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Food
Chemistry
Food Chemistry 109 (2008) 207–211
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Analytical Methods

GC-MS quantification of fatty acid profile including trans FA

in the locally manufactured margarines of Pakistan
Aftab Kandhro, S.T.H. Sherazi *, S.A. Mahesar, M.I. Bhanger, M. Younis Talpur,
Abdul Rauf
National Center of Excellence in Analytical Chemistry, University of Sindh, Jamshoro 76080, Pakistan

Received 19 August 2007; received in revised form 7 December 2007; accepted 13 December 2007

Abstract

Ten margarine brands of Pakistan were analyzed for their fatty acid composition with emphasis on trans fatty acids (TFA) using GC-
MS. Saturated, cis-monounsaturated and polyunsaturated fatty acids were present at 24.2–58.1, 5.7–35.4 and 3.8–37.4% of total fatty
acids, respectively. Among the saturated fatty acids, palmitic acid (16.9–33.8%) was dominant in all analyzed margarine brands and
its higher amount indicates that palm oil was a major contributor in the margarine manufacturing. Among samples tested only one con-
tained a low level of TFA (2.2%) while the rest contained very high amounts of TFA (11.5–34.8%) which clearly shows that hydrogenated
oils were used in the formulation of margarines. Fatty acid profiles demonstrated that all samples belong to the hard margarine category
containing high amounts of trans and saturated fatty acids which is an alarming issue for the health of consumers.
Ó 2008 Elsevier Ltd. All rights reserved.

Keywords: Margarine; Fatty acid profile; Trans fatty acids; GC-MS

1. Introduction lower in saturated fat. Margarine at that time was heavily

hydrogenated and was widespread in the food supply as the
Margarine is a butter-like product obtained from mix- major source of industrially produced TFA. The curves for
tures of various edible fats and oils. Usually margarine CHD mortality tracked fairly closely with those of TFA
contains appropriate ratios of hard vegetable fats from intake over time. Angina and myocardial infarction were
coconut, palm kernel, interesterified vegetable oils and/or unusual clinical events in the early part of the 1900s, but
hydrogenated vegetable oils. Mostly in the industrial cata- CHD increased rapidly and became the major cause of
lytic hydrogenation process some natural fatty acids are death by mid-twentieth century. Rates peaked in 1950–
destroyed and new artificial trans isomers are produced 1960, and mortality has gone down by about 50% since
that behave similar to saturated fats. These isomers lack that time. Thus, changes in TFA intake correspond
the essential metabolic activity of the parent compounds roughly with the epidemic of CHD. Therefore apprehen-
and inhibit the enzymatic desaturation of essential fatty sion in trans-fatty acids originates from their association
acids (Dimitrios, Vasilios, & Haralambos, 2003). As a with coronary heart disease (Willet et al., 1993; Willett,
result of the economic dislocations during World War II, 2006). Metabolic studies have provided unequivocal
margarine production rose rapidly as a replacement for evidence that TFA increase plasma concentrations of
butter. During the 1960s margarine became viewed as the low-density lipoprotein (LDL) cholesterol and reduce con-
healthy alternative to butter because comparatively it was centrations of high-density lipoprotein (HDL) cholesterol
relative to the parent natural fat (Ascherio & Willet,
*
Corresponding author. Tel.: +92 22 2771379; fax: +92 22 2771560. 1997). These effects of trans fatty acids were more harmful
E-mail address: tufail_sherazi@yahoo.com (S.T.H. Sherazi). than saturated fatty acids. Studies also indicated that TFA

0308-8146/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodchem.2007.12.029
208 A. Kandhro et al. / Food Chemistry 109 (2008) 207–211

raise lipoprotein level, an independent inherited factor of peratures were 240 and 260 °C, respectively. The mass
coronary heart diseases (Mensink & Katan, 1990; Saba- spectrometer was operated in the electron impact (EI)
rense & Mancini Filho, 2004). In addition to adverse effect mode at 70 eV in the scan range of 50–550 m/z.
on lipoprotein, trans fatty acids (TFAs) also reported to
influence vascular functions, cardiac arrhythmias and 2.5. Calculations and statistical analyses
SCD (Katz, 2002).
Peak identification of the fatty acids in the analyzed
2. Materials and methods margarine samples was carried out by the comparison with
retention times and mass spectra of known standards.
2.1. Samples and reagents Standard methyl esters of palmitic, stearic, oleic and lino-
leic acids were used for the confirmation of GC-MS
Ten margarine samples were purchased from local libraries result. Two samples of each brand were collected
supermarkets of Hyderabad, Pakistan. The choice of the and each sample was analyzed three times. The data
brands was based on the highest consumption among those obtained were put into Origin 7 program and reported as
available in the market. All reagents, chemicals and sol- mean (n = 2  3).
vents used were from E. Merck (Darmstadt, Germany).
Trans and cis fatty acids methyl esters (FAME) standards 3. Results and discussion
(GLC 481-B and 607) were purchased from Nu-Check-
Prep, Inc. (Elysian, MN). The results of fatty acid composition of analyzed marga-
rine samples are divided into saturated and unsaturated
2.2. Sample preparation fatty acids, shown in Tables 1 and 2, respectively. The mar-
garine brands were coded as M-A, M-B, M-C, M-D, M-E,
Approximately 1 g of margarine samples was melted in M-F, M-G, M-H, M-I and M-J. All analyzed margarine
an oven at 40–50 °C to obtain the fat phase. The upper brands contain the significant amount of C12:0, C14:0,
fat phase was removed after centrifugation at 448 g for C16:0 and C18:0. Table 1 shows that the dominant fatty
4 min (Mehmet, Ayhan, & Metin, 2003) then dried by add- acid among the saturated group is palmitic acid (C16:0)
ing anhydrous sodium sulphate to remove the moisture and its range vary from 16.9 to 33.8%. The highest amount
from margarines. Fat obtained from margarine samples of palmitic acid (33.8%) was found in sample M-C while it
was transferred into 5 ml glass vials. The decanted samples was lowest 16.9% in M-D sample. The results of palmitic
were all frozen at 18 °C until analysis. acid indicate the greater contribution of palm oil in the
margarine manufacturing. Stearic acid (C18:0) was present
2.3. Determination of fatty acid composition at 6.1– 19.0%. Meanwhile, lauric acid (C12:0) was present
at 0.1–11.2%, and myristic acid (C14:0) at 0.2–8.7%. Some
For the determination of fatty acids composition of the odd number fatty acids like pentadecanoic acid (C15:0) and
margarine samples FAMEs were prepared using standard margaric acid (C17:0) were also determined in considerable
IUPAC, 1979 method 2.301. Agilent GC-MS 5975 was amounts in some samples. Naturally these both fatty acids
used with ChemStation 6890 Scale Mode software. GC- are not very common in the vegetable oils while almost all
MS chromatogram obtained were compared with two animal fats contain their some amount (Shoji, Kazutaka, &
libraries (NIST & Wily) which provide best information Masatoshi, 2005). Saturated fatty acids with the chain
about the identification of fatty acid present in margarine length of (C12:0–C16:0) carbon atoms have been reported
samples to avoid the use of costly standards. to be atherogenic, stearic acid (C18:0) neutral, while oleic
and polyunsaturated fatty acids produced a blood lipid
2.4. GC-MS conditions lowering effect (Aro, Jauhiainen, Partanen, Salminen, &
Mutanen, 1997; Hu et al., 1999; Yu, Etherton, & Kris-Eth-
The GC-MS analysis for FAMEs was performed on erton, 1995).
Agilent 6890 N gas chromatography instrument coupled Table 2 includes the monounsaturated (MUFA) and
with an Agilent MS-5975 inert XL mass selective detector polyunsaturated fatty acids (PUFA) of margarines. Among
and an Agilent autosampler 7683-B injector (Agilent Tech- the MUFA, oleic acid (C18:1 cis-9) was the major fatty
nologies, Little Fall, NY, USA). A capillary column HP- acid present. Oleic acid is considered to be responsible
5MS (5% phenyl methylsiloxane) with dimension of for lowering the LDL (bad) cholesterol levels. Whereas
30 m  0.25 mm i.d  0.25 lm film thickness (Agilent polyunsaturated fatty acids have beneficial effects on both
Technologies, Palo Alto, CA, USA) was used for the sepa- normal health and chronic diseases, such as regulation of
ration of fatty acid methyl esters. The initial temperature of lipid levels (Mori et al., 2000) cardiovascular (Kris-Ether-
150 °C was maintained for 2 min raised to 230 °C at the ton, Harris, & Appel, 2002) and immuno functions (Hwang
rate of 4 °C/min, and kept at 230 °C for 5 min. The split 2000). The highest amount of oleic acid was found in the
ratio was 1:50, and helium was used as a carrier gas with sample M-B (34.8%) while other samples contained in the
the flow rate of 0.8 ml/min. The injector and detector tem- range of 4.2–19.4%. The other members of MUFA,
 A. Kandhro et al. / Food Chemistry 109 (2008) 207–211 209

Table 1
Saturated fatty acids composition (mean percentage FAMEs) of margarine samples
Samples C8:0 C10:0 C12:0 C14:0 C15:0 C16:0 C17:0 C18:0 C20:0 C22:0 C23:0
M-A 0.4 0.5 1.0 7.8 1.6 30.1 1.3 14.9 0.4 0.2 0.1
M-B 0.7 0.3 6.2 3.9 – 20.9 – 19.0 0.8 0.8 –
M-C – 0.1 0.3 1.5 0.1 33.8 0.1 8.2 0.4 0.1 –
M-D – – 0.1 0.2 – 16.9 – 6.1 0.4 0.5 –
M-E – 0.4 0.9 6.5 1.7 28.0 2.7 13.2 0.4 0.2 0.1
M-F 0.2 0.6 11.2 2.4 0.9 28.6 2.0 7.5 0.5 – –
M-G 0.2 0.5 8.9 2.4 0.3 30.5 0.9 8.6 0.3 0.1 0.1
M-H 0.5 0.7 10.1 4.0 – 25.4 0.1 11.6 0.4 0.1 –
M-I – 0.9 8.2 7.2 – 27.0 – 11.0 0.1 0.1 –
M-J 0.2 – 1.1 8.7 0.1 24.1 0.1 13.7 1.3 0.3 –

Table 2
Unsaturated fatty acids composition (mean percentage – FAMEs) of margarine samples
Samples C16: C18: C18: C18: C18: C18: C18: C18: C18: C20: C20: C22:
1n9 1n8 1n9t 1n9 2n10,13 2n8,11 2n9,12t–t 2n9,12 3n9,12,15 1n11 3n1,4,8t–t–t 1n13
M-A 2.3 – 24.9 5.8 1.5 1.0 1.4 2.2 0.2 0.3 0.1 1.5
M-B 0.1 – 2.2 34.8 – 9.6 – – – 0.5 – –
M-C 0.2 7.5 34.7 – – 12.3 0.1 – – 0.1 – –
M-D 0.1 5.7 32.6 – 35.3 – – 0.1 2.0 – – –
M-E 1.9 4.9 26.5 4.2 2.3 4.3 – 0.8 – 0.5 – 0.2
M-F 0.2 1.0 10.5 14.1 – 0.1 1.0 17.3 1.3 0.1 0.1 0.6
M-G 0.1 2.2 8.1 19.4 0.2 0.1 0.2 14.1 2.0 0.7 – –
M-H 0.1 – 11.5 13.6 0.1 – 0.2 19.1 2.0 0.1 – 0.1
M-I – 1.1 15.0 14.7 – – 0.5 13.7 0.5 – – –
M-J 2.3 3.0 28.8 10.0 0.7 3.1 1.5 – – 0.1 – 0.6
n indicates the position of double bond.

C16:1 cis-9, C18:1 cis-8, C20:1 cis-11 and C22:1 cis-13 was four (M-F, M-G, M-H and M-I) contained greater amount
also determined in the range of 0.1–2.3, 1.0–7.5, 0.1–0.7 of linoleic acid (C18:2 cis 9,12) at 13.7–17.3%. In some sam-
and 0.1–1.5%, respectively. The major trans fatty acid ples like M-B, M-C and M-J it was found to be totally
observed in all margarine brands was elaidic acid (C18:1 absent. While the samples M-D and M-E contained less
trans-9) in the range of 2.2–34.7%. Out of 10, three samples than 1%. Ovesen, Torben, & Hansen (1998) divided marga-
(M-C and M-D and M-J) contained elaidic acid (C18:1 rine into three categories on the basis of linoleic acid (LA)
trans-9) at 28–35% (34.7, 32.6 and 28.8%), two samples (i) hard margarines with less than 20% LA (ii) semi soft
(M-A and M-E) have 24.9 and 26.5%, and of four samples margarines with 20–40% LA and (iii) soft margarines with
(M-G, M-F, M-H, M-I) ranging from 8 to 16% trans (8.1, more than 40% LA. Therefore all analyzed margarine
10.5, 11.5 and 15.0%), respectively. Only one (M-B) marga- brands were hard margarines and none of the samples
rine sample contained 2.2% TFA which is lowest value was included in the semi soft or soft margarines. The pres-
among all analyzed margarine brands. Other trans fatty ence of different isomers of oleic and linoleic acid in marga-
acids determined in the margarine samples were C18:2 t–t rine samples identify that hydrogenated oil is used in all
9, 12 and C20:3 t–t–t 1, 4, 8 in the ranges of 0.1–1.5 and analyzed margarine brands. It is the only partial hydroge-
0.1%, respectively. None of the analyzed sample in the nation process that can change both the geometrical config-
present study shows less than 2% TFA, while the data of uration and the double bond shift (McDonald & Mossoba,
other countries indicate that each study contain few marga- 2002).
rine samples in which there were not any TFA or had less Table 3 represents the fatty acids groups and ratio
than 1% TFA (Dimitrios et al., 2003; Jiri & Jan, 2000; Kar- between them. The saturated/unsaturated FA shows the
abulut & Turan, 2006; Matsuzaki et al., 2002; Tsanev, relation between two major fatty acid groups of the marga-
Russeva, Rizov, & Dontcheva, 1998). rine fat. Its value varies from 0.32 to 1.41. Only one sample
PUFA has major importance for biological and nutri- had a ratio of 0.32 while in all other samples it varied from
tional value of margarines. C18:2 cis10,13; C18:2 cis 8,11; 0.82 to 1.41 which clearly indicates a high proportion of
C18:2 9,12 t–t; C18:2 cis 9,12, C18:3 cis 9,12,15, C20:3 t– saturated fatty acids. The prevalence of unsaturated over
t–t 1,4,8 are the members of PUFA determined in the mar- saturated fatty acids (smaller ratio) is considered to be
garine brands, in the range of 0.1–35.3, 0.01–12.3, 0.1–1.5, positive from the nutritional point of view. The satu-
0.1–19.1, 0.2–2.0 and 0.1%, respectively. Out of 10 samples, rated + TFA fraction ranged from 54.8 to 84.5% and
210 A. Kandhro et al. / Food Chemistry 109 (2008) 207–211

Table 3
Groups and ratio between the types of fatty acids from the composition of margarine samples
Groups and ratio of FA M-A M-B M-C M-D M-E M-F M-G M-H M-I M-J
SFA 58.1 52.6 44.7 24.2 54.2 53.9 52.7 52.9 54.4 49.8
cis MUFA 9.9 35.4 7.7 5.7 11.8 15.9 22.3 13.9 15.8 16.0
trans MUFA 24.9 2.2 34.7 32.6 26.5 10.5 8.1 11.5 15.0 28.8
Total MUFA 34.8 37.6 42.4 38.3 38.3 26.5 30.5 25.4 30.8 44.8
cis PUFA 4.9 9.6 12.3 37.4 7.4 18.6 16.5 21.3 14.2 3.8
trans PUFA 1.5 – 0.1 – – 1.02 0.2 0.2 0.5 1.5
Total PUFA 6.3 9.6 12.4 37.4 7.4 19.7 16.7 21.4 14.7 5.3
Total TFA 26.4 2.2 34.8 32.6 26.5 11.5 8.3 11.7 15.6 30.3
SFA + TFA 84.5 54.8 79.5 56.8 80.7 65.4 61.0 64.6 70.0 80.1
MUFA + PUFA 41.2 47.1 54.8 75.7 45.7 46.1 47.1 46.8 45.5 50.1
cis MUFA + PUFA 14.8 44.9 20.0 43.1 19.2 34.6 38.8 35.1 30.0 19.7
SFA/UFA 1.4 1.1 0.8 0.3 1.2 1.2 1.1 1.1 1.2 1.0
cis PUFA/SFA 0.1 0.2 0.3 1.6 0.1 0.4 0.3 0.4 0.3 0.1
trans/cis 1.8 0.1 1.7 0.8 1.4 0.3 0.2 0.3 0.5 1.5
cis PUFA/(SFA + TFA) 0.1 0.2 0.2 0.7 0.1 0.3 0.3 0.3 0.2 0.1
cis MUFA + PUFA/SFA + TFA 0.2 0.8 0.3 0.8 0.2 0.5 0.6 0.5 0.4 0.3

achieved a very high mean value of 69.7%. The ratio of was used to achieve more accurate peak identification.
trans/cis-FA represents the degree of formation of artificial Great variation in the fatty composition has been observed
TFA from the natural cis forms of unsaturated fatty acids among different brands of analyzed Margarine. In this
of the margarines and the ratio varies between 0.05 and study, a total of ten local margarine brands were collected,
1.79. Few samples M-B, M-F, MG and M-H had lower nine of them were found to contain very high amounts of
ratios of 0.05, 0.33, 0.21 and 0.33, respectively. While other trans fatty acids (15.6–34.8%) and only one sample con-
samples had a ratio greater than 0.50 which corresponds to tained comparatively a low level of TFA (2.2%). The higher
a higher content of TFA and showing that margarine man- amount of TFA and saturated fatty acids demonstrated the
ufactures are not serious to reduce the trans content in their poor quality of margarine in the market which is harmful
product and still using the conventional technology in the to the health of consumers. Further the amount of trans
processing and production of margarines. These results fat was not mentioned on the label of any local manufac-
also indicate that there is great variation in the quality of tured margarine brands.
local commercially available brands and even no two mar-
garine samples had similar fatty acid profiles. The cis-poly- Acknowledgements
unsaturated fatty acids (PUFA) which are nutritionally
important were found in the range of 3.8–37.4%. The mean The National Centre of Excellence in Analytical Chem-
ratio of cis PUFA/SFA recommended by the British istry, University of Sindh, Jamshoro, Pakistan is gratefully
Department of Health is 0.45 (Da et al., 2002). In this study acknowledged for the financial support. The authors also
cis PUFA/SFA ratio ranged from 0.08 to 1.55 and the would like to thank Higher Education Commission
mean value was 0.36, which is less than the recommended (HEC), Pakistan.
value. The mean values of the indices most commonly used
to express the nutritional value of edible fats cis-PUFA/ References
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