R E S E A R C H Open Access
Comparative analysis of EPA and DHA in fish oil
nutritional capsules by GC-MS
, Shuk-Man Li
, Jia-Yi Fan
, Lan-Lan Fan
, Zhi-Feng Zhang
, Pei Luo
, Xiao-Jun Zhang
, Jian-Gang Wang
, Zhong-Zhen Zhao
and Hu-Biao Chen
Background: Fish oil is a popular nutritional product consumed in Hong Kong. Eicosapentaenoic acid (EPA) and
docosahexaenoic acid (DHA) are the two main bioactive components responsible for the health benefits of fish oil.
Market survey in Hong Kong demonstrated that various fish oil capsules with different origins and prices are sold
simultaneously. However, these capsules are labelled with same ingredient levels, namely EPA 180 mg/g and DHA
120 mg/g. This situation makes the consumers very confused. To evaluate the quality of various fish oil capsules, a
comparative analysis of the contents of EPA and DHA in fish oil is crucial.
Methods: Agaschromatography–mass spectrometry (GC-MS) method was developed for identification and
determination of EPA and DHA in fish oil capsules. A comprehensive validation of the developed method was
conducted. Ten batches of fish oil capsules samples purchased from drugstores of Hong Kong were analyzed by
Results: The present method presented good sensitivity, precision and accuracy. The limits of detection (LOD) for
EPA and DHA were 0.08 ng and 0.21 ng, respectively. The relative standard deviation (RSD) values of EPA and DHA for
repeatability tests were both less than 1.05%; and the recovery for accuracy test of EPA and DHA were 100.50% and
103.83%, respectively. In ten fish oil samples, the contents of EPA ranged from 39.52 mg/g to 509.16 mg/g, and the
contents of DHA ranged from 35.14 mg/g to 645.70 mg/g.
Conclusion: The present method is suitable for the quantitative analysis of EPA and DHA in fish oil capsules. There is a
significant variation in the contents of the quantified components in fish oil samples, and there is not a linear relationship
between price and contents of EPA and DHA. Strict supervision of the labelling of the fish oil capsules is urgently needed.
Keywords: Fish oil, EPA, DHA, GC-MS, Comparative analysis
Omega-3 polyunsaturated fatty acids, which include the
fish oil components eicosapentaenoic acid (EPA) and
docosahexaenoic acid (DHA, Figure 1), are essential for
humans as cannot be synthesized by the human body
. It has been reported that omega-3 fatty acids are
very important in preventing and managing heart disease
. Findings show omega-3 fish oil may help to lower
blood pressure , reduce triglycerides accumulation ,
slow the development of plaque in the arteries , reduce
the chance of abnormal heart rhythm , reduce the
likelihood of heart attack and stroke, and lessen the
chance of sudden cardiac death in people with heart dis-
ease . Omega-3 parenteral nutrition can reduce the rate
of inflammatory complications after surgery . It is also
thought that EPA in particular may possess some bene-
ficial therapeutic potential in mental conditions, such
as schizophrenia, depression, hyperactivity and atten-
tion symptoms . DHA is essential for the growth and
functional development of the brain in infants , and
is also required for maintenance of normal brain func-
tion in adults [10,11]. The FDA states it is safe to take
up to 3 g of omega-3 per day to lower the risk for coron-
ary heart disease (CHD) and maintain health . Besides,
the American Heart Association (AHA) recommends
everyone eat fish (particularly fatty fish) at least twice a
* Correspondence: firstname.lastname@example.org;email@example.com
School of Chinese Medicine, Hong Kong Baptist University, Hong Kong
Special Administrative Region, Hong Kong, China
Full list of author information is available at the end of the article
© 2014 Yi et al.; licensee BioMed Central. This is an Open Access article distributed under the terms of the Creative Commons
Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution , and
reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain
Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article,
unless otherwise stated.
Yi et al. Lipids in Health and Disease 2014, 13:190
week. As omega-3 fatty acids are deemed important from
authoritative bodies, supplementation in addition to food
sources may need to be considered to help U.S. adults
meet recommendations .
In the past 10 years, many people have taken omega-3
fish oil supplements for their health benefits. Hong Kong
is one of the strongest markets for health supplements
in Asia , and more and more fish oil products are
being sold in the markets. These fish oils come from
different sources, and product prices vary significantly.
However, these products are labelled with same ingredi-
ent levels, namely EPA 180 mg/g and DHA 120 mg/g
(Table 1). Confronted with this variety, consumers are
eager to know whether these fish oils contain same EPA
and DHA contents? Whether there is relationship between
the EPA/DHA contents and the price or source? Are the
more expensive fish oil products really of better quality?
To answer these questions, a comparative analysis of the
contents of EPA and DHA in commercially available fish
oil is urgently needed.
The determination of EPA and DHA in fish oil has been
reported by high-performance liquid chromatography
(HPLC) [15,16] and liquid chromatography-mass spec-
trometry (LC-MS) [17,18]. However, the HPLC is not
sensitive enough and the cost of LC-MS is high. In the
present study, a new gas chromatography–mass spectrom-
etry (GC-MS) method was developed for the analysis of
fish oil. The samples bought from different drugstores in
Hong Kong were analyzed to determine DHA and EPA
contents. The results demonstrated that our method is
highly precise and accurate, and is therefore suitable for
the determination of EPA and DHA in fish oil. Significant
variation in the contents of EPA and DHA in fish oil
samples was founded, and there is not a linear relationship
between price and contents of EPA and DHA. Higher
price can not guarantee higher contents of EPA and
DHA. But because fish oil is expensive and its health
claims are so significant, this is one product the govern-
ment should seriously monitor.
The sources of the fish oil capsule samples are listed in
Table 1. Corresponding voucher specimens were deposited
in the School of Chinese Medicine, Hong Kong Baptist
Reagents and chemicals
The standard compounds of eicosapentaenoic acid methyl
ester, docosahexaenoic acid methyl ester and Supelco® 37
component fame mix were purchased from Sigma-Aldrich
(St. Louis, MO, USA). The purity of these chemical stan-
dards was more than 98% by GC-MS.
N-hexane was used as a solvent in GC-MS analysis,
which was purchased from the RCI Lab-Scan Limited
(Bangkok, Thailand). Potassium hydroxide and sodium
Figure 1 The chemical structures of EPA and DHA.
Table 1 The original sources, prices and contents of EPA and DHA in ten batches of fish oil capsule samples bought in
Hong Kong (n=3)
Labelled contents (mg/g) Determined contents (mg/g)
EPA DHA EPA DHA
2 Australia 1.68 148.05 137.15
3 USA 1.53 509.16 501.18
4 USA 1.49 418.54 645.70
5 Australia 1.02 132.44 123.26
6 USA 0.98 361.36 419.73
7 New Zealand 0.65 208.42 204.72
8 USA 0.64 166.42 125.33
9 USA 0.34 39.52 35.14
10 USA 0.23 112.19 153.11
Samples are listed in order of decreasing price; that is, the most expensive are listed first.
Yi et al. Lipids in Health and Disease 2014, 13:190 Page 2 of 6
chloride of analytical grade was purchased from Uni-Chem
(Shanghai, China). Boron trifluoride methanol complex so-
lution (13-15% BF
basis), used to carry out methyl esterifi-
cation, was purchased from Sigma-Aldrich (St. Louis, MO,
USA). Water was purified using a Milli-Q water system
(Millipore; Bedford, MA, USA).
GC–MS instrumentation and conditions
Shimadzu QP2010 GC-MS system (Kyoto, Japan) was used
for qualitative and quantitative analysis of fish oil. DB-5 ms
high resolution capillary column (Dikma Technologies.
thickness: 0.25 μm, length: 30 m, diameter: 0.25 mm)
was used for sample separation.
For temperature programming, the oven was maintained
at 80°C for one minute and then increased at a rate of 10°C
per minute to 250°C, the rate was then slowed to 8°C per
minute until 280°C was reached and maintained for 5 min.
Split injection was conducted with a split ratio of 10:1, and
helium was used as the carrier gas at a rate of 0.8 ml/min,
with the volume of injection as 1 μL. The mass spec-
trometer was operated in electron-impact (EI) mode. Pre-
column pressure: 70 kPa. Injection temperature: 250°C.
Ion source: EI (200°C). Interface temperature: 280°C. Elec-
tron energy: 70 eV. Solvent delay: 5.5 min. For qualitative
analysis, the full scan mode was used and the scan range
was 40–400 m/z. For quantitative analysis, selective ion
mode was used, and m/z 79 was chosen as the ion frag-
ment of EPA and DHA.
Preparation of standard and sample solutions
The stock solutions of EPA methyl ester (5 mg/L) and
DHA methyl ester (2.5 mg/L) were prepared in n-hexane
and stored in the refrigerator. The working solutions were
prepared by appropriate dilution of the stock solutions
with n-hexane, and the resulting concentrations of were 1,
2.5, 5, 10, 20, 25 and 30 mg/L. DHA was prepared in serial
dilutions of 2, 5, 10, 20, 40, 50 and 60 mg/L. Calibration
standard solution (1 μL) was injected for GC-MS analysis.
The preparation of sample solutions was performed as
previously described with modifications . Samples were
obtained from fish oil capsules by puncturing the capsule
with a needle syringe. Each sample of approximately 60 mg
was weighed accurately and placed in a centrifuge tube
with a ground stopper. 3 mL potassium hydroxide metha-
nol solution (0.5 M) was added. The contents were thor-
oughly mixed, and then the tube was filled with nitrogen
gas, heated in a water bath at 60°C, shaken three times in
the course of 20 min. When the oil droplets had disap-
peared completely and the solution was transparent, 3 mL
boron trifluoride methanol complex solution was added,
Figure 2 Typical GC-MS chromatograms of (A) the mix standard and (B) fish oil sample at full scan mode; and (C) fish oil sample at
selective ion mode. 1, Methyl myristate (18.4 min); 2, Methyl palmitoleinate (25.5 min); 3, Methyl palmitate (26.5 min); 4, Methyl heptadecanoate
(33.1 min); 5, Linoleic acid methyl ester (33.7 min); 6, Octadecenoic acid methyl ester (34.0 min); 7, Eicosapentaenoic acid methyl ester (40.8 min);
8, Docosahexaenoic acid methyl ester (48.5 min).
Yi et al. Lipids in Health and Disease 2014, 13:190 Page 3 of 6
and the mixture cooled. Each tube was then filled with ni-
trogen gas, and placed in a water bath at 60°C for 5 min.
Saturated sodium chloride solution of 2 mL and n-hexane
of 2 mL were added and mixed well. After centrifugation
(4000 rpm) for 10 min, the supernatant was drawn off, to
be used as sample solution. Dilution of the supernatant is
necessary in case its concentration falls out of the linear
range. An aliquot of 1 μL supernatant was injected for
Assay validation and sample determination
Linearity for standards was determined with five data
points over the concentration range of the working solu-
tions. Precision was evaluated by six injections of the sam-
ple solution (batch 1) within one day. Repeatability was
evaluated in intra- and inter-day assays of the fish oil sam-
ple FO1. The stability test was performed by analyzing the
sample solution (batch 1) over a period of 24 h. The rela-
tive standard deviation (RSD) was taken as the measures
of precision, repeatability and stability. Recovery of all the
quantified constituents was determined by sample in dif-
ferent concentration levels using a mixture of standards
with 50, 100 and 200% of the quantified levels of constitu-
ents in the samples. All fish oil samples were analyzed
using this method, and the acid/ester conversion factor
was set to 0.96.
Results and discussion
Optimization of hydrolysis and esterification conditions
EPA and DHA are present in fish oil in the form of vari-
ous triglycerides. To generate a volatile DHA methyl ester
for GC-MS analysis, EPA and DHA are released from the
triglycerides by hydrolysis, and then methyl esterified.
Various hydrolysis and methyl esterification conditions
(different time, temperature, usage of nitrogen gas) were
evaluated to obtain maximum extraction efficiency. The
results demonstrated that incubation of fish oil (c.a.
60 mg) in potassium hydroxide methanol solution (0.5 M,
3 mL) at 60°C for 20 min achieved complete hydrolysis.
Esterification with 3 mL boron trifluoride methanol solu-
tion at 60°C for 5 min provides the most methyl esters.
Moreover, nitrogen used as protection gas ensures less
oxidation and higher stability of the constituent contents.
After further optimization, the best experimental condi-
tions are shown in “Preparation of standard and sample
Chromatographic conditions and GC-MS identification
The chromatographic conditions such as temperature
gradient and carrier gas flow rate were optimized to achieve
satisfactory separation and sharp peak shape in the chro-
matograms. Full scan mode was used in qualitative tests
because it provides more peaks for identification. For
the qualitative analysis, apart from EPA (7) and DHA
(8), 6 characteristic peaks of constituent chemicals had
been successfully identified with the aid of the reference
standard. They are methyl myristate (1), methyl palmito-
leinate (2), methyl palmitate (3), methyl heptadecanoate
(4), linoleic acid methyl ester (5), and octadecenoic acid
methyl ester (6). The typical GC-MS chromatograms are
shown in Figure 2A and 2B.
In quantitative test, selective ion mode (SIM) was used
due to its higher sensitivity. Considering the abundance
of fragment ions of EPA and DHA in mass spectra
(Additional file 1: Figure S1), m/z 79 was used for calculat-
ing amount of EPA and DHA. The typical chromatogram
is shown in Figure 2C.
Validation of the analysis method
Results for assessment of the validity of the method are
summarized in Table 2, 3 and 4. The data indicate good
linearity between concentrations and peak areas of the ana-
lytes within the test ranges. The limits of detection (LOD)
for EPA and DHA were found to be 0.08 ng and 0.21 ng,
respectively. Therefore, the system was considered to be
sensitive. The relative standard deviation (RSD) values of
intra-day and inter-day variations were not more than
0.59% and 1.00% for EPA, and not more than 1.08% and
1.05% for DHA, respectively. The established method also
had acceptable accuracy with average recovery of
100.50% and 103.83% for EPA and DHA. All these re-
sults demonstrate that the developed GC-MS method
was sufficiently reliable and accurate and is therefore
suitable for quantification of EPA and DHA in fish oil.
The present method was successfully applied to the quan-
tification of EPA and DHA in fish oil capsule samples, and
the results are summarized in Table 1 and Figure 3. The
results reveal significant variation in the contents of the
quantified components in fish oil samples. Such variations
Table 3 Precision, repeatability and stability of EPA and
(RSD, %, n=3)
(RSD, %, n=3)
(RSD, %, n=6)
EPA 0.38 0.38 0.59 0.16 1.00 0.85
DHA 0.32 0.08 0.32 1.08 1.05 0.63
Table 2 Linearity calibration curve factors, LOD and LOQ
of EPA and DHA
Analyte Equation Range
LOD (ng) LOQ (ng)
EPA y = 41328.56x - 7.85 1-30 0.9999 0.08 0.15
DHA y = 11863.62x + 4.62 2-60 0.9999 0.21 0.60
Yi et al. Lipids in Health and Disease 2014, 13:190 Page 4 of 6
may be mainly due to the source and processing of the fish
Fish oils are commercially produced from cold water
fatty fish, including salmon, tuna, sardines, shellfish, and
herring. The contents of EPA and DHA in theses fishes
are significantly varied [20,21]. During the filling of soft
capsules, poor quality fish oil or even vegetable oil may
be added thereby adulterating good quality fish oil .
Adulteration can induce unstable contents of EPA and
DHA in the final products, which make the actual con-
tents do not match the label.
Figure 3 also shows that the relationship between the
price and the contents of DHA and EPA are insignificant.
In other words, according to the prices for the various fish
oil sold in markets, it was clear that the contents of EPA
and DHA did not always correlate with price. For ex-
ample, sample 3, 4 and 6 contained higher contents of
EPA and DHA but it was cheaper than samples 1, 2 and 5.
Distinctly, the classification of various prices of fish oil ac-
tually did not distinguish relative quality. This finding con-
firms the need for developing a reliable evaluation method
to ensure the quality of fish oil products.
A GC-MS method was developed for determination of
EPA and DHA content in fish oil capsules. A comprehen-
sive evaluation of the developed method was conducted,
and the method was shown to be highly sensitive, repro-
ducible and accurate. Samples of 10 commercial fish oil
capsule samples bought in Hong Kong retail stores were
tested. The results demonstrated significant variation in
the contents of EPA and DHA in the samples, and there is
not a linear relationship between price and contents of
EPA and DHA. Strict supervision of the labelling of the
fish oil capsules is urgently needed.
Additional file 1: Figure S1. The mass spectra of (a) EPA and (b) DHA
EPA: Eicosapentaenoic acid; DHA: Docosahexaenoic acid; GC-MS: Gas
chromatography–mass spectrometry; LOD: Limit of detection; LOQ: Limit of
The authors declare that they have no competing interests.
TY and HBC initiated the study; all authors contributed to designing the
study. The sample extraction was conduct by SML, JYF and LZ. The
optimization of experimental conditions was performed by LLF, ZFZ and PL.
The data analysis was conduct by XJZ, JGW and ZZZ. TY and SML drafted
the manuscript. All authors contributed to data analysis and manuscript
finalization. All authors read and approved the final manuscript.
This work was financially supported by the National Science Fund
(no.31060055) of the People’s Republic of China.
School of Chinese Medicine, Hong Kong Baptist University, Hong Kong
Special Administrative Region, Hong Kong, China.
Guangxi Botanical Garden
of Medicinal Plant, Nanning, Guangxi 530023, China.
The State Key
Laboratory of Quality Research in Chinese Medicine, Macau University of
Science and Technology, Macau, China.
School of Chinese Medicine,
Guangzhou University of Traditional Chinese Medicine, Guangzhou, China.
Received: 7 July 2014 Accepted: 9 December 2014
Published: 13 December 2014
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Cite this article as: Yi et al.:Comparative analysis of EPA and DHA in
fish oil nutritional capsules by GC-MS. Lipids in Health and Disease
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