Food Control 57 (2015) 293e301

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Food Control
journal homepage: www.elsevier.com/locate/foodcont

A quick method for routine analysis of C18 trans fatty acids in non-
hydrogenated edible vegetable oils by gas chromatographyemass
spectrometry
M. Zhang a, b, X. Yang a, b, *, H.T. Zhao a, A.J. Dong a, J. Wang a, b, **, G.Y. Liu a, P. Wang a,
C.L. Cheng a, H. Zhang a
a
School of Food Science and Engineering, Harbin Institute of Technology, 150090 Harbin, PR China
b
Key Laboratory of Agro-Product Quality and Safety, Institute of Quality Standard and Testing Technology for Agro-Product, Chinese Academy of
Agricultural Sciences, 100081 Beijing, PR China

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

Article history: A quick routine analysis has been established for quantitative determination of thirteen individual C18
Received 8 December 2014 trans fatty acids in both pressed and solvent extracted non-hydrogenated edible vegetable oils, using gas
Received in revised form chromatography-mass spectrometer (GCeMS) equipped with HP-88 capillary column following potas-
15 April 2015
sium hydroxide/methanol (KOH/MeOH) methylation. Based on optimization and appraisal of the char-
Accepted 17 April 2015
Available online 1 May 2015
acteristics of GCeMS, thirteen C18 trans fatty acid methyl ester (FAME) standards were separated and
verified in 23 min, validation experiments were also conducted. The correlation coefficient was R2
0.99
in the linear range of each FAME. At the low, medium and high fortification levels, recoveries fell within
Keywords:
GCeMS
90e119%. The relative standard deviations were below 9% for all 13 FAMEs. Low limits of detection (0.001
Routine analysis e0.002 g/100 g) and quantification (0.002e0.007 g/100 g) were readily achieved. The content of C18
C18 trans fatty acid trans fatty acid isomers in pressed and solvent extracted oils were analyzed using this method.
Non-hydrogenated edible vegetable oil © 2015 Elsevier Ltd. All rights reserved.

1. Introduction
Quick and accurate quantification of trans fatty acids (TFA) has
been an issue more important since the USA and a number of Eu-
ropean paid particular attention to the link between TFA intake and
cardiovascular disease (CVD) (Fritsche, Petersen, & Jahreis, 2010;
WHO, 2008). Major amounts (estimated 55e65%) of TFA intakes
derived from processing of vegetable oils (SACN, 2007; Filip, Fink,
Hribar, & Vidrih, 2010), while minor amounts of TFA isomers are
found in refined edible vegetable oils due to the high temperatures
used during the deodorization procedure (Ceriani & Meirelles,
2007; Santos, Cruz, & Casal, 2015; Filip, Hribar, & Vidrih, 2011),
which have been shown to induce geometrical isomerization of cis-
* Corresponding author. School of Food Science and Engineering, Harbin Institute
of Technology, No. 73 Huanghe Road, Nangang District, 150090 Harbin, Hei-
longjiang, PR China. Tel.: þ86 451 86282910; fax: þ86 451 86282906.
** Corresponding author. Key Laboratory of Agro-Product Quality and Safety,
Institute of Quality Standard and Testing Technology for Agro-Product, Chinese
Academy of Agricultural Sciences, No. 12 South Street, Zhongguancun, Haidian
District, 100081 Beijing, PR China.
E-mail address: yangxin@hit.edu.cn (X. Yang).
unsaturated fatty acids including linoleic acid (18:2 cis-9, cis-12)
and linolenic acid (18:3 cis-9, cis-12, cis-15) (Devinat, Scamaroni, &
Naudet, 1980). Tasan and Demirci (2003) observed that the trans
C18:2 isomer content was increased 13.8-fold at the end of the
refining process. The TFAs in common vegetable oils such as corn,
peanut, olive, soybean oil are mainly compounds of 18 carbon
atoms with 1e3 double bonds (Hou, Wang, Wang, Xu, & Zhang,
2012). In terms of quantitative distribution, not only the total
amount of TFA can largely vary within the same food category, but
also the C18 trans fatty acid isomeric profile can significantly be
affected by processing parameters. The development of a quick
method for quantifying the respective C18 trans fatty acid isomers
is extremely useful for clarifying the transformation in either
refinement or modification process and identifying the isomers of
C18 trans fatty acid that contribute to the risk of CVD (Hunter, 2005;
Stender, Dyerberg, Bysted, Leth, & Astrup, 2006).
Analysis of C18 trans fatty acid isomers could be achieved by
different methods. Gas chromatography (GC) represents the cur-
rent qualitative and quantitative analysis routinely used among the
major techniques such as TLC, HPLC and FTIR (AOCS 2005; Chen
et al. 2014; JOCS 2013; Yoshinaga et al. 2013). However, in

http://dx.doi.org/10.1016/j.foodcont.2015.04.027
0956-7135/© 2015 Elsevier Ltd. All rights reserved.
294 M. Zhang et al. / Food Control 57 (2015) 293e301
complex mixtures of C18 trans fatty acid, all isomers are rarely
resolved by GC equipped with usual flame ionization detector (GC-
FID), resulting in some slight overlap of positional isomers as well
as in biased data. Nevertheless, the problem could be overcome by
auxiliary fractionation techniques, such as silver nitrate thin-layer
chromatography (Agþ-TLC), silver ion solid phase extraction (Agþ-
SPE), silver ion high-pressure liquid chromatography (Agþ-HPLC)
and mass spectrometry (MS). Complex sample preparation pro-
cedures involving purification of TFA isomers by Agþ-TLC, Agþ-SPE,
Agþ-HPLC have been well-documented and used for research
purposes (Destaillats et al. 2007). However, in routine analysis of
food industry and safety supervision, time and cost constraints do
not permit multiple analytical steps. As an alternative approach
without prior silver-ion fractionation, direct quantification such as
GCeMS can be performed by the use of long and highly polar sta-
tionary phase and under optimal temperature programming con-
ditions (Bicalho, David, Rumplel, Kindt, & Sandra, 2008; Golay,
Giuffrida, Dionisi, & Destaillats, 2009; Kandhro et al., 2008;
Priego-Capote, Ruiz-Jime nez, & Luque de Castro, 2007). Therefore,
quick methods using GCeMS for routine analysis of common C18
trans fatty acids in non-hydrogenated edible vegetable oils are
necessary.
The current study was aimed to find a quick routine method
based on GCeMS to simultaneously analyze C18 trans fatty acids in
vegetable oil samples. We employed different methylation pro-
cedures as well as chromatography and mass spectrometry condi-
tions. GC was coupled with MS to achieve more accurate peak
identification than the FID detector and more simplification than
silver ion-based methods in routine analysis. The linoleic acid and
linolenic acid geometrical isomers in the pressed and solvent
extracted oil sample were analyzed with the optimized condition.
2. Experimental
2.1. Reagents
Hexane (HPLC Grade) and MeOH (HPLC Grade) were purchased
from Sigma Company. The boron trifluoride (BF3) methanol solu-
tion was acquired from Aldrich (Milwauke, WI, USA). Thirteen kinds
of fatty acid methyl ester (FAME) standards (Table 1)were pur-
chased from Sigma Company, and were of purity
99.0% (GC).
Mixed standard solution: Concentration of mixed standard solu-
tions is decided, depending upon sensitivity of each compound for
the instrument. Mixed standard solutions should be stored at
2e8  C. Working standard mixed solutions are prepared fresh,
diluted with hexane, mixed thoroughly. Ultra-pure water
(18.2 MU cm) was produced by a Millipore purification system
Table 1
Mixed standard solutions of thirteen C18 trans fatty acid methyl esters.
No. Trans fatty acid
1 18:1 trans-6
2 18:1 trans-9
3 18:1 trans-11
4 18:2 trans-9, trans-12
5 18:2 cis-9, trans-12
6 18:2 trans-9, cis-12
7 18:3 trans-9, trans-12, trans-15
8 18:3 trans-9, trans-12, cis-15
9 18:3 trans-9, cis-12, trans-15
10 18:3 cis-9, trans-12, trans-15
11 18:3 cis-9, cis-12, trans-15
12 18:3 cis-9, trans-12, cis-15
13 18:3 trans-9, cis-12, cis-15
(USA). All other chemicals and solvents were of the analytical grade
and used without further purification.
2.2. Selection of trans fatty acids
The protocol was performed for thirteen trans fatty acid of
eighteen carbon chain length: 18:1 trans-6; 18:1 trans-9; 18:1
trans-11; 18:2 trans-9, trans-12; 18:2 trans-9, cis-12; 18:2 cis-9,
trans-12; 18:3 trans-9, trans-12, trans-15; 18:3 cis-9, trans-12, trans-
15; 18:3 trans-9, cis-12, trans-15; 18:3 trans-9, trans-12, cis-15; 18:3
trans-9, cis-12, cis-15; 18:3 cis-9, trans-12, cis-15; 18:3 cis-9, cis-12,
trans-15. The selection was chosen according to the case that the
TFAs in common vegetable oils such as corn, peanut, olive, soybean
oil are mainly compounds of 18 carbon atoms with 1e3 double
bonds. These isomers were hardly separated and verified. The
names of these TFAs and their corresponding methylated pro-
ductions were presented in Table 1.
2.3. Edible oil samples
Six kinds of non-hydrogenated edible vegetable oils were pur-
chased from a local supermarket in Harbin, China and stored in
dark bottles without headspace at room temperature. The samples
were analyzed following the procedure described below, then used
as unfortified samples in the recovery study and comparison test of
solvent extracted and pressed oils.
2.4. Methylation
The oil was methyl esterified by potassium hydroxide/methanol
(KOH/MeOH) and boron trifluoride/methanol (BF3/MeOH)
methods. (1) To be a conventional derivatization procedure, KOH/
MeOH method was carried out according to the ISO official method
(ISO 12966-2, 2011) with some modifications. 1 mL 0.5 mol L1
potassium hydroxide/methanol was added to 0.03 g sample in 1 mL
n-hexane and vortexed for 30 s, then neutralized with hydrochloric
acid and centrifuged. The upper n-hexane layer was collected and
filtered, after that, 1.0 mL filtrate was injected into the GCeMS
systems for FAMEs analysis. (2) The BF3/MeOH method was based
on reported method (Araujo, Nguyen, Froyland, Wang, & Kang,
2008) with some modifications. Briefly, 0.03 g sample in 1 mL
hexane and 1 mL of 14% BF3/MeOH was heated at 100  C for 1 h
under nitrogen and cooled to room temperature. After 1 mL hexane
and 2 mL H2O were added, vortex-mixed for 15 s, centrifuged for
2 min and the methyl esters were extracted from the upper hexane
phase. Depending on the fat content the extract is either concen-
trated under nitrogen using a nitrogen evaporator (Organomation
Trans fatty acid methyl ester Concentration (mg mL1)
Trans-6-petroselaidic acid methyl ester 500.0
Methyl elaidate 435.5
Trans-11-vaccenic acid methyl ester 500.0
Trans-9, trans-12-octadecadienoic acid methyl ester 1000.0
Cis-9, trans-12-octadecadienoic acid methyl ester 400.0
Trans-9, cis-12-octadecadienoic acid methyl ester 400.0
Trans-9, trans-12, trans-15-octadecatrienoic acid methyl ester 1200.0
Trans-9, trans-12, cis-15-octadecatrienoic acid methyl ester 1800.0
Trans-9, cis-12, trans-15 -octadecatrienoic acid methyl ester
Cis-9, trans-12, trans-15-octadecatrienoic acid methyl ester
Cis-9, cis-12, trans-15- octadecatrienoic acid methyl ester 840.0
Cis-9, trans-12, cis-15-octadecatrienoic acid methyl ester
Trans-9, cis-12, cis-15-octadecatrienoic acid methyl ester
 M. Zhang et al. / Food Control 57 (2015) 293e301 295

Table 2
Parameters for determination of thirteen C18 trans fatty acids by GCeMS.

No. Trans fatty acid Retention time (min) Quantification ion Quantification ion 1 Quantification ion 2 Quantification ion 3

1 18:1 trans-6 17.91 55 (100) 296 (11) 264 (38) 222 (28)
2 18:1 trans-9 18.00 55 (100) 296 (11) 264 (34) 222 (24)
3 18:1 trans-11 18.10 55 (100) 296 (11) 264 (31) 222 (21)
4 18:2 trans-9, trans-12 19.17 67 (100) 294 (26) 263 (18) 81 (96)
5 18:2 cis-9, trans-12 19.65 67 (100) 294 (24) 263 (18) 81 (96)
6 18:2 trans-9, cis-12 19.82 67 (100) 294 (24) 263 (20) 81 (97)
7 18:3 trans-9, trans-12, trans-15 20.59 95 (100) 292 (6) 261 (10) 79 (81)
8 18:3 trans-9, trans-12, cis-15 21.12 95 (100) 292 (16) 261 (9) 79 (67)
9 18:3 trans-9, cis-12, trans-15 21.20 79 (100) 292 (11) 261 (9) 95 (77)
10 18:3 cis-9, trans-12, trans-15 21.36 95 (100) 292 (19) 261 (13) 79 (81)
11 18:3 cis-9, cis-12, trans-15 21.45 79 (100) 292 (19) 261 (13) 95 (88)
12 18:3 cis-9, trans-12, cis-15 21.83 95 (100) 292 (22) 261 (14) 79 (83)
13 18:3 trans-9, cis-12, cis-15 21.90 79 (100) 292 (20) 261 (14) 95 (84)

Associates, USA) or diluted with hexane and subsequently sub- to target abundance ratios. Quantification was based on peak area
jected to GCeMS analysis. ratio of the target ion divided by the external standard. Table 2
summarizes the trans fatty acids studied with their retention
times, the target and qualifier ions, and the qualifier to target
2.5. GCeMS analysis abundance ratios. Table 3 indicates the SIM programs used to
analyze trans fatty acids in edible oil.
An Agilent Technology 6890 gas chromatograph with an HP
5973 mass selective detector was employed. The gas chromato-
2.6. Method evaluation
graph was equipped with an Agilent 7683 autosampler (Agilent
technologies, USA). An HP-88, 100 m  0.250 mm i.d. capillary
The analytical method was validated according to the single
column with a 0.20 um film and a DB-5, 60 m  0.250 mm i.d.
laboratory validation approach. The performance of the method
capillary column with a 0.25 um film (J&W Sci. USA) were used,
was evaluated considering different validation parameters that
with helium as carrier gas at a constant flow of 1 mL min1. The
include the following parameters.
temperature of the injection port was 250  C and 1 mL volume was
The qualitative analysis of mass spectrum was based on more
injected in split mode with a split ratio of 50. The effect of tem-
than 3 ions, and the relative abundance of the ions should be within
perature program on the resolution of C18 trans fatty acid isomers
20%. The calibration curves for all of the compounds were obtained
was investigated. The following three temperature programs were
by plotting the peak area against the concentration of the corre-
used. (1) initial temperature 140  C, hold 5 min, 2.5  C/min ramp to
sponding calibration standards at six calibration levels. The iden-
240  C, hold 10 min, total analysis time 55 min; (2) initial tem-
tification of respective fatty acids was carried out by using the
perature 190  C, hold 5 min, 2  C/min ramp to 210  C, then 3  C/min
retention times of the standard samples and the contents were
to 240  C and held 5 min, total analysis time 30 min; (3) initial
calculated as grams per 100 g oil. The limit of detection (LODs) was
temperature 180  C, hold 2 min, 2  C/min ramp to 210  C and held
determined by considering a signal-to-noise ratio of 3, whereas the
5 min, then 2.5  C/min to 220  C and held 4 min, total analysis time
limits of quantification (LOQs) were determined by considering a
30 min; (4) initial temperature 180  C, hold 5 min, ramp at 2  C/
signal-to-noise ratio of 10. Trans fatty acid standards were added to
min, final temperature 216  C, total analysis time 23 min. Optimum
samples before methyl esterification and the recovery rate was the
condition was established based on the resolution of trans fatty
ratio of recovered concentration to prepared concentration. Tests
acids in the standard and used to analyze the samples.
were performed in triplicate.
The mass spectrometer was operated in electron ionization
mode with an ionizing energy of 70 eV, ion source temperature
230  C, MS Quad temperature 150  C, electron multiplier voltage 3. Results and discussion
(EM Volts) 1717 V when performing selected ion monitoring,
scanning from m/z 50 to 500 at 3.25 s per scan; solvent delay, 3.1. Selection of trans tatty acids
17.0 min.
Analysis was performed in the selected ion monitoring mode The primary components of vegetable oils are triacylglycerols,
(SIM) based on the use of one target and three qualifier ions. Target and their main intermediates during the biological activity are C18
and qualifier abundances were determined by injection of indi- fatty acids. In research of Gunawan group (Gunawan, Melwita, & Ju,
vidual trans fatty acid standards under the same chromatographic 2010), 18:1 and 18:2 fatty acids (18:1 cis-9; 18:1 trans-9; 18:2 cis-9,
conditions in full-scan mode with the mass/charge ratio ranging trans-12; 18:2 cis-9, cis-12) were verified in soybean oil rather than
from m/z 50 to 500. Trans fatty acids were identified according to 18:3 fatty acids. Under the instrumental conditions given above,
the retention times, the target and qualifier ions, and the qualifier efforts were undertaken to optimize the tuning parameters of all
the C18 trans fatty acid in the GCeMS in flow injection mode and
then their analytical performances were tested in different scan
Table 3 modes. Eventually, all the 13 kinds of C18 trans fatty acid methyl
Monitoring program of selected ions for thirteen C18 trans fatty acid methyl esters esters were proved to be applicable to GCeMS for determination.
by GCeMS.

Segment time (min) Monitored ions (m/z) Dwell time (ms)

3.2. Methylation
17.25 55, 222, 264, 296 60
18.50 67, 81, 263, 294 60 Before GC analysis, the fatty acid components of lipids are
20.35 79, 95, 261, 292 60
converted to the simplest convenient volatile derivative, usually
296 M. Zhang et al. / Food Control 57 (2015) 293e301
methyl esters. However, there is no single esterification procedure
that can be applied in all circumstances as the physical state of the
lipids can vary (Christie, 1993). Then the usual preparation of esters
with boron trifluoride-methanol (BF3/MeOH) and potassium
methoxide catalysts (KOH/MeOH) from C18 trans fatty acids in
vegetable oil were compared and discussed (Fig. 2). The Lewis acid,
boron trifluoride, in the form of its coordination complex with
methanol is a powerful acidic catalyst for the esterification of fatty
acids. But the result showed that the level of fatty acids esterifica-
tion was much lower, the peaks were unidentified and the
composition of isomers also changed when BF3/MeOH method was
carried out. As BF3/MeOH method was applied at usual reaction
temperature 100  C (boiling point of MeOH was 60  C), high tem-
perature may cause loss of solvent as the vials were not completely
gastight and transfer of double-bond. The higher reaction time also
increases the risk of side reactions. This was accordance with the
previous warning that boron trifluoride-methanol does have
serious drawbacks. Methoxy artefacts were produced from unsat-
urated fatty acids by addition of methanol across the double bond
even when normal (not higher than 50%) concentrations of boron
trifluoride in methanol were used. It has been reported that the
side-reactions are exacerbated by the presence of oxidized lipids
and sample size is critical with substantial losses sometimes
occurring with samples of less than 200 mg (Christie, 1993).
Therefore, in quick chromatography analysis, BF3/MeOH method is
inappropriate for microgram amounts only of seed oil samples. So
the peaks were complex and unidentified. Compared with the BF3/
MeOH method, the KOH/MeOH method is more effective, simpler,
less laborious, and easier to handle. The result was in accordance
with a study aiming at comparing ultrasonic and silent methylation
of vegetable oils, drawing a conclusion that BF3 is not a reliable
catalyst while giving rise to many artifacts (Stavarache, Vinatoru, &
Maeda, 2006). At the same time, as the most useful basic trans-
esterifying agents, potassium methoxide is also stable for several
months at refrigeration temperature. So the KOH/MeOH method is
Fig. 1. GCeMS-SIM chromatogram of 13 trans octadecenoic acid methyl esters in n-hexane. The retention time of each FAME was determined through the Fig. 1, and the numbered
peaks were identified as Table 1.
the suitable preparation of esters for quick C18 trans fatty acids
analysis.
3.3. GCeMS conditions
The GC conditions were optimized to enable separation of the
C18 trans fatty acids with a single GC column. Different column
types, temperature regimes, flow rates and column identities were
tested and verified in order to resolve the analytes of the standard
mixture during an acceptable run time.
3.3.1. Column types
Two capillary columns including HP-88 and DB-5 were consid-
ered in this GC/MS analysis. The difference lies in the nature of the
substituted radical in the polymer chain, which leads to different
solvation properties of the stationary phase. Huang's work
demonstrated that cis and trans fatty acids could be separated and
quantified by the gas chromatography with a highly polar AT™-
Silar-90 column (Huang, Wang, & Crenshaw, 2006). Compared with
DB-5, isothermal operation with the strongly polar column HP-88
produced the fewest overlapping peaks of 13 kinds of C18 fatty
acids, especially for octadecatrienoic acid (C18:3) methyl esters
which had most isomers in C18 trans fatty acids and most difficult
to identify, such as 18:3 cis-9, trans-12, cis-15 and 18:3 trans-9, cis-
12, cis-15; 18:3 cis-9, trans-12, trans-15 and 18:3 cis-9, cis-12, trans-
15. The retention times and elution orders of 13 FAMEs on HP-88
column following optimization gave us confirmation of the iden-
tity of those peaks (Fig. 1). However, the result of Ratnayake's work
showed that the SP-2560 capillary column has a slight advantage
over the HP-88 column for the simultaneous resolution of all the
fatty acids generally found in partially hydrogenated vegetable oils
(Yoshinaga et al. 2014). This could facilitate further studies aiming
to improve the accuracy of C18 trans fatty acid analysis.
 M. Zhang et al. / Food Control 57 (2015) 293e301 297

Fig. 2. GCeMS-SIM Chromatogram of thirteen C18 trans fatty acid methyl esters in six edible oils at high concentration spiked level and six unfortified oil matrix, as well as the
enlargements framed in gray of (i) three trans monoenoic FAMEs (C18:1), (ii) three trans dienoic FAMEs (C18:2) and (iii) seven trans trienoic FAMEs (C18:3). Chart A, B, C, D, E and F
respectively indicate SIM chromatogram of thirteen C18 trans fatty acid methyl esters at high concentration spiked level showed in Table 4 (red curve) and unfortified matrix (black
curve) of pressed sunflower seed oil, pressed corn oil, pressed olive oil, solvent extracted sunflower seed oil, extracted rice oil, extracted soybean oil. Chart G indicates the
chromatogram of 13 FAMEs in extracted soybean oil using BF3/MeOH and KOH/MeOH methods. (For interpretation of the references to color in this figure legend, the reader is
referred to the web version of this article.)
298 M. Zhang et al. / Food Control 57 (2015) 293e301

Table 4
Concentrations (g/100 g FAME), average recovery (%) (n ¼ 3), RSDs (in parentheses), limits of detection (LODs) and limits of quantification (LOQs) of thirteen C18 trans fatty
acids in pressed and solvent extracted edible oil samples.

No. Trans fatty acid Pressed edible oils

Sunflower seed oil Corn oil Olive oil

Blank Average recovery% Blank Average recovery% Blank Average recovery%

Spiked level Spiked level Spiked level

0.02 0.06 0.12 0.02 0.06 0.12 0.02 0.06 0.12

1 18:1 trans-6 ND 107 (4) 95 (2) 109 (5) ND 114 (6) 114 (1) 109 (2) ND 108 (2) 112 (6) 103 (8)
2 18:1 trans-9 0.02 (5) 106 (1) 90 (1) 94 (7) 0.02 (1) 101 (1) 108 (1) 105 (3) 0.01 (8) 114 (1) 106 (6) 97 (9)
3 18:1 trans-11 ND 115 (1) 107 (5) 109 (6) ND 114 (1) 111 (3) 102 (5) ND 100 (3) 91 (1) 95 (2)
4 18:2 trans-9, trans-12 ND 102 (6) 113 (1) 114 (4) 0.06 (3) 99 (2) 114 (3) 113 (2) ND 105 (10) 108 (1) 112 (4)
5 18:2 cis-9, trans-12 0.11 (2) 104 (1) 102 (1) 95 (1) 0.30 (5) 104 (1) 106 (1) 106 (2) 0.03 (5) 107 (4) 94 (2) 97 (3)
6 18:2 trans-9, cis-12 0.07 (1) 92 (1) 102 (3) 106 (2) 0.27 (9) 101 (1) 102 (1) 102 (1) ND 114 (2) 106 (1) 106 (8)
Total trans 18:2 0.18 0.63 0.03
7 18:3 trans-9, trans-12, trans-15 ND 114 (1) 108 (8) 110 (5) 0.52 (6) 105 (1) 112 (1) 110 (6) ND 115 (2) 103 (1) 105 (9)
8 18:3 trans-9, trans-12, cis-15 0.048,10 (6) 113 (1) 113 (1) 112 (3) 0.069 (1) 100 (2) 104 (4) 113 (1) 0.039 (2) 115 (1) 118 (1) 106 (8)
9 18:3 trans-9, cis-12, trans-15
10 18:3 cis-9, trans-12, trans-15
11 18:3 cis-9, cis-12, trans-15 0.0112,13 (4) 113 (1) 114 (1) 108 (1) 0.0511,13 (7) 113 (1) 102 (1) 112 (1) 0.0211 (4) 104 (8) 98 (4) 96 (3)
12 18:3 cis-9, trans-12, cis-15
13 18:3 trans-9, cis-12, cis-15
Total trans 18:3 0.05 0.63 0.05
Total TFA 0.25 1.29 0.09

ND: not detected.

Superscript: detected TFA No.

3.3.2. Temperature programs
In this study, different initial oven temperatures and different
ramping programs were assayed. There was a clear increase in the
total run time, as well as merged peaks of 18:3 cis-9, trans-12, trans-
15 and 18:3 cis-9, cis-12, trans-15 when initial temperature were
carried out at 140  C (temperature program (1) referred in Section
2.3). The peak resolution of 18:3 cis-9, trans-12, cis-15 and 18:3
trans-9, cis-12, cis-15 under 190  C initial temperature (temperature
program (2)) is lower than 180  C (temperature program (3) and
(4)). There was no significant difference in peak width and reso-
lution under column temperature program (3) and (4). However,
under program (4) (initial temperature 180  C, hold 5 min, ramp at
2  C/min, final temperature 216  C, total analysis time 23 min.) the
process is simpler and the total analysis time is shorter. The results
were accordance with a previous report (Huang et al., 2006) that
highly polar capillary columns exhibit temperature dependence
because the dispersion forces and polar interactions between the
stationary phase and fatty acids increase as the column tempera-
ture decreases.
3.3.3. Flow rate
In this study three different constant gas flow rates (0.8, 1.0,
1.2 mL min1) were tested being evaluated the chromatographic
separation and the ratio of each trans fatty acid. The best results
were obtained with 1.0 mL min1, thus, this flow sped was chosen.
3.3.4. MS conditions
We initially carried out a scanning test of the standard solution
of each fatty acid to be analyzed to describe its scanning mass
spectrum and retention time. Once retention times of each analyte
were determined in full scan mode, the next step involved the
determination of the optimal SIM mode conditions, in order to
obtain for each fatty acid a correct identification, a chromato-
graphic peak with enough number of points (10e15 points along
the peak), and a good signal intensity. The relevant considerations
in SIM mode parameters included a careful choice of the number of
ions selected for each fatty acid as well as for each time window,
and the dwell time (i.e. the time spent monitoring a single ion).
When the fatty acid varieties determined were either great in
numbers, or their retention times were relatively concentrated, we
selected ions based on the following four criteria: (1) choose the
molecular ion as detecting ions; (2) choose fragment ion with high
abundance such as base peaks; (3) choose characteristic ions with
selectivity to minimize the cross-interferences between different
fatty acids; (4) the monitoring ions selected, in the matrix their
signal/noise ratio should be greater than three with subtracted
background. Based on these criteria, we finally selected one target
ion and three qualitative ions for each compound (Table 2). Fig. 1
shows GCeMS chromatogram of 13 trans fatty acids in hexane
based on the SIM program.
3.4. Isolation and identification of FAMEs
The identification of FAMEs was achieved by comparing the
retention times of the sample with those of the synthesized FAMEs
standards. Overlapping of different individual FAMEs was observed
as shown in Fig. 1. The peaks of FAMEs mixtures containing (ii) trans
dienoic FAMEs (C18:2) were separated with good resolution but (i)
trans monoenoic FAMEs (C18:1) and (iii) trans trienoic FAMEs
(C18:3) moieties were not resolved so satisfactorily, especially
performed in oil samples (Fig. 2). The differences in the double
bond positions in the fatty acids and their corresponding volatility
could affect the response which has been known to be related to the
resolution. However, combined with mass spectrometry, identifi-
cation could be much better achieved than GC-FID analysis. For
example, the peaks of 18:3 trans-9, trans-12, cis-15 (Peak No. 8) and
18:3 trans-9, cis-12, trans-15 (Peak No. 9) were totally overlapped
using FID detector in previous work (Hou et al. 2012). Although GC-
FID with longer column is one of the solutions to separate FAMEs, it
is difficult for them to separate all the thirteen C18 trans fatty acids
and longer columns take longer time. The compositions of C18 trans
fatty acid methyl esters in oil samples were showed in Fig. 2. As
expected, C18 cis fatty acids are the most abundant among these
vegetable oils, which has been widely reported before. Rather 18:1
trans-9 than 18:1 trans-6 and 18:1 trans-11 were detected in all six
oil samples. Compared with fraction (ii) dienoic and (iii) trienoic
 M. Zhang et al. / Food Control 57 (2015) 293e301 299

Solvent extracted edible oils LOD LOQ

Sunflower seed oil Rice seed oil Soybean oil

Blank Average recovery% Blank Average recovery% Blank Average recovery%

Spiked level Spiked level Spiked level

0.02 0.06 0.12 0.02 0.06 0.12 0.02 0.06 0.12

ND 109 (1) 114 (2) 108 (4) ND 114 (6) 115 (3) 98 (8) ND 108 (3) 96 (1) 111 (2) 0.002 0.006
0.02 (5) 98 (4) 112 (7) 107 (7) 0.20 (6) 93 (1) 107 (1) 96 (2) 0.05 (4) 115 (2) 91 (6) 102 (9) 0.002 0.007
ND 115 (4) 102 (4) 92 (1) ND 116 (3) 94 (4) 108 (3) ND 119 (1) 114 (1) 118 (2) 0.002 0.007
ND 108 (1) 114 (3) 102 (1) 0.17 (7) 119 (1) 94 (2) 97 (5) ND 104 (8) 111 (3) 112 (2) 0.002 0.005
0.06 (5) 107 (1) 112 (1) 93 (1) 1.62 (5) 111 (1) 99 (1) 108 (1) 0.12 (8) 118 (1) 108 (1) 102 (4) 0.001 0.002
0.05 (6) 98 (1) 95 (1) 105 (1) 1.59 (3) 106 (1) 113 (1) 109 (1) 0.83 (9) 107 (1) 117 (1) 114 (2) 0.001 0.002
0.11 3.38 0.95
ND 100 (1) 101 (1) 100 (1) ND 102 (1) 92 (1) 104 (8) ND 118 (1) 102 (1) 119 (1) 0.002 0.006
0.249 (6) 101 (7) 114 (1) 105 (1) 0.368,9,10 (3) 102 (2) 115 (1) 118 (1) 0.118,10 (6) 95 (1) 100 (2) 96 (1) 0.002 0.005

0.3511,12,13 (6) 101 (1) 112 (3) 100 (8) 1.1811,12,13 (4) 116 (1) 105 (1) 116 (1) 0.8011,13 (7) 107 (1) 105 (1) 109 (2) 0.002 0.006

0.59 1.54 0.91

0.72 5.10 1.92

FAMEs, there was a peak resolution problem in fraction (i) mono-
enoic FAMEs which was similar to previous observation that the
individual peak of FAMEs with C18 moieties in bakery products
were overlapping when applying GC confirmation (Phillips, Ruggio,
& Amanna, 2010). In Stolyhwo's work (Stolyhwo & Rutkowska,
2013), FAMEs were separated by Agþ/HPLC into classes, then
trans fraction could be separated into eight structural C18:1 isomers
including 18:1 trans-9 and 18:1 trans-11. It seems to be a practical
solution to the difficult question of analysis of the composition of
monoenoic FAMEs. However, from the standpoint of much simpler
process and acceptable recovery (90e119%) of 13 trans FAMEs, this
GCeMS method was efficient to analyze TFAs in vegetable oils.
3.5. Validation procedure
3.5.1. Linearity
The external standard method was used in the quantification.
The respective C18 trans fatty acid isomer was quantified using a
six-point calibration of mixed standard solutions covering respec-
tive concentration range. Good linearity of the MS detector
response was found for all C18 trans fatty acid at concentrations
from 5.0 to 50.0 mg mL1 (take 18:1 trans-9 as reference, relative
concentrations were showed in Table 1) with linear regression
coefficients (R2) higher than 0.990.
3.5.2. Recovery, reproducibility
We validated analytical results by establishing the precision and
recovery of the analysis on quality control samples. Recoveries
(n ¼ 5) were calculated as follows: %Recovery ¼ [(cF  cU)/cS]  100,
where cF ¼ concentration of TFAs measured in the fortified sample,
cU ¼ concentration of TFAs measured in the unfortified sample (set
to zero), and cS ¼ concentration of TFAs added to the fortified
sample. The experiments were performed by spiking TFA species
into oil samples at 3 levels: low, medium and high. Recoveries of
the analytes ranged from 90 to 119%. Repeatability of peak areas for
all trans-fatty acids expressed as RSDs was below 9% (Table 4). Fig. 2
displays GCeMS chromatogram of 13 trans fatty acids in six edible
oil matrix and six spiked edible oil samples.
3.5.3. LOD and LOQ
The LODs in edible oil ranged from 0.001 to 0.002 g/100 g and
the LOQs from 0.002 to 0.007 g/100 g for 13 kinds of C18 trans fatty
acid (Table 4). Because of the similar structure, the peak identifi-
cation is extremely difficult which led to relatively higher limits
than 5 trans fatty acid methyl esters standards (LODs ranged from
0.8 to 1.2 ppm and LOQs ranged from 2.6 to 3.9 ppm) reported (Liu,
Stephen, & Chen, 2007). However, compared with the determina-
tion of FAMEs (LOD was 0.10 mg g1 and the LOQ was 0.30 mg g1)
including 2 trans fatty acid methyl esters in edible oils using GC
(Petrovic, Kezi
c, & Bolan
ca, 2010), the limits of the 13 kinds of C18
trans fatty acid methyl esters were much lower. In addition, there
were not much more papers or standards to refer to. The presented
validation data combined with others predicted the method was
suitable for the proposed analyses.
3.6. Pressed and extracted oil sample results
The TFA composition of non-hydrogenated edible vegetable oil
samples are shown in Fig. 2. The major TFA isomers found in sun-
flower seed oil, olive oil, corn oil, rice oil, soybean oil were 18:1
trans-9; 18:2 trans-9, trans-12; 18:2 trans-9, cis-12; 18:2 cis-9, trans-
12; 18:3 trans-9, trans-12, trans-15; 18:3 cis-9, trans-12, trans-15;
18:3 trans-9, cis-12, trans-15; 18:3 trans-9, trans-12, cis-15; 18:3
trans-9, cis-12, cis-15; 18:3 cis-9, trans-12, cis-15; 18:3 cis-9, cis-12,
trans-15. The TFA composition of soybean oil was compatible with
Wolff (1992), two trans isomers (18:2 trans-9, cis-12 and 18:2 cis-9,
trans-12) of linoleic acid and four trans isomers (18:3 trans-9, cis-12,
trans-15; 18:3 cis-9, cis-12, trans-15; 18:1 trans-9, cis-12, cis-15, 18:3
cis-9, trans-12, cis-15) of linolenic acid were identified in soybean
oil. The total TFAs detected in soybean oil, extracted and pressed
sunflower oil, corn oil samples were 1.92, 0.72, 0.25, 1.29 g/100 g,
respectively. They compared with the range from 0.14 g/100 g to
4.76 g/100 g reported by Hou et al. (2012) for The TFAs in edible oils.
At the same time, TFA in olive oil (0.09 g/100 g) was below the
minimum (0.14 g/100 g) and TFA in rice oil (5.10 g/100 g) was above
the maximum (4.76 g/100 g). In addition to total TFA, all the con-
tents of trans C18:1, 9t, trans C18:2 and trans C18:3 in pressed olive
300 M. Zhang et al. / Food Control 57 (2015) 293e301
Fig. 3. The contents of the C18:1, 9t (A), total trans C18:2 (B), total trans C18:3 (C), and total trans fatty acid (D) in pressed sunflower oil, pressed corn oil, pressed olive oil, extracted
sunflower oil, extracted rice oil, extracted soybean oil.
oil were lowest among the six kinds of non-hydrogenated edible
vegetable oil (Fig. 3). Though 3e12% for linoleic acid and 0.6e1.4%
for linolenic acid were ranged in Ballus's work (Ballus et al. 2014) as
well as others, much higher than other vegetable oils, cold pressing
process decrease the TFA amount. The concentrations of trans
linoleic acids in both pressed and extracted sunflower oil were just
higher than pressed olive oil, while Romero, Cuesta, and Sa nchez-
Muniz (2000) discovered the abundance of them in sunflower oil.
Obviously, the trans unsaturated fatty acids in solvent extracted rice
oil were particularly richer than others. Unlike pressed oils,
extracted oils are disposed by deodorization process during which
TFA may be formed because their activation energy (125 kJ/mol) is
low (Gunawan et al., 2010). Furthermore, TFAs formation during
refining will be inevitably higher in oils rich in either a-linoleic
acids or a-linolenic acids such as rice and soybean oil. In addition,
not all pressed oils (0.09e1.29 g/100 g) were lower in content of C18
trans fatty acid than extracted oils (0.72e5.10 g/100 g) such as
pressed corn oil. However, the TFA content of the sunflower seed oil
in different process showed that the oils being pressed maybe
healthier than extracted when the two processes were both viable
for the seeds. Some raw materials like rice were not suitable for
press process because of low oil extraction rate, while olive was just
suitable for being pressed.
4. Conclusions
Gas chromatography-mass spectrometry combined with KOH/
MeOH methylation has been proved valid for routine analysis of
C18 trans fatty acid in edible vegetable oils. Using this method, the
respective C18 trans fatty acids are identified quickly without
longer columns and standard addition which are useful but can not
meet the cost and time needs of quick routine analysis of food in-
dustry and safety supervision. As a result, this method features the
short analysis time, simple analytical steps and individual isomers.
Several limitations including the imperfect separation of three
monoenoic acids were observed. It will be of great importance if a
method to identify individual C18 trans fatty acid and evaluate the
suitability of GCeMS analysis by optimizing temperature profile in
reasonable analysis time can be established in the future. The C18
trans fatty acid isomers in the pressed and solvent extracted oil
samples including sunflower seed oil, corn oil, olive oil, rice oil and
soybean oil were analyzed and compared. The method described
here was found practicable for routine analysis of edible oil.
Compliance with ethics requirements
Min Zhang declares that she has no conflict of interest. Xin Yang
declares that he has no conflict of interest. Haitian Zhao declares
that he has no conflict of interest. Aijun Dong declares that she has
no conflict of interest. Jing Wang declares that she has no conflict of
interest. Guangyang Liu declares that she has no conflict of interest.
Pu Wang declares that she has no conflict of interest. Cuiling Cheng
declares that she has no conflict of interest. Hua Zhang declares that
she has no conflict of interest. This article does not contain any
studies with human or animal subjects.
 M. Zhang et al. / Food Control 57 (2015) 293e301
Acknowledgment
This work was financially supported by the Chun Hui Project of
the Chinese Ministry of Education (Z2005-1-23001) and the Special
Financial Aid to Postdoctoral in China (No. 201003192) during
2011e2013.
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