Food and Nutrition Sciences, 2013, 4, 1255-1259
Published Online December 2013 (
Open Access FNS
Development and Validation of a Single HPLC Method for
Analysis of Purines in Fish Oil Supplements
Liton Roy, Chad C. Harrell, Alan S. Ryan, Thorsteinn Thorsteinsson, Frederick D. Sancilio
Department of R & D, Sancilio and Company, Inc., Riviera Beach, USA.
Received September 24th, 2013; revised October 24th, 2013; accepted November 2nd, 2013
Copyright © 2013 Liton Roy et al. This is an open access article distributed under the Creative Commons Attribution License, which
permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. In accordance of
the Creative Commons Attribution License all Copyrights © 2013 are reserved for SCIRP and the owner of the intellectual property
Liton Roy et al. All Copyright © 2013 are guarded by law and by SCIRP as a guardian.
Gout is one of the most common forms of inflammatory arthritis, affecting over 8 million adults in the US. Individuals
with gout are advised to avoid the habitual intake of purine-rich foods such as meats, seafood, purine-rich vegetables,
and animal protein. An increased risk of developing or having subsequent attacks of gout is particularly associated with
the consumption of seafood. However, clinical studies have shown that certain seafood and fish oil supplements that
contain large amounts of omega-3 fatty acids provide important cardiovascular benefits. Individuals who might benefit
from omega-3 fatty acid supplementation may therefore avoid fish oil products because they contain purines. Currently,
there are no distinct high-performance liquid chromatography (HPLC) methods available in the literature that are vali-
dated as per the International Conference on Harmonisation (ICH) guidelines for the analysis of omega-3 fatty acid oils
or fish oil containing products for purine content. A robust, fast, and efficient reversed-phase high-performance liquid
chromatography (RP-HPLC) method was developed and validated for the specific analysis of the naturally occurring
purines guanine, purine, theobromine, and adenine. These purines are often found in fish oil and seafood. The analytical
method reported herein quantifies all four purines in fish oil in about 20 minutes.
Keywords: Purines; Chromatography; Gout; Uric Acid; Omega-3 Fatty Acids
1. Introduction
Gout is a type of inflammatory arthritis that is associated
with the accumulation of uric acid, also known as hype-
ruricemia [1]. If too much uric acid accumulates in the
blood, crystals (monosodium urate) are formed, and the
crystals deposit in joints, tendons, and surrounding tis-
sues. These crystals cause joints to swell and become in-
flamed [1]. Gout leads to substantial morbidity and se-
vere pain [1]. The prevalence of gout in the US has more
than doubled between the 1960s and the 1990s [2]. Based
on data from the latest nationally representative sample
of US adults (National Health and Nutrition Examination
Survey, [NHANES, 2007-2008]) the prevalence of gout
was 3.9% (8.3 million individuals) in 2007-2008 [1]. A
western diet, sedentary lifestyle, increased prevalence of
obesity and hypertension, and increased use of diuretics
and aspirin have been linked to the growing prevalence
of gout [3]. In particular, the dietary intake of purine-rich
foods such as red meats, anchovies, sardines, beans,
peas, lentils, and spinach is associated with hyperurice-
mia because the human body produces uric acid when it
breaks down purines [4]. The risk of gout associated with
seafood consumption is particularly high, especially
among men [5]. As a result, individuals who are suscep-
tible to gout are often careful to avoid dietary products
that contain large amounts of purines.
The long-chain polyunsaturated omega-3 fatty acids
eicosapentaenoic acid (EPA) and docosahexaenoic acid
(DHA) are found mainly in marine foods, particularly in
cold water fish like salmon, anchovies, sardines, and her-
ring. Some of these fish are also known to contain large
amounts of purines. However, many clinical studies have
demonstrated that EPA and DHA provide numerous car-
diovascular benefits [6]. Both EPA and DHA reduce se-
rum triglyceride and very low-density lipoprotein cho-
lesterol (VLDL-C) levels by 30% or more. Both DHA and
EPA have also been shown to modestly raise high-density
lipoprotein cholesterol (HDL-C) and lower total choles-
Development and Validation of a Single HPLC Method for Analysis of Purines in Fish Oil Supplements
terol, especially in individuals with high triglyceride lev-
els who are taking statins [6]. As a result, numerous die-
tary supplements available in the marketplace, including
cod liver oil, contain fish oil with the omega-3 fatty acids
EPA and DHA. Additionally, the Food and Drug Ad-
ministration (FDA) has recently approved two drugs (Lo-
vaza® and Vascepa®) that contain concentrated levels of
omega-3 fatty acids from fish oil. These drugs are in-
dicated for individuals with severe hypertriglyceridemia
or elevated triglyceride levels (>500 mg/dL).
Purines in fish oil used for drugs and some dietary
omega-3 fatty acid supplements are typically eliminated
during the purification and distillation process. However,
not all dietary omega-3 fatty acids supplements have
been evaluated for purine content; some may contain in-
determinate levels of purine. Other commercially avail-
able products such as sardines and cod liver oil also have
not been evaluated for purine content. The primary ob-
jective of the present study is to describe a new re-
versed-phase high-performance liquid chromatography
(RP-HPLC) method that allows the testing of purine lev-
els in omega-3 fatty acid fish oils and other fish products.
2. Experimental Methods
2.1. Chemical and Reagents
Guanine, purine, theobromine, and adenine were pur-
chased from Acros Organics, NJ (Purity: 99.9%). HPLC
grade solvents used for the sample preparations and
chromatography analyses were obtained from Fisher Sci-
entific, Pittsburgh, PA. Pharmaceutical grade, super con-
centrated omega-3 fatty acid fish oil samples (Ocean
Blue® Professional Omega-3 2100™) were obtained from
Sancilio and Company, Inc., Riviera Beach, FL. Com-
mercially available fish oil from sardines and cod liver
oil (Nature’s Way) were obtained from the local super-
market (West Palm Beach, FL).
2.2. Instrumentation
The Agilent 1100 series HPLC system equipped with
multiple wavelength detector, on-line degasser, and col-
umn compartment with temperature control was used for
method development and validation. Data acquisition,
analysis, and reporting were performed using ChemSta-
tion (Agilent) software. The HPLC columns (250 × 4.6
mm ID) and Luna 5u Silica 100A were purchased from
Phenomenex, Torrance, CA. The ODS Hypersil 5u 120A
was obtained from Thermo Scientific, Waltham, MA.
The detection wavelength of 270 nm and sample injec-
tion of 10 µL with an auto sampler was used.
2.3. Sample Preparation
Purine stock solution was prepared by transferring 5 mg
of each purine standard into a 50 mL volumetric flask.
The solution was diluted to volume with diluent (80:20
methanol: water) to achieve a stock concentration of 100
µg/mL. For method validation experiments, stock purine
solution was diluted with diluent to achieve the desired
concentration levels relative to the analytical concentra-
tion (1 µg/mL is also referred as 100% level). All sam-
ples and mobile phase were stored at ambient tempera-
Omega-3 fatty acid oil samples were prepared by
transferring 100 mg of the oil into a 50 mL volumetric
flask and then diluted with 95% methanol. Volume was
adjusted with water. The mixture was sonicated for 1
hour to obtain a homogenous mixture.
2.4. Method Development
High aqueous RC-HPLC with end capped reverse phase
columns was utilized to retain and separate polar com-
pounds. Absorbance of the eluent was measured at 270
nm. Different mobile phase gradients of water and me-
thanol were used during method development to obtain
separation between purines. Initial experiments were
conducted on the ODS Hypersil C18 250 × 4.6 mm col-
umn. However, the Phenomenex Luna C18 250 × 4.6
mm produced the desired separation with tailing factors
less than 2.0 for all purines and resolution of more than
2.0 between peaks at concentration levels ranging from
0.05 µg/mL to 100 µg/mL.
2.5. Method Validation Studies
The chromatographic method was validated for sensitiv-
ity, linearity, range, accuracy, precision, specificity, and
robustness. The sensitivity of the method was determined
by establishing the limit of detection (LOD) and limit of
quantitation (LOQ) for each purine with signal-to-noise-
ratios of 3:1 and 10:1, respectively.
3. Results
The resolutions and tailing factors for the 4 purines are
shown in Table 1. The resolutions are all greater than 2,
whereas the tailing factors are not more than 2, which are
in accordance with United States Pharmacopeia (USP)
criteria. Chromatographic conditions are summarized in
Table 2. The injection volume of 10 µL represents an
acceptable level of reproducibility while extending the
longevity of the column. The mobile phases of water and
methanol facilitate use of the HPLC.
Figure 1 shows the representative chromatograms for
each purine: guanine, purine, theobromine, and adenine
at 10 µg/mL concentration, respectively. Since the method
was conducted at 270 nm, the peaks of the chromato-
grams for each purine are easy to distinguish.
According to Figure 2(a), the omega-3 fatty acid fish
oil sample obtained from Sancilio and Company, Inc.
Open Access FNS
Development and Validation of a Single HPLC Method for Analysis of Purines in Fish Oil Supplements 1257
Table 1. Resolutions (R) and USP tailing factors (TF) for
the four purines evaluated.
Guanine Purine Theobromine Adenine
(µg/mL) R TF R TF R TF RTF
0.05 - 1.1 6.9 1.5 3.9 1.2 4.41.1
1 - 1.1 7.0 1.4 3.9 1.3 3.91.4
40 - 1.1 6.6 1.5 3.7 1.5 4.01.7
100 - 1.1 4.6 1.6 3.4 1.8 3.22.0
Table 2. Chromatographic conditions.
Time (minutes) % MPB
0.0 1
3.0 1
10.0 35
10.10 85
13.0 85
13.1 1
20.0 1
Equipment: HPLC system with degasser, autosampler, and MWD detector.
HPLC column: Phenomenex Luna Silica analytical column 250 × 4.6 mm 5
micron. Flow rate: 1.0 mL/min. Injection volume: 10 µL. Wavelength: 270
nm, 16 BW, Reference 360 nm. Mobile phase A: 100% Water. Mobile
phase B: 100% Methanol. Column temperature: Ambient. Run time: 20
Figure 1. HPLC chromatogram overlay of guanine, purine,
theobromine, and adenine.
was free of purines. Fish oil obtained from sardines (Fig-
ure 2(b)) contained 0.8 mg/g of guanine and 2.9 mg/g of
purine. Cod liver oil (Figure 2(c)) contained 4.1 mg/g of
guanine and 1.7 mg/g of purine.
3.1. Linearity and Range
Linearity of the assay was demonstrated by analyzing the
peak responses against the analytical concentrations at
various concentrations. The method was linear between
the concentration ranges of 0.05 µg/mL to 1.00 µg/mL
for all 4 purines, with the linear correlation coefficient
Figure 2. (a) HPLC overlay of purine standards and omega-
3 fatty acid fish oil; (b) HPLC chromatogram of fish oil
obtained from sardines. Fish oil from sardines contained 0.8
mg/g of guanine and 2.9 mg/g of purine; (c) HPLC chro-
matogram of fish oil obtained from cod liver. Fish oil from
cod liver contained 4.1 mg/g of guanine and 1.7 mg/g of
0.9999. For all 4 purines, the limit of quantification
(LOQ) ranged from 0.02 to 0.04 µg/mL and the limit of
detection (LOD) was determined to be 0.01 µg/mL. For
quantifying the contents of purines in the fish oil samples,
the established sensitivity appeared to be sufficient.
3.2. Accuracy and Precision
The accuracy and precision of the method were verified
by evaluating percent recovery of purines from spiked
omega-3 fatty acid fish oil samples (Table 3). The
omega-3 fatty acid fish oil samples were spiked with all
four purines at 2 µg/mL, 10 µg/mL, and 20 µg/mL con-
centration levels. The assay recovery values were ana-
Open Access FNS
Development and Validation of a Single HPLC Method for Analysis of Purines in Fish Oil Supplements
Table 3. Accuracy and precision of HPLC method.
2 µg/mL level 10 µg/mL level 20 µg/mL level
Recovery %
Recovery %
Recovery %
Guanine 104 1.7 103 0.5 102 0.4
Purine 101 3.1 100 0.0 100 0.1
Theobromine 99 2.2 101 0.1 100 0.1
Adenine 100 2.0 102 0.1 102 1.7
lyzed for the individual preparations against purine stan-
dards solutions. The average (mean) percent recovery
was between 99% and 104%, indicating excellent accu-
racy. The percent standard deviations (% RSD) of three
replicate preparations were between 0.1 and 3.1, showing
good precision of the method used.
3.3. Robustness
Small and deliberate variations in HPLC parameters were
made to verify the robustness of the analytical method.
The HPLC parameter variations studied included flow
rate (1.0 ± 0.1 mL/min), column temperature (25˚C ±
3˚C), wavelength (270 ± 2 nm), injection volume (10 ± 5
µL), and a different lot of column (Table 2). For all the
HPLC variations, the chromatography was comparable to
the procedural conditions in terms of peak resolution,
peak tailing, and injection precision. The method was,
therefore, considered to be robust.
3.4. Specificity
Method specificity was established by demonstrating that
there was no interfering peak from the diluent or omega-
3 fatty acid fish oil free of purines. Peak homogeneity of
the purine samples were evaluated using a HPLC with a
photodiode array detector. No degradation was observed
for the purine standards in solution stored at ambient
conditions and exposed to light for up to 15 days. Figure
2(a) is a representative chromatogram of the omega-3
fatty acid fish oil sample and the four purine standards.
4. Discussion
Gout management requires a comprehensive strategy that
considers the underlying causes of hyperuricemia [4].
Successful uric-acid lowering will prevent frequent goat
attacks and disease progression. Uric acid-lowering drugs
are usually prescribed concomitant with nonsteroidal
anti-inflammatory agents (NSAIDs) to reduce inflamma-
tion and pain [4]. For proper gout management, a dietary
change involving the reduced intake of purine-rich foods
is a prudent approach accompanied by regular monitor-
ing of serum uric acid levels. For those with gout, it is
important to follow a strict, low-purine diet. Knowing the
amount of purines in different food products may help
reduce the risk of gout flare-ups induced by abrupt
changes in serum uric acid levels.
This paper describes a simple, efficient, and repro-
ducible RP-HPLC method specifically for the analysis of
naturally occurring purines in fish oils and other food
products. The resulting method has been fully validated
as per ICH guidelines [7]. As the new method is a simple
and reproducible approach, it can be easily implemented
in a quality controlled laboratory. To the best of our
knowledge, this is the first fully ICH validated RP-HPLC
method that can be used as a single procedure to quantify
accurately the amount of guanine, purine, theobromine,
and adenine in fish oils in about 20 minutes. Three com-
mercially available fish oil products were analyzed: a
super concentrated omega-3 fatty acid fish oil sample
(Ocean Blue® Professional Omega-3 2100™) and fish oil
from sardines and cod liver oil. While the Ocean Blue®
omega-3 fatty acid fish oil sample was free of purines,
the fish oil from sardines and cod liver oil contained sub-
stantial amounts of guanine and purine. Notably, this
method can also be used to quantify the amount of
purines in various other matrices provided that the purine
peaks do not interfere with the matrix.
The ability to retain and separate polar and hydrophilic
molecules such as purines was very challenging. When
using conventional reverse phase liquid chromatography,
it is necessary to use ion pair reagents, mobile phase pH
modification, and concentrated buffers [8]. Such an ap-
proach may have potential detrimental effects on method
reproducibility and sample solubility. Using conventional
techniques also result in poor retentions and separations.
An optimized gradient chromatography system allows
the detection of guanine, purine, theobromine, and ade-
nine at very low concentrations [9]. As described in this
paper, chromatographic peaks were identified via the
retention times of known standards, with detection at 270
nm wavelength [10]. The Liquid Chromatography Mass
Spectrometry (LC-MS) compatible mobile phase of the
new method is useful in the identification of any future
potentially unknown and or new chromatographic peaks
that may be present in commercially available fish oil
5. Conclusion
In sum, a simple, efficient, reproducible RP-HPLC me-
thod has been developed for the analysis of naturally
occurring purines in fish oils. An LC-MS compatible
mobile phase would allow the analysis of any contami-
nates found in fish oils. Such applications are currently
underway in our research and development laboratory at
Sancilio and Company, Inc.
Open Access FNS
Development and Validation of a Single HPLC Method for Analysis of Purines in Fish Oil Supplements
Open Access FNS
6. Acknowledgements
The authors thank our colleagues in the Analytical Re-
search and Development Department at Sancilio and
Company Inc. for their support during this study.
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