Development and Validation of a Single HPLC Method for Analysis of Purines in Fish Oil Supplements
1256
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-
ture.
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.
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