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ISSN: 2157-7064

Journal of Chromatography

Ardente, et al., J Chromatogr Sep Tech 2016, 7:5
DOI: 10.4172/2157-7064.1000334

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Research Article

A Targeted Metabolomics Assay to Measure Eight Purines in the Diet ofCommon Bottlenose Dolphins, Tursiops truncatus
Ardente AJ1, Garrett TJ2, Wells RS1,3, Walsh M1, Smith CR4, Colee J5 and Hill RC*
College of Veterinary Medicine, University of Florida, Gainesville, FL, USA
Southeast Center for Integrated Metabolomics, Mass Spectrometry Core Laboratory, University of Florida, Gainesville, FL, USA
Chicago Zoological Society’s Sarasota Dolphin Research Program, C/O Mote Marine Laboratory, Sarasota, FL, USA
4
National Marine Mammal Foundation, San Diego, CA, USA
5
Department of Statistics, Institute of Food and Agricultural Sciences, University of Florida, Gainesville, FL, USA
1
2
3

Abstract
Bottlenose dolphins managed under human care, human beings and Dalmatian dogs are prone to forming
urate uroliths. Limiting dietary purine intake limits urate urolith formation in people and dogs because purines are
metabolized to uric acid, which is excreted in urine. Managed dolphins develop ammonium urate nephroliths,
whereas free-ranging dolphins do not. Free-ranging dolphins consume live fish, whereas managed dolphins consume
different species that have been stored frozen and thawed. Differences in the purine content of fish consumed
by dolphins under human care versus in the wild may be responsible for the difference in urolith prevalence.
Commercially available purine assays measure only four purines, but reported changes in purines during frozen
storage suggest that a wider range of metabolites should be measured when comparing fresh and stored fish. A
method using high performance liquid chromatography with tandem mass spectrometry was developed to quantify
eight purine metabolites in whole fish and squid commonly consumed by dolphins. The coefficient of variation within
and among days was sometimes high for purines present in small amounts but was acceptable (≤ 25%) for guanine,hypoxanthine, and inosine, which were present in high concentrations. This expanded assay identified a total purine
content up to 2.5 times greater than the total that would be quantified if only four purines were measured. Assuming
additional purines are absorbed, these results suggest that additional purine metabolites should be measured tobetter understand the associated risk when fish or other purine-rich foods are consumed by people or animals prone
to developing uroliths.

Keywords: Kidney stones; LC-MS/MS; IMP; Inosine; Hypoxanthine;
Urate; Uric acid; Fish; Purines

Abbreviations
DNA: Deoxyribonucleic acid; RNA: Ribonucleic acid; ATP:
Adenine triphosphate; GTP: Guanine triphosphate; AMP: Adenine
5’-monophosphate; IMP: Inosine 5’-monophosphate; LC: Liquid
chromatography; HPLC: High performance liquid chromatography;
MS/MS: Tandem mass spectrometry; HESI: Heated electrosprayionization; NH4: Ammonium; NH4OH: Ammonium hydroxide; MS222: Tricaine methanesulfonate; TP4: Total purine four metabolite
assay; TP8: Total purine eight metabolite assay.

Introduction
Purines contribute to the structure of deoxyribonucleic acid
(DNA) and ribonucleic acid (RNA), adenine triphosphate (ATP) andguanine triphosphate (GTP). Purines are made by the body, salvaged
and recycled, or absorbed from food. They are metabolized to uric acid,
which is excreted in the urine (Figure 1) [1-4].
Dietary purines are found in high concentrations in organ meatand seafood [1,5]. Consumption of a purine-rich diet increases the
production and excretion of uric acid. When the concentration ofuric acid in the urine reaches a threshold, uric acid precipitates out ofsolution and can aggregate to form urate-based stones in the urinary
tract. These urate uroliths can be composed of uric acid, ammonium
urate, monosodium urate, sodium calcium urate, or potassium urate.
Human beings primarily develop uric acid uroliths, and the prevalenceof these uroliths has increased internationally as a consequence ofincreased consumption of meat [6,7]. Individuals living in Taiwan,
for example, experience a high occurrence of uric acid stones as aconsequence of consuming purine-rich diets that include seafood
J Chromatogr Sep Tech, an open access journal
ISSN: 2157-7064

and specifically grass shrimp [8]. Some Dalmatian dogs have a genetic
predisposition to develop ammonium urate uroliths because of alteredpurine metabolism. For both species, consumption of a purinerestricted diet minimizes uric acid excretion and the risk of forming
urate uroliths.
Foods are currently classified according to their total purine
content, defined as the sum of the four nucleobases, adenine, guanine,hypoxanthine, and xanthine [1,8]. However, individual metabolites
have different propensities for causing hyperuricosuria and individual
metabolite concentrations may vary widely among purine-rich foods [912]. Human beings, for example, fed a diet supplemented with adenineand hypoxanthine excreted greater urinary uric acid concentrations
than when fed a diet supplemented with guanine and xanthine [13].
Thus, it may be more important to describe the individual purine
metabolite composition of food rather than total purine content, when
determining a food’s uricogenic potential.
It also may not be sufficient to describe just the nucleobase
content of foods. Additional metabolites, including nucleotides*Corresponding author: Hill RC, Small Animal Clinical Sciences, College of
Veterinary Medicine, University of Florida, 2015 SW 16th Ave., Gainesville, FL
32608, USA, Tel: +13523922235; E-mail: hillr@ufl.edu
Received August 29, 2016; Accepted September 12, 2016; Published September
19, 2016
Citation: Ardente AJ, Garrett TJ, Wells RS, Walsh M, Smith CR, et al. (2016) A
Targeted Metabolomics Assay to Measure Eight Purines in the Diet of Common
Bottlenose Dolphins, Tursiops truncatus. J Chromatogr Sep Tech 7: 334. doi:
10.4172/2157-7064.1000334
Copyright: © 2016 Ardente AJ, et al. This is an open-access article distributed
under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided theoriginal author and source are credited.

Volume 7 • Issue 5 • 1000334

Citation: Ardente AJ, Garrett TJ, Wells RS, Walsh M, Smith CR, et al. (2016) A Targeted Metabolomics Assay to Measure Eight Purines in the Diet ofCommon Bottlenose Dolphins, Tursiops truncatus. J Chromatogr Sep Tech 7: 334. doi: 10.4172/2157-7064.1000334

Page 2 of 9
adenine 5’-monophosphate (AMP), inosine 5’-monophosphate(IMP) and the nucleoside inosine (Figure 1) are absorbed by the
mammalian gastrointestinal tract and may also increase urinary uricacid concentrations [13-15]. Furthermore, the concentrations ofpurine metabolites are affected by food source and storage conditions[8,16,17]. For example, hypoxanthine concentrations progressively
increase over time in pork stored at 2°C, but this effect is delayed atlower storage temperatures [14]. Also, IMP, hypoxanthine, xanthine,
and inosine concentrations in fish fileted for human consumption havebeen demonstrated to vary with water temperature where fish werecaught, cooking methods, storage methods and duration, and among
species [8,18-20]. Thus, it would seem important to measure morepurine metabolites than the four nucleobases that are usually measured
and to report individual concentrations, when considering a fooditems’ uricogenic potential.
This is particularly relevant when considering purines in whole
fish and squid commonly consumed by dolphins. Common bottlenose
dolphins (Tursiops truncatus) managed under human care developammonium urate kidney stones, but these stones rarely occur in freeranging dolphins [21,22]. Why ammonium urate nephroliths form inmanaged dolphins is unclear, but the purine-rich whole fish diet may
contribute to nephrolith development because the diets of free-ranging
and managed bottlenose dolphins differ [5,23].
The total purine content of the fish species consumed by dolphinshas not been previously reported. The purine content of the fillets ofsome fish species has been measured using high performance liquid

chromatography (HPLC) with ultraviolet detection, but the authors
are aware of only one study that has measured purines in a whole fish
(gilted sea bream) [24]. Fish organs, including the liver and skin, arereported to be rich in purines; therefore, purines measured only in themuscle of fish may underestimate total purine content of whole fish
[5,25].
We sought, therefore, to develop an expanded assay using HPLC
with tandem mass spectrometry (MS/MS) that would accurately
identify and quantify a broader range of purine metabolites in both
fresh frozen fish commonly consumed by free-ranging dolphins andin stored frozen and thawed fish and squid commonly consumed bymanaged dolphins. We also hypothesized that it would not be possible
to predict the total purine content obtained with the new assay using anassay that only measures four nucleobases. In particular, we suspectedthat the concentration of some of the additional metabolites would belower and hypoxanthine concentrations would be higher for the frozen
stored and thawed species fed to managed dolphins.

Materials and Methods
Chemicals
The stock solutions used for the mobile phase and for purine
standards were prepared freshly at least monthly. The water used for
mixing all solutions was HPLC-grade. These stock solutions included:
0.1% acetic acid (glacial) in water, 10% formic acid in water, 10 mM
ammonium (NH4) acetate in water, and 2 mM ammonium hydroxide(NH4OH) in water. All chemicals were purchased from Fisher ScientificCompany, LLC (Suwanee, GA 30024).

Figure 1: Synthetic degradation and salvage pathways of purines compiled from several sources. Dotted lines represent salvage pathways.

AMP (Adenine monophosphate); IMP (Inosine monophosphate); XMP (xanthine monophosphate); GMP (Guanine monophosphate); APRT (Adenosine
phosphoribosyl transferase); HGPRT (Hypoxanthine-guanine phosphoribosyl transferase); PNP (Purine nucleoside phosphorylase)

J Chromatogr Sep Tech, an open access journal
ISSN: 2157-7064

Volume 7 • Issue 5 • 1000334

Citation: Ardente AJ, Garrett TJ, Wells RS, Walsh M, Smith CR, et al. (2016) A Targeted Metabolomics Assay to Measure Eight Purines in the Diet ofCommon Bottlenose Dolphins, Tursiops truncatus. J Chromatogr Sep Tech 7: 334. doi: 10.4172/2157-7064.1000334

Page 3 of 9
Individual standard solutions were freshly prepared before eachrun. Purine metabolite solubility is highly pH dependent. Thus, AMP,
IMP, adenine, and inosine were dissolved in 10 mM NH4CH3CO2 (pH6.5), whereas uric acid and xanthine were dissolved individually andhypoxanthine and guanine were dissolved together in 2 mM NH4OH(pH 12, Sigma-Aldrich, St. Louis, MO). Each solution was thensonicated in a heated (35°C) water bath for 10-15 minutes to achievecomplete dissolution. The individual standard purine solutions in
NH4CH3CO2 were combined to generate one mixed standard solution
with final concentrations of 0.01 mM for adenine, 0.02 mM for IMP,0.05 mM for AMP, and 0.15 mM for inosine. Individual standard
solutions dissolved in NH4OH were also combined to generate anothermixed standard solution with final concentrations of hypoxanthine andguanine at 0.23 mM, xanthine at 0.22 mM, and uric acid at 0.01 mM.
An internal standard, 15N2-xanthine (Cambridge Isotopes Laboratory,
Inc., Tewksbury, MA 01876), was mixed with 2 mM NH4OH to achievea concentration of 1.3 mM. The final concentrations of the individual
metabolites in the combined solutions were half their original starting
concentration.

Fish sampling and processing
Fish samples were collected by the Chicago Zoological Society’s
Sarasota Dolphin Research Program under the Mote Marine
Laboratory and University of Florida (UF) Institutional Animal Careand Use Committee approvals.
Eight fish species commonly consumed by free-ranging bottlenose
dolphins and six fish species and one squid species commonly fed
to dolphins under human care were analyzed for purine metabolite
content (Table 1) [26-28]. Free-ranging fish samples were caught
during the months of May to September 2013 from the waters off the
FL west coast by local fisherman using a rod and reel, crab trap, or
cast net, or with a purse-seine net during fish surveys conducted by the
SDRP team in Sarasota Bay. To mimic the rapid death of fish consumed
by dolphins in the wild as closely as possible, fish were euthanizedhumanely by immersion in 500 ppm tricaine methanesulfonate (MS-222, Western Chemical, Ferndale, WA 98248) in sea water [29,30].
Death was confirmed by cessation of opercula movement for 10
minutes, and then fish were weighed, length was measured, andsamples of fish were individually bagged, placed into a cooler of dry ice
and transported to the UF nutrition laboratory where fish were stored
at -80°C until further processing.
Boxes containing six fish species and one species of squid (‘managed
species’) commonly fed to dolphins were supplied by two facilities
where bottlenose dolphins are managed under human care (Table 2).
Fish and squid were caught during one commercial fishing season,
frozen stored at -18°C for 6 to 9 months, and then shipped overnighton dry ice from the dolphin management facilities to UF. Upon arrival,
fish were stored at -20°C until further processing.
Five separate samples of each species were analyzed. To provide
sufficient material to perform all the analyses on every sample, aminimum of 2 individual fish (or squid) were included in each sample;
however, the number of individual fish (or squid) included in eachsample varied depending on the size of the species so that each sample
of smaller species contained more individuals than samples of large
species. The five samples of each species were individually groundusing commercial meat grinders with 4.5 and 10 mm plates (Biro 6642,
Marblehead, OH 43440, and 1.5 HP, LEM Products, West Chester, OH
45011).
Free-ranging fish species were thawed the minimum amountneeded to allow grinding, whereas managed diet fish species were
thawed more completely using the standard operating procedureof one dolphin management facility. Free-ranging fish species were
air thawed in a temperature controlled cold room (11-12°C) for
approximately 1 hr., until fish thawed to a firm, slightly malleable
texture. Managed diet fish were removed from the cardboard boxes,
maintained wrapped in plastic, and air thawed in the cold room forapproximately 20 hrs. Fish were then removed from the plastic andrinsed with cold water (approximately 16°C). Both minimally and wellthawed fish were then transported to the grinder in a cooler containing

Table 1: Fish and squid species commonly consumed by free-ranging and managed bottlenose dolphins.
Free-ranging diet species [26,27]

Managed diet species [28]Pinfish (Lagodon rhomboids)

Pacific herring (Clupea pallasii)

Striped mullet (Mugil cephalus)

Atlantic herring (Clupea harengus)

Sheepshead (Archosargus probatocephalus)

Icelandic capelin (Mallotus villosus)

Ladyfish (Elops saurus)

Canadian capelin (Mallotus villosus)

Pigfish (Orthopristis chrysoptera)

Pacific mackerel (Scomber japonicus)

Spot croaker (Leiostomus xanthurus)

Pacific sardine (Sardinops sagax)

Spotted sea trout (Cynoscion nebulosus)West coast Loligo squid (Loligo opalescens)

Gulf toadfish (Opsanus beta)
Table 2: Columns tested for separation of purines and observed results.
Column (Manufacturer)

Chromatographic results

Acquity UPLC HSS T3 1.8 μm, 150 × 2.1 mm, with HSS T3 1.8 μm Vanguard(Waters Corporation, Milford, MA 01757)

Failed after ~ 800 injections

Symmetry C18 3.5 μm, 150 × 2.1 mm (Waters Corporation)

Poor retention

Excel C18-PFP 2 μm, 100 × 2.1 mm (ACE, Aberdeen, Scotland)

Poor separation

Excel C18-amide 2 μm 100 × 2.1 mm (ACE)Poor elution AMP*, IMP*

Excel Ultra Core Super Phenyl Hexyl 2.5 μm, 75 × 2.1 mm (ACE)Poor metabolite retention

HALO-PFP 2.6 μm, 150 × 2.1 mm (ACE)Poor elution adenine

HALO-HILIC 2.6 μm, 150 × 2.1 mm (ACE)Poor elution AMP*, IMP*

Prodigy ODS 3 μm, 100 × 2 mm (Phenomenex, Torrance, CA 90501)

Low sensitivity, except for AMP*, IMP*

Luna PFP(2) 3 μm, 150 × 3.0 mm (Phenomenex)

Sufficient retention and separation, good sensitivity†*

AMP (Adenine 5’-monophosphate); IMP (Inosine 5’-monophosphate); †Column selected for purine metabolite separation

J Chromatogr Sep Tech, an open access journal
ISSN: 2157-7064

Volume 7 • Issue 5 • 1000334

Citation: Ardente AJ, Garrett TJ, Wells RS, Walsh M, Smith CR, et al. (2016) A Targeted Metabolomics Assay to Measure Eight Purines in the Diet ofCommon Bottlenose Dolphins, Tursiops truncatus. J Chromatogr Sep Tech 7: 334. doi: 10.4172/2157-7064.1000334

Page 4 of 9ice. Grinder equipment was thoroughly rinsed between each sample
to prevent contamination between samples. Ground samples were
homogenized by hand and stored in sample bags (Whirl-Pak® bags,
Nasco, Fort Atkinson, WI 53538) at -80°C until shipped overnight on
dry ice to each laboratory for analysis.

Fish extraction
For each fish purine extraction, a 25 g sample bag was removed
from the -80°C freezer where it had been stored and immersed in waterat room temperature for 30-60 minutes until thawed. The aqueous
extraction method published by Clariana et al. was followed, but
required several modifications [17]. Thawed fish tissue was hand mixed
by massaging the sample bag, and 2 g of mixed sample was transferred
into a beaker with 20 mL of ultra-pure water and 500 µL of the internal
standard solution. Following homogenization, sonication, heating, andcooling, as described by Clariana, et al., the extract supernatant was
filtered through Whatman #1 filter paper (Whatman Inc, Clifton, NJ
07014) with a glass funnel. In all subsequent steps, extract and solvent
volumes were doubled to provide a greater final volume for standard
additions to be performed. Thus, a 10 mL aliquot of the filtrate wascombined with 10 mL of HPLC-grade hexanes (Fisher) and centrifuged
at 6,500 rpm for 7 minutes at 20°C. A 4 mL aliquot from the bottom
layer of fluid was removed and combined with 4 mL of HPLC-grade
methanol (Fisher), 4 mL of HPLC-grade acetone (Fisher), and 80 µL of
10% formic acid in water. Samples were centrifuged at 18,100 rpm for17 minutes at 15°C. Five aliquots of 1,500 µL each were pipetted into 5
mL conical snap-top tubes (Eppendorf, Fisher).
Standard additions were used to quantify individual purine
concentrations in the fish extract. Thus, for each fish sample, the first
1,500 µL aliquot was considered the blank fish extract sample. To theremaining 4 aliquots, the NH4CH3CO2 mixed purine standard solution
and the NH4OH mixed purine standard solution were added inincreasing quantities to provide standard additions of 2x, 4x, 6x, and 8x
purine concentrations. Conical tubes were centrifuged at 3,000 rpm for
5 minutes at room temperature and then dried while heating to 35°C
under a stream of nitrogen gas (MULTIVAP, Organomation, Berlin,
MA 01503) for approximately 5-6 hrs.
The dried samples were reconstituted with 500 µL of 10 mM
NH4CH3CO2. Conical tubes were centrifuged at 4,000 rpm for 15
minutes. Following centrifugation, if the extract had a distinct pellet
at the bottom of the conical tube, supernatant was transferred toa 2 mL LC vial (Thermo Scientific, Waltham, MA 02451) for HPLC
analysis. If the sample had floating particulate matter, the extract was
re-mixed, transferred to a 2 mL microcentrifuge 0.22 µm nylon filter
tube (Corning Incorporated, Corning, NY 14831), and centrifuged at
11,884 rpm for 5 minutes. The supernatant was then transferred to a 2
mL LC vial for HPLC-MS/MS analysis.

µL/min. The elution conditions for separation of AMP, IMP, inosine,
adenine, guanine, hypoxanthine, xanthine, and uric acid began with
97% A (0.1% acetic acid, pH 3.5) and 3% B (methanol) for 2 minutes,
gradually decreased to 90% A and increased to 10% B from 2 to 4
minutes, 70% A and 30% B from 4 to 5.5 minutes, 50% A and 50%
B from 6 to 8 minutes, with a final rapid return back to the original
conditions at 8.5 minutes. The column was maintained under these
conditions for re-equilibration for a further 6.5 minutes. In addition
to testing different columns, several mobile phases were tested todetermine which combination of solvents and timing of transitions
achieved the best separation of the metabolites with similar masses.
The maximum number of injections per day, including duplicate
sample injections and blanks, was 84; therefore, the maximum daily
run time was approximately 22.5 hrs. To optimize the lifespan of thecolumn, particularly considering the potential for particulate buildup on the column, the column was flushed at the end of every longrun with methanol at 500 µL/min for approximately 15-20 minutes.
Additionally, the guard column was changed after approximately 400
injections. For column storage between runs, the column was flushed
at 500 µL/min with 65% acetonitrile and 35% water for at least 10
column volumes.

Mass spectrometry analysis
Purine metabolites were identified by mass and retention time witha triple quadrupole mass spectrometer (TSQ Quantum Access Max,
Thermo) with heated electrospray ionization (HESI). The HESI source
was operated in negative-ion mode with the following settings: sprayvoltage 4000 V, vaporizer temperature 250°C, sheath gas pressure
50 arb, ion sweep gas pressure 0.0 arb, auxillary gas pressure 10 arb,
and capillary temperature 300°C. Compounds were identified bycomparing the retention time and selected reaction monitoring (SRM)
pairs (Table 3) during one scan event with a 20 msec scan time, as wellas through the addition of purine standards. Data was processed using
computer software (Xcalibur Quan Browser, Thermo).

Purine standard stock solution stability
The stability of purine standard stock solutions was assessed by firstpreparing combined stock solutions as described above and spiking thesolutions into 1000 μL of 10 mM NH4CH3CO2 to achieve equivalent 2x,
Table 3: Purine metabolite mass spectrometer identification parameters.
Product mass
(g/mol)Collisionenergy (eV*)

Tube lens(V)

Adenine134.00107.002474135.05

65.34

29

74

95.22

18

74

150.00108.001874

151.02

80.28

26

78

108.16

18

78

80.95

32

66

109.201866167.00124.2416

65

267.02108.21

41100

135.15

25

100

79.25

42

98

134.23

39

98

79.31

37

90

135.13

34

90

GuanineXanthine

Purine metabolite separation was carried out with an auto-sampler(Accela Open, Thermo) and HPLC system (Accela 1250, Thermo).
Several HPLC columns were tested (Table 2) under various conditions,manipulating column and sample stack temperature and using several
mobile phase solvent combinations, including 10 mM NH4CH3CO2 inwater (pH adjusted with acetic acid from 4-6), 10 mM NH4CH3CO2
in 90:10 acetonitrile in water, 100% acetonitrile, 90:10 acetonitrile inwater, 0.1% acetonitrile in water, 0.1% formic acid in water, 0.1% acetic
acid in water, 0.1% NH4OH in water, and 100% methanol.

J Chromatogr Sep Tech, an open access journal
ISSN: 2157-7064

Precursor
mass (g/mol)

Hypoxanthine

Chromatographic conditions

The column and compartment were maintained at ambient
temperature. The injection volume was 10 µL with a flow rate of 500

Metabolite

15

N2 - Xanthine
Uric acid
Inosine
AMP

*

IMP*

152.95345.99346.96eV (Electron volts);
5’-monophosphate)*

AMP(Adenine5’-monophosphate);

IMP(Inosine

Volume 7 • Issue 5 • 1000334

Citation: Ardente AJ, Garrett TJ, Wells RS, Walsh M, Smith CR, et al. (2016) A Targeted Metabolomics Assay to Measure Eight Purines in the Diet ofCommon Bottlenose Dolphins, Tursiops truncatus. J Chromatogr Sep Tech 7: 334. doi: 10.4172/2157-7064.1000334

Page 5 of 96x, and 8x standard addition concentrations. Samples were analyzed
in triplicate immediately then stored at -80°C for 24 hrs. Samples werethen permitted to thaw for at least 1 hr. until approximately ambient
temperature, vortexed, and re-analyzed in triplicate alongside freshly
prepared standard solutions. This procedure was repeated once more
for a total of 2 freeze/thaw cycles at 24 and 48 hrs. In addition, stability
over 24 hr while at ambient temperature was assessed for standard
stock solutions and fresh prepared Pacific herring and mullet samples,
prepared using the method described above.

Method validation
Repeatability was assessed on pooled samples of Pacific herringand striped mullet that were divided into separate bags and frozen at-80°C. Purines were analyzed in these samples over 4 separate days
to determine between-day variability. Additionally, 4 extracts wereprepared from one sample bag for each fish species and analyzed on
the same day to determine within-day variability.

Purine concentration quantification
For each sample, the ratio of peak area relative to internal standard
was regressed against the concentration of purine metabolite after
standard additions. The purine metabolite concentration (mmol/L) in
the fish extract was obtained from the absolute value of the x-interceptof the regression line. Total purine content (mmol/L) was calculated
either as the sum of adenine, guanine, hypoxanthine, and xanthine,
representing the commercially available four-metabolite assay (TP4),or as the sum of those four metabolites plus uric acid, AMP, IMP, andinosine, representing the total obtained using this expanded eightmetabolite assay (TP8).

Statistical analysis
Statistical comparisons were performed using statistical software(SAS® for Windows, version 9.4, Cary, NC, 27513). The coefficientof variation for within and between days (n=4) was calculated forindividual samples of Pacific herring and striped mullet. The ratios of
TP4 to TP8 within species were assessed visually and were found to
be normally distributed using the Shapiro-Wilk test. The correlations
between TP4 and TP8, and between the logarithms of TP4 and TP8,
were compared between groups of species using a general linear
model procedure (SAS proc glm). The ratio of TP8:TP4 and the ratio
of inosine to hypoxanthine were compared among fish species’ nested
within species group using a general linear model (SAS glimmix) andpost-hoc comparisons of least square means were performed with a
Tukey-Kramer correction applied.
The primary endpoint was to determine whether there is a 50%
increase in the concentration of hypoxanthine and other purines
during frozen storage. Based on previous reports of the variability
in concentrations of hypoxanthine and other purines in filleted fish
during storage, comparing five samples of each species gave an 80%
power to detect a 50% change in hypoxanthine concentration with atype I error of 0.05 [8,31,32].

Results
Purine metabolites were separated by mass and retention time using
this HPLC/MS-MS method (Figure 2). Several of the metabolites wereof similar mass: adenine and hypoxanthine, guanine and xanthine, and
AMP and IMP. The reverse phase 100 Å, 150 mm × 3.0 mm column(Luna 5 μm PFP(2), Phenomenex, Torrance, CA 90501) with a guard
column (SecurityGuard for PFP HPLC, Phenomenex) provided the

Figure 2: Chromatographic separation of eight purine metabolites in striped mullet with the addition of the 8x concentration standard. Time (minutes) and relative
abundance are represented along the x-axis and y-axis, respectively. Each metabolite is labeled with purine name, precursor mass, and product mass(es). The
retention time (RT) and the area under the curve (AA) are noted above each metabolite peak. AMP (Adenine 5’-monophosphate); IMP (Inosine 5’-monophosphate)

J Chromatogr Sep Tech, an open access journal
ISSN: 2157-7064

Volume 7 • Issue 5 • 1000334

Citation: Ardente AJ, Garrett TJ, Wells RS, Walsh M, Smith CR, et al. (2016) A Targeted Metabolomics Assay to Measure Eight Purines in the Diet ofCommon Bottlenose Dolphins, Tursiops truncatus. J Chromatogr Sep Tech 7: 334. doi: 10.4172/2157-7064.1000334

Page 6 of 9

There was a 15-30% decrease in signal for all metabolites after the
first 24 hr. freeze and thaw cycle. There was no signal for adenine, AMP,
and IMP following the second freeze/thaw cycle and the signal for theother metabolites decreased by an average of 25%. During a single
24 hr. run, the signal for neat standards decreased by up to 90% foradenine, between 20-40% for AMP, IMP, and uric acid, and between
5-15% for all other metabolites. The decrease in signal for metabolites
in the fish extract was less than 20% over a 24 hr. run.
The coefficient of variation, measured both within and between
days, for the Pacific herring and mullet samples were much greaterfor metabolites with minimal measured concentrations, like AMP,
adenine, IMP, but was mostly below 20% for metabolites with greatermeasured concentrations, like guanine and hypoxanthine (Table 4). The
ratio of TP8:TP4 differed among fish species and between fresh frozen
free-ranging diet species and managed species which had been frozen
stored for several months and then thawed (p < 0.0001; Figure 4). The
slope of linear correlations of TP8 to TP4 was steeper for fresh frozen
fish commonly fed to free-ranging dolphins than in stored and thawed
species commonly fed to managed dolphins (p=0.01). Regression of thelogarithm of TP8 with the logarithm of TP4 improved the fit, and thelogTP8:logTP4 regression slopes were almost identical for both groupsof fish (0.963 and 0.969, respectively, Figure 5). Values for TP8 were on
average 24% higher relative to TP40.966 in free-ranging diet species thanin managed diet species but variation was substantial. The difference
between TP8 and TP4 was largely due to a change in the ratio of inosine
to hypoxanthine, which was on average greater for the free-ranging
species (3.70) than for managed species (1.67; p < 0.0001).

Discussion
This study describes a new method that quantified eight purine
metabolites contained in whole fish and a squid commonly consumed
by bottlenose dolphins. The four metabolites in greatest concentration
were guanine, hypoxanthine, xanthine, and inosine, and themethod provided satisfactory, repeatable results for measuring theconcentrations of those purines. Inosine is not measured by the fournucleobase commercial assay, so the whole fish total purine content
would be underestimated if only the usual four nucleobases, adenine,guanine, hypoxanthine, and xanthine, were measured.
The total purine content from eight metabolites cannot be reliably
predicted by measuring just four metabolites because the ratio of thetotal obtained from eight versus four metabolites varied widely among
species. Furthermore, the ratio of the total purine content measured
by eight versus four metabolites differed between free-ranging andmanaged diet species possibly because of inherent species and handlingdifferences. For example, inosine and IMP degrade to hypoxanthineduring frozen storage and storage conditions can markedly affectpurine degradation rates [16,33]. In this study, fresh frozen fish had agreater inosine to hypoxanthine ratio than frozen, stored, and thawed
fish. This likely represents the degradation of inosine to hypoxanthineduring frozen storage for managed diet species, whereas degradation
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was probably minimal for the free-ranging fish species that werequickly frozen at -80°C.
These findings confirm our initial hypothesis that it is important
to measure more than just four purine metabolites when comparing
the purine intake of managed and free-ranging bottlenose dolphins.
All of the metabolites measured are absorbed through the mammalianintestinal tract; however, the uricogenic importance may not be
similar for all metabolites. For example, the uricogenic potential of
inosine is not well-described. In rats, it is suspected that intestinal
absorption of inosine is saturable, but the threshold for saturation
has not been established [34]. This may be important because eventhough fish concentrations of inosine are large when compared toother purine metabolites, limited absorption may diminish its impact
on urine uric acid excretion. Nevertheless, very little is known aboutpurine metabolism in dolphins. This is the first attempt to quantify thepurine content of the diet of dolphins, and the relationship between
diet and uric acid excretion has not been established [21,22]. At thistime, therefore, all purine metabolites should be considered equallyimportant in assessing the dolphin diet.
Measuring more purines with this assay may also be relevant forhuman medicine. Purine-rich foods are classified based on the total
nucleobase content. Human beings susceptible to developing goutor uric acid stones are advised to avoid foods containing increased
purines. This study shows that foods may be misclassified as to total
purine content if the additional purines that are known to be absorbedby the mammalian intestinal tract are not measured. In particular, thisstudy suggests that the total purine content may be underrepresented
by the traditional assay especially in fresh foods that are not frozen andstored for long periods.
This method of sample preparation and analysis has some
limitations. First, fish were pooled and ground for analysis, andthere was inherent sample heterogeneity of some species because
the diverse tissues of whole fish respond differently to grinding. Thiswas particularly true for some of the smaller, more boney species
Figure 3: Example of a standard addition curve that shows the relationshipbetween concentration and the ratio of peak area of xanthine to that of theinternal standard that was used to establish the xanthine concentration in
extract from a ground sample of striped mullet. Values are plotted either
when no external standard was added or when increasing concentrations (2x,4x, 6x, and 8x) of xanthine external standard were added to the fish extract.
The xanthine concentration in the fish extract was calculated as the absolutevalue of the x-intercept of the linear regression line. Thus, for this curvex=(y-32.18)/949.62 (R2=1.00) and xanthine concentration in the extract wasdetermined to be |-32.18/949.62|=0.0339 mmol/L.
160

8x

140
6x

120

y=949.62x+32.18
R2=1.00

4x

100
Peak area ratio

best metabolite separation and most stable retention times when usedwith the method conditions described above. This column also had
sufficient lifespan (~ 800 injections) for fish tissue purine identification.
All other columns did not achieve desired results (Table 2). Standard
additions verified the location of each metabolite, particularly
when concentrations in the fish extract were low. Each metabolite
concentration in the fish extract was quantified as the x-intercept of
the regression of concentration after standard additions with area ratio
relative to that of the internal standard (Figure 3).

80
2x

60
40
20
0

0.000

0.020

0.040

0.060

0.080

0.100

0.120

0.140

Xanthine concentration (mmol/L)

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Citation: Ardente AJ, Garrett TJ, Wells RS, Walsh M, Smith CR, et al. (2016) A Targeted Metabolomics Assay to Measure Eight Purines in the Diet ofCommon Bottlenose Dolphins, Tursiops truncatus. J Chromatogr Sep Tech 7: 334. doi: 10.4172/2157-7064.1000334

Page 7 of 9
Table 4: Purine metabolite concentrations and assay variability for two whole ground fish samples.
Within day

Between day

Mean ± SD*(μmol/L)

CV (%)†

Mean ± SD*(μmol/L)

CV
(%)†

AMP*

5.46 ± 3.51

64

2.97 ± 3.21

108

Adenine0.32 ± 0.09

27

0.14 ± 0.17

122

Guanine201 ± 0.02

0241 ± 40

17IMP*0.25 ± 0.36

147

0.10 ± 0.23

225

Inosine228 ± 219262 ± 63

24

Hypoxanthine

156 ± 2415

166 ± 30

18

Xanthine

31.1 ± 4.8

1534.4 ± 13.6

39

Uric acid

0.00 ± 0.00

N/A*

0.04 ± 0.13

316

AMP*

0.70 ± 0.09

13

0.77 ± 0.28

36

Adenine1.34 ± 0.19

14

1.17 ± 0.50

43

Guanine152 ± 1711

165 ± 2113IMP*0.95 ± 1.41

147

1.84 ± 2.16

117

Inosine180 ± 158191 ± 18

9

Hypoxanthine

35.95 ± 2.53

7

41.78 ± 6.65

16

Xanthine

12.18 ± 1.64

14

11.2 ± 2.5

22Uric acid

0.00 ± 0.00

N/A

0.00 ± 0.00

N/A

Metabolites
Pacific herring (Clupea pallasii)

Striped mullet (Mugil cephalus)*SD (Sandard deviation); AMP (Adenine 5’-monophosphate); IMP (Inosine 5’-monophosphate); N/A (Not applicable because concentration is zero); †Purine metabolitemean variability was calculated within day (n=4) and between days (n=4)

Figure 4: Ratios of the sum of concentrations of eight purine metabolites versus four purine metabolites in extracts of fish and squid samples of species commonly
consumed by free-ranging dolphins and dolphins under human care. Boxes represent the range, the solid line represents the median, and the dotted line is the mean.
Free-ranging diet species overall have a greater ratio than managed diet species (p<0.0001), and ratios of individual species with different letter superscripts are
significantly different (p < 0.0001; n=5).

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Citation: Ardente AJ, Garrett TJ, Wells RS, Walsh M, Smith CR, et al. (2016) A Targeted Metabolomics Assay to Measure Eight Purines in the Diet ofCommon Bottlenose Dolphins, Tursiops truncatus. J Chromatogr Sep Tech 7: 334. doi: 10.4172/2157-7064.1000334

Page 8 of 9
Figure 5: Comparison of the total concentration of eight purine metabolites(TP8) versus the total concentrations of four purine metabolites (TP4) amongfish fed to free-ranging dolphins and species consumed by managed dolphins.
Regression lines obtained by regressing the logarithm of TP8 with the logarithmof TP4 are shown. The exponents of the regression lines were approximately
the same for both groups of fish but values for TP8 were on average 24%
higher than for TP4.

like pinfish, where it was challenging to ensure ground sample waswell homogenized. Additionally, the aqueous purine extraction
method used was time-consuming, whereas acid-hydrolysis is a morecommonly used and more time-efficient method for purine extraction.
Nevertheless, acid-hydrolysis extraction of Pacific herring used withthis HPLC-MS/MS method resulted in lower MS/MS signals for somepurine metabolites, including guanine and inosine, when comparedto the aqueous extraction method. There were also some noticeable
differences in the appearance of the aqueous extract depending on
the species. For example, Pacific herring aqueous extract had a moreopaque, pink color and a more viscous consistency than striped mullet
aqueous extract, which may account for the greater variability observed
in Pacific herring purine measurements compared with mullet.
The fish matrix seemed to have a protective factor, stabilizing
purine metabolites in the extracted sample. Nevertheless, lower MS
signals for neat standards following the frozen storage and thawing
experiments meant that purine standard stock solutions had to beprepared fresh for each experiment and added to experimental samples
within 24 hrs. Thus, each analytical run had to be completed within
24 hrs. It was also necessary to use standard additions to quantify
purine concentrations, which increased the number of runs necessary
to analyze each sample and thus reduced the number of samples that
could be analyzed in any one day. Standard additions were used for two
important reasons. First, purines were soluble at different pH: AMP,
IMP, inosine, and adenine dissolved only at a pH of approximately
7 (NH4CH3CO2 solvent), whereas guanine, hypoxanthine, xanthine,
and uric acid dissolved at a pH of approximately 10 (NH4OH solvent).
Thus, the aqueous extraction conditions of this method did not provide
soluble conditions for all purine standards. Additionally, the retention
times of neat individual purine standards prepared in NH4CH3CO2 andNH4OH solutions differed from retention times of purines found in
the whole fish tissue; therefore, the matrix effect of the fish tissue did
not permit accurate use of a standard calibration curve and isotopicallylabeled internal standards for each compound were not available. Thus,
standard additions of four increasing concentrations were used toverify and quantify metabolites present in the fish.
This is an expanded assay in comparison to the commercially
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ISSN: 2157-7064

available assay but is not inclusive of all metabolites in the purine
degradation pathway that may lead to uric acid formation. The
number of purine metabolites measured by this assay was limited by
the variable solubility of individual metabolites, which was affectedby the extraction solution volume and the pH of extraction andchromatographic methods. Thus, metabolites had to be prioritized forinclusion in the expanded assay, and this decision was based on theirreported abundance in whole fish carcasses, ability to be intestinallyabsorbed in mammals, and/or uricogenic potential [1,13,34-36]. For
example, adenosine 5’-diphosphate (ADP) and adenosine triphosphate(ATP) were initially included in the assay because moderateconcentrations are reported in fish tissue; however, these metabolites
were removed from the analysis because measured concentrations
in whole fish were below the assay’s limit of detection (<7.2 μmol/L)[37]. Rapid post-mortem degradation or lack of solubility relating tophosphorylation and pH may have resulted in the low concentrationsof ADP and ATP measured by this HPLC method. We also elected tomeasure guanine rather than guanosine because of its abundance inthe metallic scales of fish, despite reports of the metabolite being lessurigogenic than other purines in human beings [13,25]. To the authors’knowledge, dolphins appear to digest fish scales, having no evidence of
undigested scales in their feces, but it is unknown whether guanine isabsorbed through the dolphins’ intestinal tract. It is plausible, however,that guanine may be an important purine metabolite for the dolphin
when compared to human beings because of their whole fish diet. In
order to more accurately determine the total purine content of thedolphin diet, future work will involve development of two different
extraction and chromatographic methods in order to accommodate
the neutral- versus alkaline-soluble metabolites and permit a greaternumber of purines to be included in the assay.
In conclusion, this current expanded method of purine analysis
enables precise and reproducible identification and quantification of
the individual purine metabolites contained in whole fish. Furthermore,
the inclusion of additional metabolites better quantifies the total purine
content of a food item and represents a foods’ uricogenic potential.
Assessing the differences in the purine content of diets consumed
by managed and free-ranging bottlenose dolphins may help explain
why managed dolphins are predisposed to forming ammonium urate
nephroliths.
Acknowledgements
The University of Florida (UF) Aquatic Animal Health Program, the US Navy
Marine Mammal Program (MMP), Sea World Parks and Entertainment, Inc., andthe Southeast Center for Integrated Metabolomics (NIH grant U24DK097209)
provided funding to support this research. The MMP and Sea World Orlando also
provided the managed diet fish samples analyzed, and the Chicago ZoologicalSociety’s Sarasota Dolphin Research Program provided the opportunity to collectthe free-ranging diet fish species. Karen Scott, PhD, Brian Vagt, and the UF Meat
Sciences Department assisted with sample processing.

References
1. Clifford AJ, Story DL (1976) Levels of purines in foods and their metabolic
effects in rats. Journal of Nutrition 106: 435-442.
2. Choi HK, Liu SM, Curhan G (2005) Intake of purine-rich foods, protein, anddairy products and relationship to serum levels of uric acid - the third national
health and nutrition examination survey. Arthritis and Rheumatism 52: 283-289.
3. Richette P, Bardin T (2010) Gout. The Lancet 375: 318-328.
4. Ho CY, Miller KV, Savaiano DA, Crane RT, Ericson KA, et al. (1979) Absorption
and metabolism of orally administered purines in fed and fasted rats. The
Journal of Nutrition 109: 1377-1382.
5. Choi H, Atkinson K, Karlson E, Willett W, Curhan G (2004) Purine-rich foods,
dairy and protein intake, and the risk of gout in men. The New England Journalof Medicine 350: 1093-1103.

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Citation: Ardente AJ, Garrett TJ, Wells RS, Walsh M, Smith CR, et al. (2016) A Targeted Metabolomics Assay to Measure Eight Purines in the Diet ofCommon Bottlenose Dolphins, Tursiops truncatus. J Chromatogr Sep Tech 7: 334. doi: 10.4172/2157-7064.1000334

Page 9 of 9
6. Serio A, Fraioli A (1999) Epidemiology of nephrolithiasis. Nephron 81: 26-30.
7. Sakhaee K, Maalouf NM (2008) Metabolic syndrome and uric acidnephrolithiasis. Seminars in Nephrology 28: 174-180.

23. Bernard J, Allen M (2002) Feeding captive piscivorous animals: Nutritional
aspects of fish as food. Nutrition Advisory Group Handbook. In: Baer D,
Crissey S, Ullrey D. Nutrition Advisory Group, p: 12.

8. Lou SN (1998) Purine content in grass shrimp during storage as related to
freshness. Journal of Food Science 63: 442-444.

24. Huidobro A, Pastor A, Tejada M (2001) Adenosine triphosphate and derivatives
as freshness indicators of gilthead sea bream (sparus aurata). Food Scienceand Technology International 7: 23-30.

9. Bartges JW, Osborne CA, Felice LJ, Allen TA, Brown C, et al. (1995) Diet effecton activity product ratios of uric-acid, sodium urate, and ammonium urate inurine formed by healthy beagles. American Journal of Veterinary Research 56:
329-333.

25. Levy-Lior A, Pokroy B, Levavi-Sivan B, Leiserowitz L, Weiner S, et al. (2008)
Biogenic guanine crystals from the skin of fish may be designed to enhancelight reflectance. Crystal growth and design 8: 507-511.

10. Hardy R, Klausner J (1983) Urate calculi associated with poral vascularanomalies, in Current veterinary therapy. In: Kirk R. WB Saunders: Philadelphia,
pp: 1073-1076.
11. Brockis JG, Levitt AJ, Cruthers SM (1982) The effects of vegetable and animal
protein diets on calcium, urate and oxalate excretion. British Journal of Urology
54: 590-593.
12. Sarwar G, Brule D (1991) Assessment of the uricogenic potential of processed
foods based on the nature and quantity of dietary purines. Progress in Foodand Nutrition Science 15: 159-181.
13. Clifford AJ, Riumallo JA, Young VR, Scrimshaw NS (1976) Effect of oral
purines on serum and urinary uric acid of normal, hyperuricemic and gouty
humans. The Journal of Nutrition 106: 428-450.
14. Spann WK, Grobner W, Zollner N (1980) Effect of hypoxanthine in meat onserum uric acid and urinary uric acid excretion. Purine metabolism in man-III
122A: 215-219.
15. Brule D, Sarwar G, Savoie L (1992) Changes in serum and urinary uric acidlevels in normal human subjects fed purine-rich foods containing different
amounts of adenine and hypoxanthine. Journal of the American College of
Nutrition 11: 353-358.
16. Fraser D, Dingle J, Hines J, Nowlan S, Dyer W (1967) Nucleotide degredation,
monitored by thin-layer chromatography, and associated postmortem changes
in relaxed cod muscle. Journal of the Fisheries Research Board of Canada 24:
1837-1841.
17. Clariana M, Gratacos-Cubarsi M, Hortos M, Garcia-Regueiro JA, Castellari M(2010) Analysis of seven purines and pyrimidines in pork meat products by ultrahigh performance liquid chromatography–tandem mass spectrometry. Journalof Chromatography A 1217: 4294-4299.
18. Aubourg SP, Piñeiro C, Gallardo JM, Barros-Velazquez J (2005) Biochemicalchanges and quality loss during chilled storage of farmed turbot (psetta
maxima). Food Chemistry 90: 445-452.
19. Lou SN, Lin CD, Benkmann R (2001) Changes in purine content of tilapia
mossambica during storage, heating, and drying. Food Science and Agricultural
Chemistry 3: 23-29.
20. Bennour M, El Marrakchi A, Bouchriti N, Hamama A, El Ouadaa M (1991)
Chemical and microbiological assessments of mackerel (scomber scombrus)
stored in ice. Journal of Food Protection 54: 789-792.

26. McCabe E, Gannon D, Barros N, Wells R (2010) Prey selection by resident
common bottlenose dolphins (tursiops truncatus) in sarasota bay, florida.
Marine Biology 157: 931-942.
27. Wells RS, Rhinehart HL, Hansen LJ, Sweeney JC, Townsend FI, et al. (2004)
Bottlenose dolphins as marine ecosystem sentinels: Developing a healthmonitoring system. EcoHealth 1: 246-254.
28. Smith CR, Poindexter JR, Meegan JM, Bobulescu IA, Jensen ED, et al. (2014)
Pathophysiological and physicochemical basis of ammonium urate stone
formation in dolphins. The Journal of Urology 192: 260-266.
29. Jacobs J (2012) NOAA/NCCOS/Oxford lab - tricaine methanesulfonate (ms222) effect on tissue mineral concentrations. A. Ardente Oxford.
30. Posner L (2012) North carolina state university college of veterinary medicine,anesthesiologist - tricaine methanesulfonate (ms-222) effect on tissue mineral
concentrations.
31. Piñeiro-Sotelo M, Rodrıg
́ uez-Bernaldo de Quirós A, López-Hernández J, SimalLozano J (2002) Determination of purine bases in sea urchin (paracentortus
lividus) gonads by high-performance liquid chromatography. Food Chemistry79: 113-117.
32. Kabacoff R (2012) Power analysis. Quick R - Accessing the power of R.
33. Aubourg SP, Quitral V, Angélica Larraín M, Rodríguez A, Gómez J, et al.
(2007) Autolytic degradation and microbiological activity in farmed coho salmon(oncorhynchus kisutch) during chilled storage. Food Chemistry 104: 369-375.
34. Salati LM, Gross CJ, Henderson LM, Savaiano DA (1984) Absorption andmetabolism of adenine, adenosine-5'-monophosphate, adenosine, andhypoxanthine by the isolated vascularly perfused rat small intestine. The
Journal of Nutrition 114: 753-760.
35. Yamamoto S, Inoue K, Murata T, Kamigaso S, Yasujima T, et al. (2010)
Identification and functional characterization of the first nucleobase transporterin mammals: Implication in the species difference in the intestinal absorption
mechanism of nucleobases and their analogs between higher primates andother mammals. The Journal of Biological Chemistry 285: 6522-6531.
36. Caulfield MJ, Munroe PB, O'Neill D, Witkowska K, Charchar FJ, et al. (2008)
Slc2a9 is a high-capacity urate transporter in humans. Plos Medicine 5: 1509-1523.
37. Hattula T (1997) Adenosine triphosphate breakdown products as a freshness
indicator of some fish species and fish products, in Biochemistry. University of
Helsinki: VTT Publications, Technical Research Centre of Finland, p: 47.

21. Venn-Watson S, Townsend F, Daniels R, Sweeney J, McBain J, et al. (2010)
Hypocitraturia in common bottlenose dolphins (tursiops truncatus): Assessing
a potential risk factor for urate nephrolithiasis. Comparative Medicine 60: 149-153.
22. Venn-Watson S, Smith C, Johnson S, Daniels R, Townsend F (2010) Clinicalrelevance of urate nephrolithiasis in bottlenose dolphins, tursiops truncatus.
Diseases of Aquatic Organisms 89: 167-177.

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Citation: Ardente AJ, Garrett TJ, Wells RS, Walsh M, Smith CR, et al. (2016) A
Targeted Metabolomics Assay to Measure Eight Purines in the Diet of Common
Bottlenose Dolphins, Tursiops truncatus. J Chromatogr Sep Tech 7: 334. doi:
10.4172/2157-7064.1000334

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ISSN: 2157-7064

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