleavage of the O-glycosidic bond, which typically
occurs at lower collision energies, and cross-ring cleavage of the
C-bound saccharide at higher collision energies [59,110].
Differentiation of isomeric acylated avonoid-glycosides is also
possible to some extent based on MS/MS spectra [178], although
limited systematic studies for this class of compounds have been
reported.
2.3.2. Flavonoid aglycones
According to the nomenclature proposed by Ma et al. [168]
(improved from that developed by Mabry et al. [140]) for avonoid
aglycones, ions are designated i,j A 0 + and i,j B 0 for fragment ions
containing intact A- and B-rings, respectively. The superscripts i
and j indicate the cleaved C-ring bonds, while the subscript 0 dis-
tinguishes these ions from carbohydrate fragments k,l A i and B i
(i 1). In general, the fragmentation pathways discussed below are
applicable to both positive and negative ionisation, although the
former mode is more commonly used in the structural analysis
of avonoids (exclusively in the case of anthocyanidins). Nega-
tive ionisation requires higher collision energies and may lack
some diagnostic ions in product spectra [59,62,179]. Fragment ions
 A. de Villiers et al. / J. Chromatogr. A 1430 (2016) 1678
Fig. 3. General fragmentation scheme and illustration of nomenclature used for the MS(n) analysis of (a) avonoid-O-glycosides (b) avonoid-C-glycosides and (c) avonoid
aglycones [167,168].
formed from cleavage of the C-ring bonds are very informative, as
they provide information on the number and nature of substituents
on the A- and B-rings. The fragmentation pathways entail retro-
DielsAlder (RDA) reactions involving the C-ring, typically at bond
positions 1/2, 0/2, 0/3, 0/4 or 2/4 [56,59,62,110,155] (Fig. 3C).
The relative prevalence of each fragmentation pathway depends
on the class and substitution pattern of the avonoid. The 1,3 A 0
ion, observed for all avonoid subclasses in positive and neg-
ative modes, is most readily formed and often constitutes the
base peak ion in second-order mass spectra [59,62]. For avones
and avonols, cleavage of the 0/2 bonds is common, leading to
the formation of the 0,2 B 0 ion, while the corresponding 0,2 A 0
ion is characteristic for avonols [155]. Further losses of 18 amu
( H2 O), 28 amu ( CO), 42 amu ( C2 H2 O) and in the case of nega-
tive ionisation 34 amu ( CO2 ) or successive losses of these groups
are also commonly observed in MS/MS spectra. In the case of O-
methylated avonoids the loss of 15 amu ( CH3 ) is prominent,
with the [M HCH3 ] radical ion generally constituting the base
peak. Direct cleavage of the bond between the B- and C-ring can
be observed for some ions in negative ionisation mode and antho-
cyanidins in positive mode [180], resulting in an [MB-ring]
fragment [179].
Chalcones and dihydrochalcones differ from the other avonoid
subclasses in that they do not possess a heterocyclic C-ring (Fig. 1).
In the case of dihydrochalcones, the saturated 3-carbon chain frag-
ments easily at low collision energies in positive ionisation mode.
Cleavage of the bond between the A-ring and the carbon atom from
the keto group, followed by loss of a ketone, yields the product ions
[C9 H9 O2 ]+ and [C7 H7 O]+ , respectively. Other ions characteristic for
the dihydrochalcones include [C8 H9 O4 ]+ and [C6 H7 O3 ]+ , produced
from the rupture of the and carbons adjacent to the carbonyl
[110]. Chalcones on the other hand have been shown to fragment
according to two pathways, depending on the number and position
of phenolic substituents on the A-ring. The rst reaction, prevalent
for compounds with a C -2 hydroxyl, involves formation of the cor-
responding avanone isomer, which then fragments according to a
RDA mechanism as outlined above. For compounds without a C -2
hydroxyl, cleavage of the bonds on either side of the carbonyl group
is common [143].
Proanthocyanidins show relatively distinct fragmentation reac-
tions, illustrated schematically in Fig. 4 for positive ionisation.
The rst of these involves elimination of a B-ring as a result of
23
RDA ssion of ring C (in essence resulting in the 0,2 A fragment
of the relevant proanthocyanidin moiety) [181183]; this path-
way is energetically favoured in the top unit of proanthocyanidins
[184] and is commonly followed by the loss of an additional water
molecule. The second fragmentation pathway, designated het-
erolytic ring ssion (HRF) [184], involves loss of the A-ring, again
primarily of the top unit (this corresponds to the 1,4 B fragment).
The loss of a 126 amu neutral fragment is indicative of a 1,3,5-
trihydroxybenzene structure of the A-ring. The third fragmentation
pathway involves quinone methide (QM) cleavage of the inter-
avan bond [181183]. This pathway is useful in the assignment
of the position of A-type interavan bonds in higher oligomers
[181,184]. A fourth mechanism, benzofuran-forming (BFF) ssion,
follows a similar mechanism to HRF to produce a C-ring benzofuran
derivative [185]. Further loss of water molecules is also common in
MS/MS spectra of proanthocyanins, which may further complicate
the spectra of high molecular weight compounds. Li and Deinzer
[185] proposed a set of rules that may be used to identify the
composition of procyanidins based on MS/MS spectra and the four
fragmentation pathways, demonstrating the applicability thereof
by assigning molecules up to a DP of six. A similar approach has also
been used in the characterisation of A-type proanthocyanins [186].
It is important to note though that assignment of stereoisomers is
not possible based on MS/MS data.
Finally, it is worth pointing out that a range of electronic
databases containing MS/MS and HR-MS(/MS) data for metabo-
lites, phenolics or avonoids are nowadays available [54,187,188],
or may be compiled in-house [189], and these are nding increasing
application in the screening and identication of plant avonoids
[190193].
3. Advances in HPLC separation
Like all forms of chromatographic separation, the performance
of HPLC methods is inherently constrained by the properties of the
target analytes, the stationary and mobile phases as well as instru-
mental limits. This also has clear implications for the analysis of
avonoids by HPLC. The two main driving forces behind improve-
ments in HPLC in general apply equally in the HPLC analysis of
avonoids: the need for decreased analysis times and increased
resolution. The former applies mainly in routine environments,
where large numbers of samples need to be analysed daily, but is
24 A. de Villiers et al. / J. Chromatogr. A 1430 (2016) 1678

OH O
HO O -H 2 HO O
HO O
BFF m/z 287
OH
OH RDA OH OH
OH OH HO O
OH
-H2O HO O
HO O
OH OH
OH OH
OH
OH HRF m/z 427
m/z 563 O
O
QM BFF

OH -H2O OH
OH OH
HO O
HO O
O O OH
m/z 297
OH
OH
OH
OH RDA
OH O
m/z 273 m/z 437
OH

HO O OH m/z 285
OH
HO O
OH
OH
m/z 291 OH

Fig. 4. General fragmentation scheme for procyanidins illustrated for a procyanidin dimer in positive ionisation mode. The fragmentation pathways involving retro-
DielsAlder (RDA), heterolytic ring ssion (HRF), quinone methide (QM) and benzofuran-forming ssion (BFF) are demonstrated.
Source: Adapted from [185].

also important from a cost-saving perspective and in cases where
analytical data is required within a short time. The latter require-
ment is important for the analysis of complex samples, especially
where numerous chemically related species which cannot be dis-
tinguished by the detector used occur in the same sample. This
is clearly the case for a wide range of avonoid-containing natu-
ral products [4] and therefore explains the incentive for improving
HPLC separation in avonoid analysis.
As a measure of the separation performance of chromatographic
systems, the concept of peak capacity has gained popularity as an
alternative to more conventional measures such as efciency and
resolution, which are limited to isocratic analyses and selected peak
pairs, respectively. The peak capacity of a separation represents the
number of compounds that can theoretically be separated under
dened experimental conditions (column, gradient, ow rate, tem-
perature, etc.) [194]. It is important to note though that no real-life
separation can resolve a number of components equal to the peak
capacity of that separation, a consequence of irregular distribu-
tion of analyte peaks across the separation window. Davis and
Giddings [195] used the statistical theory of component overlap
to demonstrate that to improve the probability of obtaining pure
chromatographic peaks, the peak capacity should exceed the num-
ber of components to be separated by a signicant factor. To use an
oft-quoted example, to resolve with 98% certainty n randomly dis-
tributed peaks, the peak capacity should be equal to 100 n [196].
For relatively simple samples containing few analytes of interest,
the experienced chromatographer can select experimental condi-
tions so as to improve the likelihood of obtaining separation (for
example through the careful selection of stationary phase, mobile
phase composition and gradient, temperature, etc.). However, for
highly complex samples, this is no longer possible for all analyte
peaks in this case, the sample components approximate a ran-
dom distribution and Davis and Giddings ndings emphasise the
importance of improved chromatographic performance.
For gradient separations, the peak capacity, nc , can be deter-
mined according to [197]:
tg
nc = 1 + 1 (1)
(1/n) n
wb
where tg refers to the gradient time (i.e. the separation window)
and wb the baseline width of analyte peaks averaged for n peaks.
Typical peak capacity values for RP-LC vary between 50 for fast
separations and in the region of a few hundred for highly opti-
mised separations. Taking cognisance of the ndings of Davis and
Giddings, and considering that many natural products contain in
excess of 100 avonoids, it is clear that continued efforts to improve
the performance of LC separations are of critical importance also in
the eld of avonoid analysis.
3.1. One-dimensional HPLC
In the last 15 years, a number of signicant developments in the
eld have extended the performance limits of (uni)-dimensional
HPLC. Several of these have gained widespread acceptance also in
the area of avonoid analysis, notably the use of UHPLC, alternative
stationary phase morphologies such as monolithic and supercially
porous phases, comprehensive multidimensional separations and
to a lesser extent the used of elevated temperature. In the following
sections, the fundamental background for each of these approaches
will be outlined briey prior to presenting an overview of their
application in the analysis of avonoids during the last 7 years.
 A. de Villiers et al. / J. Chromatogr. A 1430 (2016) 1678
3.1.1. Ultra high pressure liquid chromatography
One of the oldest and best-known ways to increase performance
in HPLC is by reducing the particle size of the packing material. In
fact, the evolution of HPLC coincides with a continuous decrease
in the size of the packing materials used, dp . This decline was rel-
atively gradual until the late 1990s, when 33.5 m particles had
begun to replace more conventional 5 m phases. Further reduc-
tion in particle size was not considered practical due to the high
pressures required to operate such columns. In a series of landmark
papers from 1997, Jorgenson and co-workers [198] demonstrated
the feasibility of using sub-2 m phases on dedicated high pres-
sure instruments, and showed the benets of this approach for fast,
high-efciency separations in what has come to be termed ultra
high pressure liquid chromatography (UHPLC). Today this term is
considered to refer to HPLC separations performed at pressures
above 400 bar (the pressure limit of conventional instruments).
Commercial instruments capable of routine operation at pressures
up to 1250 bar (18400 psi) and a range of columns packed with
particle sizes ranging from 1.3 to 2.5 m have been introduced since
2005, and in the last decade UHPLC has gained widespread accep-
tance by the separations community. Indeed, UHPLC has also found
extensive application in the analysis of avonoids, as summarised
in a number of recent reviews [6365].
The benets of UHPLC compared to conventional HPLC are com-
monly represented in terms of van Deemter curves depicting the
variation in plate height (H, dened as L/N where L is column length
and N the number of theoretical plates) and the linear velocity of the
mobile phase (u0 , related to the ow rate via the column diameter).
According to chromatographic theory, a reduction in particle size
results in lower A (eddy dispersion) and C (resistance to mass trans-
fer) terms of the van Deemter equation. The combined effect of this
is a lower minimum plate height (i.e. higher efciency for a given
column length) and a higher optimal linear velocity. A typical exam-
ple of the effect of reduction in particle size is shown for avanols
in Fig. 5 [199], where van Deemter curves are compared for 1.7 and
5 m C18 phases (the former operated at pressures up to 1000 bar).
The benets of the 1.7 m phase are evident: the minimum value
for H is much lower (4.2 compared to 11.2 m, meaning that for
the same column length, 2.7 higher efciency can be obtained),
and an increase in the optimal mobile phase linear velocity (uopt )
from 0.6 to 1.3 mm/s (i.e. separations will be 2.2 faster for the
same column length). Plate height curves such as these are com-
mon for most avonoids on such columns, implying that UHPLC
offers two principal advantages compared to conventional HPLC:
increased speed, achieved by a combination of shorter columns and
higher ow rates, and increased efciency, achieved by maintaining
column lengths.
While the benets of UHPLC are well documented, the ulti-
mate implications are less well appreciated. Inspection of plate
height curves such as presented in Fig. 5a elicits the conclusion
that columns packed with smaller particle sizes will always out-
perform those with large particles. This is however not true in all
cases. For example, if very high efciencies are required, very long
columns should be used (N is directly proportional to L, while res-
olution is proportional to the square root of N). Column length is
however limited by the maximum instrumental pressure available,
and the pressure drop across the column (P) is determined by its
permeability (Kv0 ) according to Darcys law:
u0  L
Kv0 = (2)
P
where u0 is the linear velocity and  the viscosity of the mobile
phase. Kv0 is related to the particle size via [200,201]:
dp2 3
Kv0 = (3)
180 (1 )2 T
25
where and T are the external and total porosities, respectively. It
is clear that a reduction in dp coincides with a signicant decrease
in column permeability. The somewhat counter-intuitive result of
this is that higher overall efciencies are attained with an increase
in particle size, a result of the relative importance of column per-
meability and (current) instrumental pressure constraints.
In this context, plate height curves such as the van Deemter
provide limited information due to the fact that column perme-
ability, and by extension the maximum column length, are not
taken into account (H is independent of L). A more accurate rep-
resentation of the ultimate performance of column/instrument
combinations can be obtained using so-called kinetic plots, the
origins of which can be traced to Giddings [202], but which have
been adapted by various authors [203205]. In essence, these plots
combine plate height data with column permeability and pressure
constraints to provide an accurate reection of the performance
of specic column/instrument combination as a function of analy-
sis time. The group of Desmet developed a family of kinetic plots
[200,205207] which allow facile comparison of the performance
of different columns as a function of a range of parameters; this
approach has also been extended to gradient separations, where
systems can be compared in terms of peak capacities obtainable as
a function of analysis time [208].
From the kinetic plot curves presented for catechin in Fig. 5b
it is clear that UHPLC provides considerable advantages in terms
of separation speed compared to conventional HPLC methods for
the same efciency (peak capacity). UHPLC also offers the option
to increase chromatographic efciency for conventional analysis
times (moving horizontally from 5 to 1.7 m curves for a given
analysis time in Fig. 5b), although this gain is ultimately limited
by pressure constraints. The region of most interest to researchers
in avonoid analysis coincides with that where UHPLC clearly out-
performs HPLC. In light of the above, it is no surprise that UHPLC
has been applied extensively as a means to speed up conven-
tional methods for HPLC analysis (see below); exploitation of the
improved efciency benet of UHPLC is somewhat less common in
this eld.
It is important to note that the conclusions drawn from plate
height and kinetic plot representations are analyte-dependant
[209], and therefore caution should be exercised when applying
conclusions drawn for other analytes to avonoids. Limited kinetic
data for avonoids has been reported in literature, but several
studies have indicated that avonols, avones and dihydrochal-
cones [210], procyanidins [199] and especially anthocyanins [105]
are characterised by much lower optimal linear velocities and
increased C-term contributions to plate height than (neutral) small
molecules. Practically this means that gains in terms of analysis
speed are less pronounced than for such small molecules, but also
that the benets of UHPLC are extended to higher efciencies in the
case of avonoids.
Operation at elevated pressures is associated with the risk of
frictional heating and allied band broadening due to the establish-
ment of a radial temperature gradient inside the column [211,212];
this has also been conrmed experimentally [213]. This effect can
be minimised by reducing the pressure (i.e. the ow rate or column
length), or more practically by reducing the internal diameter of the
column (which coincides with a reduction in volumetric ow rate,
thereby further reducing the effects of frictional heating). It is for
this reason that most UHPLC separations are performed on columns
of 2.1 mm internal diameter (i.d.) or lower. From the perspective of
hyphenation to MS, this is clearly benecial. Optimal ow rates on
these columns (taking into account the plate height behaviour of
these phases, cf. Fig. 5a) are in the region of 0.20.5 mL/min and are
therefore ideally compatible with ESI-MS. The much narrower peak
widths typically obtained do however place severe demands on the
acquisition rate of MS detectors. While 4.6 mm i.d. UHPLC columns
26 A. de Villiers et al. / J. Chromatogr. A 1430 (2016) 1678

25 1000000
5 m porous, 400 bar
20
5 m porous a 100000
b
1.7 m porous, 1000 bar
1.7 m porous
2.6 m superficially
2.6 m superficially porous

m)
15 10000 porous, 600 bar

H (

tR (s)
10 1000

5 100

0 10
0 1 2 3 4 5 6 7 10 100 1000
u0 (mm/s) nc
6 1000000
tetrameric procyanidin, 25C tetrameric procyanidin, 25C
tetrameric procyanidin, 50C tetrameric procyanidin, 50C
catechin, 25C c 100000 catechin, 25C d
4 catechin, 50C catechin, 50C
10000
m)

tR (s)
H (

1000
2

100
1.7 m porous, 1000 bar
0 10
0 1 2 3 4 5 6 7 10 100 1000
u0 (mm/s) nc

Fig. 5. (a) Comparison of plate height curves obtained for the avanol catechin on a 5 m porous phase (Pmax 400 bar, blue diamonds), a 1.7 m porous phase (Pmax
1000 bar, yellow circles) and a 2.6 m supercially porous phase (Pmax 600 bar, brown squares). (b) shows the corresponding peak capacity (nc ) as a function of analysis
time for catechin (as determined using the corresponding retention factors). (c) illustrates the plate height curves for catechin (MW 290) and a tetrameric procyanidin (MW
1155) at 25 and 50 C on a 1.7 m porous phase (Pmax 1000 bar), and (d) illustrates the corresponding retention time vs nc plot. (For interpretation of the references to
colour in this gure legend, the reader is referred to the web version of this article.)
Source: Data adapted from [199].

are commercially available, and have also found extensive use in
avonoid analysis, optimal ow rates on these columns (typically
1.22.5 mL/min, depending on the analytes) require ow split-
ting prior to MS detection. Furthermore, frictional heating effects
become a concern on such column dimensions (something which
is not sufciently recognised by many practitioners in avonoid
analysis).
Finally, the impact of typical UHPLC column dimensions should
be considered in the context of extra-column band-broadening. For
the short, 2.1 mm i.d. columns which are the norm in UHPLC, peak
volumes are especially small because of the reduction in ow rate
and the high column efciency. In these circumstances, dispersion
in extra-column volume can signicantly affect the measured peak
width. For example, Gritti and Guiochon showed that even the most
sophisticated modern instruments (excluding capillary and nano-
systems) lead to losses of 1525% of column efciency in the case
of the latest generation high-efciency columns in isocratic mode
[214]. Fekete et al. [215] reached similar conclusions for sub-2 m
porous, supercially porous and monolithic columns. While the
effect of extra-column band broadening is signicantly reduced in
gradient separations due to focusing on the column, dispersion in
connection tubing at the outlet of the column can still have a pro-
found effect on the measured peak width. This is often evident from
comparison of peak widths in UV and MS chromatograms obtained
by UHPLC-DAD-MS, where much broader peaks are observed in the
latter. This may be ascribed in part to the size of most MS instru-
ments, which requires relatively long connection tubing between
the exit of the UV cell and the ionisation chamber. However, small
changes in tubing length and/or internal diameter can produce
much better MS chromatograms.
UHPLC technology has matured rapidly in the last decade, and
has found widespread acceptance in the separation community to
the extent that the technique has in many elds replaced conven-
tional HPLC methods for routine analysis. That this is also the case in
avonoid analysis is conrmed by the large number of applications
reported in Tables 1, 2 and 47, also evidenced by the observa-
tion that many of the papers cited in this work report method
validation results, which amply demonstrate the suitability of
columns and instrumentation for routine analysis. We will not
attempt to address each of these, but rather to highlight some
trends concerning the application of UHPLC in this eld.
The principal application of UHPLC technology, in avonoid
analysis as in HPLC in general, is to attain faster separation
and higher throughput. This makes sense, since 100 and 50 mm
columns packed with sub-2 m particles provide roughly equiv-
alent efciencies to 250 and 150 mm columns packed with 5 m
particles, respectively. This performance is obtained in much
shorter analysis times for UHPLC columns because (i) the column is
shorter and (ii) the optimal mobile phase linear velocity is higher.
The practical consequence is that similar separation is attained as
for conventional HPLC, with commensurate sensitivity, robustness
etc., but at analysis times 29 shorter. A typical example is the
speeding up of routine methods on conventional HPLC systems to
UHPLC columns and instrumentation [107]. Another application
area is in the second dimension of LC LC separations, where sub-
2 m phases show clear benets for maximising performance in
the shortest possible analysis time here the reduced C-term band
broadening on these phases is exploited on short columns operated
at high ow rates.
A second approach is to use longer columns packed with
sub-2 m phases, in this case utilising the increased pressure capa-
bilities of UHPLC columns and instrumentation to obtain higher
resolution for complex avonoid mixtures. Examples are found in
the analysis of rooibos tea [216] and strawberry [217] phenolics and
red wine anthocyanins [218] on 200 mm UHPLC columns. These
columns provide isocratic efciencies 2.4 higher than standard
250 mm 5 m columns, which translates into an increase in reso-
lution by a factor of about 1.5.
3.1.2. Monolithic columns
An alternative approach to overcome the permeability/pressure
limitations of HPLC is to use monolithic columns. Monolithic
stationary phases are cast as single continuous phases with
interconnected skeletons and through-pores. The higher external
 A. de Villiers et al. / J. Chromatogr. A 1430 (2016) 1678 27

Table 1
Applications of advanced 1-dimensional LC methods without MS detection to avonoid analysis (20092015).

Sample Flavonoids Detector(s) Stationary phases (column Mobile Reference

dimensions), ow rates, temperature, phase(s)
analysis times

Rhus coriaria L. Myricetin, quercetin and UV C18 monolith (100 mm 4.6 mm), ACNd /10 mM [229]
kaempferol FRc = 4.0 mL/min, 40 C, <1 min KH2 PO4 (38/62,
v/v, isocratic)
Tomato Flavonol and avanone UV C18 monolith (100 mm 4.6 mm), 2 mM FAd /ACN [228]
aglycones and glycosides LITb (ESI) FR = 1.0 mL/min, 15 min (75/25 (v/v),
(6 avonoids) isocratic)
Cranberry juice Proanthocyanidins (9 UV C18 monolith A: 1% AAd , B: [186]
avonoids) QITb (ESI+) (2 mm 100 mm 4.6 mm), 1% AA in ACN
TOF FR = 3 mL/min, 30 C, 16 min
Soy Isoavone aglycones and UV C18 (100 mm 4.6 mm, 2.6 m dp SPc ), A: 1% AA, B: [300]
glycosides (12 avonoids) FR = 2.7 mL/min, 50 C, 12 min ACN
Tea Flavanols, avonol UV C18 (100 mm 2.1 mm, 1.7 m dp ), A: 0.1% FA, B: [301]
aglycones and glycosides, FR = 0.42 mL/min, 25 C, 20 min MeOHd
avanones and avones
(16 avonoids)
Orange juice Flavanone aglycones and FLb C18, monolith (100 mm 4.6 mm), A: 150 mM AA, [146]
glycosides and avonols (5 FR = 0.7 mL/min, 25 min B: ACN C:
avonoids) MeOH
Grapes Catechin and epicatechin UV C18 monolith (100 mm 4.6 mm), A: 2% AA in [149]
FL FR = 2.5 mL/min, 25 C, 7.25 min 90/10
H2 O/MeOH, B:
2% AA in 10/90
H2 O/MeOH
Reseda luteola (weld) Luteolin-7,3 -O- UV C18 (50 mm 3.0 mm, 1.8 m dp ), A: 160 mM FA, [302]
diglucoside, LIT (ESI) FR = 0.9 mL/min, 35 C, 5 min 40 mM
luteolin-7-O-glucoside and ammonium
luteolin formate,
0.04 mM
EDTAd , pH 3, B:
MeOH
Wine Flavanols, avonol UV C18 (50 mm 2.1 mm, 1.7 m dp ), A: 0.1% FA, B: [303]
aglycones and glycosides FR = 0.25 mL/min, 40 C, 8 min ACN
(6 avonoids)
Soy protein sample Isoavones aglycones and UV C18 (100 mm 4.6 mm, 2.6 m dp SP), A: 1% AA, B: [264]
glycosides (12 avonoids) FR = 1.2 mL/min, 25 C, 50 min ACN
Wine Flavan-3-ols, avonol UV C18 (100 mm 4.6 mm, 2.6 m dp SP), A: 0.1% FA, B: [304]
aglycones and glycosides, FR = 1.3 mL/min, 20 min ACN
avones and avanonol (10
avonoids)
Apple juice Flavonols and avones (8 UV C18 (100 mm 4.6 mm, 2.7 m dp SP), H2 O/ACN [305]
avonoids) FR = 0.8 mL/min, 27 C, 25 min (60/40, v/v,
isocratic)
Rooibos tea (Aspalathus Dihydrochalcones, avones UV C18 (100 mm 4.6 mm, 1.8 m dp ), A: 2% AA, B: 2% [306]
linearis) and avonols (14 FR = 1.0 mL/min, 37 C, 50 min AA in ACN
avonoids)
Cocoa Flavanols and UV C18 (50 mm 4.6 mm, 2.6 m dp SP), A: 0.1% FA, B: [199]
proanthocyanidins (9 FR = 0.4 mL/min, 50 C, 1018 min ACN
avonoids)
Plant extracts Flavonols and avones (10 UV C18 (150 mm 4.6 mm, 2.7 m dp SP), A: phosphoric [103]
avonoids) FR = 1.0 mL/min, 30 C, 45 min acid (pH 2), B:
ACN C: THFd
Red and white grape Flavonol aglycones and UV C18 (100 mm 4.6 mm, 2.6 m dp SP), A: 0.5% H3 PO4 , [95]
juice and wine, teas glycosides, avanone and FR = 2.0 mL/min, 45 and 30 C, B: 0.5% H3 PO4
and plant extracts anthocyanins (10 1823.5 min in MeOH
avonoids) PFPc (100 mm 2.1 mm, 2.6 m dp SP),
FR = 0.2 mL/min, 45 C, 40 min
Tomato, broccoli, Catechin, epicatechin, rutin UV C18 (100 mm 2.1 mm, 1.8 m dp ), A: 0.1% FA, B: [307]
onion, green and red and kaempferol FR = 0.25 mL/min, 40 C, 14 min MeOH
pepper and beetroot
Jattropha gossypifolia Flavone glycosides (6 UV C18 monolith (100 mm 4.6 mm) A: 0.1% AA, B: [234]
avonoids) Q-TOFb FR = 3.0 mL/min, 25 C, 40 min 0.1% AA in
(ESI+) MeOH
Mouse plasma and Genistein UV C18 (150 mm 2.1 mm, 2.6 m dp SP) 10 mM [308]
tissue FR = 0.25 mL/min, 40 C, 13 min NaH2 PO4 /MeOH
(55/45, v/v,
isocratic)
Food supplements Rutin, troxerutin, diosmin UV Amide (100 mm 3.0 mm, 2.7 m dp ACN/H2 O [98]
and hesperidin SP), FR = 1.0 mL/min containing AA
pH 3 (30:70,
v/v, isocratic)
28 A. de Villiers et al. / J. Chromatogr. A 1430 (2016) 1678

Table 1 (Continued )

Sample Flavonoids Detector(s) Stationary phases (column Mobile Reference

dimensions), ow rates, temperature, phase(s)
analysis times

Staghorn sumac fruit Quercetin-3-rhamnoside, UV C18 (100 mm 2.1 mm, 1.7 m dp ), A: [309]
(Rhus hirta L.) quercetin and peonidin-3- QIT FR = 0.35 mL/min, 30 C, 14 min MeOH/H2 O/FA,
O-galloyl-hexose (ESI+/) 95:2:3 (v/v/v),
B:
MeOH/H2 O/FA,
2:95:3 (v/v/v)
Hops (Humulus lupulus Xanthohumol and UV C18 (100 mm 2.1 mm, 1.7 m dp ), A: 0.1% FA, B: [310]
L.) isoxanthohu-mol FR = 0.25 mL/min, 30 C, 13 min ACN
Copaba, copaiva or Quercitrin and afzelin UV C18 monolith (100 mm 4.6 mm), A: H2 O, B: ACN [226]
pa-de-leo FR = 1.0 mL/min, 60 min
(Copaifera
langsdorfi)
Juices of strawberry, Anthocyanins, avonols, UV C18 (100 mm 4.6 mm, 1.8 m dp ), A: 0.5% TFA, B: [107]
American cranberry, avanones, FL FR = 1.0 mL/min, 25 C, 40 min TFAd /ACN/H2 O
bilberry, sour cherry, dihydrochalcones and Qb (ESI+) (0.5/50/49.5,
black grape, orange avan-3-ols (48 avonoids) v/v/v)
and apple
Natural dyestuffs Apigenin, luteolin and UV C18 (150 mm 2.1 mm, 1.7 m dp ), A: H2 O/MeOH [266]
genistein FR = 0.2 mL/min, 40 C, 40 min (10/90, v/v), B:
MeOH C: 0.1%
FA
Chokeberry (Aronia Anthocyanins, avonol Q (ESI+/) PFP (50 mm 2.1 mm, 1.7 m dp SP), A: 5% FA, B: [94]
melanocarpa, Aronia glycosides (7 avonoids) FL FR = 0.2 mL/min, 18 min MeOH/H2 O
arbutifolia and Aronia UV (30/70, v/v)
prunifolia)
Green, oolong and Catechins (8 compounds) UV C18 monolith (100 mm 4.6 mm), H2 O/ACN/MeOH [227]
black teas FR = 1.4 mL/min, 7 min (83/6/11, v/v/v,
isocratic)
Red, white, black, and Flavan-3-ol derivatives and UV C18 (50 mm 2.1 mm, 1.7 m dp ) A: 1% FA, B: [150]
green teas, cocoa quercitin (8 avonoids) FL FR = 0.5 mL/min, 35 C, 2 min MeOH
LIT-
Orbitrap
(ESI+)
Barringtonia racemosa Rutin, quercetin and UV C18 (50 mm 2.1 mm, 1.8 m dp ), A: 0.1% TFA, B: [142]
kaempferol FR = 0.6 mL/min, 13.4 min ACN
Standards Flavones, isoavones, UV C18 (50 mm 3.0 mm, 2.6 m dp SP), A: 0.05 mM [90]
avanones, avan-3-ols FR = 3.0 mL/min, 2 min ammonium
and avonols (15 acetate, B: ACN
avonoids)
Chia seeds (Salvia Isoavones (5 avonoids) UV C18 (50 mm 2.1 mm, 1.8 m dp ), A: 2% AA, B: [141]
hispanica L.) FR = 0.4 mL/min, 18 min H2 O/ACN/AA
(68/30/2, v/v/v)
Herbal dietary Flavonol, chalcone and UV C18 monolith (50 mm 2.0 mm), A: 0.05% TFA [311]
supplements avanone aglycones and LIT FR = 1.21.3 mL/min, 20 C, 5 min (UV) or 0.1% FA
glycosides and avan-3-ols (MS), B: ACN
(14 avonoids)
Zhi-Zi-Da-Huang Isoavone and avanone Q (ESI) C18 (150 mm 4.6 mm, 2.6 m dp SP), A: 0.1% FA, B: [312]
decoction (TCMa ) glycones and glycosides, FR = 0.8 mL/min (0.2 mL/min to MS), ACN
avonol glycoside and 35 C, 55 min
ava-3-ols (13 avonoids)
Soy Isoavones (20 Q (ESI) PFP (100 mm 4.6 mm, 2.6 m dp SP), A: 1% AA, B: [93]
compounds) UV FR = 0.8 mL/min, 25 C, 37 min ACN
Forsythia owers Rutin UV UV: C18 (75 mm 4.6 mm, 2.7 m dp A: [313]
QIT (ESI) SP), FR = 1.4 mL/min, 30 C, 22 min H2 O/orthophosphoric
QIT: C18 (150 mm 2.1 mm, 1.9 m acid (99.5:0.5,
dp ), FR = 0.2 mL/min, 25 C, 68 min v/w), B: ACN
A:
H2 O/ACN/FA,
95:5:0.1
(v/v/v), B: 0.1%
FA in ACN
Grape pomaces Flavonols and avan-3-ols UV C18 (100 mm 3.0 mm, 2.6 m dp SP) A: 0.1% FA, B: [314]
(8 avonoids) FR = 0.8 mL/min, 35 C, 20 min ACN
Soy bean, black bean, Isoavones (14 UV C18 (100 mm 2.0 mm, 2 m dp ), A: 0.5% FA, B: [315]
red bean and three compounds) FR = 0.3 mL/min, 40 C, 25 min ACN
soybean pastes
a
TCM, traditional Chinese medicine.
b
LIT, linear ion trap; QIT, quadrupole ion trap; FL, uorescence detection; Q, (single) quadrupole; Q-TOF, quadrupole-time-of-ight.
c
FR, ow rate; SP, supercially porous; PFP, pentauorophenyl.
d
ACN, acetonitrile; FA, formic acid; AA, acetic acid; MeOH, methanol; EDTA, ethylenediaminetetraacetic acid; THF, tetrahudrofuran; TFA, triuoroacetic acid.
 A. de Villiers et al. / J. Chromatogr. A 1430 (2016) 1678 29

Table 2
Applications of comprehensive 2-dimensional LC to avonoid analysis (20092015).

Sample(s) Flavonoids Hyphenation Stationary phases (column Mobile phases Detector(s) Reference(s)
mode(s), dimensions), ow rates,
separation temperature, analysis times
modes,
sampling
time(s)

Apple and cocoa Procyanidins and Off-line, 1

D: Diol (250 mm 1 mm, (1 D) A: 1% AAd in UV [147]
avonol glycosides HILIC RP-LC, 5 m dp ), FRc = 0.05 mL/min, ACNd , B: FLe
1 b
(41 compounds) ts = 1 min 70 min MeOHd /H2 O/AA Q-TOFe (ESI)
2
D: C18 ((2) (94.05/4.95/1
50 mm 4.6 mm, 1.8 m (v/v/v))
dp ), FR = 1.8 mL/min, 50 C, (2 D) A: 0.1% FAd ,
4/30 min B: ACN
1
Mate extracts Flavonol glycosides On-line, D: RP-Amide (1 D) A: H2 O (pH UV [367]
(3 compounds) RP-LC RP-LC, (250 mm 1.0 mm, 5 m 3), B: ACN (pH 3) ITe -TOF
1
ts = 2 min dp ), FR = 0.01 mL/min, 25 C, (2 D) A: H2 O (pH (ESI+/)
70 min 3), B: ACN (pH 3)
2
D: C18 (30 mm 4.6 mm,
2.7 m dp , SPc ),
FR = 4 mL/min, 45 C
Longdan Xiegan Flavone and On-line, 1
D: ILCc (150 mm 4.6 mm), (1 D) 10 mM UV [368]
Decoction (TCMa ) isoavone ILCb RP-LC, FR = 0.05 mL/min, 400 min ammonium APCI-Q-MSe
aglycones and 1
ts = 10 min* 2
D: C18 (150 mm 4.6 mm), acetate (pH 6.8, ()
glycosides, FR = 2.0 mL/min (0.8 mL/min isocratic)
avanone to MS) (2 D) A: 0.1% AA, B:
glycoside (8 ACN
avonoids)
Standards Flavan-3-ols, On-line, 1
D: PEGc (150 mm 2.1 mm, (1 D, 2 D) A: 10 mM UV [357]
avones, avanone RP-LC RP-LC, 5 m dp ), FR = 0.25 mL/min, ammonium
aglycones and 1
ts = 0.58 min 40 C, 35 min, acetate, B: ACN
glycosides, avonol 2
D: C18 (30 mm 3 mm,
aglycones and 2.7 m dp SP), FR = 4 mL/min
glycosides, (0.2 mL/min to MS), 60 C
isoavone (13
avonoids)
Fructus aurantii (TCM) Polymethoxylated Off-line, 1
D: C8 (250 mm 4.6 mm, (1 D, 2 D) A: water, UV [374]
avones (42 RP-LC RP-LC, 5 m dp ), FR = 1 mL/min, B: ACN IT-MS (ESI+)
1
avonoids) ts = 1 min 30 min
2
D: C18 (250 mm 4.6 mm,
5 m dp ), FR = 1 mL/min,
30 min
Red wines Flavan-3-ols and On-line, 1
D: Phenyl (250 mm 1 mm, (1 D) A: H2 O (pH UV [344]
avonol glycosides RP-LC RP-LC, 5 m dp ), FR = 0.01 mL/min, 3), B: ACN (pH 3)
(7 avonoids) 1
ts = 2 min 25 C, 80 min (2 D) A: H2 O (pH
2
D: C18 (30 mm 4.6 mm, 3), B: ACN (pH 3)
2.7 m, dp SP),
FR = 4 mL/min (0.2 mL/min
to MS), 45 C
Red wine Flavan-3-ols, On-line, 1
D: PEG (150 mm 2.1 mm, (1 D, 2 D) A: 10 mM UV [355]
avones, avonols, RP-LC RP-LC, 5 m dp ) FR = 40, 70, and ammonium
avonol glycosides, 1
ts = 1.5 min 240 L/min, 35, 110, and acetate, B: ACN
avanone 140 min, 40 C
aglycones and 2
D: C18 (50 mm 3.0 mm,
glycosides, 2.7 m dp SP), FR = 3.3 and
isoavones (14 4.8 mL/min, 0.42, 1.4,
avonoids) 2.0 min, 50 C
Green tea Flavan-3-ols, Off-line, 1
D: Diol (250 mm 1 mm, (1 D) A: 1% AA in UV [126]
proanthocyanidins, HILIC RP-LC, 5 m dp ), FR = 0.05 mL/min, ACN, B: FL
1
avonol aglycones ts = 1 min 70 min MeOH/H2 O/AA Q-TOF (ESI)
and glycosides and 2
D: C18 (50 mm 4.6 mm, (94.05/4.95/1
avone glycosides 1.8 m dp ), FR = 0.8 mL/min, (v/v/v))
(48 avonoids) 50 C, 30 min (2 D) 0.1% FA, B:
ACN
1
Stevia rebaudiana Flavone and On-line, D: PAc -G (1 D) A: ACN (pH UV [366]
extracts avonol glycosides HILIC RP-LC, (250 mm 1.0 mm, 5 m 3), B: H2 O (pH 3)
1
(5 avonoids) ts = 20 s dp ), FR = 0.02 mL/min, (2 D) A: H2 O (pH
101 min 3), B: ACN (pH 3)
2
D: C18 (30 mm 2.1 mm,
1.8 m dp ), FR = 3.4 mL/min,
70 C
Citrus juices Flavanones and On-line, 1
D: PEG (250 mm 2.1 mm, (1 D, 2 D) A: 0.1% UV [343]
avones (11 RP-LC RP-LC, 5 m dp ), FR = 50 L/min, FA, B: H2 O/ACN/ Q-MS (ESI)
1
avonoids) ts = 1 min 80 min isopropanol/FA
2
D: C18 (50 mm 4.6 mm, (39.9:20:40:0.1
2.7 m dp SP), (v/v))
FR = 3.0 mL/min, 40 C
30 A. de Villiers et al. / J. Chromatogr. A 1430 (2016) 1678

Table 2 (Continued )

Sample(s) Flavonoids Hyphenation Stationary phases (column Mobile phases Detector(s) Reference(s)
mode(s), dimensions), ow rates,
separation temperature, analysis
modes, times
sampling
time(s)

Scutellaria barbata D. Flavone and Off-line, HILIC HILIC: HILIC HILIC: Q-TOF (ESI+) [375]
Don (TCM) avanone HILIC HILIC, 1
D: Silica (1 D, 2 D) A: UV
aglycones and HILIC RPLC, (250 mm 4.6 mm, 5 m ACN/water (95:5
1
glycosides (22 ts = 1 min dp ), FR = 1.0 mL/min, 30 min (v/v)) with 5 mM
2
avonoids) D: Amide ammomium
(150 mm 4.6 mm, 5 m formate, B: 5 mM
dp ), FR = 1.0 mL/min, 30 min ammonium
HILIC RP: formate
1
D: Amide HILIC RP:
(150 mm 4.6 mm, 5 m (1 D, 2 D) A: 0.1% FA,
dp ), FR = 1.0 mL/min, 30 min B: 0.1% FA in ACN
2
D: C18 (150 mm 2.1 mm,
5 m dp ), FR = 0.2 mL/min,
50 min
1
Standards Flavan-3-ols, On-line, D: DioEDMA, monolith (1 D) A: ACN, B: UV [362]
avones, avonol HILIC RP-LC, (180 mm 0.53 mm), 10 mM
and avanone 1
ts = 1.5 min FR = 0.01 mL/min, 40 C ammonium
2
aglycones and D: RP-Amide, C18 and acetate (pH 3.1)
glycosides, phenyl hexyl (50 and (2 D) A: 10 mM
isoavone (13 30 mm 3.0 mm, 2.6 and ammonium
avonoids) 2.7 m dp SP), acetate (pH 3.1),
FR = 34 mL/min, 50 C, B: ACN
1.5 min
1
Green and black teas Flavonol- Off-line, D: SECc (1 D) A: H2 O, B: ELSDe [81]
(Camellia sinenis) glycosides, SEC RP-LC, (300 mm 7.8 mm, ACN UV
avone-glycosides, 1
ts = 1 min 5 103 Da size exclusion), (2 D) A: 0.1% FA, B: QqQe (ESI)
avan-3-ol FR = 1.0 mL/min, 60 C, MeOH
derivatives (54 60 min
avonoids) 2
D: C18 (50 mm 2.1 mm,
1.7 m dp ), FR = 1.0 mL/min
(0.1 mL/min to MS), 80 C,
10 min
Rooibos tea (Aspalathus Dihydrochalcones, Off-line/on- 1
D: Diol (250 mm 1 mm, (1 D) A: 2% AA in UV [127]
linearis) avanones, line, 5 m dp ), FR = 0.05 mL/min, ACN, B: Q-TOF (ESI+/)
avones, and HILIC RP-LC, 100 min MeOH/H2 O/AA
avonols (20 1
ts = 1/2 min 2
D: C18 (50 mm 4.6 mm, (93.05/3.95/2
avonoids) (off-line/on- 1.8 m dp ), (v/v/v))
line) FR = 1.0 mL/min, 48 C, (2 D) A: 1% AA, B:
29 min ACN
1
Six TCMs Isoavone, avone Off-line, D: Click CDc 1
D) A: H2 O, B: Q-TOF (ESI+) [376]
and avanone HILIC RP-LC, (150 mm 4.6 mm, 5 m ACN, C: 100 mM UV
1
aglycones and ts = 2 min* dp ), FR = 1.0 mL/min, 30 min ammonium
glycosides (25 2
D: C18 (150 mm 2.1 mm, formate
avonoids) 5 m dp ), FR = 0.3 mL/min, (2 D) A: 0.2% FA, B:
15 min or Click OEGc 0.2% FA in ACN
(150 mm 2.1 mm, 5 m
dp ), FR = 1 mL/min, 30 min
Qingkailing (TCM) Baicalin and On-line, 1
D: SEC (20 mm 2.1 mm), (1 D) A: 10 mM TOF (ESI) [350]
wogonoside SEC RP-LC, FR = 0.02 mL/min, 25 C ammonium
1
ts = 5 min** 2
D: C18 (100 mm 2.1 mm, formate (isocratic)
1.7 m dp ), (2 D) A: 0.1% FA, B:
FR = 0.7 mL/min, 80 C ACN
1
Standards Flavan-3-ols, Online, D: PEG (1 D, 2 D) A: 10 mM UV [377]
avones, avonols RP-LC RP-LC, (150 mm 2.1 mm, 5 m ammonium
1
and avanones (14 ts = 1.5 min dp ), FR = 0.066 mL/min, acetate (pH 3.1),
avonoids) 40 C, 75 min B: ACN
2
D: PFPc (50 mm 3.0 mm,
2.6 m dp SP),
FR = 2.5 mL/min, 40 C
1
Standards Flavones, avonols, On-line, D: Zwitterionic monolith (1 D, 2 D) A: 0.01 M UV [231]
avanones, HILIC RP-LC, (210 mm 0.53 mm), FR = 2 ammonium
avan-3-ols and 1
ts = 1.5 min and 3 L/min, 60 C, 60, acetate, B: 0.01 M
isoavones (15 100 and 140 min ammonium
avonoids) 2
D: C18 (30 mm 3.0 mm) acetate in ACN
FR = 4.5 mL/min, 50 C, 1.0
and 1.5 min
 A. de Villiers et al. / J. Chromatogr. A 1430 (2016) 1678 31

Table 2 (Continued )

Sample(s) Flavonoids Hyphenation Stationary phases (column Mobile phases Detector(s) Reference(s)
mode(s), dimensions), ow rates,
separation temperature, analysis times
modes,
sampling
time(s)

Apple Procyanidins, On-line, 1

D: Diol (150 mm 1.0 mm, (1 D) A: ACN/AA IT-MS (ESI) [369]
dihydrochalcones HILIC RP-LC, 5 m dp ), FR = 21 L/min, (98/2 (v/v)), B: UV
1
and avonols (64 ts = 78 s 50 min MeOH/H2 O/AA
avonoids) 2
D: C18 (50 mm 4.6 mm, (95/3/2 (v/v/v))
2.7 m dp SP), (2 D) A: 0.1% FA, B:
FR = 3.0 mL/min ACN and A: 0.1%
FA, B: ACN/MeOH
(50/50 (v/v))
Cocoa Procyanidins (38 On-line, 1
D: Diol (250 mm 1 mm, (1 D) A: 1% AA in UV [340,342]
compounds) off-line, 5 m dp ), FR = 0.025 mL/min ACN, B: FL
stop-ow, (on-line) and 0.05 mL/min MeOH/H2 O/AA Q-TOF (ESI)
HILIC RP-LC, (stop-ow and off-line), (94.05/4.95/1
1
ts = 1 min 50 min (stop-ow and (v/v/v))
(off-line and off-line) and 100 min (2 D) A: 0.1% FA, B:
stop ow), (on-line) ACN
1 2
ts = 2/3 min D: C18 column
(on-line) (50 mm 4.6 mm, 1.8 m
dp ), FR = 1.5 mL/min, 50 C,
12.6 min
Grape seed Procyanidins (78 On-line, 1
D: Diol (250 mm 1 mm, (1 D) A: 1% AA in Q-TOF (ESI) [148]
single peaks, HILIC RP-LC, 5 m dp ), FR = 0.025 mL/min, ACN, B: FL
2
species of DP up to ts = 2 min 100 min MeOH/H2 O/AA
16 detected) 2
D: C18 (50 mm 4.6 mm, (94.05/4.95/1
2.6 m dp ), FR = 1.5 mL/min, (v/v/v))
50 C, 2 min (2 D) 0.1% FA, B:
ACN
Grape seeds Procyanidins (43 On-line, 1
D: Diol (150 mm 1.0 mm, (1 D) A: 2% AA in UV [230]
avonoids) HILIC RP-LC, 5 m dp ), FR = 0.015 mL/min, ACN, B: IT-MS (ESI)
1
ts = 1.3 min 85 min MeOH/H2 O/AA
2
D: C18 (50 mm 4.6 mm, (95/3/2 (v/v/v))
2.7 m dp SP), FR = 3 mL/min (2 D) A: 0.1% FA, B:
(0.6 mL/min to MS), 1.3 min ACN/MeOH (50/50
2
D: C18 monolith (v/v))
(100 mm 4.6 mm), 1.3 min (2 D) A:0.1% FA, B:
MeOH or
ACN/MeOH (50/50
(v/v))
Cocoa and red grape Proanthocyanidins Off-line/on- 1
D Diol (250 mm 1 mm, (1 D) A: 1% AA in UV [363]
seeds (42 compounds) line, 5 m dp ), ACN, B: Q-TOF (ESI)
HILIC RP-LC, FR = 0.050.025 mL/min MeOH/H2 O/AA ABTSe assay
1
ts = 1 min 2
D C18 (50 mm 4.6 mm, (94.05/4.95/1
(off-line), 2 min 2.6 m dp SP), (v/v/v))
(on-line) FR = 1.5 mL/min, 50 C, (2 D) A: 0.1% FA, B:
15/2 min (off-line/on-line) ACN
1
Fenugreek seeds Flavone glycosides Off-line D: C18/propylphenyl (1 D) A: 0.1% TFAd , UV [378]
(15 avonoids) (heart-cutting) (100 mm 1 mm, 5 m dp ), B: MeOH/H2 O/TFA Q-MS (ESI+/)
and on-line, FR = 0.017 mL/min, 20 C (50/50/0.1 (v/v/v))
RP-LC RP-LC, 2
D: C18 (100 mm 2.1 mm, (2 D) A: 0.1% TFA,
1
ts = 1.54 min 2.6 m dp SP) (off-line) or B: ACN/H2 O/TFA
(on-line)* C18 (75 mm 2.10 mm, (33/4/8/0.05
3 m dp ) (on-line), (v/v/v))
FR = 2.3 mL/min (1:1 split
before MS), 20 C
1
Blueberries, black Anthocyanins (73 Off-line, D: Amide (1 D) A: 0.4% TFA in UV [248]
beans, red radish, red compounds) HILIC RP-LC, (150 mm 4.6 mm, 2.5 m ACN, B: 0.4% TFA Q-TOF (ESI+)
grape skins, red 1
ts = 1 min dp ), FR = 0.2 mL/min, 50 C, (2 D) A: 7.5% FA, B:
cabbage. 79113 min 7.5% FA in ACN
2
D: C18 (50 mm 4.6 mm,
2.6 m dp SP),
FR = 0.5 mL/min, 50 C,
40 min
1
Ge-Gen (kudzu root, Isoavones (19 Heart-cutting, D: CSHc C18 (1 D) A: 0.1% FA, B: UV [364]
TCM) avonoids) on-line, (100 mm 2.1 mm, 1.7 m MeOH IT-MS (ESI)
RP-LC RP-LC, dp ), FR = 0.1 mL/min, 50 C, (2 D) A: 0.1% FA, B:
1
ts = 0.5 min 35 min ACN
2
D: Phenyl-hexyl
(50 mm 3.0 mm, 2.7 m dp
SP), FR = 2.5 mL/min
(0.25 mL/min to MS), 50 C
32 A. de Villiers et al. / J. Chromatogr. A 1430 (2016) 1678

Table 2 (Continued )

Sample(s) Flavonoids Hyphenation Stationary phases (column Mobile phases Detector(s) Reference(s)
mode(s), dimensions), ow rates,
separation temperature, analysis
modes, times
sampling
time(s)

Sugarcane (Saccharum Flavones (15 On-line, 1

D: CNc (250 mm 1 mm, (1 D) A: 0.1% AA, B: UV [356]
spp.) avonoids) RP-LC RP-LC, 5 m dp ), FR = 0.02 mL/min, 0.1% AA in EtOHd Q-MS (ESI+/)
1
ts = 2 min 172 min (2 D) A: 0.1% AA, B:
2
D: C18 (30 mm 4.6 mm, 0.1% AA in MeOH
2.7 m dp SP),
FR = 3 mL/min (0.4 mL/min
to MS)
1
Radix Astragali (TCM) Isoavones (34 Off-line, D: SEC (1 D) A: H2 O, B: UV [370]
avonoids) SEC RP-LC, (300 mm 4.6 mm), ACN Orbitrap (ESI+)
1
ts = 2 min FR = 1 mL/min, 30 min (2 D) A: 0.1% AA, B: IT-MS (ESI+)
2
D: C18 (250 mm 4.6 mm, 0.1% AA in ACN
3 m dp ), FR = 0.8 mL/min
(0.2 mL/min to MS)
1
Licorice (Glycyrrhiza Flavanone On-line, D: CSHc C18 (1 D) A: 0.1% FA, B: UV [379]
uralensis) aglycones and RP-LC RP-LC, (100 mm 2.1 mm, 1.7 m MeOH IT-MS (ESI)
glycosides, 1
ts = 0.5 min dp ), FR = 0.1 mL/min, 50 C, (2 D) A: 0.1% FA, B:
chalcone 40 min ACN
2
glycosides, D: Phenyl-hexyl
isoavones, (50 mm 3.0 mm, 2.7 m
avonols and dp SP), FR = 2 mL/min
avones (39 (0.2 mL/min to MS), 50 C
avonoids)
Hedyotis diffusa (TCM) Flavonol glycosides On-line, 1
D: CN (150 mm 2.0 mm, (1 D) A: H2 O, B: UV [365]
(10 avonoids) RP-LC RP-LC, 3 m dp ), MeOH Q-TOF (ESI+)
1
ts = 1 min FR = 0.025 mL/min, 50 C, (2 D) A: H2 O, B:
150 min 0.05% FA in ACN
2
D: C18 (50 mm 3.0 mm,
2.6 m dp SP),
FR = 2.0 mL/min
(0.25 mL/min to MS), 50 C
a
TCM, traditional Chinese medicine.
b 1
ts , sampling time; ILC, immobilised liposome chromatography.
c
FR, ow rate; ILC, immobilised liposome chromatography; PEG, polyethylene glycol; PA, polyamine; SEC, size exclusion chromatography; CD, cyclodextrin; OEG,
oligo(ethylene glycol); PFP, pentauorophenyl; SP, supercially porous; CN, cyanopropyl; CSH, charged surface hybrid.
d
AA, acetic acid; ACN, acetonitrile; FA, formic acid; MeOH, methanol; EDTA, ethylenediaminetetraacetic acid; TFA, triuoroacetic acid; EtOH, ethanol.
e
ABTS, 2,2 -azino-bis(3-ethylbenzothiazoline)-6 sulphonic acid; ELSD, evaporative light scattering detector; FL, uorescence detector; Q, (single) quadrupole; IT, ion trap;
TOF, time-of-ight; QqQ triple quadrupole.
*
Not truly comprehensive due to the use of excessive sampling times.
**
Not truly comprehensive due to fraction transfer protocol.

porosity of these phases, resulting from the large through-pores, is
responsible for the higher permeability of monolithic columns (cf.
Eq. (3), where values are 0.57 for monoliths compared to 0.4
for silica packings).
Two types of monolithic columns can be distinguished, namely
polymeric and silica monoliths [219]. The former are produced
by in situ polymerisation using a suitable monomer (such as
polymethacrylates, polystyrene-divinylbenzene, etc.), free radical
initiator and porogenic solvent [220]. In contrast, silica monoliths
are produced using solgel processes involving polycondensation
of tetra-alkyloxysilanes with polyethylene glycol (PEG) as poro-
gen, followed by derivatisation of the phase [221]. Control of the
silica skeleton and macro- and meso-pore sizes is possible through
the selection of the silane starting material and PEG concentration
[222].
The increased permeability of monolithic phases may be
exploited in one of two ways. First, very high ow rates can be used,
which in combination with the favourable mass transfer proper-
ties of monoliths allows for fast analyses. This is the tactic most
often used in avonoid analysis. Fast analyses using monolithic
columns have also been used in the second dimension of LC LC
separations [223]. Alternatively, high-permeability silica mono-
liths can be used as very long columns to provide high efciency
[224]; this approach has not been utilised in avonoid analysis to
date.
Comparison of the kinetic performance of monolithic columns
with porous silica phases is challenging due to the wide range
of monolithic phases of different properties available. Neverthe-
less, kinetic studies on silica monoliths, which are mostly used
in avonoid analysis, generally show the benet of these phases
to primarily lie in the high-efciency regime, with conventional
phases outperforming monoliths for fast analyses [200,222]. A
second generation of commercial silica monoliths (Chromolith
High-Resolution) with macropores of 1.2 m and mesopores of
15 nm was introduced in 2011. These columns have closed the
gap to porous phases in terms of minimum plate heights of 7 m
[225]. However, this performance comes at the cost of lower perme-
ability; the net result being that their optimal kinetic performance
shifts to lower efciencies and faster analysis, yet these columns are
still outperformed by sub-2 m and supercially porous phases in
this region [44]. Increasing the maximum pressure above the cur-
rent 200 bar dictated by the column cladding might improve the
competitiveness of monolithic columns [44].
Monoliths have found some application in avonoid analysis,
primarily in two areas. The rst is for the fast separation of a
limited number of target avonoids, where typically commercial
(Chromolith ) C18 silica monoliths of 100 mm length (or two of
these coupled in series [186,226]) are used at high ow rates to
increase analysis speed. For simple mixtures, isocratic elution is
often used [149,227229]. The second main application area of
 A. de Villiers et al. / J. Chromatogr. A 1430 (2016) 1678
monoliths is in comprehensive 2-dimensional LC analyses (refer
to Section 3.2), where the high-ow compatibility of these phases
are benecial for fast second dimension analyses [230], or alterna-
tive retention mechanisms of polymeric monoliths are exploited in
the rst dimension [231].
Considering that monolithic columns have been around since
the late 1980s [232,233], they have found relatively limited appli-
cation in the LCMS analysis of avonoids, especially compared
to UHPLC (only 5% of the applications listed in the tables, refer
to Fig. 7). One reason for this may lie in the fact that the very
high ow rates typically used for fast analyses on commercially
available monolithic column dimensions of i.d.s 4.6 or 3 mm are a
serious drawback from the perspective of solvent consumption and
hyphenation to MS. For example, in typical studies utilising mono-
liths, ow rates between 2.5 and 4 mL/min [149,229,234] were
used, with ow splitting required prior to MS detection [186]. Cer-
tainly the familiarity and experience with conventional phases of
most practitioners, coupled with the range of high-performance
silica-based columns available nowadays, also plays a role in this
regard, reected also in the relatively sparse use of monoliths in
general.
3.1.3. Supercially porous stationary phases
Supercially porous (also referred to as coreshell, shell
or fused-core) phases were originally introduced in the 1960s
[235,236], but it has only been with the introduction of second gen-
eration supercially phases since 2006 [237,238] that an upsurge
in interest in these phases has occurred. This is a consequence
of the remarkable performance of these columns: in contrast to
conventional porous silica phases, minimum plate heights (Hmin )
as low as 1.11.8dp have been measured for supercially porous
phases, where for years Hmin = 2dp was considered a practical limit
for HPLC columns. Initially marketed as providing reduced diffusion
distances due to the smaller porous layer surrounding a solid core,
and therefore a reduced stationary phase mass transfer term, exten-
sive subsequent fundamental research has demonstrated that the
improved performance of these phases is mostly due to reduced
A-term [239242] (ascribed to a narrower particle size distribu-
tion [238,239] and/or better packing efciency [243]) and B-term
[239,242,244247] contributions to plate height, in addition to a
lower C-term [242,247]. The latter advantage in terms of the C-term
behaviour of supercially porous phases is particularly pronounced
for high MW compounds due to the slow diffusion of these com-
pounds [238,241,242]. From the perspective of avonoid analysis,
this is pertinent in that it implies particular advantages of these
phases for the analysis of oligomeric proanthocyanidins [199] and
highly glycosylated and acylated avonoids such as anthocyanins
[128,248].
The primary benet of supercially porous phases is therefore
that performance similar to fully porous phases of smaller dp can
be obtained, however at lower pressures due to the larger particles
(cf. Eqs. (2) and (3)) [247] the same applies for larger particle size
supercially porous phases [201]. This reduces the need for ultra
high pressure operation and the risk of frictional heating (super-
cially porous phases have also been shown to be characterised
by higher heat conductivity, further reducing this risk [249]). The
only potential drawback of these phases is reduced loadability asso-
ciated with the increase in the phase ratio [250], although this
remains a point of discussion and is more relevant in the case of
ionisable analytes [251253] than for avonoids. Several recent
reviews on supercially porous phases can be consulted for details
on the theoretical aspects, applications, stationary phase manufac-
turing and available chemistries [254257].
Fig. 5a and b illustrates the advantages of supercially porous
phases for avanols: for a 2.6 m supercially porous phase,
Hmin and uopt values of 3.9 m and 1.2 mm/s were measured for
33
catechin, compared to 4.24 m and 1.27 mm/s for a fully porous
1.7 m phase [199]. In fact, this column outperforms fully porous
phases of various particle sizes across the entire range of practical
efciencies (Fig. 5b).
Since 2006 a range of supercially porous columns of different
(mostly RP-LC) chemistries, column dimensions and particle sizes
varying between 1.3 and 5 m have become commercially avail-
able from several manufacturers [257]. HILIC supercially porous
phases are nowadays also available [251], although to the best of
our knowledge these have not to date been applied in the LCMS
analysis of avonoids.
Mirroring the contemporary situation in HPLC, application
of these phases has grown rapidly in both one- and multi-
dimensional HPLC analysis of avonoids during the past seven
years (Tables 1, 2 and 47). These phases are increasingly being
used as alternatives to sub-2 m porous phases, since they gen-
erally provide similar performance at lower operating pressures.
Accordingly, supercially porous phases are also utilised to achieve
two principal goals: to obtain faster separations, and, to a lesser
extent, to maximise resolution for complex mixtures. The major-
ity of applications therefore use relatively short columns to benet
from speed gains, although column lengths up to 150 mm have been
used to provide maximum resolution [96,124,258263] these
provide much higher resolution than conventional columns of 150
or 250 mm length and packed with 5 m phases.
Several studies have compared the performance of coreshell
and fully porous phases. For example, Ruiz et al. [260] compared
a conventional (250 mm 4.6 mm, 5 m), a supercially porous
(150 mm 4.6 mm, 2.6 m) and 2.2 m (150 mm 2.0 mm) C18
phases for the analysis of glycosylated avonols, and found the
supercially porous phase to be superior for this application. For the
analysis of polyphenols in lentil seed coats, two types of coreshell
2.6 m columns (Kinetex C18 and PFP) where compared to a fully
porous 4 m phase. The coreshell columns outperformed the fully
porous particles, with the PFP phase providing better separation
of avone and avonol glycosides than the C18 coreshell col-
umn [91]. This phase has also been found to be ideally suited for
the analysis of hydrophobic isoavones [92,93] and some antho-
cyanins [95]. Manchn et al. similarly reported better performance
for a 2.6 m supercially porous C18 phase compared to 3.5 m
fully porous C18 phase and a monolithic column for the separa-
tion of isoavones [264]. Zhang et al. [265] found a 150 mm 1.6 m
coreshell column to provide better peak capacity and symmetry
than two 100 mm sub-2 m phases for the separation of rhubarb
constituents. An example of the performance obtained on this col-
umn in gradient mode is presented in Fig. 6. Note that this column
should, based on a minimum plate height of 1.8dp , provide an iso-
cratic efciency of 52,000 plates the increased resolving power is
clearly evident from Fig. 6. This performance is only attainable on
UHPLC instrumentation though. It should be noted that compari-
son between column performance in gradient mode for complex
samples is far from straightforward due to the effect of differ-
ent particle sizes, column dimensions and selectivity differences
between phases. For example, Serrano et al. found a polar embed-
ded 1.7 m fully porous phase to be better suited than supercially
porous phases for the separation of natural avonoid dyes [266]. In
line with the characteristics of supercially porous phases, they
have also found growing application in LC LC separations (see
Section 3.2).
It is worth pointing out that although the reduced pressure-drop
for supercially porous columns allows their utilisation on con-
ventional HPLC instruments a benet which is widely heralded
by manufacturers and in literature special care should be taken
regarding extra-column band broadening on such instrumentation.
Most conventional HPLC instruments are characterised by large
delay- and extra-column volumes, and even allowing for careful
34 A. de Villiers et al. / J. Chromatogr. A 1430 (2016) 1678
Fig. 6. Representative RP-LCTOF-MS chromatogram illustrating 104 common peaks identied in rhubarb using a 150 mm 2.1 mm 1.6 m supercially porous phase.
Analysis conditions: 0.1% formic acid/ACN gradient; 0.3 mL/min; 40 C; detection: ESI-Q-TOF-MS.
Source: Reproduced with permission from [265].
Fig. 7. Overview of the sub-2 m porous, supercially porous and monolithic phases
used in the advanced LCMS analysis of avonoids (data based on references listed
in Tables 1, 2, 47), and grouped according to particle size (SP indicates supercially
porous phases). Less than 2% of the total number are HILIC phases, the rest are RP-LC
phases.
modication to reduce these volumes does not allow attainment
of the potential performance of short, narrow-bore high-efciency
supercially porous columns [215,253,257].
A graphical representation of the stationary phases used in the
reports listed in Tables 1, 2 and 47 is presented in Fig. 7. It is
clear that fully porous sub-2 m phases are still used in the bulk of
advanced LC separations of avonoids (75% of all analyses cited
here), although supercially porous phases have found growing
application especially in the last few years. Considering the perfor-
mance of these phases, this trend is likely to continue. Furthermore,
the fact that supercially porous phases with pressure capabilities
of 1000 bar or above and particle sizes of 1.31.6 m have recently
been introduced will translate into increased use of these phases
on UHPLC instrumentation to provide further speed and efciency
gains [267].
3.1.4. High temperature liquid chromatography (HTLC)
Somewhat analogously to supercially porous phases, the rst
use of high temperature in HPLC was demonstrated many years
ago [268], although the technique has been largely neglected until
the last decade. In contrast to coreshell particles, however, HTLC
currently still nds relatively limited application in mainstream
literature (there are some exceptions, such as for example HTLC
analysis of polymers). The term HTLC is somewhat vaguely dened
as any LC separation performed at temperatures above room tem-
perature, typically spanning the range of 40200 C [269]; in this
work we will dene high temperature applications as those per-
formed above 50 C.
The primary effect of an increase in temperature in HPLC is to
reduce the viscosity of the mobile phase, with an important con-
comitant increase in analyte diffusion in the mobile phase. The
former effect of elevated temperature results in lower pressures
when the same column lengths and ow rates are used (Eq. (2),
reduction in ); this effect can be exploited to increase efciency
by using longer columns, provided that separation is not performed
under pressure-constrained conditions (i.e. when operating at the
maximum pressure of the column/instrument) [270].
At elevated temperature, an increase in the diffusion coefcient
of the analyte in the mobile phase, Dm , leads to higher B-term and
lower C-term contributions to the plate height, and therefore a shift
in uopt to higher values (illustrated in Fig. 5c and d for the RP-LC
separation of avonols). There is also an important reduction in
the slope of the plate height curve at elevated temperature (due to
a lower C-term), which is clearly benecial from the perspective of
fast analyses. Under pressure-constrained conditions the increase
in optimal mobile phase ow rate roughly cancels the reduction
in pressure due to lower mobile phase viscosity, and the primary
benet of elevated temperature operation is therefore an increase
in analysis speed (in fact, the maximum efciency obtainable on a
given stationary phase/instrument combination decreases slightly
with an increase in temperature [269]). An added benet is that the
use of high ow rates allowed by elevated temperature operation
signicantly reduces the time required to re-equilibrate the column
in gradient analysis [271275], thereby further increasing through-
put. This is especially benecial in LC LC [271,273,275,276].
Further benets of HTLC include a reduction in solvent consump-
tion due to the fact that less organic modier or even pure water
can be used at elevated temperatures [277], and potential increases
in selectivity [278].
Despite the theoretical benets of HTLC, the technique has found
limited application in general [43], and in the analysis of avonoids
in particular. One reason for this is the lack of commercial instru-
ments and columns suitable for high temperature operation. Most
silica-based columns are susceptible to thermal degradation above
60 C (although there are some notable exceptions), and many
dedicated HTLC columns show relatively poor chromatographic
performance. Furthermore, effective HTLC operation requires ded-
icated ovens, efcient mobile phase pre-heating and for most
detectors post-column cooling devices [279]; these require-
ments are not met by most commercial instruments dedicated
 A. de Villiers et al. / J. Chromatogr. A 1430 (2016) 1678
HTLC ovens have only relatively recently become commercially
available.
In the case of avonoid analysis, the well-known thermal
instability of these molecules raises concern about the risk of on-
column degradation occurring, and is likely the main reason for the
restricted application of HTLC in the eld. However, to assess the
risk of on-column degradation it is important to consider the resi-
dence time of the analyte in the heated column relative to the rate of
its thermal degradation at this temperature [268,272]. Simply put, if
the analyte spends less time in the heated column than is required
for its thermal degradation, HTLC at that temperature is a viable
option. For example, anthocyanins are notoriously thermally labile
[280,281]. Yet, it has been demonstrated that operation at tempera-
tures up to 80 C are feasible for anthocyanin analysis provided that
analysis times are kept to less than an hour [105,282]. Grata et al.
[283] similarly reported that no thermal degradation was observed
for metabolites, including avonoids, in Aridopsis thaliana based on
a detailed analysis of TOF-MS data for separations performed at 30
and 90 C.
From Tables 1, 2 and 47, it is clear that the use of temperatures
above 40 C in avonoid analysis is relatively rare. Nevertheless,
a number of studies employed column temperatures of 50 C
[98,143,180,199,218,284293], 60 C [80,217,294], 70 C [282,295],
80 C [81,296] or 90 C [283] in one- and two-dimensional analy-
ses. Qi et al. [297] implemented a temperature gradient between
22 and 35 C for the analysis of avones and avonols in traditional
Chinese medicine (TCM) in order to limit pressure restraints on a
1.8 m RP column operated on conventional instrumentation.
Two particular applications of HTLC are worth mentioning.
Reichelt et al. [99] used a polymeric PRP-1 column operated at
120 C for the fractionation of natural products prior to the sen-
sory evaluation of the fractions (an approach termed LC Taste). The
mobile phase contained only water and ethanol, and fractions could
be directly tasted after dilution with a basic tastant solution. High
temperature operation is essential to accommodate the high vis-
cosity of the ethanol-based mobile phase. The authors conrmed
the avour modulating activities of several avonoids using this
approach [99].
High temperature operation also provides a particular benet in
instances where on-column conversion between multiple species
of an analyte occurs [298,299]. A pertinent example is the case of
anthocyanins, where several different chemical forms may occur in
the mobile phase. It has been shown [105] that the inter-conversion
reaction between the avylium cation and carbinol pseudoba-
sic species is directly responsible for additional band broadening
in the separation of these compounds under conventional con-
ditions (ow rate and temperature). An increase in the analysis
temperature results in faster kinetics of the relevant equilibria,
and therefore a reduction in band broadening [105]. Operation at
elevated temperature and optimal ow rates result in signicant
increases in chromatographic performance for anthocyanin analy-
sis, without the risk of analyte degradation [105,218].
Even a modest increase in temperature to 80 C can have a large
impact on mobile phase viscosity and therefore chromatographic
behaviour. For pure water for example, the viscosity will decrease
by about 60% compared to room temperature (the correspond-
ing values are about 40% and 35% for methanol and acetonitrile,
respectively). This implies that the benets of elevated temperature
operation can be exploited with even a modest increase in tem-
perature in the range where commercial column ovens operate. It
has to be stressed though that for high-ow operation (for example
4.6 mm i.d. columns and above), efcient mobile phase pre-heating
is critical even if the column can effectively be thermostatted at
these temperatures.
While the use of reasonably high temperatures in avonoid
analysis will likely remain limited to certain niche elds such as
35
anthocyanin analysis and LC LC separations, increasing aware-
ness of the benets of elevated temperature and the means to assess
the risk of analyte degradation, coupled to the commercial avail-
ability of growing numbers of columns and instruments suitable
for HTLC operation may see a concomitant increase in the number
of reports utilising modest increases in temperature (in the range
3080 C) for improved avonoid analysis in the future.
Guillarme et al. [43] provided an informative comparison of the
kinetic benets of UHPLC, monolithic columns, supercially porous
phases, HTLC, and combinations of these, compared to conventional
HPLC for the avonol rutin (MW 610). Fig. 8 presents a graphical
overview of their ndings, where the x-axis shows the highest pos-
sible peak capacity (P in this gure) for a gradient time of 3 h, and
the y-axis the time required for a peak capacity of 100. The benets
of these technologies for both high throughput and high resolution
separations are evident, as is the complementary nature of UHPLC
and HTLC approaches.
Table 1 lists the applications of UHPLC, monolithic and super-
cially porous columns without MS detection to avonoid analysis
during the last 7 years (applications with MS detection are summ-
arised in Tables 47 and will be discussed below).
3.2. Comprehensive multi-dimensional LC
Fundamental aspects
Multidimensional liquid chromatography (MD-LC) provides a
powerful means to signicantly improve the performance of LC
separations. This is achieved by combining the separation power
and selectivity of multiple 1-dimensional HPLC methods. MD-LC
approaches can be divided in heart-cutting methods, where only
selected part(s) of a sample are subjected to multiple separations,
and comprehensive separations, where all constituents of the sam-
ple are analysed in several dimensions. In this report, we will limit
our attention to comprehensive MD-LC separations, as these are
more generic in nature and provide much higher resolving power.
(As an aside, although comprehensive systems where the number
of separation dimensions exceeds two have been demonstrated
[316], this has not yet been achieved for avonoid analysis). The
subsequent discussion will therefore focus on comprehensive 2-
dimensional liquid chromatography (LC LC) only. For detailed
overviews of the eld, the reader is referred to several dedicated
LC LC reviews [4549,317320], as well as more specic reviews
covering hyphenation of LC LC with MS [321], column selectiv-
ity in LC LC [322] and multidimensional chromatography in food
analysis [323].
The incentive behind the growing interest in LC LC lies in the
exceptional performance gains that may be realised when combin-
ing two HPLC separations under ideal conditions: the peak capacity
of such a separation is theoretically the product of peak capacities
of both 1-dimensional separations [324328]. LC LC is therefore
capable of providing separation performance an order of magnitude
higher than obtainable by even highly optimised 1-dimensional
HPLC methods. Attaining such performance is however more chal-
lenging in practice. Two particular aspects should be addressed
to meet the criteria for methods to be considered comprehensive
[329] and exploit the potential of these separations.
The rst pertains to the requirement that different separa-
tion mechanisms be used in both dimensions. This is essential
to effectively utilise the two-dimensional separation space pro-
vided by LC LC. The degree to which two separations provide
complementary information is referred to as their orthogonality.
A combination of highly orthogonal separations will result in the
spread of analytes across the two-dimensional space, and therefore
maximise resolution, whereas weakly orthogonal separations (i.e.
where retention of sample constituents between the two separa-
tions are correlated to a signicant degree) provide limited benet.
36 A. de Villiers et al. / J. Chromatogr. A 1430 (2016) 1678
Fig. 8. Comparison of the performance achievable (x-axis: peak capacity, P, for a gradient time of 3 h, y-axis: gradient time required for a peak capacity of 100) by HPLC,
UHPLC, monolithic phases, HTLC and combinations of these. Data obtained for the avonol rutin.
Source: Reproduced with permission from [43].
Deriving a commonly accepted means of quantifying orthogonality,
as is required to determine the practical peak capacity of an LC LC
separation, has however proven difcult. Nevertheless, in recent
years the so-called surface coverage metrics, which are based on
determining the fraction of the two-dimensional space accessi-
ble to sample components, has gained popularity. A variety of
approaches can be used to determine the fractional surface cov-
erage of the two-dimensional space, fc [330,331]. This value can
then be used to quantitatively account for lack of orthogonality in
determining the practical peak capacity (Eq. (4)).
The second important criterion addresses the requirement that
the separation attained in the rst dimension (1 D) should be essen-
tially maintained during the modulation or transfer of fractions to
the 2 D column. This implies that fraction collection times, called
the rst dimension sampling time (1 ts ), should be less than the
rst dimension peak width (typically 1/3rd [332]) failure to do so
would result in the recombination of peaks (partially) separated
in the rst dimension, and therefore sacrice performance. The
effect of this so-called under-sampling can (and should) also be
taken into account when the practical 2-dimensional peak capac-
ity is determined by using the under-sampling correction factor,
[276,333,334]:
1n
c 2 nc fc
nc,2D =
where 1 nc and 2 nc are the peak capacities of the rst- and second-
dimension separations, respectively, and nc,2D the practical peak
capacity of the LC LC separation. From this equation it is clear
that the potential performance gains of LC LC are heavily reliant
on both the degree of orthogonality and under-sampling.
In practice, LC LC can be performed in one of three ways. The
rst, and most simple, is the off-line approach, where fractions elu-
ting from the 1 D column are collected (manually or automatically).
These can then be concentrated, dissolved in a suitable solvent
or directly injected onto the second dimension column. Off-line
LC LC can therefore be performed on a single HPLC instrument,
since fractions can be injected after their collection.
On-line LC LC involves the direct transfer of fractions to the
second dimension (2 D) column, typically using a switching valve
equipped with loops. Analysis in 2 D is then performed while the
next fraction is collected an important consequence is therefore
that the 2 D analyses must be very fast to meet rst-dimension
sampling criteria. Alternative congurations are also possible, such
as for example using trapping columns and an added make-up ow
to trap analytes eluting from the rst dimension. This approach
has the benet of essentially removing all or the majority of the
rst-dimension mobile phase prior to injection on the second
dimension, and can be used to minimise dilution (see below) and
remove unwanted mobile phase components such as salts (the
approach is often used in ion exchange (IEC) RP-LC-MS for this
reason). Furthermore, multiple 2 D columns can be used [335,336]
to effectively provide longer second dimension analysis times and
therefore increase the overall resolution, at the cost of increased
instrumental and method complexity [337,338].
The third mode, stop-ow LC LC, entails direct transfer of frac-
tions to the 2 D column, followed by stopping of the 1 D ow while
separation of the transferred fraction is concluded; this process
is continued until the 1 D separation is completed. Both on-line
and stop-ow LC LC separations have as minimum instrumen-
tal requirements one complete HPLC instrument, a transfer device
such as a valve and a second pump to provide the 2 D ow. Examples
of the different experimental congurations used are presented in
Fig. 9 [48].
Off-line operation is the simplest form of LC LC and provides
(4) the best performance, but requires very long analysis times (in the
order of a few hours up to a few days). In contrast, analysis times
in on-line LC LC are typically commensurate with 1-dimensional
HPLC, and the performance is lower than off-line operation due to
very fast second dimension analyses. Stop-ow provides similar
performance (provided that rst-dimension band broadening dur-
ing stop-ow periods is not excessive [339,340]) and analysis times
to off-line LC LC [340342].
LC LC raw data is typically acquired either as a series of sepa-
rate 2 D chromatograms (in the case of off-line or stop-ow LC LC),
or in the form of a 1-dimensional chromatogram (i.e. detector signal
versus time) comprising a string of sequential 2 D chromatograms
in which the 1 D separation information is implicitly contained (on-
line LC LC). Visualisation is then performed by slicing the raw data
into consecutive 2 D chromatograms and placing them next to each
other in a matrix format which can be viewed as a surface plot or
more commonly as a contour plot (see for example Figs. 1113).
Quantication can also be performed in LC LC by combining the
 A. de Villiers et al. / J. Chromatogr. A 1430 (2016) 1678 37

Fig. 9. Schematic representations of instrumental congurations used for on-line LC LC using (a) empty storage loops, (b) trapping columns and (c) multiple 2D columns;
and (d) for stop-ow LC LC.
Source: Reproduced with permission from [48].
38 A. de Villiers et al. / J. Chromatogr. A 1430 (2016) 1678
contribution of each individual 2 D slice of a 1 D chromatographic
peak, or by using more advanced algorithms [343,344]. Advanced
data analysis techniques can also be applied to LC LC data [345],
although multivariate data analysis is necessarily more compli-
cated than in 1-dimensional separations since alignment of data
in two dimensions present several challenges [346].
Compared to one-dimensional HPLC, method development and
optimisation is much more complex in LC LC [347349]. This topic
is much too complex to be addressed fully in the context of this con-
tribution, and we will therefore focus on providing some general
guidelines for parameter selection in LC and the implications for
performance and hyphenation to MS. The rst choice involved in
LC LC method development is the selection of separation modes,
where orthogonality, performance (speed and efciency) and com-
patibility with the detection strategy to be used are important
criteria. Considering the chemical properties of avonoids, LC LC
methods for their analysis have almost exclusively involved RP-LC
and/or HILIC (with a few notable exceptions [81,296,350]). RP-LC
is most often used in the second dimension because of compatibil-
ity with MS and the fact that good performance is obtained in this
mode for fast separations [351], although HILIC is in principle also
suited to fast 2 D analysis [352,353] prior to MS detection and has
been used for this purpose in proteomics research. The combination
of HILIC and RP-LC promises higher orthogonality than RP-LC RP-
LC, although hyphenation of the former modes is more complicated
due to the relative elution strengths of the mobile phase used in
each dimension (see below). The orthogonality of RP-LC RP-LC
systems can be improved through judicious selection of the mobile
phase composition (typically the organic modier used, since pH in
the range used for avonoid analysis has little effect on the selec-
tivity) and especially the stationary phase chemistries used in both
dimensions. Furthermore, in the case where gradient separation is
performed in both dimensions the most common approach which
provides this highest overall performance [347] the 2 D gradi-
ent can be varied throughout the 1 D separation to maximise the
utilisation of the 2-dimensional space [354356]. This may require
correction of 2 D retention times when multiple slices of the same
compound elute in consecutive gradients [357].
The choice of hyphenation mode, assuming availability of suf-
cient instrumentation, depends primarily on the performance
requirements and time considerations: for maximum resolution
off-line or stop-ow approaches should be used, whereas on-line
is preferable for faster separations. Regarding column dimensions,
the selection is governed largely by mobile phase considerations.
RP-LC and HILIC mobile phases are perfectly miscible, but the
solvent composition of most fractions generally implies high
elution strength in the second dimension. In HILIC RP-LC (or RP-
LC HILIC) this is because of the disparate percentages of organic
modier used in each of these modes, whereas in RP-LC RP-LC
this follows from the fact that compounds are eluted from the 1 D
column in a solvent composition where their retentivity is low. (It is
for this reason that RP-LC columns with lower retention are mostly
used in 1 D for RP-LC RP-LC). The practical implication of this is
that the injection volume on the 2 D column should be minimised
to avoid injection band broadening. The volume of each 1st dimen-
sion fraction (Vfrac ) is related to the 1 D ow rate, 1 F, and sampling
time, 1 ts , according to:
Vfrac = 1 ts 1 F (5)
The most common approach to minimise injection effects in on-
line LC LC is therefore to reduce the i.d. of the 1 D column, leading
to a concomitant reduction in 1 F. Other options to address this lim-
itation include the use of trapping columns [358] or incorporating
a split between the two dimensions [342,359]. The latter approach
is simpler, but reduces overall sensitivity, whereas the former has
the effect of effectively de-coupling fraction volumes from the 1 D
ow rate. In off-line LC LC, this limitation can be circumvented
by injection of a portion of each fraction, diluting fractions (both
resulting in lower sensitivity) or by evaporating the solvent fol-
lowed by dissolution in a weaker injection solvent.
Second dimension column stationary phases are selected based
on performance for fast separations technologies such as UHPLC
[360], supercially porous phases [357] and HTLC [147] have there-
fore all found application in the second dimension of LC LC
systems. For the same reason, relatively short columns are gen-
erally used, typically of i.d. 2.14.6 mm to increase capacity and
minimise injection problems. While monolithic columns are ide-
ally suited for fast analyses at high ow rates, the fact that splitting
is required prior to MS detection represents a signicant drawback.
Finally, the dilution of analytes occurring in both dimensions
should be considered. For passive modulation (i.e. where analytes
are not trapped and focussed after the 1 D separation), such as
mostly used in LC LC, total dilution is generally much higher than
is the case for 1-D HPLC. Dilution in each dimension, and the total
dilution in LC LC, can be determined according to [347,361]:
1 1F
1
DF = 2 1V
(6)
inj
2F 2  SR
2
DF = 2 (7)
1F 1t
s
2D
DF = 1 DF 2 DF (8)
where DF is the dilution factor,  the standard deviation in time
units and F the ow rate for each dimension, as specied by the
superscript, 1 Vinj is the 1 D injection volume and SR is the split ratio
to accommodate cases where only a portion of each fraction may
be injected. Eqs. (6)(8) clearly illustrate that analytes are diluted
to a far greater extent following passage through two columns than
is the case for 1-dimensional HPLC; this has clear implications for
the detector requirements in LC LC.
LC LC has found considerable and growing application in
avonoid analysis, especially during the last ve years (Table 2).
An overview of the applications listed in this table is pre-
sented in Fig. 10. Several different combinations of separation
modes have been reported, including RP-LC RP-LC, HILIC RP-LC,
HILIC HILIC and SEC RP-LC. HILIC RP-LC has found the most
widespread application in the LC LC analysis of avonoids (47%
of applications listed in Table 2), followed by RP-LC RP-LC (38%)
(Fig. 10a). Compared to LC LC in general, where according to Li
et al. [351] RP-LC RP-LC is the most common combination of
modes (32% vs. 22% for NP-LC/HILIC RP-LC), this relative prefer-
ence for HILIC in avonoid analysis is likely associated with the
unique and complementary retention order obtained in this mode
compared to RP-LC. Noteworthy examples include glycosylated
avonoids, where compounds are separated according to their
degree of glycosylation and/or acylation [126,248], and proantho-
cyanidins, where HILIC separation according to size is extensively
used to study this family [147]. Most reports (two thirds during
the last 7 years, Fig. 10b) use on-line LC LC, followed by off-line
LC LC (31%); stop-ow HILIC RP-LC has also only been used in
two reports for the separation of procyanidins [340,342]. For high
speed separations in the second dimension, it is noteworthy that
the use of supercially porous phases exceeds that of fully porous
UHPLC phases; monoliths have found limited application in LC LC
separation of avonoids (with some exceptions [230,231,362]).
Furthermore, the elevated temperature has found extensive use to
facilitate fast separations in the second dimension, with temper-
atures of 50 C [147,231,248,340,342,362365], 60 C [357], 70 C
[366] and 80 C [81,350] being used.
 A. de Villiers et al. / J. Chromatogr. A 1430 (2016) 1678
Fig. 10. Overview of the LC LC analysis of avonoids (20092015) summarising the different separation modes (a), hyphenation modes (b), column formats (c) and mass
spectrometers (d) used in the reports listed in Table 2.
Virtually all classes of avonoids, including avonols [344,367],
avanols and procyanidins [81,147,344], chalcones [127],
(iso)avones [357,368], avanones [357,368] and anthocyanins
[248] have been separated successfully in LC LC. Furthermore,
the suitability of LC LC-UV for quantitative analysis of avonoids
has been demonstrated [343,344]. The authors noted that LC LC
provided higher limits of detection than 1-dimensional HPLC, a
consequence of greater dilution occurring in two dimensions (cf.
Eqs. (6)(8)), but that in some cases improved chromatographic
resolution provided more accurate results.
Hyphenation of LC LC separation with MS is clearly benecial
from the perspective of compound identication and selectivity.
The improved separation offered by LC LC separation is also ben-
ecial from the point of view of MS, since reduced co-elution
minimises the number of potential interfering ions and thereby
matrix effects [321]. However, especially on-line LC LC also places
strict demands on the speed of MS detection due to the very nar-
row peak widths commonly obtained for fast 2 D separations. This
is reected in the fact that most (48%) of LC LCMS methods for
avonoid analysis during the review period were performed using
TOF (or Q-TOF) instruments (Table 2). IT detectors have also been
used [230,369], while Zhang et al. [370] recently used an Orbitrap
instrument operated at a resolving power of 50,000 and scan speed
of 2 Hz in combination with off-line SEC RP-LC analysis.
Qiao et al. [364] reported an interesting heart-cutting/RP-
LC RP-LC approach for the analysis minor compounds in herbal
medicines. Major compounds were removed following 1 D separa-
tion using a time-activated heart-cutting method, whereas minor
compounds were separated in comprehensive mode using shifted
gradients in the second dimension to improve the orthogonality
of the RP-LC RP-LC system. The instrumental conguration and
an example contour plot obtained in this work are presented in
Fig. 11. Separated compounds were identied based on MS/MS
spectra obtained using IT-MS.
SEC and RP-LC have also been combined for avonoid analysis,
rst in heart-cutting mode by de Souza et al. [80] for the separation
of avonol glycosides. In a follow-up report, the same group used an
off-line SEC RP-LC method for the analysis of green and black tea
avonoids [81]. The SEC mobile phase was collected automatically
and evaporated between the two dimensions. The authors reported
39
surprisingly good size-based separations in the rst dimension,
with tetra-, tri-, di- and monoglycosylated avonoids eluting in
the order of decreasing size. The separation mechanism is not
clearly dened, however, with isomeric avonol glycosides also
being separated to some extent in this dimension. In the sec-
ond dimension, a conventional RP-LC column operated at 60 C
[80] or a UHPLC column at 80 C [81] was used. The SEC RP-LC
combination showed good orthogonality, and by using an off-line
hyphenation strategy involving fraction concentration, good sensi-
tivity was obtained. Thirty six avonoid-glycosides were identied
in the heart-cutting 2D LC analysis of Maytenus ilicifolia, whereas
54 avonoids were identied in green and black teas using the off-
line comprehensive approach with detection performed by means
of a triple quadrupole MS. An example of the contour plot obtained
for the off-line SEC RP-LC analysis of a black tea is presented in
Fig. 12.
Alternative separation modes have also found application in the
2-dimensional analysis of avonoids, for example the combination
of CPC [371] or CCC [372] with RP-LC and RP-LC with micellar elec-
trokinetic chromatography (MEKC) [87], although these fall outside
the scope of the current report.
While many of the applications of LC LC report similar num-
bers of compounds than are nowadays also identied using
advanced LCMS methods (Section 4), the benets of improved
chromatographic resolution cannot be overstated. Especially in
qualitative analyses of avonoids, the main application area of
LC LC to date, the quality of mass spectral data is greatly improved
as a consequence of less co-elution provided by the combination of
complementary separation modes [321].
A eld where LC LC demonstrates clear benets is in the
analysis of proanthocyanidins. The complexity of these com-
pounds, with the number of isomeric structures increasing
exponentially with DP, means that they cannot be resolved using
any available HPLC method [373]. Furthermore, because of the
large number of isomeric structures, high resolution MS and
MS/MS data are also of limited utility. LC LC operation com-
bining HILIC separation according to MW and RP-LC separation
according to isomeric composition has been shown to offer a
powerful approach for the elucidation of procyanidin content
in a variety of natural products, especially when hyphenated to
40 A. de Villiers et al. / J. Chromatogr. A 1430 (2016) 1678

Fig. 11. (A) Instrumental conguration used for the on-line heart-cutting/RP-LC RP-LC analysis of Puerarialobata herbal medicine. Valve 1 was used to selectively cut the
major constituents (underlined peak numbers in (B)), and valve 2 for the comprehensive LC LC analysis of the remainder of the constituents. (B) shows the UV contour plot
obtained for this analysis, with a zoom of the separation area in the red block on the right. Numbered compounds except for 12, 13 and 19 are isoavones. Analysis conditions:
1
D: 100 mm 2.1 mm CSH C18 column, 50 C, 0.1 mL/min 0.1% formic acid/methanol gradient; 2 D: 50 mm 3.0 mm 2.7 m supercially porous C18 phase, shifted gradients,
50 C, 2.5 mL/min (split 1:9 before ESI-IT-MS detection).
Source: Reproduced with permission from [364].

MS [126,147,148,230,340,342,363,369]. As an example, Fig. 13
demonstrates the information obtained for the on-line HILIC RP-
LC-FL-ESI-TOFMS analysis of grape seed procyanidins [148]. Mass
spectral data allowed extraction of information for particular
classes of compounds, as illustrated in Fig. 13D for the mono-
galloylated procyanidins. While this methodology provides much
more detailed molecular information than could be obtained using
1-dimensional LCMS, the complexity of the high MW fraction
(Fig. 13C) also serves to emphasise the challenges associated
with proanthocyanidin analysis. For such applications, LC LC is
expected to play an increasingly important role in the future.
4. Advances in LCMS analysis of avonoids
From the perspective of LCMS, technological advances in MS
instrumentation over the last fteen years have largely over-
shadowed those in HPLC. Since the beginning of the century, the
commercial availability of more robust and more advanced LCMS
instruments, coupled to the reduction in the price of these instru-
ments, has certainly contributed to the much more widespread use
and utility of the technique in avonoid analysis. The extent of
these improvements, as well as the most important developments,
are clearly highlighted when comparing current practice with the
state of affairs at the time of several important reviews. By the end
of the previous century, the majority of LCMS applications utilised
fast atom bombardment (FAB) and thermospray (TSP); ESI and APCI
were recent additions to the ion sources used for avonoid analysis
[55,380382]. Furthermore, most analyses were performed using
quadrupole (single stage or triple quadrupole, QqQ) or ion trap
instruments [383]. By the middle of the next decade [59,62] ESI
and APCI had become the norm in avonoid analysis, while time-
of-ight (TOF) and quadrupole-time-of-ight (Q-TOF) instruments
were only just nding application in avonoid analysis [384].
As will be shown in the following sections, the contemporary sit-
uation is signicantly different. In addition to very signicant gains
in the sensitivity and speed of MS systems which have been pro-
gressively attained with newer generation instruments, a range of
new high resolution analysers and tandem MS congurations have
also found application in avonoid analysis. We will not attempt to
address in any detail instrumental developments or the operation
of all instruments, but rather to highlight the potential benets of
the different types of mass analysers and the types of analyses for
which they are suited on the hand of recent applications.
From the perspective of hyphenation of MS to high-performance
separations, several pertinent aspects should be considered. First,
the fact that optimal ESI performance is attained at ow rates of
0.20.4 mL/min is entirely compatible with the trend in UHPLC of
a reduction in column diameter from conventional 4.62.1 mm i.d.
columns dictated by frictional heating considerations [199,213]. On
the other hand, commercial monolithic columns are mostly used in
4.6 mm i.d. formats, which, coupled to the higher ow rates used
for fast analysis on these columns, implies ow splitting prior to
MS detection. Supercially porous phases, due to their relatively
high permeability and favourable thermal conductivity properties
[249] are viable to use in both 4.6 and 2.1 mm formats, although
clearly the latter is preferred from the perspective of hyphenation
to MS. Indeed, of the applications listed in Tables 47, the majority
employ columns of internal diameter 2.1 mm.
 A. de Villiers et al. / J. Chromatogr. A 1430 (2016) 1678
Fig. 12. Contour plot (325 nm UV detection) obtained for the off-line SEC RP-LC separation of a black tea hydro-alcoholic extract (note that the rst dimension SEC separation
is on the y-axis). Compounds 2463 are avonoids, as well as 5, 910, 12, 1518 and 21 and 22. Analysis conditions: 1 D: 300 mm 7.8 mm SEC column, 60 C, 1 mL/min
water/ACN gradient; 2 D: 50 mm 2.1 mm 1.7 m porous C18 phase, 80 C, 1 mL/min.
Source: Reproduced with permission from [81].
Lower column volumes however also place stricter demands on
instrumentation in terms of extra-column volume. For example,
Wolfender and co-workers [283] showed that peak capacities of
UHPLC separations are typically reduced by between 15% and 30%
upon connection to MS detection, even if care is taken to reduce
the volume of the connection tubing. Indeed, comparison of UV
and MS chromatograms in many literature reports visually reveals
the effect of this phenomenon. Finally, the scan speed is of obvious
importance for hyphenation to very fast separations this aspect
will be addressed in more detail for the different mass spectrome-
ters below.
4.1. Ionisation modes
Early ionisation techniques applied for the direct analysis
of avonoid analysis include chemical ionisation (CI) [385] and
electron impact (EI) [386] (these also in combination with GC sep-
aration), eld desorption [387], plasma-desorption-MS (PD-MS)
[388,389], FAB [169,390392] and matrix-assisted laser desorp-
tion/ionisation (MALDI) [393,394].
However, it is only with the development of ionisation sources
that allowed coupling of HPLC with MS that the technique found the
pre-eminence it enjoys today in avonoid analysis. Initial ionisation
techniques used for this purpose such as TSP [395,396], moving belt
(MB) [397,398], continuous-ow FAB [399] have now largely been
replaced by API techniques such as ESI [400403], APCI [404406]
and to a lesser extent APPI.
Of these, ESI is undoubtedly the most important. Indeed, in
the advanced LCMS analysis of avonoids during the period cov-
ered by this review, only three reports utilised APCI [163,368,407],
mostly involving analysis of isoavones, and only one used APPI
41
[293]; the rest without exception used ESI. Compared to the global
picture for LCMS reported in 2012 [41] (82% ESI, 16% APCI, 2%
APPI), the higher prevalence of ESI is a consequence of the fact that
most avonoid classes are more efciently ionised using this mode.
ESI was used in negative ionisation mode in 48% of all applications,
and both positive and negative ionisation modes in 33% of applica-
tion during this time (refer to Section 2.3 for a general discussion
of the MS behaviour of avonoids).
APCI is generally considered an alternative to ESI for relatively
non-polar compounds which are less efciently ionised in ESI.
While APCI shows comparable to better sensitivity compared to
ESI for avonoid aglycones [161], and therefore is well suited to the
analysis of hydrolysed samples, the latter typically outperforms the
former for natural glycosylated species [165,407,408]. Two poten-
tial advantages of APCI over ESI are better compatibility with high
ow rates, and lower susceptibility to matrix effects during the ioni-
sation process [383]. APCI continues to nd application in avonoid
analysis, and indeed has been used in recent years in combination
with UHPLC-(HR)MS [163,407] and LC LC [368], especially in the
analysis of more apolar isoavonoids and isoavones. Nevertheless,
the better performance of ESI for a wide range of avonoid species
is likely an important contributing factor to the dominance of this
form of ionisation in contemporary avonoid analysis.
APPI presents an alternative API source which is complementary
to ESI or APCI, especially for the analysis of non-polar compounds.
Similar to APCI, the liquid sample is vaporised by a modied heated
nebuliser, but instead of using a corona discharge to establish ioni-
sation, the process relies on charge transfer from dopant or solvent
molecules ionised by 10 eV photons produced by a discharge lamp
[409]. Rauha et al. [165] compared ESI, APCI and APPI with a vari-
ety of mobile phases in positive and negative ionisation modes for
42 A. de Villiers et al. / J. Chromatogr. A 1430 (2016) 1678

Fig. 13. Total ion chromatogram (TIC) (A) and uorescence (B) contour plots for the HILIC RP-LCFLESI-MS separation of a grape seed extract. (C) A detailed section of the
TIC and (D) the extracted ion contour plot for monogalloylated procyanidins. Analysis conditions: 1 D: 250 mm 1 mm Diol column, 100 min gradient at 25 L/min; sampling
time 2 min (split 1:24 between two dimensions); 2 D: 50 mm 4.6 mm 1.8 m porous C18 phase, 50 C, 1.5 mL/min (split 1:1.2 before ESI-QTOF-MS detection). Peak labels:
PCX GY refers to a procyanidin of degree of polymerisation X and galloylation Y. F denotes the z-axis scale.
Source: Adapted from [148].

the analysis of ve selected avonoids, and concluded that nega-
tive mode ESI with ammonium acetate containing mobile phases
provided the lowest limits of detection (LODs). While this was espe-
cially true for avonoid-glycosides, similar LODs were obtained
between all three modes for avonoid aglycones in both positive
and negative ionisation, although different optimal mobile phase
compositions were used for each ionisation mode [165]. APPI has
also more recently been used in avonoid analysis. Riffault et al.
chose this ionisation method for the phytochemical characterisa-
tion of rose owers, although the choice of ionisation mode was
primarily based on its performance for more apolar compounds
such as fatty acids and sesquiterpenoids [293].
The ability to perform swift polarity switching between posi-
tive and negative ionisation modes has proven benecial in several
research elds due to the complementary information that may be
gained for compound classes preferentially ionised in either mode.
In the case of avonoid analysis, this is however less important
since most avonoids are sufciently ionised in either mode (the
exception being anthocyanins, where mobile phase considerations
for efcient separation in any case preclude the use of nega-
tive ionisation). Nevertheless, when avonoids are determined
together with other classes of compounds, such as for example in
metabolomic research or studies aimed at comprehensive char-
acterisation of natural product constituents, polarity switching
becomes a valuable tool. A common example is where complete
phenolic analysis is the goal and avonoids are analysed together
with phenolic acids. However, Wolfender and co-workers showed
that polarity-switching, using a switch time of 0.3 s on a TOF
instrument, leads to an additional 30% loss in peak capacity for
highly efcient separations [283]. Therefore this option should be
employed with care, especially for ultra-fast separations.
It is relevant to note that in addition to the extensive use of
MALDI, a range of ionisation sources suitable for the direct analysis
of avonoids from liquid or solid samples have been developed and
found application in avonoid analysis. The most important of these
are desorption electrospray ionisation (DESI) [40] and direct anal-
ysis in real time (DART) [410412]. Since these ionisation sources
are not applicable in LCMS, they will not be discussed here.
4.2. Mass analysers
Mass analysers can broadly be divided into two main classes,
namely high and low resolution (or nominal mass) instruments,
based on their ability to distinguish ions with small mass-to-
charge (m/z) differences. The critical parameter here is the resolving
power, dened as the ratio of an ions mass to the width of its
peak at half height (full width at half maximum, FWHM). The
resolving power is normally specied with the mass at which it
is determined, as resolving power decreases with ion mass. High
resolution mass spectrometers (HR-MS) are generally considered
to provide resolving power greater than 10,000, while for ultrahigh
resolving power instruments this value is above 100,000 [41]. The
former class includes TOF analysers, while the Fourier transform
(FT) instruments Orbitrap and ion cyclotron resonance (ICR) fall in
the latter class. Mass accuracy is the relative difference between
measured and theoretical m/z values, reported in parts per million
 A. de Villiers et al. / J. Chromatogr. A 1430 (2016) 1678
Table 3
Overview of the specications of various mass analysers used in the LCMS analysis of avonoids. The table is based on the work of Holcapek et al. [41], updated using the
latest specications for QqQ, Q-LIT and Orbitrap instruments.
Mass analysera Resolving power (FWHM) [103 ] Mass range m/z (upper limit)
QqQ 20003000
ITa 20486000
TOFa 1060 100,000
ICR 7502500 400010,000
Orbitrapa 100500 20006000
a
Includes hybrid congurations.
b
Calculated for a mass range of m/z 1000.
(ppm) units. Sufcient mass accuracy provides the key benet of
allowing determination of elemental composition [413]. The mass
accuracy required for this however depends on the mass of the ion,
with mass accuracy of <5 ppm typically sufcient for compounds
of m/z < 300; for higher MW ions, this degree of mass accuracy is no
longer sufcient because of the much larger number of potential
combinations of elements [41].
HR-MS instruments such as double-focusing sector and modi-
ed magnetic sector instruments used in the early LCMS analysis
of avonoids [108,171] have nowadays largely been replaced by
TOF, Orbitrap and to a lesser extent FT-ICR systems.
A second classication of MS instruments is based on whether
they are single or multistage systems. Multistage instruments allow
MSn (where n > 1) operation either in time, as in ion trap (IT)
instruments, or by combining multiple mass analysers in space.
Especially in combination with LCMS using soft ionisation tech-
niques such as ESI, where limited fragmentation information is
obtained, tandem MS (MS/MS) or MSn capabilities are essential for
structural elucidation purposes. An array of very powerful hybrid
instruments is nowadays commercially available, including QqQ,
quadrupole-linear ion trap (Q-LIT), Q-TOF [414,415], ion-trap-TOF
(IT-TOF) [416418], quadrupole- and linear ion trap-Orbitrap [419]
and quadrupole- and linear ion trap-ICR instruments. With the
exception of the ICR instruments, all of these have found increasing
application in avonoid analysis during the last decade.
From the perspective of hyphenation to advanced LC separa-
tions such as UHPLC and LC LC, a critical requirement of any mass
analyser is a sufciently high acquisition speed [420]. Peak widths
in the order of a few seconds are often encountered, which implies
that fast acquisition rates, in the order of 510 Hz and higher, are
required to obtain the requisite 15 data points across each peak.
The latest generation of quadrupole and ion trap based analysers
are mostly capable of meeting this requirement, while TOF systems
provide the best scanning speeds of all analysers.
Another important characteristic of mass analysers is the mass
range they are capable of covering. This value is relatively limited
for quadrupole, ion trap and Orbitrap instruments, with TOF sys-
tems providing the highest mass range of all systems.
An overview of the performance characteristics of mass anal-
ysers currently used in LCMS analysis of avonoids is presented
in Table 3. It should be noted that these are generalised specica-
tions, with some variation occurring between instruments and the
mode in which each instrument is operated. For example, the scan
speed is inversely related to the resolving power in FT instruments
(Orbitrap and ICR) and the mass range in scanning instruments (Q
and IT) as well as the sensitivity of all analysers to varying degrees.
For a relatively recent overview of the performance of the differ-
ent instruments, the reader is referred to the excellent reviews by
Holcapek et al. [41] and Forcisi et al. [42], while more dedicated
reviews may be consulted for details on particular instruments such
as Q-TOF [415], Orbitrap [421,422] and FT-ICR [423] systems.
Flavonoid analyses may broadly be divided into targeted and
untargeted applications. In the case of the former, a nite number
of known target molecules are determined, mostly quantitatively,
43
Mass accuracy (ppm) Scanning speed (Hz)b Dynamic range
1030 106
1060 106
<25 10100 104 105
<0.251 2 104
<13 120 40005000
whereas for untargeted or screening analyses the goal is to iden-
tify as many compounds as possible, often only qualitatively. The
nature of the analysis being performed will determine the best
suited mass analyser for the given application.
In the case of targeted analyses, selectivity and quantica-
tion specications (linearity, dynamic range, sensitivity) are of
paramount importance. Currently the most widely used approach
for targeted analysis relies on the high selectivity inherent to tan-
dem MS operation, most commonly exploited by utilising QqQ
instruments operated in multiple reaction monitoring (MRM)
mode. While IT instruments are also capable of this measurement
mode, the quantitative performance of these detectors is generally
inferior to QqQ. An alternative strategy involves exploiting the ben-
ets of HR-MS data to increase selectivity, with the added benet
that full-scan data are also available. Especially Orbitrap instru-
ments are increasingly being used for this purpose, where the high
resolving power of these instruments is benecial.
For untargeted analysis, resolving power is critical. In combi-
nation with high efciency or fast separations, however, the duty
cycle of the mass spectrometer is equally important. Currently,
TOF-MS instruments still provide the best compromise between
these parameters, although developments in Orbitrap systems
show promise to obtain higher resolving power for similar scan
times in the future. Secondly, tandem MS measurements are highly
benecial, especially so in the case of ESI-MS analysis of avonoids
due to the amount of information regarding substitution patterns
of isomeric compounds that may be obtained (cf. Section 2.3). The
combination of both MS/MS and high resolving power is ideal, and
this explains the contemporary popularity of Q-TOF systems for the
non-targeted analysis of avonoids. On the other hand, the latest
generation of Orbitrap instruments are capable of providing the
same information at high resolving power (although lower scan
speeds), and have gained more widespread acceptance in the last
few years. In the following sections, the applications of advanced LC
separation with each of the different types of mass spectrometers
for avonoid analysis during the last seven years will be outlined.
4.2.1. Nominal mass instruments: ion trap (IT) and triple
quadrupole (QqQ)
Ion trap instruments use oscillating electric elds to trap and
store ions in two or three dimensions, based on which 3D (QIT or IT,
also called Paul traps) and 2D (linear (quadrupole) ion trap (LQIT or
LIT) systems can be distinguished. The latter have higher trapping
efciencies and are less susceptible to space-charge effects, pro-
viding higher sensitivity and an increased dynamic range [413].
Accordingly, most recent applications in avonoid analysis utilise
LIT instruments (Table 4). The main advantage of ion trap analysers
is that they enable the true MSn operation by allowing selective
trapping and fragmentation of parent and/or daughter ions as a
function of time [161]. The primary application of these instru-
ments has therefore been in qualitative analysis.
Ion trap instruments are extremely versatile and played a cru-
cial role in the early years of avonoid analysis by LCMS, primarily
in terms of structural elucidation by MSn experiments. Nowadays,
44 A. de Villiers et al. / J. Chromatogr. A 1430 (2016) 1678

Table 4
Applications of advanced LC hyphenated to ion trap MS detectors to avonoid analysis (20092015).

Sample Flavonoids Ionisation Detector(s) Stationary phase (column Mobile phase(s) References
source dimensions), ow rate,
temperature, analysis time

Licorice (Glycyrrhiza Prenylated and ESI+/ LITa C18 (150 mm 2.1 mm, A: 0.1% AAc , B: 0.1% [428]
glabra) glycosylated avanones, UV 1.7 m dp ), AA in ACNc
chalcones, isoav-3-enes, FRb = 0.3 mL/min, 25 min
isoavones and
isoavanes (18
avonoids)
Peanut skins A- and B-type ESI LIT C18 (150 mm 2.1 mm, A: 0.1% AA, B: 0.1% [286]
proanthocyanidins (83 UV 1.7 m dp ), FR = 0.6 mL/min AA in ACN
avonoids) (0.3 mL/min to MS), 50 C,
27 min
Berry skins (Vitis Anthocyanins (18 ESI+ QITa C18 (250 mm 4.6 mm, A: 2% FAc , B: 2% FA [284]
vinifera L.) avonoids) 5 m dp ), FR = 1.0 mL/min, in ACN
50 C, 45 min
Black currant juice Flavone, avonol and ESI Q-LITa C18 (50 mm 2.1 mm, A: 0.1% FA, B: 0.1% [430]
avanone aglycones and 1.8 m dp ), FA in ACN
glycosides (13 avonoids) FR = 0.3 mL/min, 45 min
Rooibos (Aspalathus Flavonol and avone ESI QIT C18 (150 mm 4.6 mm, A: 1% FA in [424]
linearis) aglycones and glycosides, TOFa 1.8 m dp ), FR = 0.5 mL/min H2 O/ACN (90/10,
avanone and chalcone (0.2 mL/min to MS), 25 C, v/v), B: ACN
glycosides (26 avonoids) 35 min
Asian plant dyes Flavone aglycones ESI+/ LIT C18 (150 mm 2.1 mm, A: 0.03% TFAc , B: [431]
(Arthraxon hispidus glycosides (9 avonoids) UV 1.8 m dp ), 0.024% TFA in ACN
and Miscanthus FR = 0.1 mL/min, 40 min
tinctorius)
Pomegranate (Punica Flavan-3-ols, avonols, ESI+/ LIT C18 (50 mm 2.0 mm, A: 0.1% FA, B: 0.1% [429]
granatum L.) juice avanones, 1.8 m dp ), FA in ACN
dihydrochalcones, and FR = 0.2 mL/min, 30 C,
avones (14 avonoids) 20 min
Eucalyptus wood Flavan-3-ols, avonol ESI QIT C18 (50 mm 2.1 mm, A: H2 O/ACN (99:1, [432]
aglycones and glycosides, UV 1.9 m dp ), v/v) containing
avanones and FR = 0.6 mL/min, 25 C, 0.1% FA, B: 0.1% FA
avanonols (18 37 min in ACN
avonoids)
Pomegranate (Punica Flavonol glycosides, ESI+/ LIT C18 (50 mm 2.0 mm, A: 0.1% FA, B: 0.1% [433]
granatum L.) juice anthocyanins, taxifolin 1.8 m dp ), FA in ACN
hexoside and naringenin FR = 0.2 mL/min, 30 C,
(11 avonoids) 20 min
Rat serum Isoavones (4 avonoids) ESI+ Q-LIT C18 (50 mm 4.6 mm, A: 0.1% FA, B: 0.1% [92]
2.6 m dp SPb ), FA in ACN
FR = 0.5 mL/min, 9 min
Halimione portulacoides Flavonol and avone ESI QIT C18 (50 mm 2.1 mm, A: H2 O:ACN (99:1) [434]
glycosides (13 avonoids) UV 1.9 m dp ), containing 0.1% FA,
FR = 0.6 mL/min, 25 C, B: 0.1% FA in ACN
37 min
Eastern Teaberry Flavan-3-ols, ESI+/ QIT C18 (150 mm 2.1 mm, A: H2 O/ACN/FA [435]
(Gaultheria procyanidins and avonol UV 1.7 m dp ), (95:5:0.1, v/v), B:
procumbens L.) aglycones and glycosides FR = 0.3 mL/min, 25 C, 0.1% FA in ACN
(22 avonoids) 80 min
Viola yedoensis Makino Flavone glycosides (35 ESI+/ LIT C18 (50 mm 2.1 mm, A: H2 O, B: ACN [154]
avonoids) UV 1.9 m dp ),
FR = 0.25 mL/min, 30 C,
33 min
Green tea, oolong tea Flavonol glycosides (18 ESI QIT C18 (100 mm 2.1 mm, A: 2% AA, B: ACN [436]
and black tea avonoids) UV 1.8 m dp ),
FR = 0.4 mL/min, 20 C,
20 min
Forsythia owers Rutin ESI QIT C18 (150 mm 2.1 mm, A: H2 O/ACN/FA [313]
UV 1.9 m dp ), (95:5:0.1, v/v/v), B:
FR = 0.2 mL/min, 25 C, 0.1% FA in ACN
68 min
Dietary supplements Flavonol and avanones ESI+ QIT C18 (100 mm 0.075 mm, Isocratic: [437]
and food matrices (5 avonoids) UV <2 m dp ), FR = 450 nL/min, ACN/MeOHc /H2 O/FA
10 min (32.5:10:57:0.5,
v/v/v/v).
Ros wines Flavonols, avan-3-ols, ESI+/ QIT C18 (150 mm 1.0 mm, A: 1% FA, B: 1% FA [438]
dihydrochalcones and ESI+/ QqQ 1.8 m dp ), in MeOH
anthocyanins (113 UV FR = 0.08 mL/min, 40 C,
avonoids) 46 min
a
LIT, linear ion trap; Q-LIT, Quadrupole-linear ion trap; QIT, quadrupole ion trap; TOF, time of ight; QqQ, triple quadrupole.
b
FR, ow rate; SP, supercially porous.
c
AA, acetic acid; ACN, acetonitrile; FA, formic acid; TFA, triuoroacetic acid; MeOH, methanol.
 A. de Villiers et al. / J. Chromatogr. A 1430 (2016) 1678
however, IT instruments are less commonly used on their own, but
are often combined with HR analysers in powerful hybrid congu-
rations such as IT-TOF and IT-Orbitrap instruments, which have
largely superseded conventional IT-MS in avonoid analysis. In
such instruments, the ion storage and sequential fragmentation
capabilities of IT instruments are highly benecial, particularly in
structural elucidation studies. Alternatively, IT multi-stage frag-
mentation is often used in combination with separate LCHR-MS
analysis for structural elucidation [424426]. From the perspec-
tive of hyphenation to ultra-fast and high resolution separations,
several latest generation IT and especially LIT instruments provide
sufcient acquisition speeds of up to 60 Hz (Table 3).
Ion trap instruments are generally considered inferior to
quadrupole or triple quadrupole instruments for quantitation. This
is because of the need to account for variable trapping times and
also the lower dynamic range of these instruments (the latter
limited by the capacity and need to avoid space-charge effects).
Nevertheless, Stanoeva et al. [427] have demonstrated the quanti-
tative analysis of avonoids in full-scan MS and MS2 modes using
an IT instrument, providing similar results but better sensitivity
than UV detection. Gavina et al. [92] also utilised an IT instrument
in MRM mode for the quantitative analysis of isoavones in rat
serum, reporting a dynamic range of 3 orders of magnitude and
LODs of 0.013 pg on-column. An overview of the application of
IT instruments in combination with advanced separation methods
is presented in Table 4; some noteworthy reports are discussed
briey below.
A good example of the utility of IT instruments in the struc-
tural elucidation of avonoids is the work of Cao et al. [154],
who reported an extensive study aimed at characterising avone-
di-C-glycosides using UHPLC-ESI-MSn spectra obtained on an IT
instrument. Negative ionisation was found to provide more diag-
nostic information, and the authors were able to show that the
rst sugar cleavage in MS2 spectra occurs at the C-6 position, fol-
lowed by the C-8 sugar in MS3 spectra. Based on this, information
about the nature of the sugars can be derived. Furthermore, the
0,2 X 0,2 X ion could be used to identify the aglycone species. The
1 2
utility of the approach was demonstrated for the analysis of a
butanol extract of Viola yedoensi, where 26 avone-di-C-glycosides
could be identied.
The group of Gruppen [286] used both QIT and LIT instruments
to study in detail the proanthocyanidins present in peanut skins.
NP-LC fractionation of the sample was followed by RP-HPLC and
UHPLC to simplify the analysis due to the extreme complexity of
the sample. The authors found that A-type oligomers were pre-
dominant in this sample, and were able to assign the position of
the A-linkages in 43 trimeric and tetrameric procyanidins based on
MS/MS fragments resulting from QM ssion (Fig. 4) [182,184]. The
same group reported a UHPLC-LIT method for the rapid screening
of prenylated avonoids [428]. The methodology is based on the
detection of neutral losses of 56 and 42 amu associated with prenyl
and pyran ring moieties. The ratio of these two neutral losses could
then be used to distinguish between these two classes of com-
pounds.
He et al. [284] used a conventional HPLC column operated at
50 C in combination IT detection to conrm for the rst time
the presence of pelargonidin-3-O-glucoside in Vitis vinifera L.
grape skins. Mena et al. [429] proposed an UHPLC-IT method-
ology using three different sets of MS conditions (scan times
0.190.38 s) for the screening of phenolics in pomegranate juice.
Flavonoids and hydolyzable tannins were identied in nega-
tive ionisation mode, and anthocyanins in positive ionisation
mode.
Rak and co-workers used a hybrid triple quadrupole-LIT instru-
ment to devise a method for untargeted screening of avonoids
[430]. Two separate experiments were used: in the rst instance,
45
MRM scanning was used to detect the presence of selected agly-
cones following in-source fragmentation of glycosylated avonoids
using a high declustering potential. This was followed by a second
full scan detection mode in the same analysis to allow tenta-
tive assignment of the molecular weight of the suspected target
analytes. Finally, a second LC analysis was performed with the
detector used in product ion scan mode (with the third quadrupole
used as a linear ion trap) for the identication of avonoid
derivatives.
Within the scope of this review, we will limit our atten-
tion to triple quadrupole (QqQ) instruments. These combine two
quadrupoles with a RF collision chamber in-between to allow
tandem MS operation in space. Since both quadrupoles may be
operated in selected ion monitoring (SIM) or scan modes, a range
of different modes can be used on QqQ instruments. Scan mode,
where either of the quadrupoles is scanned, is less commonly used
in avonoid analysis, in part because better performance in terms
of sensitivity and accurate mass information can be obtained on
other instruments. For structural studies, product ion scan (Q1 in
SIM, Q3 in scan), neutral loss (Q1 and Q3 in SIM mode at a xed off-
set) and precursor ion scan (Q1 scan, Q3 SIM) modes may be used
[439]. QqQ instruments are however mostly utilised as selective
and sensitive detectors in selected or multiple reaction monitoring
mode (SRM or MRM), where Q1 is used in SIM mode to isolate par-
ent ions, and Q3 in SIM to monitor selected daughter ions. For such
targeted analysis, QqQ instruments provide very high sensitivity
and selectivity as a consequence of the combination of two mass
lters in SIM mode.
The latest generation quadrupole systems can achieve scan rates
as high as 1015,000 amu/s [321,420], and using this technology in
QqQ format, dwell times as low as 5 ms can be achieved in MRM
mode [420,440], which are mostly sufcient for quantication of
narrow peaks commonly obtained in UHPLC separations [420,441].
Because of the operation principles of quadrupole instruments,
however, a compromise between speed and sensitivity has to be
made; this is especially relevant when relatively large numbers of
MRM transitions are to be monitored.
Indeed, in the majority (87%) of the studies listed in Table 5,
QqQ instruments are used in MRM mode for targeted analysis.
Two examples of the power of this approach for avonoid anal-
ysis will be highlighted. Vrhovsek et al. [442] reported a targeted
metabolomic approach based on UHPLC-QqQ for the quantication
of 135 phenolics, including 66 avonoids, in fruits and beverages
within 15 min. Quantication limits varied between 0.5 and >50 pg,
while linear responses spanning more than four orders of mag-
nitude were reported. Lambert et al. [438] used a 1 mm UHPLC
column and QqQ detection in MRM mode to quantify 152 pheno-
lics in Ros wines (Fig. 14). In this work, dwell times were varied
for each transition to obtain 1015 points across each peak (most
values were in the order of 5 ms).
QqQ instruments are nowadays less often used in the other
operational modes for structural elucidation or untargeted analy-
sis, with more advanced HR-MSn instrumentation having surpassed
QqQ systems for such applications. In fact, triple quadrupole instru-
ments are often used together with HR-MS systems such as TOF or
Orbitrap instruments in the study of avonoids, where the latter is
used for compound identication and the former for quantication
[425,443,444]. Product ion scans are also used to assist in tentative
compound identication [96,180,445] and to determine suitable
CID conditions and transitions for MRM operation [446,447]. Some-
what surprisingly, Spcil et al. [447] reported a 10-fold higher
sensitivity in positive ionisation mode, although negative ionisa-
tion provided better selectivity. A similar observation (23 times
higher sensitivity in positive ionisation) was made by Nagy et al.
[408], and might be due to reduced background noise in QqQ instru-
ments operated in MRM mode.
46 A. de Villiers et al. / J. Chromatogr. A 1430 (2016) 1678

Table 5
Applications of advanced LC hyphenated to triple quadrupole MS detectors to avonoid analysis (20092015).

Sample Flavonoids Ionisation Detector(s) Sensitivity Stationary phase Mobile References

source (injection (column dimensions), phase(s)
volume) ow rate, temperature,
analysis time

Vitaavan and cocoa Flavanol ESI QqQb (MRMb ) 1.7692 g C18 (100 mm 2.1 mm, A: 2% AAd , B: [452]
nibs monomers and (not specied) 1.8 m dp ), ACNd
procyanidins (4 FRc = 0.4 mL/min, room
avonoids) temp, 13 min
Milk-based food Anthocyanins and ESI+ QqQ (MRM) 33000 pg C18 (50 mm 2.1 mm, A: 10% FAd , B: [408]
products avonols (26 on-column 1.7 m dp ), ACN
avonoids) (10 L) FR = 0.4 mL/min, room
temp, 13 min
Cocoa Procyanidins (10 ESI QqQ (MRM) 130 pg/L C18 (100 mm 2.1 mm, A: 0.2% AA, B: [453]
avonoids) 1.8 m dp ), ACN
FR = 0.4 mL/min, 30 C,
12.5 min
GegenQinlian Puerarin, ESI+ QqQ (SIMb ) 12.751 pg C18 (100 mm 2.1 mm, A: 0.1% FA and [454]
decoction (TCMa ) daidzein, baicalin, on-column 1.7 m dp ), 5 mM AA, B:
wogonoside and (5 L) FR = 0.2 mL/min, 30 C, MeOHd
liquiritin 6 min
Black tea Proanthocyanins ESI QqQ (MRM) LODs not C18 (100 mm 2.1 mm, A: 0.1% FA, B: [88]
(8 avonoids) UV specied 1.7 m dp ), 0.1% FA in ACN
FR = 0.5 mL/min, 30 C,
7 min
Rat plasma Procyanidins and ESI+/ QqQ (MRM) 1.5606 g C18 (100 mm 2.1 mm, A: 0.2% AA [455]
anthocyanins (21 (not specied) 1.8 m dp ), (procyanidins),
avonoids) FR = 0.4 mL/min, 10% AA
12.5 min (anthocyanins),
B: ACN
Tea Catechins (8 ESI+/ QqQ (MRM) LODs C18 (100 mm 2.1 mm, A: 0.05% AA [447]
compounds) 4.57.2 pg 1.7 m dp ), (+mode) or
(+mode) and FR = 0.45 mL/min, 35 C, 0.01% FA
4572 pg 5.5 min (mode), B:
(mode) MeOH
on-column
(1.5 L)
Chamomile owers Flavones and ESI+/ QqQ (MRM) LODs C18 (100 mm 2.1 mm, A: 0.1% FA, B: [456]
and Chamomile tea avonols (9 2.612.9 pg 1.7 m dp ), MeOH
extracts avonoids) (mode), FR = 0.45 mL/min, 30 C
4.9408 pg
(+mode)
on-column
(3 L)
Radix puerariae Isoavones ESI QqQ (MRM) C18 (50 mm 2.1 mm, A: 0.2% FA, B: [457]
aglycones and 2.6 m dp SPc ), MeOH
glycosides (13 FR = 0.5 mL/min, 35 C,
avonoids) 12 min
Red wine Anthocyanins ESI+ QqQ (Neutral Qualitative C18 (100 mm 2.1 mm, A: 7.5% FA, B: [180]
(121 avonoids) loss, survey 1.7 m dp ), ACN
scan) FR = 0.1 mL/min, 50 C,
30 min
Mung bean (Vigna Flavanones, ESI+ QqQ (MRM) LODs C8 (150 mm 2.1 mm, A: 10 mM FA, [83]
radiate) avones and UV 0.043173 pg 1.7 m dp ), B: MeOH
isoavones (24 on-column FR = 0.2 mL/min, 40 C,
avonoids) (2 L) 17 min
Apple, berries, green Dihydrochalcones, ESI+/ QqQ (MRM) LODs C18 (100 mm 2.1 mm, A: 0.1% FA, B: [442]
tea and red wine isoavones, 0.550 pg 1.8 m dp ), 0.1% FA in ACN
avones, on-column FR = 0.4 mL/min, 40 C,
avanones, (2 L) 15 min
avan-3-ols and
avonols (63
avonoids)
Sangiovese wines Pyranoanthocyanins ESI QqQ (MRM) LODs 2860 pg C18 (150 mm 2.1 mm, A: 5% FA, B: 5% [458]
and anthocyanin UV on-column 1.7 m dp ), FA in ACN
avanol adducts (2 L) FR = 0.4 mL/min, 40 C,
(92 avonoids) 18 min
Cherry tomatoes, Flavonols and ESI QqQ (MRM, LODs C18 (50 mm 2.1 mm, A: 0.1% FA, B: [445]
tomato sauce and avanones (3 product ion 1001540 pg 1.7 m dp ), 0.1% FA in ACN
juice avonoids) and neutral on-column FR = 0.4 mL/min, 30 C,
loss scan) (10 L) 3 min
Cocoa beans Procyanidins (3 ESI QqQ (MRM, Semi- C18 (150 mm 2.1 mm, A: 0.1% FA, B: [446]
avonoids) product ion quantitative 1.7 m dp ), 0.1% FA in ACN
scan) FR = 0.3 mL/min, 30 C,
UV 35 min
 A. de Villiers et al. / J. Chromatogr. A 1430 (2016) 1678 47

Table 5 (Continued )

Sample Flavonoids Ionisation Detector(s) Sensitivity Stationary phase Mobile References

source (injection (column dimensions), phase(s)
volume) ow rate, temperature,
analysis time

Tomato, broccoli, Isoavones, ESI+/ QqQ (MRM) 5100 g/kg C18 (100 mm 2.1 mm, A: 30 mM AA [440]
carrot, eggplant and avones and 1.8 m dp ), adjusted to pH
grape avonols (22 FR = 0.2 mL/min, 30 C, 5 with FA, B:
avonoids) 11 min MeOH
Fermented orange juice Flavanones (9 ESI QqQ (MRM) 6.2544.08 pg C18 (150 mm 2.1 mm, A: 0.1% FA, B: [459]
compounds) on-column 1.7 m dp ), 0.1% FA in ACN
(10 L) FR = 0.32 mL/min,
12 min
Huang-Lian-Jie-Du Flavones and ESI QqQ (MRM) 0.021.0 pg/L C18 (50 mm 2.1 mm, A: 0.1% FA, B: [444]
decoction (TCM) avanones (24 (injection 1.7 m dp ), 0.1% FA in ACN
avonoids) volume not FR = 0.45 mL/min, 35 C,
specied) 12 min
Apple peel Flavonol ESI QqQ LODs not C18 (100 mm 2.1 mm, A: 0.1% FA, B: [460]
glycosides, ESI TOF specied 1.8 m dp ), 0.1% FA in ACN
avan-3-ols and FR = 0.3 mL/min, 30 C,
chalcone (8 14 min
compounds) and
procyanidins
(DP110)
Citrus juices and Flavanones (13 ESI+ QqQ (MRM) 5002900 pg PFPc (50 mm 2.1 mm, A: 0.1% FA, B: [461]
beverages avonoids) on-column 2.6 m dp SP), ACN
(10 L) FR = 0.3 mL/min, 16 min
Standards and urine Flavone ESI+ QqQ (MRM) LODs not C18 (50 mm 0.3 mm, A: 2% AA, B: 2% [462]
metabolites ESI+ IMb -LIT specied 2.7 m dp SP), AA in MeOH
(diosmetin-3-O- (FAIMSb ) FR = 35 L/min, 50 C,
glucoronide, 6 min
-7-O-glucoronide
and -3,7-O-
diglucoronide)
Grape berries Flavonols, ESI+ QqQ (MRM) LODs not Non-anthocyanins: Non- [463]
avan-3-ols, specied C18 (100 mm 2.1 mm, anthocyanins:
dihydrochalcones 1.8 m dp ), A: 0.1% FA, B:
and anthocyanins FR = 0.4 mL/min, 40 C, 0.1% FA in ACN
(37 avonoids) 15 min Anthocyanins:
Anthocyanins: C18 A: 5% FA, B: 5%
(150 mm 2.1 mm, FA in MeOH
1.7 m dp ),
FR = 0.4 mL/min, 40 C,
14 min
Tomato, carrot, grape, Flavonols, ESI+/ QqQ (MRM) LODs not C18 (100 mm 2.1 mm, A: 30 mM [464]
eggplant and brocolli isoavones and specied 1.8 m dp ), ammonium
avones (16 FR = 0.2 mL/min, 30 C, acetate, pH 5,
avonoids) 12 min B: MeOH
Olive oil Flavonol and ESI QqQ (MRM) LODs 5137 pg C18 (50 mm 2.1 mm, A: 0.2% AA, B: [465]
avones (3 on-column 1.8 m dp ), ACN
avonoids) (10 L) FR = 0.4 mL/min, 30 C,
5 min
Gingko biloba tablets Flavonol ESI QqQ (MRM) LODs C18 (50 mm 4.6 mm, A: 1% FA, B: [450]
aglycones and 0.0355.1 pg 1.8 m dp ), MeOH
glycosides (13 on-column FR = 0.5 mL/min, 30 C,
avonoids) (5 L) 28 min
Gingko biloba seeds Flavan-3-ols, ESI+/ QqQ (MRM) LODs C18 (100 mm 2.1 mm, A: 0.1% FA, B: [466]
avonol aglycones 0.15.7 pg 1.7 m dp ), 0.1% FA in ACN
and glycosides on-column FR = 0.4 mL/min, 35 C,
and avones (19 (2 L) 11 min
avonoids)
Sausages with added Flavonol- ESI QqQ (MRM) 15510 g/kg C18 (150 mm 1.0 mm, A: ACN/0.1% FA [467]
cocoa and grape seed glycosides and 1.7 m dp ), (5/100, v/v), B:
extracts proanthocyani- FR = 0.13 mL/min, 35 C, ACN/0.1% FA
dins (10 15 min (60/100, v/v)
avonoids)
Artichoke, garlic and Flavonols, ESI+/ QqQ (MRM) 150 g/kg C18 (100 mm 2.1 mm, A: 30 mM [451]
spinach isoavonols and 1.8 m dp ), ammonium
avones (22 FR = 0.2 mL/min, 30 C, acetate, pH 5,
avonoids) 11 min B: MeOH
Sweet cherry (Prunus Anthocyanins, ESI+/ QqQ (MRM, full LODs 376 pg C18 (50 mm 2.1 mm, A: 1% FA, B: 1% [294]
avium L.) avonols and scan and on-column 1.8 m dp ), FA in ACN
avan-3-ols (18 product ion (1.1 L) FR = 0.5 mL/min, 60 C,
avonoids) scan) 15 min
UV
48 A. de Villiers et al. / J. Chromatogr. A 1430 (2016) 1678

Table 5 (Continued )

Sample Flavonoids Ionisation Detector(s) Sensitivity Stationary phase Mobile References

source (injection (column dimensions), phase(s)
volume) ow rate, temperature,
analysis time

Lentil seed coats Anthocyanins, ESI+ QqQ (MRM) Qualitative PFP (100 mm 2.1 mm, A: 1% FA, B: [91]
avan-3-ols, UV 2.6 m dp SP), H2 O/ACN/FA
proanthocyani- FR = 0.4 mL/min, 50 min (5:90:5, v/v/v)
dins, avanones,
avones and
avonols (21
avonoids)
Wild Berberis species Flavonol ESI QqQ (product LODs not C18 (150 mm 4.6 mm, A: 0.1% FA, B: [260]
glycosides (16 ion) specied 2.6 m dp SP), ACN
avonoids) UV FR = 1.0 mL/min, 30 C
15.1 min
Belamcanda chinensis Isoavone ESI+ QqQ (product 0.141.02 pg C18 (100 mm 2.1 mm, A: 0.1% FA, B: [443]
(TCM) aglycones and ESI+ ion) on-column 1.7 m dp ), 0.1% FA in ACN
glycosides (26 Q-TOF (2 L) FR = 0.4 mL/min, 40 C,
avonoids) 17 min
Brazilian pepper Anthocyanins and ESI QqQ (product Qualitative C18 (150 mm 2.1 mm, A: 1% FA [96]
(Schinus biavones (7 ion scan, 1.8 and 1.7 m dp ), (anthocyanins),
terebinthifolius Raddi) avonoids) neutral loss, FR = 0.4 mL/min, 40 C, 0.1% FA
exocarp CID) 18 and 23.5 min (nonantho-
ESI+ Q-TOF cyanins), B:
UV 0.1% FA in ACN
Grape (V. vinifera, V. Flavonol ESI QqQ (MRM, Semi- C18 (100 mm 2.1 mm, A: 5% FA, B: [449]
riparia, V. labrusca, glycosides (24 precursor-ion quantitative 1.7 m dp ), ACN
Vitis lincecumii and V. compounds) and FR = 0.45 mL/min, 40 C,
rupestri) species neutral-loss 10 min
scan)
ESI Q-TOF
Mulberry (Morus alba Flavan-3-ols, ESI QqQ (MRM) 0.1477.28 mg/kg C18 (100 mm 2.1 mm, A: 0.2% FA, B: [425]
L.) anthocyanins and ESI+/ LIT-Orbitrap 1.7 m dp ), ACN
avonol UV FR = 0.4 mL/min, 40 C,
glycosides (34 15 min
avonoids)
Ros wines Flavonols, ESI+/ QqQ (MRM) 1050,000 pg C18 (100 mm 1.0 mm, A: 1% FA, B: 1% [438]
avan-3-ols, ESI+/ QIT on-column 1.8 m dp ), FA in MeOH
dihydrochalcones UV (2 L) FR = 0.17 mL/min, 40 C,
and anthocyanins 30 min
(113 avonoids)
Green, white and black Flavan-3-ol ESI QqQ (MRM) 7.537.5 pg C18 (100 mm 2.1 mm, A: 0.1% FA, B: [468]
teas derivatives (8 on-column 1.7 m dp ), 0.1% FA in
compounds) (5 L) FR = 0.3 mL/min, 40 C, MeOH
4 min
Gentiana cruciata L. Flavone ESI+ QqQ (MRM) LODs not C18 (50 mm 2.1 mm, A: 0.1% FA, B: [469]
roots glycosides (4 UV specied 1.9 m dp ), 0.1% FA in ACN
compounds) FR = 0.4 mL/min, 30 C
a
TCM, traditional Chinese medicine.
b
QqQ, triple quadrupole; MRM, multiple reaction monitoring; SIM, selected ion monitoring; IM, ion mobility; FAIMS, eld-asymmetric waveform ion mobility spectrometry.
c
FR, ow rate; PFP, pentauorophenylpropyl; SP, supercially porous.
d
AA, acetic acid; ACN, acetonitrile; FA, formic acid; MeOH, methanol.

Nevertheless, neutral loss and precursor ion scans are use-
ful in group-type identication of avonoids, for example by
studying glycosylated and/or acylated avonoid derivatives, prior
to HR-MSn identication of compounds [180,448]. As an exam-
ple, Alberts et al. [180] applied UHPLC-QqQ in neutral loss and
survey scan (neutral loss-triggered product ion scan mode)
for the analysis of red wine anthocyanins (Fig. 15). In neu-
tral loss mode, class-type detection of anthocyanidin-glycosides,
-acetylglycosides, -coumaroylglycosides, -caffeoyglycosides and
-di-glycosides was performed. Compound identity was then con-
rmed based on characteristic high-energy CID spectra obtained
for the anthocyanidin aglycones in survey scan mode. Signicantly,
the characteristic product ion spectra showed consistent behaviour
also for the derived anthocyanins encountered in wine, which
allowed the authors to identify 121 compounds in red wine using
this approach.
De Rosso et al. [449] used UHPLC-QqQ in various modes for
the detailed analysis of red wine avonols. First of all, precursor
ion analysis of the six known wine avonol aglycones was used
to identify compounds of this class. Neutral loss mode (monitor-
ing losses of 146, 162, 176, 308 and 324 for pentosides, hexosides,
glucoronides, rutinosides, di-hexosides and hexosyl-glucoronides,
respectively) was used to conrm putative compounds of interest.
The authors reported that the latter operation mode was less sen-
sitive and relied on slower scan times compared to precursor ion
scan mode, such that closely eluting isobaric compounds were not
resolved. Further conrmation of the tentatively identied com-
pounds was obtained using high resolution MS and MS/MS data
acquired on a Q-TOF instrument [449].
Obtaining a general sense of the sensitivity of QqQ detection
in MRM mode is complicated by the fact that sensitivity varies
between instruments and as a function of the number and nature of
target analytes, ionisation mode and chromatographic conditions.
Nevertheless, reported limits of detection (LODs) fall in the range
of 0.02 pg3 ng on-column (Table 5), with higher values reported
for aglycones [83,408]. Usual LOD values for glycosylated species
are in the region of 0.110 pg on-column, and typical linear ranges
are in the order of 25 orders of magnitude [408,442,450], which
 A. de Villiers et al. / J. Chromatogr. A 1430 (2016) 1678 49

Fig. 14. Overlay of MRM traces acquired for the UHPLCESI-QqQ-MS analysis of 152 phenolics in ros wine. Analysis conditions: 100 mm 1 mm 1.7 m porous C18 phase,
40 C, 0.17 mL/min.
Source: Reproduced from [438].

Fig. 15. (A) Chromatogram obtained for the UHPLCMS/MS analysis of red wine anthocyanins on a QqQ instrument in neutral loss mode for the detection of anthocyanin-
glycosides (neutral loss of m/z 162). The insert shows the fragmentation pattern at low collision energies (25 V). (B) The characteristic product ion spectrum of malvidin-3-
O-glucoside obtained in survey scan mode (neutral loss-triggered product ion scan) at 50 V collision energy. The insert shows the fragmentation scheme for the aglycone
following the loss of the neutral sugar moiety. Analysis conditions: 100 mm 2.1 mm 1.7 m porous C18 phase, 50 C, 0.1 mL/min.
Source: Adapted from [180].
50 A. de Villiers et al. / J. Chromatogr. A 1430 (2016) 1678
explains the widespread use of these detectors for targeted quan-
titative analysis. It is relevant to note that matrix effects remain
a concern for such applications using ESI. This can be minimised
by using standard addition quantication the increased number
of injections is then offset by the shorter analysis times offered by
UHPLC separation [440,451].
4.2.2. Time-of-ight (TOF) instruments
Time-of-ight mass spectrometers [470] work on the principle
that lighter ions travel faster than heavier ions following an initial
acceleration by an electric eld. All ions acquire the same kinetic
energy during this initial acceleration period, and are separated in
the eld-free ight tube according to their different velocities. The
physical property that is measured is ight time, which is directly
related to the mass-to-charge ratio of the ion. Due to this mode of
operation, TOF instruments offer very high mass ranges (without
compromising sensitivity, as is the case in scanning mass spectro-
meters), very high acquisition rates and for the latest generation of
instruments, relatively high resolving power and good sensitivity
(although the latter decreases with high acquisition rates) (Table 3).
Several important developments have improved the per-
formance of TOF instruments [471]; from the perspective of
hyphenation to LC with ionisation sources such as ESI or APCI the
introduction of orthogonal acceleration to transform continuous
ion beams into a pulsed source is especially relevant [472,473]. Fur-
thermore, reectron instruments improve the resolving power and
mass accuracy of TOF instruments through the use of an electro-
static reector (ion mirror) to correct for kinetic energy dispersion
of ions leaving the ion source [474], although this comes at the cost
of somewhat reduced sensitivity and dynamic range [42].
In addition, several hybrid TOF instruments are nowadays com-
mercially available, which provides the option of tandem MS
operation with high resolving power acquisition. The most com-
mon of these, the quadrupole-TOF (Q-TOF) [414,415] has found
extensive use in avonoid analysis (Table 6). The Q-TOF congu-
ration allows collimation of the ion beam as well as the option of
mass selection in Q1 and fragmentation in a RF only quadrupole. A
further variant of the Q-TOF system equipped with an ion mobility
cell between the quadrupole and the TOF analyser is available (see
below for a discussion on ion mobility) [475]. In another hybrid
instrument, the ion trap TOF (IT-TOF) [416], the IT is used to store
ions from a continuous source such as ESI, from where they are
ejected via a dc pulse to the TOF, delivering enhanced sensitivity
through the storage of ions in the IT. This conguration can also
be used to perform MSn experiments prior to high resolution TOF
detection.
The high acquisition speed of TOF instruments (already high-
lighted for GC in the 1980s [476]) makes this an ideal MS detector
for non-targeted analysis in combination with very fast UHPLC sep-
arations [42,420] and therefore also on-line LC LC systems [321].
Combined with the relatively high resolving power and excellent
mass range of TOF-MS, this characteristic is responsible for the
extensive application of the detector in contemporary LCMS anal-
ysis of avonoids. QqQ instruments operated in MRM mode still
provide lower limits of detection than TOF systems for targeted
analyses, although at the cost of loss of information about the com-
plete ion population. Similar quantitative performance (although
not sensitivity) has been reported for TOF and QqQ instruments by
exploiting the selectivity of high resolution mass data [477].
An illustrative example of the advantage of single-stage TOF
analysis may be found in the work of Abrank et al. [193], who
reported a systematic bottom-up approach for the screening
analysis of avonoid derivatives. The methodology is based on
the use of two slightly different LCMS analyses with in-source
CID used to produce characteristic fragments (aglycones for
avonoid-O-glycosides and [Y0 +C2 H2 O+H]+ and [Y1 +C2 H2 O+H]+
ions for avonoid-C-glycosides). Further putative identication of
the parent ions was then obtained based on extracted ion chro-
matograms (using a 10 mDa window) of the characteristic ion and
an automated screening approach where known adducts such as
hexosides, pentosides, etc. are searched for in the full scan spectral
data acquired. Sodium adducts were used as added conrmation
of the proposed structure (based on the observation that fragment
ions do not form sodium adducts).
A trend in the last few years has been increasing use of hybrid
TOF instruments, with single stage TOF analysers increasingly being
replaced by especially Q-TOF systems in avonoid analyses. While
in-source CID can be used to obtain fragmentation information
needed to identify compounds [164,193,297,478], hybrid systems
are much more exible in allowing dual scan modes, true MS/MS
and also pseudo MS3 operation by utilising in-source CID [479,480].
The most used conguration for avonoid analysis is the Q-TOF
(two-thirds of the papers listed in Table 6 used this instrument). In
addition to the tandem MS capabilities of Q-TOF systems, alterna-
tive acquisition modes are also possible, such as recording spectra
at both high and low collision energy settings within a single analy-
sis (referred to as MSE by one vendor). An example of the application
of this approach for the identication of phenolic compounds in
apple products by UHPLCQ-TOF-MS analysis is presented in Fig. 16
[481]. Fig. 16B shows the extracted ion chromatograms for two
partially co-eluting avonoids, with the corresponding MSE spec-
tra obtained using a collision energy ramp of 1040 eV presented
in Fig. 16C. While this approach is useful for the fast screening of
unknown avonoids, providing as it does useful information for
tentative compound identication based on the molecular ion and
major fragments in a single analysis, it also lacks the selectivity
offered by the MS/MS mode. This is evident from Fig. 16C where MSE
spectra contain fragment ions from co-eluting compounds, which
may hamper structural elucidation of co-eluting avonoids of the
same class.
UHPLC-Q-TOF has also been used in the detailed analysis of
the MS/MS behaviour of avonoids. For example, Guo et al. [177]
reported a systematic approach to differentiate between isomeric
6-C and 8-C avone glycosides based on negative and positive
ionisation MS/MS spectra of the deprotonated and protonated
molecular ions, with the latter proving to be more discriminative.
Ma et al. [482] reported a MS/MS method on a Q-TOF instrument to
allow differentiation of regioisomers of methylated avonols based
on the position-specic [M+HCH4 ]+ ion and methylated RDA frag-
ments, as well as complementary information obtained from the
RP-LC elution order.
On the other hand, the utility of Q-TOF-MS for targeted analysis
of selected avonoids is illustrated by the work of Delcambre and
Saucier [483], who were able to demonstrate the presence of 14
avanol-glucosides in Merlot grapes and wine for the rst time. To
do this, the authors exploited the MS/MS capabilities of the Q-TOF
to conrm the identity of the parent ion based on the fragments
observed and their chemical formula obtained from accurate mass
data.
Ion trap time-of-ight (IT-TOF) instruments have found rela-
tively limited application in avonoid analysis compared to Q-TOF
systems [484486], especially in combination with advanced LC
separation methods. Nevertheless, the work of Dugo et al. and Som-
mella and co-workers illustrates the utility of such an approach,
where supercially porous phases were combined with the MS/MS
and high resolution capabilities of the IT-TOF for the characteri-
sation of avonoids in mulberry leave extracts [258], Mate [367],
citrus juices [487] and apples [124].
Li et al. [488] reported an interesting approach based on
UHPLC separation and Q-TOF detection for the identication of
antioxidants in licorice. To do this, the authors compared chro-
matographic proles before and after spiking the sample with the
 A. de Villiers et al. / J. Chromatogr. A 1430 (2016) 1678 51

Table 6
Applications of advanced LC hyphenated to time-of-ight MS detectors to avonoid analysis (20092015).

Sample Flavonoids Ionisation Detector(s) Stationary phase Mobile References

source (column dimensions), phase(s)
ow rate, temperature,
analysis time

Rose species (Rosa Flavonol ESI+/ Qb -TOFb C18 (100 mm 2.1 mm, A: 0.05% TFAd , [489]
damascene, Rosa aglycones and UV 1.7 m dp ), B: ACNd
bourboniana and glycosides (14 FRc = 0.3 mL/min, 30 C,
Rosa brunonii) avonoids) 4.5 min
Plant extracts Kaempferol, ESI+/ TOF C18 2 A: 0.1% FAd , B: [283]
(Arabidopsis thaliana quercetin, rutin (150 mm 2.1 mm, 0.1% FA in ACN
and Ginkgo biloba) and silibinin 1.7 m dp ),
FR = 0.35 mL/min, 90 C,
120 min
Buyang Huanwu Flavan-3-ols, ESI+/ TOF C18 (50 mm 4.6 mm, A: 0.3% FA, B: [490]
decoction (TCMa ) chalcones, UV 1.8 m dp ), ACN
avonols FR = 0.8 mL/min, 25 C,
glycosides and 30 min
isoavones (24
avonoids)
Flos Lonicerae Japonicae Flavone and ESI+/ TOF C18 (50 mm 4.6 mm, A: 0.1% FA, B: [297]
(TCM) avonol UV 1.8 m dp ), FR = 0.7 and ACN
glycosides (10 0.8 mL/min (0.35 and
avonoids) 0.4 mL/min to MS),
temperature gradient
2235 C, 25 min
Morus alba leaves Flavonol ESI ITb -TOF C18 (150 mm 4.6 mm, A: H2 O (pH 3, [258]
glycosides (13 UV 2.7 m dp SPc ), FA), B: ACN (pH
avonoids) FR = 1.0 mL/min 3, FA)
(0.2 mL/min to MS),
25 C, 50 min
Hops (Humulus lupulus Flavan-3-ols, ESI+ Q-TOF C18 (50 mm 2.1 mm, A: 0.1% FA and [491]
L.) avonol UV 1.8 m dp ), 50 M
aglycones and FR = 0.25 mL/min, 45 C, ammonium
glycosides 36 min formate, B: 95%
ACN containing
0.1% FA and
50 M
ammonium
formate
Radix paeoniae rubra Catechin ESI TOF C18 (50 mm 4.6 mm, A: 0.3% FA, B: [492]
(TCM) UV 1.8 m dp ), 0.3% FA in ACN
FR = 0.8 mL/min, 25 C,
24 min
Durum wheat Anthocyanidins, ESI TOF C18 (150 mm 4.6 mm, A: 0.5% AAd , B: [493]
avone UV 1.8 m dp ), ACN
aglycones and FR = 0.5 mL/min
glycosides, (0.125 mL/min to MS),
isoavones and 40 C, 62 min
proanthocyani-
dins (42
avonoids)
Radix Puerariae (TCM) Isoavones (13 ESI TOF C18 (50 mm 4.6 mm, A: 0.1% FA, B: [494]
compounds) UV 1.8 m dp ), MeOHd
FR = 2.0 mL/min
(0.5 mL/min to MS),
46 C, 8 min
Wild Mexican Lupine Flavone and ESI+/ TOF C18 (100 mm 2.1 mm, A: 0.5% FA, B: [495]
(Lupinus reexus) isoavone ESI+/ IT 1.8 m dp ), 0.5% FA in ACN
conjugates (62 FR = 0.5 mL/min
avonoids) (0.2 mL/min to MS),
60 min
Granule decoctions of Flavone ESI+/ Q-TOF C18 (100 mm 2.1 mm, A: 50 mM [496]
TCM aglycones and UV 1.7 m dp ), ammonium
glycosides, FR = 0.5 mL/min, 35 C, acetate, B: ACN
avonols and 12 min
avanones (13
avonoids)
Buckwheat Flavonols, ESI TOF C18 (150 mm 4.6 mm, A: 1% AA, B: [497]
avan-3-ols 1.8 m dp ), MPd A/ACN
and FR = 0.5 mL/min (60/40, v/v)
procyanidins (0.125 mL/min to MS),
(28 avonoids) 25 C, 70 min
Rock rose (Cistus Flavan-3-ols, ESI TOF C18 (150 mm 4.6 mm, A: 0.5% AA, B: [498]
ldanifer) avonols and ESI IT 1.8 m dp ), ACN
avones (13 UV FR = 0.8 mL/min
avonoids) (0.25 mL/min to MS),
70 min
52 A. de Villiers et al. / J. Chromatogr. A 1430 (2016) 1678

Table 6 (Continued )

Sample Flavonoids Ionisation Detector(s) Stationary phase Mobile References

source (column dimensions), phase(s)
ow rate, temperature,
analysis time

Erigeron injection Flavonol, ESI Q-TOF C18 (50 mm 4.6 mm, A: 0.2% FA, B: [499]
avone and ESI QIT 1.8 m dp ), FR not ACN
avanone specied, 40 C, 30 min
glycosides (11
avonoids)
Tempranillo red wine Proanthocyanidins ESI+/ TOF C18 (100 mm 2.1 mm, A: 5% FA, B: [500]
(9 compounds) UV 1.7 m dp ), ACN
FR = 0.2 mL/min, 40 C,
13.6 min
Tomato fruit Aglycones and ESI+/ TOF C18 (150 mm 4.6 mm, A: 0.5% AA, B: [501]
glycosides of ESI+/ IT 1.8 m dp ), ACN
avonols, UV FR = 1.8 mL/min
avanones and (0.2 mL/min to MS),
avones (20 37 C, 46 min
avonoids)
Fructus Aurantii, TCM Flavanone ESI+ Q-TOF C18 (100 mm 2.1 mm, A: 0.1% FA, B: [502]
extracts and rat glycosides and UV 1.7 m dp ), ACN
plasma methoxylated FR = 0.4 mL/min, 25 C,
avones (21 15 min
avonoids) and
4 metabolites
Standards Flavonols, ESI+/ TOF C18 (100 mm 4.6 mm, A: 0.5% FA, B: [164]
avones, 1.8 m dp ), 0.5% FA in ACN
anthocyanins FR = 0.5 mL/min, 40 C,
and avanone 45 min
aglycones and
glycosides (19
avonoids)
Yinhuang granules Flavone ESI+ TOF C18 (50 mm 4.6 mm, A: 0.2% FA, B: [478]
(TCM) aglycones and UV 1.8 m dp ), ACN
glycosides (7 FR = 0.6 mL/min, 25 C,
avonoids) 17 min
Rooibos tea (Aspalathus Flavones, ESI+ Q-TOF C18 (200 mm 2.1 mm, A: 2% AA, B: [216]
linearis) avanone dihy- UV 1.7 m dp ), ACN
drochalcones FR = 0.3 mL/min, 30 C,
and avonols 31 min
(28 avonoids)
Avocado fruit Flavan-3-ols, ESI+/ TOF C18 (100 mm 2.1 mm, A: 0.1% FA, B: [285]
avanones and UV 1.8 m dp ), 0.1% FA in ACN
avonols (6 FR = 0.6 mL/min, 50 C,
avonoids) 45 min
Wheat varieties Flavone-O,C- ESI TOF C18 (150 mm 4.6 mm, A: 0.5% AA, B: [503]
diglycosides, 1.8 m dp ), ACN
avones, FR = 0.5 mL/min
isoavones, (0.125 mL/min to MS),
anthocyanins 40 C, 72 min
and proantho-
cyanidins (67
avonoids)
Rosemary (Rosmarinus Flavone ESI+/ TOF C18 (150 mm 4.6 mm, A: 0.1% FA, B: [504]
ofcinalis) aglycones and UV 1.8 m dp ), ACN
glycosides, FR = 0.8 mL/min
avonol (0.2 mL/min to MS),
glycosides, 55 min
avanones and
avonols (24
avonoids)
Licorice (Glycyrrhiza) Flavones, ESI Q-TOF C18 (50 mm 4.6 mm, A: 0.2% FA, B: [488]
isoavones, UV 1.8 m dp ), ACN
isoavane, FR = 0.7 mL/min, 30 C,
chalcones and 36 min
avanones (21
avonoids)
Medicago truncatula Flavones, ESI+ Q-TOF C18 (100 mm 2.1 mm, A: 0.5% FA, B: [479]
root samples isoavones and 1.8 m dp ), 0.5% FA in ACN
avanones (13 FR = 0.5 mL/min
avonoids) (0.2 mL/min to MS),
19 min
Red wine Anthocyanins ESI+ Q-TOF C18 A: 7.5% FA, B: [218]
and derived UV (2 100 mm 2.1 mm, 7.5% FA in ACN
pigments and 1.7 m dp ),
proantho- FR = 0.06 mL/min, 50 C,
cyanins (137 98 min
avonoids)
 A. de Villiers et al. / J. Chromatogr. A 1430 (2016) 1678 53

Table 6 (Continued )

Sample Flavonoids Ionisation Detector(s) Stationary phase Mobile References

source (column dimensions), phase(s)
ow rate, temperature,
analysis time

Rooibos tea (Aspalathus Flavonol and ESI TOF C18 (150 mm 4.6 mm, A: 1% FA in [424]
linearis) avone IT 1.8 m dp ), H2 O/ACN
aglycones and FR = 0.5 mL/min (90/10, v/v), B:
glycosides, (0.2 mL/min to MS), ACN
avanone and 25 C, 35 min
chalcone
glycosides (26
avonoids)
Hop cones (Humulus Flavonol ESI+/ TOF C18 (150 mm 2.0 mm, A: 0.1% FA, B: [259]
lupulus L.) aglycones and 2.6 m dp SP), 0.1% FA in ACN
glycosides, FR = 0.2 mL/min, 45 C,
avan-3-ols, 78 min
chalcones and
naringenin (13
avonoids)
Polygonaceae herbs Epicatechin ESI Q-TOF C18 (50 mm 4.6 mm, A: 0.1% FA, B: [505]
(TCM) UV 1.8 m dp ), ACN
FR = 0.8 mL/min
(0.4 mL/min to MS),
25 C, 32 min
Plant material Flavonol and ESI+ TOF C18 (100 mm 4.6 mm, A: 0.5% FA, B: [193]
avone 1.8 m dp ), 0.5% FA in ACN
glycosides, FR = 0.5 mL/min, 45 min
anthocyanins
and avanones
(17 avonoids)
Merlot (Vitis vinifera L.) Flavan-3-ol ESI Q-TOF C18 (50 mm 2.1 mm, A: 0.1% FA, B: [483]
seeds and wine monoglyco- UV 1.8 m dp ), 0.1% FA in ACN
sides (14 FR = 0.4 mL/min, 30 C,
compounds) 27 min
Fructus aurantii (TCM) Flavanone ESI+ Q-TOF C18 (50 mm 2.1 mm, A: 1% AA, B: [506]
glycosides and UV 1.7 m dp ), ACN
methylated FR = 0.2 mL/min, 40 C,
avones (6 12 min
avonoids)
Rooibos tea (Aspalathus Flavones, ESI+/ Q-TOF C18 (100 mm 4.6 mm, A: 2% AA, B: [210]
linearis) avanones UV 1.8 m dp ), ACN
dihydrochal- FR = 1.0 mL/min, 38 C,
cones and 45 min
avonols (26
avonoids)
Hops (Humulus lupulus Chalcones, ESI+/ Q-TOF C18 (100 mm 1.0 mm, A: 0.1% FA, B: [507]
L.) avanones and 1.8 m dp ), 0.1% FA in ACN
avonols (12 FR = 0.15 mL/min,
avonoids) 20 min
Galium spurium (TTMa ) Isoorientin, ESI+/ TOF C18 (100 mm 2.1 mm, A: 0.1% FA, B: [508]
rutin, 1.7 m dp ), 0.1% FA in ACN
isoquercitrin FR = 0.25 mL/min, 40 C,
and quercetin 26 min
Wheat (Triticum Flavone ESI+/ Q-TOF C18 (100 mm 2.1 mm. A: 0.5% FA, B: [480]
aestivum L.) glycosides (41 2.7 m dp SPc ), 0.5% FA in ACN
compounds) FR = 0.6 mL/min
(0.2 mL/min to MS),
15 min
Blueberries, red Anthocyanins ESI+ Q-TOF Amide A: 0.4% TFA in [128]
cabbage, red radish, (64 UV (150 mm 1.0 mm, ACN, B: 0.4%
grape skins and black compounds) 1.7 m dp ), TFA
beans FR = 0.025 mL/min,
50 C, 2740 min
Potato extracts Anthocyanins ESI+/ Q-TOF C18 (100 mm 2.1 mm, A: 0.5% FA, B: [292]
and avonol 1.8 m dp ), 90% MeOH
derivatives (11 FR = 0.4 mL/min, 50 C,
avonoids) 14 min
Hops (Humulus lupulus Proanthocyanidins ESI TOF C18 (50 mm 2.0 mm, A: 0.1% FA, B: [509]
L.) (40 1.7 m dp ), 0.1% FA in ACN
compounds) FR = 0.4 mL/min, 35 C,
9 min
Artichoke leaf Flavones ESI Q-TOF C18 (100 mm 1.0 mm, A: 0.1% FA, B: [510]
aglycones and ESI IT 1.8 m dp ), 0.1% FA in ACN
glycosides and UV FR = 0.15 mL/min,
hesperetin 16 min
glycoside (12
avonoids)
54 A. de Villiers et al. / J. Chromatogr. A 1430 (2016) 1678

Table 6 (Continued )

Sample Flavonoids Ionisation Detector(s) Stationary phase Mobile References

source (column dimensions), phase(s)
ow rate, temperature,
analysis time

Lettuce (Lactuca sativa) Flavonol, ESI Q-TOF C18 (150 mm 4.6 mm, A: 0.5% AA, B: [511]
avone and 1.8 m dp ), ACN
avanone FR = 0.8 mL/min, 40 min
glycosides (23
avonoids)
Trollius species (TCM) Flavone ESI Q-TOF Phenyl A: 0.05% FA, B: [89]
glycosides (34 (100 mm 2.1 mm, ACN
avonoids) 1.7 m dp ),
FR = 0.4 mL/min, 25 C,
30 min
Tea plant (Camellia Methylated ESI+ Q-TOF C18 (100 mm 2.1 mm, A: 0.5% FA [482]
sinensis) kaempferol and 1.8 m dp ), 2 mM
quercetin FR = 0.2 mL/min, 30 C, ammonium
regioisomers 30 min formate, B:
(10 avonoids) ACN
Peppers (Capsicum Flavonol, ESI TOF C18 (150 mm 4.6 mm, A: 0.5% AA, B: [191]
annuum L.) avone and 1.8 m dp ), ACN
avanone FR = 0.8 mL/min, 62 min
glycosides and
phloretin
dihexoside (23
avonoids)
Dan-Lou tablets (TCM) Isoavone and ESI+/ Q-TOF C18 (100 mm 4.6 mm, A: 0.1% FA, B: [288]
avanone UV 1.8 m dp ), ACN
glycosides (28 FR = 0.4 mL/min, 50 C,
avonoids) 40 min
Canola (Brassica napus Flavonol ESI+/ Q-TOF C18 (100 mm 1.0 mm, A: 0.1% FA, B: [512]
L.) organs aglycones and UV 1.8 m dp ), 0.1% FA in ACN
glycosides and FR = 0.15 mL/min,
naringenin (26 20 min
avonoids)
Whole lemon powder Flavanone, ESI TOF C18 (150 mm 4.6 mm, A: 0.5% AA, B: [513]
avonol and UV 1.8 m dp ), ACN
avone FR = 0.8 mL/min
aglycones and (0.2 mL/min to MS),
glycosides (16 25 C, 35 min
avonoids)
Halophyte Zygophyllum Isorhamnetin- ESI TOF C18 (50 mm 2.0 mm, A: 0.1% FA, B: [514]
album Desf. 3-O-rutinoside, UV 1.8 m dp ), ACN
malvidin FR = 0.4 mL/min, 23 C,
3-rhamnoside 70 min
and quercetin
3-sulphate
Cranberrybush Procyanidins (6 ESI TOF C18 (50 mm 2.1 mm, A: 0.1% FA, B: [515]
(Viburnum Opulus L.) compounds) UV 1.7 m dp ), MeOH
FR = 0.4 mL/min, 25 C,
8 min
Citrus bergamia juice Flavanone ESI IT-TOF C18 (100 mm 2.1 mm, A: 0.1% FA, B: [487]
glycosides (17 UV 1.7 m dp SP), 0.1% FA in ACN
compounds) FR = 0.7 mL/min
(0.35 mL/min to MS),
45 C, 6.1 min
Licorice (Raddix Liquiritin, ESI+ Q-TOF C18 (50 mm 2.1 mm, A: 0.5% AA, B: [516]
Glycyrrhizae) liquiritin UV 1.8 m dp ), ACN
apioside and FR = 0.2 mL/min, 30 C,
liquiritigenin 10 min
Apple, apple juice and Flavanols, ESI+/ Q-TOF C18 (100 mm 2.1 mm, A: 0.1% AA, B: [481]
apple cider avonols, dihy- 1.7 m dp ), 0.1% AA in
drochalcones FR = 0.35 mL/min, 40 C, MeOH
and 22 min
anthocyanins
(42 avonoids)
Licorice (Glycyrrhiza Flavanones, ESI Q-TOF C18 (100 mm 2.1 mm, A: 0.1% FA, B: [143]
species) avones, UV 1.8 m dp ), ACN
isoavones and FR = 0.6 mL/min, 50 C,
chalcones (45 9 min
avonoids)
Melon (Cucumis melo Eriodictoyl and ESI Q-TOF C18 (150 mm 4.6 mm, A: 0.5% AA, B: [517]
L.) diosmetin 1.8 m dp ), ACN
rutinosides and FR = 0.8 mL/min
hesperidin (0.2 mL/min to MS),
24 min
 A. de Villiers et al. / J. Chromatogr. A 1430 (2016) 1678 55

Table 6 (Continued )

Sample Flavonoids Ionisation Detector(s) Stationary phase Mobile References

source (column dimensions), phase(s)
ow rate, temperature,
analysis time

Green beans (Phaseolus Flavonols, ESI Q-TOF C18 (150 mm 4.6 mm, A: 0.5% AA, B: [518]
vulgaris L.) avones, 1.8 m dp ), 0.5% AA in ACN
avanones, FR = 0.8 mL/min
avan-3-ols (0.2 mL/min to MS),
and isoavones 25 C, 35 min
(60 avonoids)
Standards Flavone ESI+/ Q-TOF C18 (100 mm 2.1 mm, A: 0.1% FA [177]
glycosides (4 1.8 m dp ), (mode) or
compounds) FR = 0.2 mL/min, 0.1%
analysis time not ammonium
specied formate
(+mode), B:
0.1% FA
(mode) or
0.1%
ammonium
formate
(+mode) in
MeOH
Knotgrass (Polygonum Flavonol ESI Q-TOF C18 (150 mm 2.1 mm, A: H2 O/ACN/FA [407]
aviculare L.) glycosides (24 ESI/APCI+/ IT 1.9 m dp ), (95:5:0.1,
compounds) UV FR = 0.2 mL/min, 25 C, v/v/v), B: 0.1%
100 min FA in ACN
Shenqi Fuzheng Flavone and ESI+/ Q-TOF C18 (100 mm 2.1 mm, A: 0.1% FA, B: [519]
Injection (TCM) isoavone 1.8 m dp ), MeOH
glycosides (12 FR = 0.3 mL/min, 25 C,
avonoids) 40 min
Siwu decoction (TCM) Flavonols and ESI+/ Q-TOF C18 (100 mm 2.1 mm, A: 0.1% FA, B: [520]
avone UV 1.7 m dp ), ACN
glycosides (18 FR = 0.4 mL/min, 35 C,
avonoids) 25 min
Waterlemon (Citrullus Flavonol and ESI Q-TOF C18 (150 mm 4.6 mm, A: 0.5% AA, B: [521]
lanatus) extract avone 1.8 m dp ), ACN
glycosides (23 FR = 0.8 mL/min
avonoids) (0.2 mL/min to MS),
25 C, 35 min
Chemlali Olive Flavone, ESI TOF C18 (150 mm 4.6 mm, A: 0.5% AA, B: [426]
cultivar avonol ESI IT 1.8 m dp ), ACN
aglycones and UV FR = 0.8 mL/min, 25 C,
glycosides and 35 min
taxifolin (10
avonoids)
Sprouts (Lepidium Flavonol ESI Q-TOF C18 (50 mm 2.1 mm, A: 4.5% FA, B: [522]
sativum L.) glycosides (4 UV 1.7 m dp ), ACN
compounds) FR = 0.45 mL/min, 30 C,
15 min
Wine Flavan-3-ols, ESI TOF Serially coupled C18 C18: A: 10 mM [132]
avonols and (50 mm 3.0 mm, ammonium
eriodictyol-7- 2.7 m dp SP), acetate/ACN
glucoside (5 FR = 0.050.1 mL/min (90:10, v/v), B:
avonoids) and HILIC A: 10 mM
(150 mm 2.1 mm, ammonium
5 m dp ), acetate/ACN
FR = 0.4 mL/min, 27 min (10:90, v/v)
HILIC: A: ACN,
B: H2 O
Spanish Cistus species Flavan-3-ols, ESI TOF C18 (150 mm 4.6 mm, A: 0.5% AA, B: [523]
avonol 1.8 m dp ), ACN
aglycones and FR = 0.8 mL/min, 60 min
glycosides,
avanone and
isoavone
glycosides (29
avonoids)
Apple peel Flavonol ESI TOF C18 (100 mm 3.0 mm, A: 0.1% FA, B: [460]
glycosides, ESI QqQb 2.7 m dp SP), 0.1% FA in ACN
avan-3-ols FR = 0.3 mL/min, 50 C,
and 40 min
procyanidins
Gegen-Qinlian-Wan Flavones, ESI Q-TOF C18 (100 mm 2.1 mm, A: 0.1% FA, B: [524]
(TCM) isoavones, UV 1.8 m dp ), ACN
chalcones and FR = 0.3 mL/min, 40 C,
avanones (42 30 min
avonoids)
56 A. de Villiers et al. / J. Chromatogr. A 1430 (2016) 1678

Table 6 (Continued )

Sample Flavonoids Ionisation Detector(s) Stationary phase Mobile References

source (column dimensions), phase(s)
ow rate, temperature,
analysis time

Citrus grandis Flavanones, ESI+/ Q-TOF C18 (100 mm 2.1 mm, A: 0.1% FA, B: [525]
Tomentosa (TCM) avones and 2.6 m dp SP), MeOH
avonols (19 FR = 0.3 mL/min, 40 C,
avonoids) 16 min
Horehound Flavone ESI Q-TOF C18 (150 mm 4.6 mm, A: 0.5% AA, B: [526]
(Marrubium vulgare) aglycones and 1.8 m dp ), ACN
leaves glycosides (9 FR = 0.5 mL/min
avonoids) (0.167 mL/min to MS),
25 C, 45 min
Brazilian pepper Anthocyanins ESI+/ Q-TOF C18 (150 mm 2.1 mm, Anthocyanins: [96]
exocarp (Schinus and biavones QqQ 2.6 m dp SP), A: 1% FA, B:
terebinthifolius Raddi) (7 avonoids) UV FR = 0.4 mL/min, 40 C, 0.1% FA in ACN;
28 min non-
anthocyanins:
A: 0.1% FA, B:
0.1% FA in ACN
Orris root (Iris Flavonol ESI+/ TOF C18 (100 mm 2.1 mm, A: 5 mM [527]
rhizomes) aglycones and UV 1.8 m dp ), ammonium
glycosides and FR = 0.4 mL/min, 45 C, formate, pH
isoavones (18 12 min 3.8, B:
avonoids) MeOH/ACN
(50:50, v/v)
with 5 mM
ammonium
formate and
0.1% FA
Rhizome of Belamcanda Isoavone ESI+ Q-TOF C18 (100 mm 2.1 mm, A: 0.1% FA, B: [443]
chinensis (TCM) aglycones and ESI+ QqQ 1.7 m dp ), 0.1% FA in ACN
glycosides (26 FR = 0.4 mL/min, 20 C,
avonoids) 25 min
Mugwort (Artemisia Flavonol ESI Q-TOF C18 (150 mm 4.6 mm, A: H2 O/ACN [528]
vulgaris) leaves glycosides and 1.8 m dp ), (90:10, v/v)
luteolin FR = 0.5 mL/min, room with 0.1% FA, B:
rutinoside (5 temp, 50 min ACN
avonoids)
Raw and Isoavones and ESI+/ Q-TOF C18 (50 mm 2.1 mm, A: 0.1% FA, B: [109]
honey-processed isoavanes (12 1.8 m dp ), ACN
astragalus (TCM) avonoids) FR = 0.3 mL/min, 25 C,
23 min
Wu-tou decoction Isoavone, ESI+/ Q-TOF C18 (50 mm 2.1 mm, A: 0.1% FA, B: [529]
(TCM) chalcones and 1.7 m dp ), ACN
avanones (9 FR = 0.3 mL/min, 35 C,
avonoids) 25 min
Leaves of Myrtus Flavan-3-ol ESI Q-TOF C18 (150 mm 4.6 mm, A: 0.5% AA, B: [530]
communis L. derivatives and 1.8 m dp ), ACN
avonol FR = 0.8 mL/min, 25 C,
glycosides (10 32 min
avonoids)
Taselgha (Globularia Flavone, ESI Q-TOF C18 (150 mm 4.6 mm, A: 0.5% AA, B: [531]
alypum L.) avanone and 1.8 m dp ), ACN
avonol FR = 0.5 mL/min
glycosides, (0.167 mL/min to MS),
avan-3-ol 25 C, 45 min
derivatives,
avonols and
avanonols (11
avonoids)
Goji berry (Lycium Flavonol ESI Q-TOF C18 (100 mm 2.1 mm, A: 0.1% FA, B: [289]
barbarum) extracts glycosides (8 1.8 m dp ), 0.1% FA in
avonoids) FR = 0.3 mL/min, 50 C, MeOH
19.5 min
Commercial extra Apigenin and ESI TOF C18 (100 mm 4.6 mm, A: 0.1% FA, B: [532]
virgin olive oil luteolin 1.8 m dp ), ACN
FR = 0.5 mL/min, 30 min
Da-Huang-Gan-Cao- Flavan-3-ols, ESI+/ Q-TOF C18 (50 mm 4.6 mm, A: 0.1% FA, B: [533]
Tang chalcones, ESI+/ IT 1.8 m dp ), ACN
(TCM) isoavones and UV FR = 0.6 mL/min, 30 C,
avones (25 50 min
avonoids)
Hedyotis diffusa Willd. Flavonol and ESI Q-TOF C18 PEc A: 0.1% FA, B: [97]
(TCM) avone UV (100 mm 2.1 mm, ACN
glycosides (7 1.7 m dp ),
avonoids) FR = 0.25 mL/min, 25 C,
6577 min
 A. de Villiers et al. / J. Chromatogr. A 1430 (2016) 1678 57

Table 6 (Continued )

Sample Flavonoids Ionisation Detector(s) Stationary phase Mobile References

source (column dimensions), phase(s)
ow rate, temperature,
analysis time

Danggui San (TCM) Flavone ESI+/ Q-TOF C18 (100 mm 2.1 mm, A: 0.1% FA, B: [534]
aglycones and 1.7 m dp ), 0.1% FA in ACN
glycosides (18 FR = 0.3 mL/min, 35 C,
avonoids) 18 min
Fabaceae (Vicia faba L.) Flavan-3-ols, ESI Q-TOF C18 (150 mm 4.6 mm, A: 0.5% AA, B: [535]
proanthocyani- 1.8 m dp ), ACN
dins, avonols, FR = 0.8 mL/min
avanonols, (0.2 mL/min to MS),
avones and 25 C, 40 min
avanones (86
avonoids)
Black cumin seeds Flavonol ESI Q-TOF C18 (100 mm 1.0 mm, A: 0.1% FA, B: [536]
(Nigella sativa) glycosides (10 1.8 m dp ), 0.1% FA in ACN
avonoids) FR = 0.15 mL/min,
20 min
Hawthorn fruits Epicatechin, ESI Q-TOF C18 (50 mm 2.1 mm, A: 0.1% FA, B: [537]
hyperoside, 1.8 m dp ), ACN
isoquercitrin FR = 0.4 mL/min, 35 C,
and quercetin 12 min
Annurca apple (Malus Dihydrochalcones, ESI IT-TOF C18 (150 mm 2.1 mm, A: 0.1% AA, B: [124]
pumila Miller cv anthocyanins, 2.6 m dp SP), 0.1% AA in ACN
Annurca) avonols, FR = 0.5 mL/min, 40 C,
avanones, 27 min
avan-3-ols
and
procyanidins
(57 avonoids)
Leaves of Adhatoda Flavone and ESI Q-TOF C18 (150 mm 4.6 mm, A: 0.1% FA, B: [261]
vasica avonol UV 2.7 m dp SP), MeOH
aglycones and FR = 0.6 mL/min, 20 C,
glycosides (29 30 min
avonoids)
Temri and Tounsi Flavonols, ESI+/ Q-TOF C18 (150 mm 4.6 mm, A: 0.5% AA, B: [538]
(Ficus carica L.) fruits avones, UV 1.8 m dp ), ACN
avanones, FR = 0.5 mL/min, room
avanonols, temp, 40 min
avanols,
isoavones and
anthocyanins
(39 avonoids)
Scutellariae species Flavone ESI+/ Q-TOF C18 (100 mm 2.1 mm, A: 0.1% FA, B: [539]
(TCM) aglycones and QqQ 1.7 m dp ), ACN
glycosides, UV FR = 0.4 mL/min, 20 min
avanonol
glycosides,
isoavones,
avanones and
avonols (28
avonoids)
Araceae (Arum Flavone, ESI+/ Q-TOF C18 (150 mm 4.6 mm, A: 0.5% AA, B: [540]
palaestinum) avonol, UV 1.8 m dp ), ACN
avanone and FR = 0.8 mL/min, 25 C,
avanonol 70 min
glycosides,
anthocyanins,
isoavones,
avonol-
phenolic acid
derivatives (57
avonoids)
Euphorbiaceae Flavanone, ESI+/ Q-TOF C18 (100 mm 2.1 mm, A: 0.1% FA, B: [541]
(Phyllanthus amarus) avonol, ESI+/ IT 1.7 m dp ), ACN
avone UV FR = 0.3 mL/min, 30 C,
aglycones and 57 min
glycosides (18
avonoids)
Egyptian chickpea Flavonols, ESI Q-TOF C18 (150 mm 4.6 mm, A: 0.5% AA, B: [262]
cultivars (Cicer isoavones, UV 2.7 m dp SP), ACN
arientinum L.) isoavanones FR = 0.5 mL/min, 35 min
and
avan-3-ols
(47 avonoids)
58 A. de Villiers et al. / J. Chromatogr. A 1430 (2016) 1678

Table 6 (Continued )

Sample Flavonoids Ionisation Detector(s) Stationary phase Mobile References

source (column dimensions), phase(s)
ow rate, temperature,
analysis time

Berries (Rhodomyrtus Flavones and ESI+ Q-TOF C18 (100 mm 2.1 mm, A: H2 O, B: ACN [542]
tomentosa (Ait.) avonols (6 1.7 m dp ),
Hassk) avonoids) FR = 0.3 mL/min, 30 C,
30 min
Jinqi Jiangtang tablet Rutin, ESI+/ Q-TOF C18 (50 mm 2.1 mm, A: 0.2% [543]
(TCM) luteoloside and 1.7 m dp ), phosphoric
isoquercitrin FR = 0.3 mL/min, 35 C, acid, B: MeOH
21 min
Rhubarb species Flavan-3-ol ESI Q-TOF C18 (150 mm 2.1 mm, A: 0.1% FA, B: [265]
derivatives, 1.6 m dp SP), ACN
procyanidins FR = 0.3 mL/min, 40 C,
and avanone 29 min
glycosides (15
avonoids)
Ginkgo biloba Isoavone and ESI+ Q-TOF C18 (150 mm 2.1 mm, A: 0.1% FA, B: [544]
avonol UV 2.7 m dp SP), 0.1% FA in ACN
aglycones and FR = 0.2 mL/min, 21 min
glycosides (22
avonoids)
Mentha pulegium and Flavanols, ESI Q-TOF C18 (150 mm 4.6 mm, A: 0.5% AA, B: [545]
Origanum Majorana avones, 1.8 m dp ), ACN
species avanonols, FR = 0.8 mL/min, 25 C,
avanone and 35 min
avonols (33
avonoids)
Shuang-Huang-Lian Flavone ESI Q-TOF C18 (100 mm 2.1 mm, A: 0.1% FA, B: [546]
oral liquid (TCM) aglycone and ELSDb 1.7 m dp ), 0.1% FA in ACN
glycosides (4 FR = 0.4 mL/min, 40 C,
avonoids) 24 min
a
TCM, traditional chinese medicine, TTM, traditional Turkish medicine.
b
Q, quadrupole; TOF, time-of-ight mass spectrometer; IT, ion trap; QqQ, triple quadrupole mass spectrometer; ELSD, evaporative light scattering detector.
c
FR, ow rate; SP, supercially porous; PE, polar embedded.
d
TFA, triuoroacetic acid; ACN, acetonitrile; FA, formic acid; AA, acetic acid; MeOH, methanol; MP, mobile phase.

Fig. 16. (A) UV chromatogram obtained at 280 nm for the UHPLCQ-TOF-MS analysis of an apple extract. (B) The extracted ion chromatograms for phloretin-2 -O-xylosyl-
glucoside (m/z 569) and quercetin-3-O-xyloside (m/z 435) and (C) the corresponding MSE spectra for the compounds obtained using the simultaneous (non-selective)
acquisition of spectral data using a collision energy ramp of 1040 eV. Analysis conditions: 100 mm 2.1 mm 1.7 m porous C18 phase, 40 C, 0.35 mL/min; ESI+ detection
at 10 Hz.
Source: Reproduced with permission from [481].
 A. de Villiers et al. / J. Chromatogr. A 1430 (2016) 1678
1,1-diphenyl-2-picrylhydrazyl (DPPH) radical to determine the
most active antioxidants. This methodology proved to be more
sensitive than the conventional on-line DPPH assay.
Q-TOF-MS has also been hyphenated with HILIC [128], with
an amide column operated at 50 C being used for the analysis of
diverse anthocyanins in blueberries, grape skins, black beans, red
cabbage and red radish.
The widespread use of TOF, and especially Q-TOF detectors in
contemporary LCMS analysis of avonoids makes sense in light of
the high resolution mass data and high acquisition rates offered by
these detectors and their relative cost compared to higher resolv-
ing power instruments. The mass accuracy of the latest generation
TOF detectors are sufcient to allow determination of molecular
formulae for avonoids of MW up to 1200 [128] and to distinguish
between isobaric compounds based on this data. Furthermore, the
MS/MS capabilities of Q-TOF systems are critical in tentative struc-
tural elucidation of avonoids. Wojakowska et al. [480] found a
resolving power of higher than 15,000 sufcient for molecular for-
mula determined for avonoids of MW up to 700 in rst order
spectra, but estimated that a resolving power of 40,000 would be
needed to attain the same level of mass accuracy of MS/MS spec-
tra. This is within the reach of several commercial instruments.
While the mass accuracy of Orbitrap and especially FT-ICR sys-
tems are certainly much better than TOF systems, and therefore
hold clear benets for the separation of high MW compounds such
as proanthocyanins, the performance of TOF systems for ultra-fast
separations currently remains unmatched between the high reso-
lution instruments.
4.2.3. Fourier transform ion cyclotron resonance (FT-ICR) MS
FT-ICR [547,548] is the mass spectrometric technique with the
highest mass resolution (up to greater than 106 ) and sub-femtomol
sensitivity. The technique works on the principle of exciting all
ions in the cyclotron simultaneously and measuring the ion image
resulting from their combined trajectories, which is amplied
and translated to the frequency domain using FT [423]. The res-
olution depends on the observation time, and as a consequence
FT-ICR instruments are too slow to allow on-line hyphenation to
UHPLC separation, and are generally used only in infusion mode for
avonoid analysis [507]. An alternative is to use at-line or off-line
coupling to HPLC separations, which essentially involves collection
of fractions from the chromatographic system and subsequent infu-
sion into the FT-ICR. While time consuming and labour intensive,
this is currently the most practical way to exploit the high resolving
power of FT-ICR in combination with HPLC separation, and shows
promise for example in metabolomic analysis [42]. By 2006 FT-ICR
had not been used in the LCMS analysis of avonoids [145], and
to the best of our knowledge the only application of LCFTICR-MS
to avonoid analysis since then was reported by Suzuki et al. [549].
These authors used conventional HPLC (5 m ODS phase) with posi-
tive mode ESI to characterise avonoids in Lotus japonicas. The mass
spectrometer was operated with a resolving power of 100,000 (at
m/z 400), although no information was provided on the scan time
and the TIC was not presented. On-going developments aimed at
extending the resolving power and reducing the time domain tran-
sient length of FT-ICR instruments may in future allow hyphenation
of the detector to fast HPLC separations [42].
4.2.4. Orbitrap instruments
The Orbitrap mass analyser, rst described by Makarov
[550,551], was introduced on the market in 2005. This system relies
on the orbital trapping of ions around a spindle-like central elec-
trode and an outer ared barrel-like electrode using electrostatic
elds. The frequency of the harmonic ion oscillations, which are
inversely proportional to the square root of the m/z ratios, is mea-
sured as an image current transient which is converted to frequency
59
spectrum using FT [421]. In the rst commercial conguration
(LTQ-Orbitrap), ions are initially stored in a linear ion trap, from
where they are co-axially ejected into a bent quadrupole or C-trap
to focus and quickly inject them into the Orbitrap. The resolving
power of these instruments can be as high as 500,000, depending
on the particular instrument, scan mode and scan time.
Various forms of bench-top Orbitrap mass spectrometers have
been utilised in the analysis of avonoids. In the LTQ-Orbitrap XLTM
a collision cell is added after the C-Trap to perform CID for MS/MS
analyses; the LTQ-Orbitrap Velos shows increased MS/MS sensi-
tivity due a more effective ion guide. The Orbitrap EliteTM uses a
smaller Orbitrap with an increased electric eld, increasing the
resolving power of the instrument to 240,000 at m/z = 400. A mod-
ied version of this instrument utilising a compact analyser has
been shown to attain a resolving power of 960,000 at m/z 400 for a
3 s detection time [552]. In the Orbitrap ExactiveTM , the ion source
is directly connected to the C Trap followed by a collision cell
before the Orbitrap analyser. Ions that underwent fragmentation
in the collision cell are transferred back into the C-Trap before
being injected into the Orbitrap for mass analysis. The latest edi-
tion is the non-hybrid Q-ExactiveTM , which has a lower resolving
power (140,000 at m/z 200) but provides higher scan speeds than
previous versions (12 Hz at a resolving power of 17,500), making it
compatible with high speed separations.
The utility of high resolution MS and MS/MS data obtained
through utilisation of Orbitrap instruments in infusion mode has
been demonstrated for the identication of polyphenols [553,554],
while a number of reports utilising conventional HPLC with Orbit-
rap detectors have also been reported [150,192,555560]. For
example, Lamuela-Ravents and co-workers used conventional
HPLC methods in combination with an LTQ-Orbitrap Velos to study
the phenolic composition of tomatoes [448], beer [561], walnut
[562] and sofrito [563], while Cahill et al. [564] developed and
validated an HPLC-Orbitrap method for quantifying isoavones in
waste water following APCI ionisation. In the following discussion
pertaining to Table 7, the focus will however be on selected appli-
cations of advanced LC methods hyphenated to Orbitrap detection
for avonoid analysis.
The majority of these have to date been performed using the
LTQ-Orbitrap XLTM instrument. Li et al. [565] used this instru-
ment coupled to a UHPLC separation to identify 15 avonoids in
the traditional Chinese medicine (TCM) Sarcandra glabra, including
glycosylated avanones, avanonols and a avanol, based on neg-
ative ESI MSn spectra and accurate mass information. Analysis of
Serbian honeys [566568] was achieved on a 1.9 m dp column,
while Orbitrap MS/MS was used to identify up to 31 avonols,
avanonols, avanones and avones. An example of the benet
of high resolution MS data is demonstrated for these samples in
Fig. 17, where four peaks are observed in the EIC obtained at 269.06
(Fig. 17(b), 200 ppm mass precision). Extraction of masses 269.0459
and 269.0822 at 1 ppm mass precision (Fig. 17(c) and (d), respec-
tively) shows the peak pairs observed for closely eluting apigenin
and galangin (m/z 269.0459) and alpinetin and pinostrobin (m/z
269.0822), respectively, as conrmed by data-dependent MS/MS
operation [566].
Lebel et al. [290] used a LTQ-Orbitrap XLTM instrument for the
quantitative analysis of erectile dysfunction ingredients, including
9 natural avonoids. The authors used full scan mode for deter-
mination of accurate mass and data-dependent MS/MS of targeted
precursors. Limits of detection for the avonoids varied between 3
and 23 pg on-column.
Tchoumtchoua et al. [163] reported a comprehensive structure-
oriented approach based on UHPLCESI-MS/MS using an LTQ-
Orbitrap DiscoveryTM instrument for the characterisation of
isoavonoids (isoavones, dihydroisoavones and their cor-
responding glycosides) in natural products. The proposed
60 A. de Villiers et al. / J. Chromatogr. A 1430 (2016) 1678

Table 7
Applications of advanced LC hyphenated to Orbitrap and ion mobility mass spectrometry to avonoid analysis (20092015).

Sample Flavonoids Ionisation Detector(s) Stationary phase Mobile References

source (column dimensions), phase(s)
ow rate, temperature,
analysis time

Sarcandra glabra Flavanonols, ESI LITb -Orbitrap C18 (100 mm 2.1 mm, A: 0.1% FAd , B: [565]
(TCMa ) avanones, avonones UV 1.9 m dp ), ACNd
and avonols (15 FRc = 0.6 mL/min,
avonoids) 20 min
Red mustard greens Anthocyanins and ESI+/ LIT-Orbitrap C18 (200 mm 2.1 mm, A: 0.1% FA, B: [189]
avonol-glycosides UV 1.9 m dp ), 0.1% FA in ACN
(169 avonoids) FR = 0.3 mL/min, 65 min
Blueberry, Hongcaitai Anthocyanins and ESI LIT-Orbitrap C18 (200 mm 2.1 mm, A: 0.1% FA, B: [287]
and red radish avonol aglycones and 1.9 m dp ), 0.1% FA in ACN
glycosides (64 FR = 0.3 mL/min, 50 C,
avonoids) 65 min
Huang-Lian-Jie-Du Flavones and ESI+/ Qb -Orbitrap C18 (100 mm 2.1 mm, A: 0.05% FA, B: [444]
decoction (TCM) avanones (24 QqQb 1.7 m dp ), 0.05% FA in
avonoids) FR = 0.45 mL/min, 35 C, MeOHd
12 min
Vitus vinifera L. juice Flavonoids ESI+/ Q-Orbitrap C18 (100 mm 2.1 mm, A: 0.1% FA, B: [572]
(unspecied) 2.6 m dp SPc ), 0.1% FA in
FR = 0.3 mL/min, 40 C, MeOH
20 min
Apple Flavonol, avone, ESI Orbitrap C18 (150 mm 3.0 mm, A: 0.1% FA, B: [112]
avanone aglycones (single-stage) 1.7 m dp ), 0.1% FA in ACN
and glycosides, UV FR = 0.5 mL/min, 40 C,
chalcones, avanonols, 23 min
avanols and
anthocyanins (27
avonoids)
Whole coffee fruit Procyanidins, ESI Orbitrap C18 (150 mm 3.0 mm, A: 0.1% FA, B: [263]
avan-3-ols and (single-stage) 2.6 m dp SP), ACN
avonol glycosides (12 UV FR = 0.7 mL/min
avonoids) (0.2 mL/min to MS),
40 C, 20 min
Euterpe oleracea palm Flavonol aglycones and ESI LIT-Orbitrap C18 (100 mm 2.1 mm, A: 0.1% FA, B: [575]
juice glycosides, avone 1.8 m dp ), 0.1% in MeOH
aglycones and FR = 0.4 mL/min, 30 C,
glycosides, 35 min
anthocyanins,
avanonols and
avan-3-ols (29
avonoids)
Berry extracts Flavonols, avanones ESI LIT-Orbitrap C18 (50 mm 2.1 mm, A: 0.1% FA, B: [576]
and avones (18 1.9 m dp ), 0.1% FA in ACN
avonoids) FR = 0.4 mL/min, 9 min
Olive oil Flavone aglycones and ESI LIT-Orbitrap C18 (100 mm 2.1 mm, A: 0.1% AA, B: [577]
glycosides and 2.7 m dp SP), ACN
quercetin-3-O- FR = 0.4 mL/min, 33 min
rutinoside (7
avonoids)
Serbian unioral Flavonols, avanonols, ESI LIT-Orbitrap C18 (50 mm 2.1 mm, A: 0.1% FA, B: [566]
honeys avanones and 1.9 m dp ), 0.1% FA in ACN
avones (31 FR = 0.4 mL/min, 9 min
avonoids)
Stem bark of Amphimas Isoavones, ESI+/ LIT-Orbitrap C18 (50 mm 2.1 mm, A: 0.1% FA, B: [163]
pterocarpoides isoavanones and APCI+/ UV 1.9 m dp ), MeOH
avonols (19 FR = 0.2 mL/min, 12 min
avonoids)
Brassica species Anthocyanins and ESI+/ LIT-Orbitrap C18 (200 mm 2.1 mm, A: 0.1% FA, B: [569]
microgreens avonol glycosides UV 1.9 m dp ), 0.1% FA in ACN
(135 avonoids) FR = 0.3 mL/min, 65 min
Rorippa indica (Linn.) Flavonol glycosides ESI LIT-Orbitrap C18 (200 mm 2.1 mm, A: 0.1% FA, B: [570]
Hiern. (Cruciferae) and acylated glycosides UV 1.9 m dp ), 0.1% FA in ACN
(46 avonoids) FR = 0.3 mL/min, 65 min
Strawberry (Fragaria Dihydroavonols, ESI+/ LIT-Orbitrap C18 (200 mm 2.1 mm, A: 0.1% FA, B: [217]
vesca) avonol aglycones and UV 1.9 m dp ), 0.1% FA in ACN
glycosides, FR = 0.3 mL/min, 60 C,
proanthocyanidins and 65 min
anthocyanins (19
avonoids)
Lithocarpus Dihydrochalcones, ESI+/ LIT-Orbitrap C18 (100 mm 4.6 mm, A: 0.1% FA, B: [578]
polystachyus (TCM) avan-3-ols, 1.8 m dp ), MeOH
avanones and FR = 0.6 mL/min, 50 min
avanonols (46
avonoids)
 A. de Villiers et al. / J. Chromatogr. A 1430 (2016) 1678 61

Table 7 (Continued )

Sample Flavonoids Ionisation Detector(s) Stationary phase Mobile References

source (column dimensions), phase(s)
ow rate, temperature,
analysis time

Lime honey Flavonol glycosides (15 ESI LIT-Orbitrap C18 (100 mm 2.1 mm, A: 0.1% FA, B: [568]
avonoids) 1.9 m dp ), 0.1% FA in ACN
FR = 0.3 mL/min, 13 min
Rhizoma Smilacis Flavanonols, ESI LIT-Orbitrap C18 (100 mm 3.0 mm, A: 0.1% FA, B: [579]
glabrae avan-3-ols and UV 1.7 m dp ), 0.1% FA in ACN
procyanidins (23 FR = 0.3 mL/min, 30 C,
avonoids) 10 min
Serbian lime honey Flavones, avonols and ESI LIT-Orbitrap C18 (50 mm 2.1 mm, A: 1% FA, B: [567]
avanones (24 1.9 m dp ), ACN
avonoids) FR = 0.3 mL/min, 15 min
Soy based nutraceutical Isoavones, avones, ESI+/ Orbitrap C18 (100 mm 2.1 mm, A: 30 mM [574]
products avonols, avanones (single-stage) 1.7 m dp ), ammonium
and chalcones (49 FR = 0.2 mL/min, 30 C, acetate, B:
avonoids) 47.5 min MeOH
Adulterated or Flavone aglycones and ESI+ LIT-Orbitrap C18 (100 mm 2.1 mm, A: 0.1% FA, B: [290]
counterfeit erectile glycosides, isoavones 2.6 m dp SP), 0.1% FA in ACN
dysfunction products and prenylated FR = 0.35 mL/min, 50 C,
avonols (9 avonoids) 10 min
Jujubu, fuji apple, Proanthocyanidins (76 ESI LIT-Orbitrap C18 (200 mm 2.1 mm, A: 0.1% FA, B: [571]
litchi, mangosteen, avonoids) UV 1.9 m dp ), 0.1% FA in ACN
chocolate, grape and FR = 0.3 mL/min, 65 min
cranberry extracts
Serbian poplar type Flavonols, avanonols, ESI LIT-Orbitrap C18 (50 mm 2.1 mm, A: 1% FA, B: [580]
propolis avones and 1.9 m dp ), ACN
avanones (56 FR = 0.3 mL/min, 15 min
avonoids)
Fufang decoction Flavanones aglycones ESI+/ LIT-Orbitrap C18 (50 mm 2.1 mm, (+mode) A: [581]
(TCM) and glycosides and 1.9 m dp ), 0.1% FA, B:
avones (10 FR = 0.3 mL/min, 30 C, MeOH;
avonoids) 31 min (mode) A:
H2 O, B: MeOH
Mulberry (Morus alba Anthocyanins, avonol ESI+/ LIT-Orbitrap C18 (100 mm 2.1 mm, (mode) A: [425]
L.) aglycones and ESI QqQ 1.9 m dp ), 0.1% FA, B: 0.1%
glycosides, avan-3-ol UV FR = 0.3 mL/min, 40 C, FA in ACN;
derivatives and 12 min (+mode) A: 1%
avanone glycosides FA, B: ACN
(35 avonoids)
Olive tree (Olea Flavone aglycones and ESI LIT-Orbitrap C18 (100 mm 2.1 mm, A: 0.1% FA, B: [291]
europaea) organs glycosides, avonol UV 2.7 m dp SP), ACN
aglycones and FR = 0.8 mL/min, 35 min
glycosides, avanols
and avanonols (20
avonoids)
Ragweed (Ambrosia Flavonol aglycones and ESI LIT-Orbitrap C18 (50 mm 2.3 mm, A: 0.1% FA, B: [582]
artemisiifolia L.) glycosides (16 1.8 m dp ), 98% ACN
pollen avonoids) FR = 0.3 mL/min, 9 min
Nepeta species Flavonols, avones and ESI LIT-Orbitrap C18 (100 mm 2.1 mm, A: 1% FA, B: [583]
avanones (18 QqQ 1.7 m dp ), ACN
avonoids) UV FR = 0.3 mL/min, 30 C,
15 min
Green tea nutraceutical Flavan-3-ol derivatives, ESI+/ Orbitrap C18 (100 mm 2.1 mm, A: 30 mM [573]
products avonol aglycones and (single-stage) 1.7 m dp ), Ammonium
glycosides, avone FR = 0.2 mL/min, 30 C, acetate
aglycones and 16 min acidied to pH
glycosides (33 5 with FA, B:
avonoids) MeOH
Standards and urine Flavone metabolites ESI+ IMb -LIT PFPc A: 2% AAd , B: [462]
(diosmetin-3-O- (FAIMSb ) (100 mm 2.1 mm, 2% AA in MeOH
glucoronide, ESI+ QqQ (MRM) 5 m dp ),
-7-O-glucoronide and FR = 0.4 mL/min, room
-3,7-O-diglucoronide) temp
Cauliower waste Flavonol-glycosides ESI Q-IM-TOF C18 (150 mm 2.1 mm, A: 0.1% FA, B: [584]
and acylated (TWIMSb ) 1.7 m dp ), 0.1% FA in
avonol-glycosides (24 FR = 0.25 mL/min, 40 C, MeOH
avonoids) 45 min
Black tea thearubigins Theasinensins, B-type ESI Q-IM-TOF C18 (100 mm 2.1 mm, A: 0.1% AA, B: [585]
proanthocyanidin (TWIMS) 1.7 m dp ), 0.1% AA in ACN
dimers and rutin (5 FR = 0.3 mL/min, 35 C,
avonoids) 35 min
a
TCM, traditional Chinese medicine.
b
LIT, linear ion trap; Q, single quadrupole; QqQ, triple quadrupole; IM, ion mobility; FAIMS, eld-asymmetric waveform ion mobility spectrometry; TWIMS, travelling
wave ion mobility spectrometry.
c
FR, ow rate; SP, supercially porous; Pentauorophenylpropyl.
d
FA, formic acid; ACN, acetonitrile; MeOH, methanol; AA, acetic acid.
62 A. de Villiers et al. / J. Chromatogr. A 1430 (2016) 1678
Fig. 17. UHPLCESI-Orbitrap analysis of an ethyl acetate extract of Serbian honey. (a) depicts the TIC obtained in negative ionisation mode, (b) the extracted ion chromatogram
for m/z 269.09 at 200 ppm mass precision, and (c) m/z 269.0459 and (d) 269.0822 at 1 ppm mass precision, respectively. Apigenin and galangin (c) and pinetin and pinostrobin
(d) were identied based on MS/MS spectra.
Source: Reproduced with permission from [566].
methodology was illustrated for the determination of isoavones
in an Amphimas pterocarpoides crude extract. Chromatographic, UV
and MS information was combined to prioritise potential com-
pounds of the target class, which were further characterised based
on accurate mass fragmentation information. The structures of
selected tentatively identied compounds were further conrmed
by NMR, and investigated using APCI and ESI MS/MS following
infusion of the isolated compounds. For methylated isoavones,
positive mode APCI was found to provide useful information for
structural elucidation purposes.
The utility of UHPLCDAD-ESI-HR-MSn in the proling of
avonoids in foods is amply illustrated by a series of papers
from the group at the US Department of Agriculture. These
authors used an LTQ-Orbitrap XLTM (operated at a resolving
power of 15,000) with positive and negative ESI and a 1.9 m
RP-LC column to screen a range of phenolics in various sam-
ples. For example, 30 anthocyanins and 105 avonol glycosides
were identied in microgreens [569], 67 anthocyanins and 102
avonol glycosides in red mustard greens [189], 19 avonoids
in strawberries [217], 46 avonol glycosides and acylated gly-
cosides in Rorippa indica [570] and 247 proanthocyanidins in
seven different food materials [571]. In the latter report, the
authors derived relative molar response factors for ion counts in
SIM operation from UV data, and used these to quantify (sepa-
rated) proanthocyanidins in grape seeds. Despite the assumptions
inherent to this approach, the methodology represents a signif-
icant contribution in the eld of proanthocyanin analysis, since
quantication of these compounds is impossible using UV detection
only due to extensive co-elution and the lack of suitable commercial
standards.
In another interesting paper, the group investigated the use of
negative ESI behaviour of anthocyanins [287]. These compounds
are normally detected in positive ionisation mode due to the preva-
lence of the avylium cationic species in solution, but may be hard
to distinguish from avonol-O-glycosides due to identical nominal
masses and (low-energy) fragmentation patterns. The motivation
here was to use negative ionisation to discriminate between these
avonoid classes. Indeed, unusual [M2H] and [M2H+H2 O]
ions were observed for the anthocyanins, for which the authors pro-
posed a possible mechanism of formation. However, a more likely
explanation is that these ions are due to the deprotonated quinoidal
and carbinol pseudobasic forms of anthocyanins, which are present
in the column due to the low acidity of the mobile phase (0.1%
formic acid) these species are not separated chromatographically
due to their relatively fast inter-conversion [105]. In our opinion
negative ionisation of anthocyanins makes little sense in light of
the fact that these compounds can normally be distinguished from
other classes based on UV characteristics, and failing that based
on distinct high collision energy CID spectra of anthocyanidins
[180]. Furthermore, analysis of anthocyanins using mobile phases
containing only 0.1% formic acid leads to signicant and evident
band-broadening for these compounds, thereby exacerbating co-
elution and providing an additional criterion to distinguish them
from avonols, if needed.
 A. de Villiers et al. / J. Chromatogr. A 1430 (2016) 1678
The Q-ExactiveTM Orbitrap has also found application in
avonoid analysis. Palermo et al. [557] used this instrument in
combination with HPLC separation to identify 40 avonoids in arti-
chokes. The resolving power of the instrument was set to 25,000
FWHM for a scan time of 1 s. Yang et al. used this instrument in
combination with an HPLC method for the qualitative analysis of
the TCM Huang-Lian-Jie-Du decoction (HLJDD). 24 avonoids were
identied, some of which were then used as markers for quality
control and quantied in selected samples using a UHPLCQqQ-
MS method utilising polarity switching [444]. Tarr et al. [572] used
a supercially porous phase and Q-ExactiveTM Orbitrap MS in a
metabolomics study to investigate the effect of terroir and variety
on Vitis vinifera L. grape juices. The metabolic signatures in negative
and positive ionisation showed clustering according to varietal and
terroir properties, respectively, while avonoid proles obtained
from identication of 466 compounds using a avonoid database
were found to be good indicators of varietal character indepen-
dent of terroir. Garrido Frenich and co-workers reported UHPLCMS
methods based on the Q-ExactiveTM Orbitrap for the qualitative and
quantitative analysis of avonoids in green tea [573] and soy based
nutraceutical products [574]. MS data were acquired in full MS (no
fragmentation, resolving power 25,000 FWHM; scan time = 0.25 s)
and all-ion fragment (collision energy 30 eV, 10,000 FWHM; scan
time = 0.10 s) modes in both positive and negative ionisation. The
overall scan rate for the four acquisition functions was 0.56 Hz. LOD
values varying between 1 and 50 g/L were reported for a range of
different avonoids. Mullen et al. [263] described an HPLC method
employing a supercially porous phase and an ExactiveTM Orbitrap
mass spectrometer for the (semi)-quantitative analysis of procyani-
dins, avonols and hydroxycinnamic acids in whole coffee fruits,
beans and husks from China, India, and Mexico. The instrument
resolving power was set to 60,000, although the scan speed was
not specied.
It should be noted that although the Orbitrap is a true high
resolution instrument (providing for example resolving powers of
60,000100,000 FWHM for ow injection analysis [190,553,554]),
this performance is highly dependent on the scan time and mode.
In LCMS applications where fast or high-resolution separation is
performed, the instrument is commonly used at a resolution set-
ting of between 7500 and 30,000 to acquire a sufcient number of
spectra across each peak, and lower values for MS/MS acquisition
[422]. (In fact, depending on the number of scan modes utilised,
scan speeds are often insufcient for high-speed separations on
older Orbitrap instruments). In practice therefore Orbitrap instru-
ments provide little gain in resolving power compared to the latest
generation TOF systems for such applications. On the other hand,
the compromise between sensitivity and scan time is lower than
for other HR mass analysers [422].
The compromises involved in parameter selection for quanti-
tative UHPLC-Orbitrap analysis have been elegantly addressed by
De Paepe et al. [112]. These authors found a scan speed of 2 Hz,
obtained at a resolving power of 50,000 (WFHM at m/z 200) in MS
mode, to be optimal using an ExactiveTM instrument (for an anal-
ysis time of 23 min; for fast gradients resolution would need to be
adapted to increase the scan speed). Furthermore, mass accuracy
performance is also related to the dynamic range, which in turn
is related to the scan range. It is therefore clear that careful opti-
misation of MS (and MS/MS) parameters is required for optimal
performance of Orbitrap instruments.
Current generation Orbitrap instruments do not compete with
TOF systems in terms of scan speed, which explains the more
widespread use of the latter in very fast separations and LC LC,
for example. However, considering the extensive developments
in Orbitrap technology which have in a relatively short time
resulted in improved mass resolution on the one hand and
scan speeds on the other, it is possible that this will become
63
a real alternative to TOF analysers for such analyses in the
future.
4.2.5. Ion mobility spectrometry
Ion mobility spectrometry (IMS) measures the drift-time of
ions through a buffer gas in the presence of an electric eld. The
drift-time of an ion depends on its size/charge ratio ions can
therefore be separated based on differences in their net charge
or collision cross-sections, or by interactions with the gas. The
technique has found extensive application in the detection of explo-
sives, drugs and chemical warfare agents. IMS is ideally compatible
with mass spectrometry and provides complementary informa-
tion, which explains the growth in popularity of ion mobility-mass
spectrometry (IM-MS) [586]. IM-MS is especially used in the sep-
aration of isobaric compounds and isomers, conformers and in
the measurement of ion size. Interestingly, because of the rela-
tive timeframes of chromatographic separations (s), IMS (ms) and
MS (s), the technique may be considered a form of comprehen-
sive 3D separation, since the separation obtained in each previous
dimension is essentially maintained. A range of different IMS
methods may be used in IM-MS, including traditional drift-time ion
mobility (DTIMS), aspiration ion mobility (AIMS), eld-asymmetric
waveform ion mobility (FAIMS) and travelling wave ion mobility
(TWIMS). A diverse range of IM-MS instrumentation is furthermore
available, with IMS having been hyphenated to magnetic sector,
TOF, quadrupole, IT and FT-ICR instruments. For further details
on the various instrumental congurations available, the reader is
referred to [587].
IM-MS has found relatively sparse application in avonoid anal-
ysis to date (Table 7), with most work done in infusion mode (i.e.
without chromatographic pre-separations). For example, Clowers
et al. [588] described a method using a dual gate IM-LIT mass
spectrometer to separate cation adducts of avonoid-diglycosides.
This type of mass spectrometer can operate either in a dual gate
scanning (DGS) or single mobility monitoring (SMM) mode. In
DGS mode a complete mobility spectrum was obtained. Once the
drift-time(s) were obtained using DGS, ions can be selectively
transmitted to accumulate a specic ion population to improve
efciency. Troc et al. [589] demonstrated the (travelling wave) IM-
MS separation of the epimers (+)-catechin and ()-epicatechin by
complexation with chiral modiers and transition metals.
In contrast, avone-glycosides were separated under ambi-
ent pressures using a differential ion-mobility spectrometer [462].
This method included a chromatographic separation using a PFP
column. For the identication of diosmetin-7-O-glycoside and
diosmetin-3-O-glycoside an IMS coupled to a Q-LIT analyser was
used.
More recently, IM-MS has also been used in combination with
UHPLC separation for avonoid applications. Yassin et al. [585]
described a method employing UHPLC coupled to a Q-IM-TOF
mass spectrometer to separate isomeric avanols unresolved by
chromatographic separation. The authors showed that isomeric
structures of B-type prodelphinidins and theasinensins as well
as the isobaric compound rutin could be resolved based on their
mobility drift times in the negative ion mode. Gonzales et al. [584]
used the additional selectivity offered by ion mobility separation
to obtain cleaner mass spectra to assist in the identication of acyl-
ated and non-acylated avonol-glycosides. In both of these works,
a commercial instrument equipped with a TWIMS cell [475] was
used.
In TWIMS, the continuous low electric eld of traditional IMS
is replaced by sweeping a high eld sequentially through the cell
[587]; this approach provides low IMS resolution, but high sensitiv-
ity and is ideally suited for IMS-MS. This system is equipped with
trap and transfer cells before and after the IMS cell, and manip-
ulation of collision energy in these can be used for example to
64 A. de Villiers et al. / J. Chromatogr. A 1430 (2016) 1678

Fig. 18. Methodology for assignment of daughter ions based on two separate ion mobility-MS experiments and classical least squares data modelling. Data shown for
isovitexin (apigenin-6-C-glucoside).
Source: Reproduced with permission from [590].

allow parent ions to enter the IMS cell (low collision energy in
trap cell) and fragment the separated ions before transfer to the
TOF (high transfer cell collision energy) [585]. Garmn-Lobato et al.
[590] reported an interesting chemometric method for using IMS-
MS measurements to assign daughter ions formed in the transfer
cell (i.e. after IMS separation) to specic pre-cursor ions. Since
CID fragmentation is also performed in the trapping cell, this
approach provides equivalent information to MS3 experiments, but
for all precursor ions simultaneously. The procedure is illustrated
schematically in Fig. 18.
4.2.6. Overview of mass spectrometers used in avonoid analysis
Fig. 19 summarises graphically the relative proportion of appli-
cations listed in Tables 1, 2 and 47 which utilised different MS
detectors (single quadrupole applications are not included). It
should be pointed out that this is not an accurate reection of the
global picture in avonoid analysis because of the focus in the cur-
rent work on advanced LC separations (and inevitable bias based
on the incomplete data set). Nevertheless, for applications falling
within the scope of this work, the relative popularity of TOF sys-
tems is clear (50% of all applications) and not unexpected based on
 A. de Villiers et al. / J. Chromatogr. A 1430 (2016) 1678 65

trace-level targeted analysis of avonoids in complex mixtures in

Fig. 19. Overview of the application of different MS instruments in the advanced
LCMS analysis of avonoids (data based on references listed in Tables 1, 2, 47,
20092015).
performance characteristics such as speed, resolving power and
cost. Furthermore, Q-TOF instruments in particular have found
extensive application due to the combination of tandem MS and
high resolution data provided by these systems, which is especially
useful for structural elucidation of avonoids. Orbitrap instruments
currently represent the only alternative high resolution systems for
use in combination with advanced LC separation, but are still rel-
atively new additions to the market; it is likely that these systems
will nd increasing application in avonoid analysis in the near
future due to similar advantages as TOF instruments (although cur-
rently still lower scan speeds). Triple quadrupole instruments are
the second most utilised, and will likely nd continued applica-
tion in the eld due to their suitability for trace-level and targeted
analyses.
5. Conclusions and perspectives
Advances in LCMS analysis of avonoids largely mirror, and
to a degree build on those in elds such as proteomics and
metabolomics, which are underpinned by signicant developments
in both HPLC and MS instrumentation. Most inuential have been
technological advances in MS, and translation of these to commer-
cial instruments which are becoming more pervasive in routine
laboratories. Notable are the performance of the latest genera-
tion of QqQ instruments, which makes these systems ideal for the
combination with ultra-fast and high resolution separations. For
structural elucidation and non-targeted analyses, however, high
resolution and hybrid HR-MS systems have proven indispensable.
The popularity of TOF, and especially Q-TOF instruments in com-
bination with advanced LC separation is due to the performance of
these systems in terms of acquisition speed, sensitivity, mass range
and tandem MS capabilities. In recent years, Orbitrap instruments
have gained increasing popularity in avonoid analysis, although
the resolving power-scan speed compromise currently still favours
TOF systems for hyphenation to advanced separations.
In terms of chromatographic separation, the widespread
contemporary availability of UHPLC instrumentation and the per-
formance characteristics of the latest generation sub-2 m porous
and supercially porous phases have made faster and more efcient
separation of large numbers of avonoids possible without sac-
ricing robustness. The recent availability of supercially porous
phases of particle sizes of 1.3 m promises to further extend perfor-
mance gains. The complementary benets of elevated temperature
operation is increasingly being exploited in the modest range
of 5080 C; higher temperatures are unlikely to become com-
mon due to column/instrument limitations and concerns regarding
the thermal stability of avonoids. LC LC has been shown to
deliver high-resolution separation of highly complex mixtures of
avonoids. The relative complexity of the technique has limited its
use to date to that of primarily being a research tool, but the recent
availability of commercial instruments will entice further applica-
tions to avonoid analysis. One of the primary application areas
of LC LC has been in the analysis of complex proanthocyanidin
mixtures, where the demonstrable benets of the technique will
ensure its continued application.
All of these developments have ensured that many more
avonoids can now be identied in a single analysis through
judicious selection of separation conditions and suitable MS instru-
mentation, although it is worth noting that these assignments
remain tentative in the absence of reference standards; NMR there-
fore remains an essential tool in avonoid characterisation.
The extent of the advances in LCMS and their implications in
terms of the quality of analytical data obtainable are often not fully
appreciated. Fig. 20 presents an attempt to highlight the evolution
of avonoid analysis, using anthocyanins as an example. Compar-
ison of the contemporary situation with the rst HPLC separation
of wine anthocyanins in 1978 shows a clear increase in separation
performance coinciding with improvements in column technology
and LC LC operation. Concurrent progress in hyphenation to MS

Fig. 20. Evolution of the HPLC analysis of anthocyanins. (A) One of the rst applications of RP-LC-UV to the analysis of grape anthocyanins. Conditions: C18 column
(250 mm 4.6 mm, 5 m dp ), 10% formic acid/methanol gradient at 2.5 mL/min, UV detection at 520 nm. Reproduced with permission from [29] 1978 American Society for
Enology and Viticulture. AJEV 29:4249. (B) UHPLCESI-QqQ-MS analysis of ros wine showing the MRM transitions for 152 phenolic compounds. Conditions: C18 column
(150 mm 1 mm, 1.8 m dp ), 1% formic acid/methanol gradient at 0.17 mL/min, 40 C. Reproduced from [438]. (C) depicts the high efciency UHPLCESI-Q-TOF-MS analysis
of red wine anthocyanins. Conditions: C18 column (200 mm 2.1 mm, 1.7 m dp ), 7.5% formic acid/acetonitrile gradient at 0.06 mL/min, 50 C. Reproduced with permission
from [218]. (D) presents a UV contour plot obtained for the off-line HILIC RP-LC analysis of grape anthocyanins. Conditions: 1 D: amide column (150 mm 4.6 mm, 2.5 m
dp ), 0.4% TFA/acetonitrile gradient at 0.2 mL/min, 50 C, 1 ts = 0.5 min; 2 D, supercially porous C18 column (50 mm 4.6 mm, 2.6 m dp ), 7.5% formic acid/acetonitrile gradient
at 0.5 mL/min, 50 C, ESI-Q-TOF-MS and UV detection at 500 nm. Reproduced with permission from [248].
66 A. de Villiers et al. / J. Chromatogr. A 1430 (2016) 1678

detection now allows selective analysis of large numbers of target DESI desorption electrospray ionisation
compounds by tandem MS, whereas HR-MS hyphenated to high- DGS dual gate scanning
resolution separations enables identication of large numbers of DP degree of polymerisation
unknown compounds. In fact, comparison of the reports cited in DPPH 1,1-diphenyl-2-picrylhydrazyl radical
this work with literature from only ten years ago clearly highlights DTIMS drift-time ion mobility spectrometry
the performance gains in terms of both analysis time reduction and ED electrochemical detection
the number of compounds that can be separated and tentatively EI electron (impact) ionisation
identied allowed by the hyphenation of advanced separation and ELSD evaporative light scattering detector
MS technologies. Considering the pace of developments in HPLC ESI electrospray ionisation
and MS, any prediction of future progress is necessarily fraught FAB fast atom bombardment
with uncertainty; it is however clear that the growing application FAIMS eld-asymmetric waveform ion mobility spectrometry
of advanced LCMS methods to avonoid analysis will increasingly FL uorescence
play an important role in support of avonoid research. FR ow rate
FT Fourier transform
Symbols and abbreviations GC gas chromatography
HILIC hydrophilic interaction chromatography
HPLC high performance liquid chromatography
Symbols HRF heterolytic ring ssion
under-sampling correction factor HR-MS high resolution mass spectrometry
xD xth dimension HTLC high temperature liquid chromatography
Dm diffusion coefcient of the analyte in the mobile phase i.d. internal diameter
x DF dilution factor in the xth dimension ICR ion cyclotron resonance
P pressure drop IEC ion exchange chromatography
Pmax maximum pressure IM-MS ion mobility-mass spectrometry
dp particle size IMS ion mobility spectrometry
external porosity IT ion trap
T total porosity LC liquid chromatography
fc fractional surface coverage LCMS liquid chromatographymass spectrometry
xF volumetric ow rate in the xth dimension LC LC
comprehensive two-dimensional liquid
H height equivalent of a theoretical plate (typically in m) chromatography
Hmin minimum height equivalent of a theoretical plate LOD limit of detection
Kvo column permeability (typically in m2 ) LIT linear ion trap
L column length MALDI matrix-assisted laser desorption ionisation
max wavelength of maximum absorbance MB moving belt
N number of theoretical plates (dimensionless) MD-LC multidimensional liquid chromatography
 mobile phase viscosity MRM multiple reaction monitoring
n c,2D (practical) peak capacity of a two-dimensional separation MS mass spectrometry
xn peak capacity of separation in the xth dimension MS/MS tandem mass spectrometry
c
x standard deviation in dimension x MSn multistage mass spectrometry (where n 2)
1t rst dimension sampling time MW molecular weight
s
xt gradient time in the xth dimension NMR nuclear magnetic resonance
g
uopt optimal mobile phase linear velocity (typically in mm/s) NP-LC normal phase liquid chromatography
u0 mobile phase linear velocity (determined using unre- ODS octadecyl-silica
tained marker) OEG oligo-ethylene glycol
Vfrac fraction volume PD plasma-desorption
xV injection volume in the xth dimension PEG polyethylene glycol
inj
wb peak width at baseline PFP pentauorophenyl
ppm parts per million
Abbreviations Q quadrupole
1-D one-dimensional QM quinone methide fragmentation pathway
2-D two-dimensional QqQ triple quadrupole
ABTS 2,2 -azino-bis(3-ethylbenzothiazoline)-6 sulfonic acid RDA retro-DielsAlder
AIMS aspiration ion mobility spectrometry RP-LC reversed phase liquid chromatography
amu atomic mass units SEC size exclusion chromatography
APCI atmospheric pressure chemical ionization SIM selected ion monitoring
API atmospheric pressure ionization SMM single mobility monitoring
APPI atmospheric pressure photoionisation SR split ratio
BFF benzofuran-forming ssion SRM selected reaction monitoring
CCC countercurrent chromatography TCM traditional Chinese medicine
CD cyclodextrin TFA triuoroacetic acid
CE capillary electrophoresis TLC thin layer chromatography
CI chemical ionisation TOF time-of-ight
CID collision induced dissociation TSP thermospray
CPC centrifugal partition chromatography TWIMS travelling wave ion mobility spectrometry
DAD diode array detection UHPLC ultra high pressure liquid chromatography (pressures
DART direct analysis in real time >400 bar)
 A. de Villiers et al. / J. Chromatogr. A 1430 (2016) 1678
Acknowledgements
The authors gratefully acknowledge Stellenbosch University,
Sasol, the National Research Foundation (NRF, South Africa, grant
81830 to AdV), Restek (Academic Support Program Grant to AdV)
and the International Foundation of Science (IFS, Sweden, Grant
F/4904-1 to AdV) for nancial support.
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