Optimization of solid-phase extraction and liquid chromatography-tandem mass spectrometry for the determination of domoic acid in seawater, phytoplankton, and mammalian fluids and tissues.

We previously reported a solid-phase extraction (SPE) method for determination of the neurotoxin domoic acid (DA) in both seawater and phytoplankton by liquid chromatography-tandem mass spectrometry (LC-MS/MS) with the purpose of sample desalting without DA pre-concentration. In the present study, we optimized the SPE procedure with seawater and phytoplankton samples directly acidified with aqueous formic acid without addition of organic solvents, which allowed sample desalting and also 20-fold pre-concentration of DA in seawater and phytoplankton samples. In order to reduce MS contamination, a diverter valve was installed between LC and MS to send the LC eluant to waste, except for the 6-min elution window bracketing the DA retention time, which was sent to the MS. Reduction of the MS turbo gas temperature also helped to maintain the long-term stability of MS signal. Recoveries exceeded 90% for the DA-negative seawater and the DA-positive cultured phytoplankton samples spiked with DA. The SPE method for DA extraction and sample clean-up in seawater was extended to mammalian fluids and tissues with modification in order to accommodate the fluid samples with limited available volumes and the tissue extracts in aqueous methanol. Recoveries of DA from DA-exposed laboratory mammalian samples (amniotic fluid, cerebrospinal fluid, plasma, placenta, and brain) were above 85%. Recoveries of DA from samples (urine, feces, intestinal contents, and gastric contents) collected from field stranded marine mammals showed large variations and were affected by the sample status. The optimized SPE-LC-MS method allows determination of DA at trace levels (low pg mL(-1)) in seawater with/without the presence of phytoplankton. The application of SPE clean-up to mammalian fluids and tissue extracts greatly reduced the LC column degradation and MS contamination, which allowed routine screening of marine mammalian samples for confirmation of DA exposure and determination of fluid and tissue DA concentrations in experimental laboratory animals.

Localization of aquaporin-2, renal morphology and urine composition in the bottlenose dolphin and the Baird's beaked whale.

This study examined the distribution pattern of aquaporin-2 (AQP2), relative medullary thickness (RMT) and urine properties in the bottlenose dolphin Tursiops truncatus and Baird's beaked whale Berardius bairdii. Immunohistochemical studies revealed that AQP2 was localized in the collecting tubules/ducts of both species' renicules, as in terrestrial mammals. The collecting ducts with AQP2 were thinner and arranged more densely in the dolphin than in the whale. RMT values in the renicule were moderate in both species, but were significantly higher in the dolphin (6.0 +/- 0.9) than the whale (4.9 +/- 0.7). Urine of the bottlenose dolphin is comparatively concentrated (osmolality: 1715.7 +/- 279.4 mOsm kg(-1), Na(+): 490.1 +/- 87.9 mmol l(-1), Cl(-): 402.7 +/- 79.6 mmol l(-1), K(+): 80.7 +/- 25.8 mmol l(-1), urea nitrogen: 703.5 +/- 253.9 mmol l(-1)), while urine of the dead Baird's beaked whale is less concentrated (osmolality: 837.5 +/- 293.8 mOsm kg(-1), Na(+): 192.9 +/- 81.5 mmol l(-1), Cl(-): 159.9 +/- 71.4 mmol l(-1), K(+): 44.3 +/- 29.5 mmol l(-1), urea nitrogen: 270.7 +/- 120.3 mmol l(-1)). These data suggest it is possible that the differences in these renal morphological features may be related in some way to the difference in urine composition between the species, although further studies are necessary.

Analysis of selenium metabolites in urine samples of minke whale (Balaenoptera Acutorostrata) using ion exchange chromatography.

1. To study the distribution of selenium metabolites in whale urine, an analytical methodology for separation and determination has been developed. 2. From whale urine, five selenium components, including trimethylselenonium ion were separated and determined by a combination of ion exchange chromatography and fluorometry. 3. The mean urinary selenium level of five minke whales was 1500 ng/ml, with a standard deviation of 400 ng/ml, i.e., about 30 times as high as that for humans.

Urinary concentrations of ovarian steroid hormone metabolites and bioactive follicle-stimulating hormone in killer whales (Orcinus orchus) during ovarian cycles and pregnancy.

Reproductive hormone profiles of six captive killer whales (Orcinu orcus) from three Sea World aquaria were studied for intervals up to 2 yr. Daily urine samples and bimonthly blood samples were collected and analyzed for hormone concentration. Immunoreactive estrone conjugates, pregnanediol-3-glucoruonide, 20-alpha-hydroxyprogesterone as well as bioactive follicle-stimulating hormone (FSH) were measured in urine samples and indexed by creatinine concentrations of the same sample. In selected cases, serum progesterone concentrations were also measured. Three of the animals in the study became pregnant during the study period and two of these animals were evaluated during the time of conception and throughout most of gestation. From the data of the three animals that conceived, hormone profiles of the complete ovarian cycle, early pregnancy, and mid- to late gestation are described. The remaining three animals did not conceive and only one of these demonstrated hormone changes that indicated regular ovarian activity. The female reproductive pattern of the killer whale is characterized by a gestation of 17 mo and an ovarian cycle of 6-7 wk in duration. The hormone changes associated with the ovarian cycle of the killer whale are similar to those of most other mammalian species. A bimodal pattern of bioactive FSH with a pronounced rise of estrogen predominates the preovulatory hormone profile. After ovulation, increased progesterone production is observed for approximately 4 wk in the nonconceptive ovarian cycle. During the luteal phase and early pregnancy, when progesterone metabolites are elevated, estrogen metabolite excretion remains low. These data extend the application of urine collections for longitudinal studies involving hormone changes, particularly those involving nondomesticated species.(ABSTRACT TRUNCATED AT 250 WORDS)