Microchemical Journal 118 (2015) 124–130

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Microchemical Journal
journal homepage: www.elsevier.com/locate/microc

Determination of toxic substances, pesticides and mycotoxins, in ginkgo

biloba nutraceutical products by liquid chromatography
Orbitrap-mass spectrometry
Gerardo Martínez-Domínguez, Roberto Romero-González, Antonia Garrido Frenich ⁎
Research Group “Analytical Chemistry of Contaminants”, Department of Chemistry and Physics, Research Centre for Agricultural and Food Biotechnology (BITAL), University of Almeria,
Agrifood Campus of International Excellence, ceiA3, E-04120 Almeria, Spain

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

Article history: More than 250 toxic substances, including pesticides and mycotoxins, have been identified and quantified in
Received 17 July 2014 ginkgo biloba nutraceutical products using liquid chromatography (LC) coupled to high resolution mass
Received in revised form 3 September 2014 spectrometry (Exactive-Orbitrap). A “dilute and shoot” procedure, using acetonitrile with formic acid (1% v/v),
Accepted 4 September 2014
was applied for the extraction of the target compounds. In this case, after this procedure, a clean-up step was
Available online 16 September 2014
necessary using a mixture of sorbents (PSA, GBC, C18 and Z-Sep+). Parameters such as trueness, repeatability, in-
Keywords:
termediate precision, limits of detection and quantification were evaluated. Recoveries between 70 and 120%
Nutraceutical with relative standard deviation (RSD) values lower than 20% were obtained for 260 of the compounds. Limits
Ginkgo biloba of detection and quantification below 5 and 10 μg kg−1 were obtained, respectively. Nine samples of ginkgo
Pesticides biloba nutraceutical products were analyzed, finding pesticides in 8 out of 9 samples, such as hymexazol
Mycotoxins (10 μg kg−1) and tebufenozide (55–459 μg kg−1). In the case of mycotoxins, aflatoxin B1 (5–54 μg kg−1), aflatoxin
Ultra high performance liquid chromatography B2 (4–300 μg kg−1) and T-2 toxin (18–20 μg kg−1) were found in 6 samples.
High resolution mass spectrometry © 2014 Elsevier B.V. All rights reserved.

1. Introduction
Current lifestyle has provoked that society is looking for food alterna-
tives in order to obtain a good nutritional diet in a fast way [1]. Moreover,
until 2010 nutrition related factors in developing nations are responsible
for 40% of mortality worldwide [2]. Dietary supplements and functional
foods, also known as nutraceutical products, represent a perfect option
to reduce this lack of a good nutrition, and therefore, the nutraceutical
market has been growing since the last decade, providing different alter-
natives to prevent and treat modern diseases [1].
The term nutraceutical, as defined by DeFelice, is a combination of the
words “nutrition” and “pharmaceutical” referring to the foods intended to
impart a physiological or medicinal effect after delivered ingestion in pre-
sentations that differ from conventional foods or beverages, such as
capsules, tablets, liquid extracts [1]. The National Nutraceutical Center in
the United States defines three types of nutraceuticals: dietary supple-
ments (e.g. vitamins, minerals and plant extracts), functional foods
(e. g. essentials oils) and medicinal foods (e.g. energy bars) [3]. As can
be seen, a large variety of products can be considered as nutraceuticals
and defining a global legislation that includes all these products is not a
straightforward procedure.
⁎ Corresponding author. Tel.: +34 950015985; fax: +34 95005008.
E-mail address: agarrido@ual.es (A. Garrido Frenich).
Among the plants that can be used for the preparation of
nutraceuticals, ginkgo biloba is widely used, obtaining the extract
from ginkgo leaves. Different medicinal properties have been given to
this nutraceutical, such as preventing and treating memory loss,
Alzheimer sickness, vascular disease, vertigo and glaucoma [4], mainly
because the presence of flavonoids and terpene lactones found in
ginkgo leaves [5].
Keeping in mind that a plant extract is a concentrated form of this
product and that the plant itself can be exposed to different toxic sub-
stances involved in its agricultural process, pesticides and/or myco-
toxins could be detected in the final nutraceutical presentation. There
are some pesticide analysis performed in dietary supplements, such as
Scutellaria baicalensis and Acacia catechu [6], ginseng and dandelion [7,
8]. For mycotoxins, soy isoflavones [9] green coffee beans [10], ginger
[11], herbs [12] wheat and oat [13] have been studied. In the case of gink-
go biloba, few works have been made to detect pesticides [14–16], not
reporting positives in any of them. Regarding mycotoxins, aflatoxins
[17,18] and ochratoxin A, zearalenone, deoxynivalenol, T-2 toxin and
fumonisins [18] were studied in ginkgo leaves, finding concentrations be-
tween 9.1 (zearalenone) and 134.1 μg kg−1 (deoxynivalenol) [18] in the
analyzed samples. It is important to mention that these studies have
been carried out only in the raw material and not in nutraceutical
products. As can be seen, pesticides and mycotoxins can be found in
ginkgo biloba, so analytical methodologies that offer multi-class studies

http://dx.doi.org/10.1016/j.microc.2014.09.002
0026-265X/© 2014 Elsevier B.V. All rights reserved.
 G. Martínez-Domínguez et al. / Microchemical Journal 118 (2015) 124–130
are necessary in order to ensure food safety in this particular nutraceutical
product.
Regarding European legislation, there exist regulations that define
maximum residue limits (MRLs) of these contaminants. The Regulation
EC 396/2005 [19] defines MRLs for pesticides in every food and feed,
while the Regulation EC 1881/2006 [20] establishes MRLs for myco-
toxins in foodstuffs. However, these MRLs are set in the raw material
and they are not applied to nutraceutical products. Therefore, method-
ologies that can determine and quantify these toxic substances in
ginkgo biloba nutraceutical products at low levels are necessary.
Bearing in mind the lack of analytical methodologies to determine
these toxic substances in ginkgo biloba, the revised literature also in-
cluded methods used for Chinese herbs and other extracts. Concerning
the extraction procedure for pesticides, mainly the QuEChERS (quick,
easy, cheap, effective, rugged and safe) [16,21,22], pressurized liquid
extraction (PLE) [23,24], liquid–solid extraction (LSE) [25] and “dilute
and shoot” [26] procedures were used. In the case of mycotoxins, specific
separation with immunoaffinity columns [17,18,27] is mainly used,
although there are other procedures that can be applied, such as the
micro solid phase extraction [28].
To achieve the chromatographic separation of pesticides, gas chro-
matography (GC) [21,23–26] and liquid chromatography (LC) [16,22]
are basically used. Regarding mycotoxins, LC [17,27,28] is commonly
utilized. These chromatographic techniques have been coupled to dif-
ferent detectors, including low and high resolution mass spectrometers.
Single quadropole (Q) [21,26] and triple quadrupole (QqQ) [16,22,23]
have been the detectors most widely used for the detection of pesti-
cides, whereas for mycotoxins, detectors such as QqQ [17,27] and time
of flight (QTOF) [28] have been used. Nowadays, high resolution mass
spectrometry is becoming more important because it allows the deter-
mination and quantification of a large quantity of compounds from dif-
ferent classes and families, obtaining high resolving power, accurate
mass measurement and high full scan sensitivity [29]. Furthermore, a
large number of compounds can be determined at the same time, and
more than 300 compounds can simultaneously be analyzed by the
Exactive-Orbitrap [30] and QTOF [31].
For these reasons, the use of high resolution mass spectrometry is
needed in order to provide a comprehensive analysis of multi-class
toxic substances in ginkgo biloba nutraceutical products. Therefore, in
this work a methodology has been developed to identify more than
250 toxic substances, including pesticides and mycotoxins, in ginkgo
biloba nutraceuticals, studying different extraction procedures and
using ultra high performance liquid chromatography (UHPLC) coupled
to Exactive-Orbitrap for determination and quantification.
2. Materials and methods
2.1. Reagents and chemicals
Pesticide reference standards (purity higher than 99%) were pur-
chased from Dr. Ehrenstofer (Augsburg, Germany) and Riedel-de-
Haën (Seelze-Hannover, Germany). Mycotoxins reference standards
were purchased from LGC Standards (Wesel, Germany). Individual
stock standard solutions of 200 mg L− 1 (pesticides and mycotoxins)
were prepared by exact weighing of powder or liquid and dissolved in
50 mL of HPLC-grade acetone (Sigma-Aldrich, Madrid, Spain) for pesti-
cide solutions or 50 mL of acetonitrile LC–MS (Scharlab, Barcelona,
Spain) for mycotoxins. Multicompound working standard solutions
(344 compounds) for pesticides and mycotoxins (2 mg L−1 concentra-
tion of each compound) were prepared by appropriate dilutions of the
stock solutions with acetone or acetonitrile, respectively, and stored
under refrigeration at 4 °C. Water LC–MS (Scharlab, Barcelona, Spain)
was used for the preparation of buffers and other solutions. Formic
acid (Optima LC–MS) was obtained from Fisher Scientific (Geel,
Belgium). Ammonium formate was obtained from Fluka (St. Gallen,
Switzerland). All solvents were pesticide residue grade. Primary
125
secondary amine (PSA) and graphitized black carbon (GBC) were ob-
tained from Scharlab (Barcelona, Spain). Bondesil-C18 was obtained
from Agilent Technologies (Santa Clara, CA, USA). Anhydrous magne-
sium sulfate, sodium acetate, zirconium oxide (Z-Sep+) and methanol
LC–MS were obtained from Sigma-Aldrich.
2.2. Instrument and apparatus
A Consul centrifuge from Orto Alresa (Madrid, Spain) was used for
centrifugation. To achieve end-over-end agitation, a Reax 2 overhead
shaker from Heidolph (Schwabach, Germany) was used. In order to
homogenize the samples, a WX vortex from Velp Scientifica (Usmate,
Italy) was used.
For chromatographic analysis, a Transcend 600 LC (Thermo Scientific
Transcend™, Thermo Fisher Scientific, San Jose, CA, USA) coupled to a
single stage Orbitrap mass spectrometer (Exactive™, Thermo Fisher
Scientific, Bremen, Germany) was used. The system operated with a
heated electrospray interface (HESI-II, Thermo Fisher Scientific, San Jose,
CA, USA) in positive (ESI+) and negative ionization mode (ESI−). A
Hypersil GOLD aQ column (100 × 2.1 mm, 1.9 μm particle size) from Ther-
mo Scientific (San Jose, CA, USA) was used for compound separation.
In order to process the data, the Xcalibur™ program version 2.2.1
from Thermo Fisher Scientific (Les Ulis, France) with Qual and
Quanbrowser was used. In this case, Genesis peak detection was
applied. The ToxID™ program 2.1.1 and the LCQuan™ 2.6 software,
also from Thermo Scientific, were used for screening and quantification
of the target compounds, respectively.
2.3. Samples
A ginkgo biloba nutraceutical product was obtained from a local
store and was used for blank, fortified samples for recovery assays and
for matrix-matched standards during calibration purposes. For sample
study, three products were obtained from local supermarkets in
Almeria, Spain, other three from pharmacies in Krakow, Poland and
other three ordered from the United States. From these samples, most
of them are gingko leaves extracts, except for two samples, one that is
a mixture of ginkgo biloba and whitethorn (Crataegus oxyacantha)
(sample 6) and another one that also contains brown rice (sample 7).
Sample preparation was carried out by homogenizing the contents
of twenty capsules or tablets with a coffee grinder and stored with a
desiccant at 5 °C in order to eliminate variations between them.
2.4. Extraction procedures
2.4.1. Procedure I: acetate QuEChERS
First, acetate QuEChERS [32] was studied following the next steps:
(1) 2 g of sample was weighed in a 50 mL centrifuge tube and fortified
with the appropriate volume of the multicompound standard solutions
(pesticides and mycotoxins) to achieve a final concentration of
100 μg kg−1; (2) 8 mL of water was added to the mixture and it was
shaken vigorously for 30 s; (3) 10 mL of acetonitrile acidified with acetic
acid at 1% (v/v) was added and the tube was shaken vigorously for
1 min; (4) 4 g of magnesium sulfate and 1 g of sodium acetate were
added to the mixture and shaken vigorously for 1 min, and (5) the
tube was centrifuged at 3700 rpm (2755 g) for 10 min and 1 mL of the
organic phase was transfer to a vial for injection into the LC–Orbitrap-
MS.
2.4.2. Procedure II: “dilute and shoot”
A “dilute and shoot” method tested in a previous work [30] was
applied following the next steps: (1) 2.5 g of sample was weighed in a
50 mL centrifuge tube and fortified with the appropriate volume of
the multicompound standard solutions (pesticides and mycotoxins) to
achieve a final concentration of 100 μg kg− 1; (2) 2.5 mL of water
was added to the mixture and it was shaken vigorously for 30 s;
126 G. Martínez-Domínguez et al. / Microchemical Journal 118 (2015) 124–130
(3) 7.5 mL of acetonitrile acidified with formic acid (1% v/v) was added
to the mixture and the tube was shaken end-over-end for 1 h, and
(5) the tube was centrifuged at 3700 rpm (2755 g) for 10 min
and 1 mL of the extract was transferred to a vial for injection into the
LC–Orbitrap-MS.
2.4.2.1. Clean up step. A clean-up step was tested using a mixture of dif-
ferent sorbents (PSA, GBC, C18, and Z-Sep+). After the centrifugation
step described previously, 1.5 mL of the extract was transferred to an
Eppendorf microtube containing 200 mg of magnesium sulfate and
50 mg of each of the sorbents previously defined and the tube was shak-
en vigorously for 1 min. Then, the tube was centrifuged at 3700 rpm
(1438 g) for 10 min and 1 mL of the organic phase was transferred to
a vial for injection into the LC–Orbitrap-MS.
2.5. UHPLC–Orbitrap-MS
A mixture of an aqueous solution of ammonium formate 4 mM and
formic acid 0.01% (v/v) (eluent A) and methanol solution with ammoni-
um formate 4 mM and formic acid 0.01% (v/v) (eluent B) was used as
mobile phase at a constant flow rate of 0.3 mL min−1. The gradient for
the analysis of the compounds was applied as follows: the analysis
started with 95% of eluent A. After 1 min, this percentage was linearly
decreased to 0% in 7 min. This composition was held during 4 min and
increased again up to 95% in 0.5 min, followed by a re-equilibration
time of 1.5 min. The total running time was 14 min. Column tempera-
ture was set at 30 °C and the injection volume was 10 μL.
The Orbitrap mass spectrometer parameters were as follow: spray
voltage, 4 kV; skimmer voltage, 18 V (−18 V in ESI−); capillary voltage,
35 V (−35 V in ESI−); heater temperature, 305 °C; capillary tempera-
ture, 300 °C; sheath gas (N2, N95%), 35 (adimensional); auxiliary gas
(N2, N 95%), 10 (adimensional); and tube lens voltage, 95 V (−95 V in
ESI−). For full scan experiments, a mass range of m/z 100–1000 was de-
fined without lock mass. Mass accuracy was monitored as follows: the
use of multi-compound standards everyday to check performance;
mass accuracy standards evaluation at least once a week and calibration
every two weeks. For the automatic gain control (AGC), a target value of
1 × 106 was set. To obtain the mass spectra, four alternating acquisition
functions were used: (1) full MS, ESI +, without fragmentation (the
higher collisional dissociation (HCD) collision cell was switched off),
mass resolving power = 25000 FWHM; scan time = 0.25 s; (2) full
MS, ESI− using the same settings; (3) MS/MS, ESI+, with fragmenta-
tion (HCD on, collision energy = 30 eV), mass resolving power =
10000 FWHM; scan time = 0.10 s; and (4) MS/MS, ESI− using the set-
tings explained before. An overall scan rate of 0.56 Hz was obtained con-
sidering the scan time of the four acquisition functions and the polarity
switching (0.27 s).
2.6. Validation procedure
The method was validated in order to ensure that the results were
reliable before its application in samples. The parameters studied were
linearity, trueness, intraday precision (repeatability), interday precision
(intermediate precision), limits of detection (LODs) and quantification
(LOQs).
For linearity, matrix matched standard calibration was used by
analyzing spiked blank samples of ginkgo biloba nutraceuticals at five
concentration levels (2.5, 5.0, 12.5, 25.0 and 50.0 μg L−1). For trueness,
recovery studies were evaluated by spiking blank samples with the
corresponding volume of the multi-compound working standard
solutions (pesticides and mycotoxins). Concentrations of 10, 50
and 100 μg kg− 1 were studied by spiking five blank samples at each
concentration.
Repeatability and intermediate precision, expressed as relative stan-
dard deviation (RSD), were studied from the injection of five spiked
samples at 10, 50 and 100 μg kg−1 for repeatability, and three spiked
samples in five different days at the same concentration levels for inter-
mediate precision.
Finally, to determine LODs and LOQs, five fortified samples were
injected at lower concentration levels, being 0.5, 1.0, 2.0, 5.0 and
10.0 μg kg− 1. In this case, the LOD was defined as the minimum con-
centration where the molecular ion can be identified (mass error
value below 5 ppm) and the LOQ as the minimum concentration
where it can be observed a linear response (mass error value below
5 ppm).
3. Results and discussion
The Orbitrap-MS parameters were optimized in a previous work
[33]. The exact mass database for all the studied compounds, including
elemental composition, retention time windows (RTWs), theoretical
masses and accurate mass errors for the detected ions is presented in
the supplementary data (Table S-1).
3.1. Optimization of the extraction procedure
First, the acetate QuEChERS [32] and the “dilute and shoot” [34]
methodologies were compared following Procedures I and II, re-
spectively, fortifying blank samples at 100 μg kg− 1. These two pro-
cedures were chosen because they are quick, cheap and robust.
Also, the solvent quantities used in the experiments are very low.
In particular, the QuEChERS methodology has been used to deter-
mine pesticides in different matrices as mentioned before, whereas
the “dilute and shoot” procedure has been applied to extract pesti-
cides and mycotoxins from different nutraceuticals, such as green
tea and royal jelly [33], as well as it has been successfully applied
for the simultaneous extraction of hormones and veterinary drugs
from complex matrices [34]. The procedures were tested for 344
compounds included in the working standard solution. The results
can be seen in Fig. 1a.
It can be observed that similar results were obtained using both pro-
cedures. The “dilute and shoot” methodology extracted 77% of the com-
pounds with recoveries between 70 and 120%, while the QuEChERS
procedure extracted 64% of the compounds with suitable recoveries. Be-
cause the amount of solvents and salts is used in the QuEChERS method,
the “dilute and shoot” procedure represents a better option for analyti-
cal laboratories. Therefore, the “dilute and shoot” methodology was
selected for further experiments.
Although good results were achieved at a fortified level of
100 μg kg− 1, when lower concentrations were tested (50 μg kg− 1)
only 27% of the compounds could be extracted with recoveries
between 70 and 120%. Taking into account the complex matrix that
represents ginkgo biloba nutraceuticals, an additional clean up step
after the “dilute and shoot” procedure is recommended in order to
obtain better results and minimize matrix effect.
In this case, a mixture of sorbents (PSA, GBC, C18, and Z-Sep+) was
used following the steps presented in Section 2.4.2.1. The results can
be observed in Fig. 1b. It can be observed that the use of a clean-up
step greatly improves the results, extracting 77% of the compounds
with suitable recoveries at 50 μg kg−1. Although good results were
achieved with this clean up step, 23% of the compounds were still unde-
tected, probably because these compounds need a specific method for
their determination and quantification. Therefore, for the validation
procedure a clean-up step was included using a mixture of sorbents
(PSA, GBC, C18, and Z-Sep+) in order to improve the results at lower
concentrations.
3.2. Analytical method validation
The optimized method was validated for 260 compounds using the
best conditions defined previously.
 G. Martínez-Domínguez et al. / Microchemical Journal 118 (2015) 124–130 127

Fig. 1. Recoveries obtained for all the compounds studied using: (a) the QuEChERS (Procedure I) and “dilute and shoot” methodologies (Procedure II); and (b) the “dilute and shoot” meth-
odology without clean-up and with a clean-up step using a mixture of different sorbents (PSA, GBC, C18 and Z-Sep+). Samples were fortified at 100 μg kg−1 (a) and 50 μg kg−1 (b).

The matrix effect was studied for all the compounds analyzed, and it
was estimated using the following equation:
 
Am
ME ¼ −1  100
As
where As is the peak area for the solvent calibration point and Am the
peak area for the matrix calibration point. When the obtained value is
between −20 and +20% it can be concluded that matrix effect can be
considered negligible. From this study, only 3% of the compounds
analyzed do not present matrix effect, whereas 94% of them got values
below − 20%, presenting matrix suppression. Furthermore, matrix
enhancement was observed for 3% of the compounds (values above
20%). As mentioned before, the complexity of the matrix studied is the
main reason why this matrix effect is still presented even after a
clean-up step was applied.
Therefore, in order to avoid the mentioned matrix effects, matrix-
matched calibration standards were used for all experiments. For linear-
ity evaluation, least-squares regression of peak area versus concentra-
tion of the calibration standards in the whole range was applied. Good
linearity was observed, finding that determination coefficients (R2)
were higher than 0.98 in all the samples, with deviation ≤ 20% for
each individual level from the calibration curve.
Trueness, repeatability, intermediate precision, LODs and LOQs were
also evaluated. A summary of the results can be seen in Table 1, whereas
in Table S2, specific information for each compound is shown.
were also observed at this same concentration level for aflatoxin G2 and
demethon-S-methyl (128% for both), and hymexazol and methomyl
(125% for both) (see Table S2). Finally, few compounds also obtained
low recoveries at 100 μg kg−1, as disulfoton sulfone and iprovalicarb
ð1Þ
(65%), and chlorbenside and isofenphos methyl (60%). Ethoxyquine
obtained a recovery of 125% at this level (see Table S2).
It has to be mentioned that 73 compounds could not be extracted at
10 μg kg−1 (see Table S2). This can be explained because the Exactive-
Orbitrap does not have enough sensitivity to suitably detect these
specific toxic substances at these low concentrations. Some mycotoxins
could not be detected at the defined levels, as deoxynivalenol, fumonisin
B1, fumonisin B2 and HT-2 toxin.
In the case of repeatability, RSD values were below 20% for all the
compounds studied, whereas for intermediate precision, RSD were
below 25%. LODs were found between 0.5 and 5 μg kg− 1 while for
LOQs between 1 and 10 μg kg−1 for most of the compounds studied,
except for those that could not be extracted at 10 μg kg−1. For these
compounds, LODs and LOQs were 10 and 20 μg kg−1, respectively (see
Table S2).
Comparing this study to other previous works in ginkgo biloba
leaves, it can be seen that the number of compounds analyzed is greatly
increased. In the revised literature, between 74 [15] and 236 [16] com-
pounds were analyzed, while in this study 260 toxic substances were
Table 1
Summary of the validation results for ginkgo biloba nutraceuticals.

In the case of trueness, recoveries between 70 and 120% were Concentration Number of compounds with suitable
obtained for most of the compounds at the three concentration levels recoveries (between 70 and 120%)
proposed before (10, 50 and 100 μg kg−1), except for diazinon, 10 μg kg−1 187
fenamiphos sulfoxide, fenpropimorph, fluazifop buthyl, flutolanil, 50 μg kg−1 260
iprovalicarb and thiazopyr, with recoveries between 60 and 64% at 100 μg kg−1 260
Repeatability b20%a
10 μg kg−1 (see Table S2). On the other hand, clodinafop propargyl, di-
Intermediate precision b25%a
sulfoton sulfone, hexazinone, mevinphos and oxydemeton methyl pro- LODsb 0.5–10.0 μg kg−1
vided recoveries above 120% (between 125 and 128%). At 50 μg kg−1 LOQsc 1.0–20.0 μg kg−1
there were also some compounds with low recoveries (between 60 and a
RSD values (n = 5).
65%), as acrinathrin, atrazine desisopropyl, diflufenican, dimethomorph, b
LOD: Limit of detection.
hexythiazox, methamidophos, phosmet and triasulfuron. High recoveries c
LOQ: Limit of quantification.
128 G. Martínez-Domínguez et al. / Microchemical Journal 118 (2015) 124–130

Table 2 the possible matrix effect. Overall, the use of high resolution mass spec-
Toxic substances, mycotoxins and pesticides detected and found in ginkgo biloba nutra- trometers gives advantages in terms of precision, robustness and com-
ceutical analyzed samples.
pounds quantity that can be analyzed at the same time. It has to be
Concentration (μg/kg) MRLa (μg/kg) mentioned that not only pesticides were analyzed, but also mycotoxins
Compounds S1 S2 S3 S4 S5 S6 S7 S8 as well using the same procedure. Therefore, for multicompound stud-
ies, the use of high resolution mass spectrometers is recommended,
Aflatoxin B1 – 5 – – – – 54 – NRb
Aflatoxin B2 – – – 4 – – – 300 NR such as the Exactive-Orbitrap used in this work.
Alachlor 207 41 – – – – – – 50
Atrazine desisopropyl – – – – – – 18 – 100
Azoxystrobin – – – – – 14 – – 50000 3.3. Application to real samples
Carbendazim – – – – – – – 25 100
Cyanofenphos – – – – 122 – – 46 NR
Etridiazole – – 22 – – – – – 50 As explained in Section 2.3, 9 ginkgo biloba nutraceuticals were an-
Fenbuconazole – – 75 – – – – – 50 alyzed with the validated method in order to determine pesticides and
Hymexazol – – 10 – – – – – 50 mycotoxins. The results can be seen in Table 2.
Propargite 22 – – – – – – – 20
From this study, pesticides were found in eight samples. In particu-
Prosulfocarb – – 23 – – – – – 2000
Pyrethrin I – – 114 – – – – – 500 lar, tebufenozide was found in three different samples (55, 67 and
T-2 toxin 18 – – – 20 – – – NR 459 μg kg− 1), while alachlor (41–207 μg kg− 1) and cyanofenphos
Tebufenozide – – 55 67 – 459 – – 100 (46–122 μg kg− 1) were found in two samples. Overall, the pesticide
Thiofanox sulfone – – – – – 42 – – NR concentrations found in these samples ranged from 10 μg kg− 1
Thiofanox sulfoxide – – 23 – – – – – NR
(hymexazol) to 459 μg kg− 1 (tebufenozide) (see Table 2). The MRLs
a
MRL: maximum residue limits for each toxic substances obtained from Regulation EC for these pesticides in ginkgo leaves defined by the European Union
396/2005 (pesticides) and Regulation EC 1881/2006 (mycotoxins).
b [19] are also presented in Table 2, finding that some concentrations
None reported.
were higher than these limits (alachlor, fenbuconazole, propargite and
tebufenozide). However, it has to be taken into account that these limits
determined and quantified. This can be explained because in other are only defined for the raw material and not for the nutraceutical prod-
works only low resolution mass spectrometers (QqQ) are used. All uct obtained from ginkgo biloba. Also, in one particular sample, seven
studies recommend the use of a clean-up step in order to minimize different pesticides were found, as can be seen in Table 2. As an

Fig. 2. Chromatograms and mass spectra obtained for: (a) tebufenozide (459 μg kg−1) and (b) aflatoxin B2 (300 μg kg−1) in ginkgo biloba nutraceuticals.
 G. Martínez-Domínguez et al. / Microchemical Journal 118 (2015) 124–130
example, the chromatogram and spectra for tebufenozide in sample 6
(459 μg kg−1) can be observed in Fig. 2a.
Regarding mycotoxins, aflatoxin B1 (5–54 μg kg− 1), aflatoxin B2
(4–300 μg kg−1) and T-2 toxin (18–20 μg kg−1) were found in different
samples. It has to be mentioned that these levels are very high, especially
the value obtained for aflatoxin B2 in sample 8 (300 μg kg−1), keeping in
mind that some of the MRLs defined in the European legislation [20] for
other matrices are below 2 μg kg−1. Also, this particular aflatoxin is con-
sidered one of the most toxic mycotoxin [20], showing the chromatogram
and spectra for aflatoxin B2 in sample 8 in Fig. 2b.
Bearing in mind the potential presence of mycotoxins, these com-
pounds should also be monitored in these nutraceutical products in
order to minimize potential health risks. It is important to mention
that two samples contain different toxic substances, detecting at the
same time pesticides and mycotoxins. Therefore, multi-class methods
are necessary in order to identify simultaneously different toxic
substances, like the one proposed in this article.
4. Conclusions
A multi-class method to determine and quantify more than 250 toxic
substances in ginkgo biloba nutraceutical products, including pesticides
and mycotoxins, was developed. A “dilute and shoot” procedure was ap-
plied for extraction of the compounds, followed by a clean-up step using
a mixture of different sorbents (PSA, GBC, C18 and Z-Sep+). Trueness, in-
termediate precision and repeatability parameters were evaluated for
validation, obtaining suitable results. Ginkgo biloba nutraceutical prod-
ucts were studied with the validated method, finding 17 different toxic
substances (14 pesticides and 3 mycotoxins) in most of them. It has to
be mentioned that, at the time of this investigation, there were few
methods that investigate these compounds in ginkgo biloba and none
in nutraceutical products, meaning that this is the first methodology
in this field, covering multi-class pesticides and mycotoxins. Taking
into account the different classes of these toxic substances, methods
that allow multi-class studies are necessary in order to ensure better
quality controls for these particular products and, therefore, health
safety for the consumers.
Acknowledgments
The authors gratefully acknowledge the Spanish Ministry of Economy
and Competitiveness (MINECO) and FEDER (project ref. CTQ2012-
34304). GMD acknowledges the Health Secretary from Veracruz,
Mexico and the Mexican Senate for the financial support. RRG is also
grateful for personal funding through the “University Research Plan”
(Almería University) and Cajamar.
Appendix A. Supplementary data
Supplementary data to this article can be found online at http://dx.
doi.org/10.1016/j.microc.2014.09.002.
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