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Comparison of derivatization methods for the

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quantitative gas chromatographic analysis of oils†
Eliise Tammekivi, *a Signe Vahur,a Ott Kekišev, a
Inez D. van der Werf,b
Lauri Toom, a Koit Herodesa and Ivo Leito a

The determination of fatty acid composition using quantitative gas chromatographic (GC) analysis is
a common method of characterizing fats and oils. A wide variety of derivatization methods have been
developed to enable the GC analysis of non-volatile oil components. However, there has been no
systematic comparison of these derivatization procedures in truly quantitative terms, i.e. with absolute
amounts of fatty acids, not just ratios. In this paper, for the first time, a comprehensive quantitative
comparison of four derivatization methods is presented: (1) m-(trifluoromethyl)
phenyltrimethylammonium hydroxide (TMTFTH) methylation, (2) two-step derivatization with sodium
ethoxide (NaOEt) and N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA), (3) two-step derivatization with
KOH and BSTFA and (4) acid-catalyzed methylation (ACM). The comparison of the results obtained with
both a mass spectrometric (MS) detector and a flame ionization detector (FID) is mainly based on
Received 6th May 2019
Accepted 15th June 2019
derivatization efficiency (absolute quantification) and intermediate precision (within-lab reproducibility)
over several weeks. The overall results indicate that out of the four examined methods the TMTFTH
DOI: 10.1039/c9ay00954j
derivatization was the least work-intensive and the most accurate – both in terms of reproducibility and
rsc.li/methods derivatization efficiency.

physical and chemical properties as well as the type of oil are

1. Introduction
Over the centuries oils have been used in diverse areas, e.g.
nutrition, art, cosmetics and medical purposes.1 Depending on
the requirements of their physical and chemical properties,
various oils are employed in each sector. A great deal of research
has been devoted to the qualitative2–6 as well as quantitative
analysis of oil composition.4,7–12 Quantitative determination of
fatty acid composition is preferred since it determines the oil's
properties and enables differentiation between oils.
The quantitative analysis of oil components is complicated
by the fact that their composition and degradation products are
very similar. Oils consist of triacylglyceride (TAG) molecules,
which, in turn, consist of glycerol esteried with three fatty acid
molecules. The ve most abundant fatty acids in oils are pal-
mitic (C16:0), stearic (C18:0), oleic (C18:1), linoleic (C18:2) and
linolenic (C18:3) acids. Some oils also contain characteristic
fatty acids that facilitate the determination of the type of oil. For
example, (Z)-eicos-11-enoic (gondoic) and (Z)-docos-13-enoic
(erucic) acids are markers for canola oil.4 Usually, however,
there are no characteristic fatty acids and, in general, the
a
University of Tartu, Institute of Chemistry, Ravila 14a, 50411 Tartu, Estonia. E-mail:
eliise.tammekivi@ut.ee; Tel: +372 562 34 473
b
Cultural Heritage Agency of the Netherlands, Cultural Heritage Laboratory,
Hobbemastraat 22, 1071 ZC, Amsterdam, The Netherlands
† Electronic supplementary information (ESI) available.
10.1039/c9ay00954j
determined by the relative quantities of the main ve fatty
acids.13
Gas chromatography (GC) is a widely used selective and
sensitive technique for the analysis of volatile organic
compounds.14 Since TAGs are not volatile enough for direct GC
analysis, it is necessary to hydrolyze and derivatize these
molecules to obtain sufficiently volatile compounds. The most
commonly used derivatives are methyl, ethyl or silyl esters of the
corresponding fatty acids.1 Nowadays, mostly in situ derivati-
zation methods are used, where the sample preparation treat-
ments are combined into one step. In this way less reagents,
work, and sample are needed, and at the same time, the effi-
ciency and reliability of the methods improve. Therefore, mostly
silylation reagents (BSA: N,O-bis(trimethylsilyl)acetamide,
BSTFA: N,O-bis(trimethylsilyl)triuoroacetamide, hexame-
thyldisilazane, etc.) and transesterication reagents (basic
quaternary salts of ammonia, acid- or base-catalyzed alkylation
reagents) are used for the derivatization of TAGs.15 In spite of
the extensive use, so far, only a few studies have focused on the
comparison of different derivatization methods.8,9,16,17 There-
fore, in this study, the following four reagents are compared,
and their advantages and limitations are evidenced: (1) m-(tri-
uoromethyl)phenyltrimethylammonium hydroxide (TMTFTH)
derivatization, (2) two-step derivatization with sodium ethoxide
(NaOEt) and BSTFA, (3) two-step derivatization with KOH and
See DOI: BSTFA and (4) acid-catalyzed methylation (ACM). These

3514 | Anal. Methods, 2019, 11, 3514–3522 This journal is © The Royal Society of Chemistry 2019
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derivatization procedures were selected to include the most
frequently applied methods in the analysis of cultural heritage
studies (BSTFA18–21) and archaeology (ACM22–24). TMTFTH
derivatization25–27 was chosen because it is a more novel deriv-
atization method compared to the others. Since most labora-
tories do not possess pyrolysis GC equipment, we excluded
derivatization reagents that require pyrolysis-GC like TMAH
(tetramethylammonium hydroxide) from this study.
To the best of our knowledge, no study has been carried out
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dried on 3 Å molecular sieves) and isooctane (Lachner, purity
$99.9%).
Derivatization reagents – methylation reagent TMTFTH
(ALLTECH Meth-Prep II: 5% TMTFTH solution in methanol),
silylation reagent BSTFA (derivatization grade), NaOEt 21%
solution in ethanol, 99% triuoroacetic acid (TFA) and KOH
(purity min 85%) – were purchased from Sigma-Aldrich.
Concentrated sulfuric acid (purity 98%) was purchased from
VWR Chemicals.

where the comparison of these derivatization reagents is based
on the determination of the absolute quantities of fatty acids in
oils. Some studies8,17,28 have applied absolute quantication while
using only one or two reagents or have claimed absolute quan-
tication without presenting convincing data.12 The main aim of
most of those studies was the analysis of certain samples, not
evaluating the used derivatization procedure. This paper aims at
lling this gap. The four above-mentioned hydrolysis/derivatiza-
tion and transesterication methods for oil analysis by GC-MS
and GC-FID, not requiring pyrolysis, are compared in quantita-
tive terms. The following performance parameters were used in
comparison: (1) derivatization efficiency (yield); (2) within-labo-
ratory reproducibility; and (3) robustness/ruggedness. The overall
results of this study show the advantages and drawbacks of the
methods as well as the least work-intensive and the most accurate
derivatization procedure. This could facilitate the selection of the
most suitable derivatization reagent for the study of oil-contain-
ing materials in future studies.
2. Materials and methods
2.1. Materials
Commercial solvents were used: HPLC grade dichloromethane
(DCM, Sigma-Aldrich, purity $99%), HPLC grade methanol
(Sigma-Aldrich, purity $99%), GC grade hexane (Sigma-Aldrich,
purity $99%), toluene (Reakhim, purity 99.5%), ethanol
(Premium, purity 96.7%), GC grade diethyl ether (Sigma-
Aldrich, purity $99.5%), HPLC grade acetonitrile (MeCN,
Sigma-Aldrich, purity $99.9%, reuxed with CaH2 and further
Hexadecane (Sigma-Aldrich, purity $99%) was used as the
internal standard in GC analysis. Stock solution of the internal
standard was prepared in DCM with a concentration of 0.374
mg g 1. K2CO3 and NH4Cl (both with purity 99.5%) were
purchased from Reakhim. Glass wool was purchased from
Supelco. For quantitative analysis, mixtures of fatty acid methyl
esters (FAME – including methyl palmitate 9.9%, methyl stea-
rate 6%, methyl oleate + methyl elaidate (Z + E) 34.7%, methyl
linoleate 33.9% and methyl linolenate 5.0%, purity 99.8–99.9%)
and fatty acid ethyl esters (FAEES – including ethyl palmitate
0.9966 mg ml 1 and ethyl stearate 0.9998 mg ml 1, purity 99.0–
99.9%) were also purchased from Supelco. Glyceryl tripalmitate
(purity $99%), glyceryl tristearate (purity $99%), glyceryl tri-
oleate (purity $99%) and glyceryl trilinoleate (purity $98%)
(Sigma-Aldrich) were used for the validation of the quantitative
method. A mixture containing these TAGs was prepared in
toluene (respectively 0.43 mg g 1, 0.43 mg g 1, 0.87 mg g 1, and
0.51 mg g 1) to mimic the composition of natural oils. For the
synthesis of trimethylsilyl fatty acid standards, palmitic acid
(Sigma-Aldrich, purity $99%), stearic acid (Sigma-Aldrich,
purity ca. 99%) and oleic acid (Alfa, purity 99%) were used as
starting materials.
An analytical grade canola oil standard (Supelco) was used as
the quality control sample for the testing of the developed
quantitative analysis. The certied contents of fatty acids in
canola oil were given as peak area percentages of the methylated
fatty acids and are reported in Table 1. Commercial art claried
linseed oil (Lefranc & Bourgeois, France) and Olivia canola
cooking oil (Olivia, Estonia) were used as typical oil samples.

Table 1 Relative peak area percentages (with respect to the sum of peak areas of all fatty acid esters in the respective chromatogram) of
derivatized canola oil standard components (%)a

NaOEt– NaOEt– KOH– KOH–

TMTFTH TMTFTH BSTFA BSTFA BSTFA BSTFA ACM ACM
Sample Fatty acid GC-MS GC-FID GC-MS GC-FID GC-MS GC-FID GC-MS GC-FID Certicateb

Canola C16:0 4.3 (0.1) 4.4 (0.1) 4.1 (0.1) 4.2 (0.1) 4.5 (0.2) 4.6 (0.4) 4.9 (0.1) 4.8 (0.1) 4.31
oil C18:0 1.9 (0.2) 2.2 (0.05) 2.3 (0.4) 2.0 (0.1) 2.2 (1.0) 2.6 (1.1) 2.2 (0.1) 2.3 (0.1) 1.86
standard C18:1 66.1 (1.6) 64.7 (0.1) 66.1 (1.0) 65.4 (0.8) 71.2 (0.9) 69.2 (0.9) 64.9 (0.3) 64.4 (0.3) 60.63
C18:2 17.4 (0.7) 17.7 (0.1) 16.5 (0.7) 17.1 (0.3) 15.0 (1.1) 15.9 (1.6) 17.6 (0.1) 17.6 (0.1) 20.26
C18:3 6.0 (0.5) 6.8 (0.1) 6.8 (1.4) 6.9 (1.1) 2.8 (0.6) 3.4 (0.6) 6.1 (0.1) 6.6 (0.1) 8.62
Pooled 0.6 0.7 0.9 0.2
standard
deviation
a
Fatty acid peak area percentages of the derivatized canola oil standard and the corresponding values from the certicate. Every measured value is
an average of three determinations made during a time period of at least four weeks. Standard deviations are in parentheses. Pooled standard
deviations are calculated over 30 results. b Certicate values (GC-FID) were obtained with the AOAC 969.33 method where NaOH and BF3 in
methanol were used as derivatization reagents.

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2.2. Derivatization methods
All four derivatization methods were modied to enable the
quantitative measurement of absolute amounts (i.e. expressed
in grams or moles) of specic fatty acids. To minimize the error
from weighing, at least 10 mg of oil was weighed on an
analytical balance (GENIUS Sartorius with a resolution of 0.01
mg) and dissolved in toluene to make stock solutions with oil
concentrations in the range of 1.9 to 2.5 mg g 1. To minimize
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the uncertainty of the sample concentrations all the following
derivatization solutions and dilutions were prepared by weigh-
ing and volumes are given only as guidance.
Derivatization with the TMTFTH reagent was conducted by
using a procedure initially developed by Manzano et al.29 and
modied by Sutherland.30 TMTFTH 5% solution in methanol
(50 ml) was added to the stock solution (450 ml). The reaction
mixture was sonicated for 30 min and was thereaer kept at
room temperature for 24 h to achieve complete derivatization
(as recommended in ref. 30). Then, 140 ml of internal standard
stock solution was added and the solution was diluted with 1.5
ml of DCM and analyzed by GC.
Two-step derivatization with NaOEt and BSTFA was based on
the method used by van den Berg et al.31 This derivatization
produces two derivatives for every fatty acid: the corresponding
ethyl and trimethylsilyl esters. However, to the best of our
knowledge, the impact of the quantity of 0.01 M NaOEt in this
two-step derivatization method has not yet been veried.
Therefore, this was investigated, and the analysis is described in
detail in the ESI.† The results showed that when using lower
volumes of 0.01 M NaOEt, ethyl esters are mostly produced
(approx. 95%). Therefore, in the following analyses, the peaks of
ethylated fatty acids were used. In the rst step, 0.01 M NaOEt
solution was prepared and added (40 ml or 60 ml, depending on
the concentration of the analyte's stock solution) to the stock
solution (450 ml). Air was purged from the vial with dry nitrogen
and the vial was crimped with a PTFE-lined cap. The vial was
thereaer heated at 70  C for 1.5 h. Once the solution was
cooled to room temperature, it was treated for 20 min with
saturated NH4Cl solution in ethanol (60 ml) to remove the excess
of the derivatization reagent. The solvents were evaporated, and
the residue was dissolved in hexane (200 ml). In the second step
BSTFA (15 ml) was added, and the vial was again crimped with
a PTFE-lined metal cap and heated for 30 min at 70  C. Aer
cooling, the solution was quantitatively transferred to a 4 ml vial
(the original vial was washed three times with 200 ml of hexane).
Finally, the solution was evaporated to dryness and redissolved
in DCM (2.5 or 2 ml, depending on the concentration of the
stock solution). Once again, 140 ml or 200 ml of internal standard
stock solution was added prior to the analysis by GC.
The two-step derivatization with KOH and BSTFA was based
on the method described by Lluveras et al.32 The stock solution
(250 ml) was evaporated to dryness. For the rst saponication
reaction 250 ml of a 10 wt% KOH solution in EtOH was added to
the residue. The vial was crimped with a PTFE-lined cap and
heated at 60  C for 2 h. Aer the solution had cooled to room
temperature, 300 ml of Milli-Q water (Milli-Q® Advantage A 10,
Millipore) was added. Aer dilution, 60 ml of TFA solution in
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Milli-Q water (1:1 in volume) was added. The amount of TFA
solution was determined in a trial derivatization by adding the
acidic solution to the sample mixture until the basicity was
neutralized. Then the slightly acidic solution was extracted
three times with hexane (200 ml each time) and three times with
diethyl ether (200 ml each time). The combined extracts were
evaporated to dryness. For the second reaction, 250 ml of
isooctane and 30 ml of BSTFA were added and the mixture was
heated at 60  C for 30 min. Aer cooling, the solution was again
evaporated to dryness. Finally, the residue was dissolved in
isooctane (0.95 ml or 0.65 ml, depending on the concentration
of the stock solution) and respectively 55 ml or 30 ml of internal
standard solution was added. The obtained mixtures were
sonicated for 30 min prior the GC analysis to improve the
solubilization of the derivatized fatty acids.
Acid-catalyzed methylation was performed essentially as
described by Craig et al.33 The stock solution (250 ml) was
evaporated to dryness. Methanol (1 ml) was added and the
mixture was sonicated for 15 min. Then concentrated sulphuric
acid (200 ml) was added, and the vial was sealed (with a PTFE-
lined screw cap) and heated for 4 h at 70  C. Aer cooling, the
mixture was extracted (vortexing for a few seconds) three times
with hexane (2 ml each time). The hexane layers were carefully
separated and ltered through a K2CO3 layer, and then through
a layer of glass wool. Thereaer they were combined and
evaporated to dryness. The residue was redissolved in hexane (1
ml) and 80 ml of internal standard stock solution was added
prior to the GC analysis.
2.3. GC parameters
An Agilent 5975C inert XL MSD connected to an Agilent 7890A
GC system with a G4513A autosampler (Agilent) was used with
an Agilent DB-225MS capillary column (50% cyanopropyl-
phenyl–50% dimethyl-polysiloxane). For the quantitative anal-
ysis a MS detector and FID were used. The full parameters are
presented in the ESI.†
2.4. Quantitative analysis by GC
The aim of the quantitative analysis was to determine the fatty
acid contents in the oils expressed in grams or moles per 100 g
of oil. Peak areas were used for quantication. Quantication
was carried out via a calibration graph with the internal stan-
dard, where peak area ratios (analyte/internal standard) were
plotted against concentration ratios.
In order to conduct accurate absolute quantication, the
calibration and validation processes are the key factors. A FAME
standard mixture was used to make 7 calibration solutions for
the TMTFTH and ACM procedures. In NaOEt–BSTFA derivati-
zation fatty acids are mainly ethylated. Therefore, a FAEES
standard mixture was used to make 7 alternative calibration
solutions. It was found that the analysis of two fatty acids was
sufficient enough to describe the derivatization efficiency of the
NaOEt–BSTFA method.
For the KOH–BSTFA method the fatty acid trimethylsilyl
esters were commercially not available. Therefore, the trime-
thylsilyl esters (TMSE) of palmitic, stearic and oleic acid were
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Fig. 1 Example calibration graphs: one of the best (A: GC-FID calibration graph for methylated linoleic acid) and one of the worst (B: GC-FID
calibration graph for trimethylsilylated palmitic acid).
synthesised as described by Noda and Bode.34 The purities of
the synthesised palmitic acid TMSE (synthesis yield 79%; purity
99.1%), stearic acid TMSE (yield 47%; purity 95.4%) and oleic
acid TMSE (yield 37%; purity 99.2%) were determined with the
quantitative NMR (qNMR) method. The synthesis of the tri-
methylsilyl ester standards and the qNMR method are
described in detail in the ESI.†
Commercial claried linseed oil and canola oil (Olivia) were
used as typical real-life oil samples. In order to validate the
quantitative analysis two samples with known fatty acid
contents were used: (1) a mixture of TAGs (tripalmitin, triolein,
tristearin and trilinolein) with known contents of components
prepared by weighing and (2) a canola oil analytical standard
(see Table 1). In addition, a blank sample with only solvent was
derivatized each day the experiment was carried out.
Aer the derivatization step, the internal standard (hex-
adecane) solution was added to the calibration samples and
blank solutions. The resulting solutions were transferred to
chromatography vials and analyzed on the same day with GC-
MS and GC-FID. A calibration graph was constructed for every
methyl, ethyl or trimethylsilyl derivative of the fatty acids in the
corresponding calibration solution (Fig. 1). The peak areas of
the internal standard (SIS) and derivatized analytes (SAD) were
obtained from the chromatograms. Based on the peak areas and
internal standard concentration in the sample solution, the
concentration of the corresponding derivatized fatty acid was
Fig. 2 Total Ion Current (TIC) chromatogram of commercially available clarified linseed oil derivatized with TMTFTH and the internal standard.
a
Geometric isomer of C18:3.
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calculated through unweighted linear regression. Finally, the
obtained value was recalculated to a concentration of a TAG
molecule, where all the bonded fatty acids are the same.
2.4.1. Validation of derivatization and separation methods.
The key performance parameters in the comparison of the
derivatization methods are derivatization efficiency (yield) and
intermediate precision (within-lab reproducibility) over several
weeks.35 The combination of these two characteristics expresses
the ruggedness36 of the method (ability to deliver reliable results
under suboptimal conditions). Repeatability (within a day or
within run precision35) is a more commonly reported precision
parameter, but as it is determined within a day it fails to
account for effects that are systematic within a day but random
in the long term. The latter effects are accounted for by inter-
mediate precision, making it a more insightful parameter.
3. Results and discussion
3.1. Quantitative analysis with TMTFTH, NaOEt–BSTFA,
KOH–BSTFA and ACM methods
The developed quantitative analysis procedures were applied to
commercially available claried linseed and canola oils (Olivia)
as well as to the analytical standard canola oil. Fig. 2 displays
a typical chromatogram of linseed oil derivatized with TMTFTH.
The chromatograms obtained with GC clearly showed that
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Fig. 3 TIC chromatogram of the TAG mixture (derivatized with TMTFTH) and internal standard.

canola and linseed oils contain mainly the same fatty acids but
in different quantities (as judged by the relative peak heights).
3.1.1. Initial evaluation – analysis of the canola oil stan-
dard. The analytical grade canola oil standard with certied
peak area ratios was used for the initial evaluation that the
derivatization methods perform adequately, and that the GC
separation is satisfactory. For this purpose, the obtained peak
areas of the derivatized canola oil standard were compared with
the fatty acid area percentages reported in the certicate of the
same standard (Table 1).
As can be seen in Table 1, the area percentages of the fatty
acids obtained with the TMTFTH, NaOEt–BSTFA, KOH–BSTFA
and ACM derivatization methods are all comparable to the
certicate values, although not always strictly in agreement.
Furthermore, the pooled standard deviations remain within the
range of 0.2–0.9, which shows the good repeatability of the
methods. However, the comparison of relative peak areas does
not give information about the loss of the sample or the
completeness of the derivatization for the corresponding
procedure.
3.1.2. Derivatization efficiency and reproducibility. To
determine the derivatization efficiencies of the procedures, the
mixture of TAGs with known composition was quantitatively
analyzed as explained in Section 2.4. Fig. 3 presents a typical
chromatogram of the TAG mixture derivatized with the
TMTFTH method.
Table 2 shows the derivatization efficiencies of the four
derivatization procedures as well as the within-lab reproduc-
ibilities (intermediate precisions), expressed as pooled standard
deviations. Pooling of the individual standard deviations is
justied because the standard deviations corresponding to the
individual fatty acids are not expected to differ signicantly and
pooling enables to obtain a signicantly larger number of
degrees of freedom.
The NaOEt–BSTFA and ACM derivatizations clearly showed
lower efficiency and worse reproducibility than the TMTFTH
derivatization. The losses in the case of NaOEt–BSTFA and ACM
may be mainly caused by the laborious multi-step sample
preparation. ACM derivatization has slightly higher efficiency;
however, the pooled standard deviation values are higher (in
contrast to the pooled standard deviations of peak area
percentages, see Table 1), indicating lower reproducibility.
With the TMTFTH methylation the derivatization efficiency
for palmitic (C16:0), stearic (C18:0) and oleic (C18:1) acids is
near 100%. For these three fatty acids the observed derivatiza-
tion efficiencies were within the range of twice the

Table 2 Derivatization efficiency (%)a

TMTFTH TMTFTH NaOEt–BSTFA NaOEt–BSTFA KOH–BSTFA KOH–BSTFA ACM ACM

Sample Fatty acid GC-MS GC-FID GC-MS GC-FID GC-MS GC-FID GC-MS GC-FID

TAG mixture C16:0 102.3 (1.4) 101.7 (1.0) 67.3 (1.5) 66.7 (2.4) 97.0 (6.0) 98.0 (7.3) 96.8 (3.2) 91.8 (1.7)
C18:0 93.7 (2.7) 93.9 (2.7) 60.3 (2.3) 62.0 (2.6) 87.9 (7.8) 88.5 (8.1) 74.2 (3.1) 74.8 (4.2)
C18:1 98.3 (2.3) 96.9 (1.7) — — 100.3 (7.8) 96.1 (3.3) 85.5 (3.0) 85.5 (2.7)
C18:2 90.1 (1.3) 91.9 (1.8) — — — — 77.4 (0.7) 80.9 (2.2)
Pooled 2.0 2.2 6.9 2.8
standard
deviation
a
Fatty acid content calculated using the internal standard method in relation to the content calculated from solution preparation (%). Every value is
an average of three determinations made during a time period of at least four weeks. Standard deviations are in parentheses. The sign “—” indicates
that the calibration solution did not contain the corresponding derivatized fatty acid. Pooled standard deviations are calculated over 12 (NaOEt–
BSTFA), 18 (KOH–BSTFA) or 24 (ACM and TMTFTH) individual determinations.

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reproducibility pooled standard deviation from 100%. However,

Fatty acid TMTFTH GC-MS TMTFTH GC-FID NaOEt–BSTFA GC-MS NaOEt–BSTFA GC-FID KOH–BSTFA GC-MS KOH–BSTFA GC-FID ACM GC-MS ACM GC-FID

Fatty acid content in mmol per 100 g of oil. See the footnote of Table 2. Pooled standard deviations are calculated over 36 (NaOEt–BSTFA), 54 (KOH–BSTFA) or 90 (ACM and TMTFTH) analyses and
involved both GC-FID and GC-MS results for all reagents. b Reference contents: C16:0 16.0 mmol/100 g; C18:0 6.3 mmol/100 g; C18:1205.4 mmol/100 g; C18:2 69.1 mmol/100 g; C18:3 29.6 mmol/
132.6 (23.1)

173.2 (18.0)
141.9 (4.5)
in the TMTFTH derivatization the derivatization efficiency is

19.0 (1.1)
11.9 (0.7)
49.6 (2.5)
45.8 (2.4)

12.0 (1.1)

50.1 (6.5)
22.3 (4.4)
14.3 (1.2)

50.3 (5.4)
18.8 (1.4)
4.2 (0.4)

6.5 (0.8)
slightly lower for the more reactive linoleic acid – the average
value is 90%. For the KOH–BSTFA derivatization the derivati-

3.6
zation efficiency for palmitic (C16:0) and oleic (18 : 1) acids is

132.3 (22.7)

175.5 (19.3)
also nearly 100%. However, the reproducibility is the poorest,
134.2 (4.9)
18.4 (2.6)
11.4 (1.1)
48.2 (3.9)
41.2 (0.6)

10.2 (1.9)

48.0 (8.5)
21.8 (3.9)
14.4 (1.2)

51.2 (4.9)
18.6 (1.3)
4.1 (0.4)

6.4 (0.6)
although the procedure was optimized for quantitative analysis
in this study. This can be caused by the fact that this derivati-
zation still occasionally produced ethyl, methyl and hydroxyl
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derivatives in small quantities besides the trimethylsilyl esters.

From these results, it can be concluded that TMTFTH has the
highest derivatization efficiency and the best reproducibility
129.8 (10.1)

117.9 (16.7)
57.8 (12.9)

and is thus best suited for the quantitative analysis of fatty acids
13.5 (1.8)

11.1 (1.4)

11.8 (1.9)
9.4 (2.1)

7.3 (2.8)

9.3 (5.2)
of different oils.
3.1.3. Determination of the absolute quantities of the main
—
—

—
—

—
—
8.4 ve fatty acids. The amounts of the ve main fatty acids – pal-
mitic (C16:0), stearic (C18:0), oleic (C18:1), linoleic (C18:2) and
linolenic (C18:3) acids – in the canola oil analytical standard
135.8 (10.2)

123.8 (19.4)

and in commercial claried linseed and canola oils as deter-

56.2 (12.1)
13.3 (1.6)

10.9 (1.4)

11.4 (1.8)
8.6 (2.2)

7.2 (2.1)

9.3 (5.0)

mined with the different derivatization procedures and GC

detectors are shown in Table 3, where the reference contents of
—
—

—
—

—
—

the canola oil standard, expressed in mmol/100 g, are given in

the footnote.
This approach allows the comparison of the ethylation, tri-
methylsilylation and methylation methods. Overall, the results
show that the highest contents and the values that are closest to
12.3 (1.5)
7.5 (1.0)

9.7 (1.3)
3.2 (0.1)

9.2 (0.8)
4.1 (0.1)

the certied values are obtained with the TMTFTH derivatiza-

tion. Comparing the results of NaOEt–BSTFA, KOH–BSTFA and
—
—
—

—
—
—

—
—
—

ACM derivatization of saturated fatty acids shows that the

0.9

second best is acid-catalyzed methylation. The standard devia-

tions for the KOH–BSTFA derivatization are the highest, which
indicates that this method has the lowest repeatability.
Quantitative analysis of linseed and canola oils in mmol per 100 g of oila

While the contents of saturated fatty acids and oleic acid

11.8 (1.3)
7.1 (0.6)

8.9 (1.1)
3.3 (0.1)

8.6 (0.6)
4.3 (0.5)

found with TMTFTH derivatization agree well with the values on

the certicate, the contents of the polyunsaturated fatty acids
—
—
—

—
—
—

—
—
—

(C18:2 and C18:3) are seriously biased, the average contents

being 59.3 and 22.6 mmol/100 g, as opposed to 69.1 and 29.6
mmol/100 g from the certicate. This is not unexpected. This
175.5 (1.7)

196.9 (2.5)

203.6 (1.6)

could be caused by the high reactivity of the polyunsaturated

20.4 (0.1)
12.8 (0.2)
62.4 (0.6)
54.5 (1.4)

16.2 (0.2)

69.6 (1.6)
31.9 (0.5)
15.1 (0.1)

58.8 (0.5)
23.1 (0.4)
5.8 (0.7)

7.0 (0.3)

fatty acids. Also, as can be observed in Table 2, the average

TMTFTH derivatization efficiency of C18:2 was 90%. When
1.7

correcting the C18:2 result, a value of 65.9 mmol/100 g is found.

This value is close to the certicate value, considering that both
100 g (see the Materials section for comments).

the analysis and recovery have uncertainties, but it is impos-

163.7 (5.3)

197.5 (3.9)

203.4 (2.6)
20.9 (0.1)
12.2 (0.2)
59.0 (3.3)
49.5 (1.4)

16.3 (0.3)

69.7 (1.9)
30.9 (1.1)
15.6 (0.1)

59.8 (0.3)
22.0 (0.2)

sible to rigorously judge the agreement because the uncertainty

5.6 (0.5)

6.8 (0.2)

of the certicate value is not known.

3.1.4. Determination of the relative quantities of the main
ve fatty acids. In addition, the fatty acid content was calculated
in relation to all the quantied ve fatty acids. This kind of data
C16:0
C18:0
C18:1
C18:2
C18:3
C16:0
C18:0
C18:1
C18:2
C18:3
Canola oil standardb C16:0
C18:0
C18:1
C18:2
C18:3
Pooled standard deviation

presentation is common in the literature and gives the oppor-

tunity to compare the results of linseed and canola oils with
Claried linseed oil

those of other studies. However, it does not provide information

Olivia canola oil

about the completeness of the derivatization. For the BSTFA

derivatizations only the amounts of some fatty acids (C16:0 and
C18:0 for NaOEt–BSTFA and C16:0, C18:0 and C18:1 for KOH–
Sample
Table 3

BSTFA) could be calculated and therefore, the amounts of these

fatty acids in relation to the sum of all fatty acids would not be
a

This journal is © The Royal Society of Chemistry 2019 Anal. Methods, 2019, 11, 3514–3522 | 3519
 View Article Online

Analytical Methods Paper

Table 4 Quantitative analysis of linseed and canola oils (g/100 g)a

Sample Fatty acid TMTFTH GC-MS TMTFTH GC-FID ACM GC-MS ACM GC-FID Literature4,37,38

Claried linseed oil C16:0 6.3 (0.03) 5.8 (0.04) 6.7 (1.0) 6.6 (0.4) 4–10
C18:0 4.1 (0.1) 4.0 (0.1) 4.6 (0.4) 4.5 (0.3) 2–8
C18:1 19.6 (1.1) 19.4 (0.2) 19.3 (1.6) 18.7 (1.0) 10–24
C18:2 16.3 (0.5) 16.8 (0.4) 16.4 (0.2) 17.2 (1.0) 12–19
C18:3 53.6 (1.8) 53.9 (0.5) 53.0 (2.0) 53.0 (1.7) 48–60
Olivia canola oil C16:0 4.7 (0.1) 4.7 (0.1) 4.3 (0.8) 5.0 (0.5) 4.3–4.5
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C18:0 1.8 (0.2) 1.8 (0.2) 1.9 (0.2) 1.9 (0.2) 2.1–2.2
C18:1 62.2 (1.2) 61.9 (0.8) 61.6 (10.6) 60.4 (10.5) 61.7–64.1
C18:2 21.8 (0.6) 21.7 (0.5) 22.2 (4.0) 22.7 (3.0) 18.6–19.8
C18:3 9.6 (0.3) 9.9 (0.1) 10.0 (1.8) 10.0 (2.0) 9.1–9.5
Pooled standard deviation 0.6 3.6
a
Fatty acid content as g per 100 g of all quantied fatty acids. Every value is an average of three determinations made during a time period of at least
four weeks. Standard deviations are in parentheses. Pooled standard deviations are calculated over 60 results (involving GC-MS and GC-FID results
for both reagents).

comparable to the results obtained with the TMTFTH and ACM
methods. As a consequence, in Table 4 only the values of the
latter two methods are reported and it was shown that they are
in agreement with the results of other studies. This indicates
that derivatization efficiency may be different for various
derivatization methods even when the relative quantication
seems to be in order.
3.2. Comparison of derivatization procedures
A concise comparison of TMTFTH, NaOEt–BSTFA, KOH–BSTFA
and ACM derivatization methods is presented in Table 5.
Quantitative analysis showed that TMTFTH derivatization is
the preferred method for the determination of the absolute
quantities of the fatty acids in oil samples. This method
required the least amount of time and showed the highest
derivatization efficiency. Even though the TMTFTH method
requires the most expensive derivatization reagent, it is the least
labor-intensive and fatty acid methyl esters that are needed for
calibration are readily available. Examining the chromatograms
obtained with the four different derivatization procedures
showed that if more than one type of derivate is formed, then
the peaks can overlap and affect the integration. This can at
times occur with the NaOEt–BSTFA derivatization procedure.
With TMTFTH and ACM procedures (where methyl esters were
produced) the retention times were approximately 0.25–0.5
minutes shorter than for the other methods. Therefore, for the
methylation methods, the chromatographic run could be made
shorter.
In addition to the comparison of the derivatization methods,
the results of the quantitative analysis with GC-MS and GC-FID
were compared. Both techniques were found suitable for the
analysis of absolute quantities. However, GC-MS is preferable

Table 5 Comparison of the four derivatization methods

TMTFTH (Meth-Prep II) NaOEt–BSTFA KOH–BSTFA Acid-catalyzed methylation (ACM)

Sample preparation  One-step derivatization  Two-step derivatization  Two-step derivatization  One-step derivatization
 No sample transfer  Evaporation  Evaporation  Extractions
necessary
 Easy procedure  Labor-intensive  Labor-intensive  Evaporation
 Labor-intensive
Time required for sample 1 h 4h 4h 7h
preparation a
Approximate cost of 145V 65V 70V 60V
chemicals for 100
derivatizations
Interpretation of results Simple interpretation – one Possible to distinguish Interpretation may be Simple interpretation –
peak for every fatty acid between free and bound complicated if several one peak for every fatty acid
fatty acids; interpretation is derivatives appear
complicated by two peaks
for every fatty acid
Average derivatization 96 (2)% 64 (2)% 95 (7)% 83 (3)%
efficiency (SD)b
a
The times are clock times and do not include the time required for the synthesis of TMSEs or the NMR analysis for the quantitative analysis (KOH–
BSTFA method) or the preparation of calibration solutions (all derivatization methods). b The average derivatization efficiency value was calculated
by averaging the results obtained with one derivatization method using GC-MS or GC-FID (see Table 2). SD is the between-day pooled standard
deviation.

3520 | Anal. Methods, 2019, 11, 3514–3522 This journal is © The Royal Society of Chemistry 2019

Paper
for overall oil analysis, since it also conrms the identities of the
analytes in the sample.
4. Conclusions
In this paper, for the rst time, a comprehensive comparison of
TMTFTH, NaOEt–BSTFA, KOH–BSTFA and ACM derivatization
procedures is presented for the analysis of the fatty acid
composition of oils in terms of absolute quantities. For this
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investigation three different oils were used – analytical grade
canola oil standard, commercial claried linseed oil and
commercial canola oil as real-life samples for testing the
quantitative approach. Also, a mixture of TAGs with known
contents was prepared for the validation of the quantitative
procedures.
TMTFTH derivatization turned out the most accurate – both
in terms of reproducibility and derivatization efficiency (96 
2)%. The KOH–BSTFA method also exhibited high derivatiza-
tion efficiencies (95  7)%; however, the sample preparation
takes a longer time (two-step derivatization) than with the
TMTFTH derivatization and the reproducibility is clearly infe-
rior. An important drawback of the KOH–BSTFA method is that
for the absolute quantication TMSE standards must be
synthesized (they are not commercially available). Acid-cata-
lyzed methylation (ACM) employs readily available chemicals,
but is labor-intensive and time-consuming and has lower
derivatization efficiency (83  3)% than TMTFTH and KOH–
BSTFA derivatizations. The NaOEt–BSTFA method showed the
lowest derivatization efficiency (64  2)% and the sample
preparation takes a longer time (two-step derivatization). The
comparison of these derivatization procedures revealed differ-
ences and, so far unknown, information including advantages,
limitations, and obstacles described in this paper.
Conflicts of interest
There are no conicts of interest to declare.
Acknowledgements
This research was supported by the Personal Research Funding
PUT1521 and the Institutional Funding IUT20-14 and IUT20-15
from the Estonian Research Council and by the EU through the
European Regional Development Fund (TK141 “Advanced
materials and high-technology devices for energy recuperation
systems”). We are thankful to Dr Siim Salmar for helping with
the GC-MS/FID system, also to Dr Ester Oras and Dr Agnes Kütt
for their advice on the practical aspects and to Prof. Klaas Jan
van den Berg for the useful discussions. This work was carried
out using the instrumentation at the Estonian Center of
Analytical Chemistry (AKKI, www.akki.ee).
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