Development and validation of a high-throughput online solid-phase extraction-liquid chromatography-tandem mass spectrometry method for the detection of gonyautoxins 1&4 and gonyautoxins 2&3 in human urine.

Paralytic shellfish toxins (PSTs), including gonyautoxins and saxitoxins, are produced by multiple species of microalgae and dinoflagellates, and are bioaccumulated by shellfish and other animals. Human exposure to PSTs typically occurs through ingestion of recreationally harvested contaminated shellfish and results in nonspecific symptomology. Confirmation of exposure to PSTs has often relied on the measurement of saxitoxin, the most toxic congener; however, gonyautoxins (GTXs), the sulfated carbamate derivatives of saxitoxin, may be present in shellfish at higher concentrations. To improve identification of PST exposures, our group has developed an online solid phase extraction hydrophilic interaction liquid chromatography method to identify GTX1-4 in human urine with tandem mass spectrometry. The reportable range varied for each analyte, with all falling within 0.899 and 250 ng/mL in urine with precision <15% and >85% accuracy as determined for all quality control samples. This new online method quantitates GTX1-4 following exposures to PSTs, supporting the work of public health authorities.

Quantification of saxitoxin and neosaxitoxin in human urine utilizing isotope dilution tandem mass spectrometry.

Saxitoxin and neosaxitoxin are potent neurotoxins that can cause paralytic shellfish poisoning when consumed. A new assay is presented here to quantify saxitoxin (STX) and neosaxitoxin (NEO) in human urine samples. Sample preparation of 500-microL samples included the use of weak-cation-exchange solid-phase extraction in a multiplexed 96-well format. Extracts were preconcentrated and analyzed via 10-min hydrophilic interaction liquid chromatography followed by electrospray ionization. Protonated molecular ions were quantified via multiple reaction monitoring mode in a Qtrap mass spectrometer. The method uses novel 15N7-isotopically enriched STX and NEO internal standards. Method validation included the characterization of two enriched urine pools. The lowest reportable limits for STX and NEO were 4.80 and 10.1 ng/mL, respectively, using both quantification and confirmation ions. These two toxins were not detected in a reference range of humans who consumed seafood in the preceding 72 h, suggesting that few false positives would occur when trying to identify people exposed to STX or NEO.

Fast and quantitative determination of saxitoxin and neosaxitoxin in urine by ultra performance liquid chromatography-triple quadrupole mass spectrometry based on the cleanup of solid phase extraction with hydrophilic interaction mechanism.

Saxitoxin (STX) and neosaxitoxin (NEO) are water-soluble toxins and their cleanup in bio-matrix is a hot topic but difficult problem. A fast and quantitative determination method for STX and NEO in urine was developed using ultra performance liquid chromatography-triple quadrupole mass spectrometry (LC-MS/MS) based on the cleanup of solid phase extraction (SPE) with hydrophilic interaction (HILIC) mechanism. Acetonitrile/methanol/water mixture was used to extract the toxins in urine. Polyamide (PA) was used as HILIC SPE material to clean the toxins in sample matrix. The limits of detection were 0.2ngmL-1 for STX and 1ngmL-1 for NEO in urine. The linear ranges were 0.5ngmL-1-99.2ngmL-1 with the correlation coefficient of r=0.9992 for STX and 2.1ngmL-1-207ngmL-1 with r=0.997 for NEO in urine matrix. The recoveries at three spiking levels were 81.5%-117% with the relative standard deviations (RSDs) of 5.4%-8.5% for STX and 89.0%-118% with the RSDs of 6.7%-9.1% for NEO. STX was found in all the 6 patients' urines while NEO was only found in one sample from an intoxication case.

Saxitoxin Exposure Confirmed by Human Urine and Food Analysis.

A case of an elderly female with suspected paralytic shellfish poisoning (PSP) is presented. The patient shared a meal of recreationally-harvested shellfish with her family and soon began to experience nausea and weakness. She was taken to the local emergency department and then transported to a larger hospital in Anchorage where she was admitted to the intensive care unit with respiratory depression and shock. Her condition improved, and she was discharged from the hospital 6 days later. No others who shared the meal reported symptoms of PSP. A clam remaining from the meal was collected and analyzed for paralytic shellfish toxins (PST) by the Alaska Department of Environmental Conservation Environmental Health Laboratory; the clam tested positive for saxitoxin (STX; 277 µg/100 g), neosaxitoxin (NEO; 309 µg/100 g), multiple gonyautoxins (GTX; 576-2490 µg/100 g), decarbamoyl congeners (7.52-11.3 µg/100 g) and C-toxins (10.8-221 µg/100 g) using high-pressure liquid chromatography with post-column oxidation (AOAC Method 2011.02). Urine from the patient was submitted to Centers for Disease Control for analysis of selected PSTs and creatinine. STX (64.0 µg/g-creatinine), NEO (60.0 µg/g-creatinine) and GTX1-4 (492-4780 µg/g-creatinine) were identified in the urine using online solid phase extraction with HPLC and tandem mass spectrometry. This was the first time GTX were identified in urine of a PSP case from Alaska, highlighting the need to include all STX congeners in testing to protect the public's health through a better understand of PST toxicity, monitoring and prevention of exposures.

Quantitation of saxitoxin in human urine using immunocapture extraction and LC-MS.

AIM: An immunomagnetic capture protocol for use with LC-MS was developed for the quantitation of saxitoxin (STX) in human urine. MATERIALS & METHODS: This method uses monoclonal antibodies coupled to magnetic beads. STX was certified reference material grade from National Research Council, Canada. Analysis was carried out using LC-MS. RESULTS: With an extraction efficiency of 80%, accuracy and precision of 93.0-100.2% and 5.3-12.6%, respectively, and a dynamic range of 1.00-100 ng/ml, the method is well suited to quantify STX exposures based on previously reported cases. CONCLUSION: Compared with our previously published protocols, this method has improved selectivity, a fivefold increase in sensitivity and uses only a third of the sample volume. This method can diagnose future toxin exposures and may complement the shellfish monitoring programs worldwide.

Rapid and Sensitive ELISA Screening Assay for Several Paralytic Shellfish Toxins in Human Urine.

Paralytic shellfish poisoning is caused by a group of paralytic shellfish toxins that are produced by dinoflagellates. Toxins in this group include saxitoxin, neosaxitoxin and gonyautoxins. A rapid diagnostic test to identify poisoning by these toxins can be helpful in guiding the appropriate treatment of victims. Additionally, quick receipt of diagnostic results can provide timely proof that shellfish harvesting should be stopped in a given area, thereby preventing additional exposures. We have developed and validated a rapid urinary enzyme-linked immunosorbent assay-based screening test to diagnose exposure to several major paralytic shellfish toxins. The lower limit of detection (LLOD) for multiple paralytic shellfish toxins was characterized as 0.02, 0.10, 0.10, 1.0, 1.0 and 15 ng/mL for saxitoxin, gonyautoxin 2,3, decarbamoyl gonyautoxin 2,3, decarbamoyl saxitoxin, neosaxitoxin and gonyautoxin 1,4, respectively. No interferences were identified in unspiked pooled urine or in specimens collected from unexposed individuals indicating that this method is specific for the paralytic shellfish toxins tested. The accuracy of this test was demonstrated in 10 individual urine specimens with osmolalities ranging from 217 to 1,063 mOsmol/kg and pHs ranging between 5.06 and 7.45. These specimens were spiked with toxins at their LLODs and the presence of toxins at these concentrations was accurately identified in all cases. These results indicate that this diagnostic test can be used to rapidly and accurately screen urine for paralytic shellfish toxins.

Detection and effects of harmful algal toxins in Scottish harbour seals and potential links to population decline.

Over the past 15 years or so, several Scottish harbour seal (Phoca vitulina) populations have declined in abundance and several factors have been considered as possible causes, including toxins from harmful algae. Here we explore whether a link could be established between two groups of toxins, domoic acid (DA) and saxitoxins (STXs), and the decline in the harbour seal populations in Scotland. We document the first evidence that harbour seals are exposed to both DA and STXs from consuming contaminated fish. Both groups of toxins were found in urine and faeces sampled from live captured (n = 162) and stranded animals (n = 23) and in faecal samples collected from seal haul-out sites (n = 214) between 2008 and 2013. The proportion of positive samples and the toxins levels measured in the excreta were significantly higher in areas where harbour seal abundance is in decline. There is also evidence that DA has immunomodulatory effects in harbour seals, including lymphocytopenia and monocytosis. Scottish harbour seals are exposed to DA and STXs through contaminated prey at potentially lethal levels and with this evidence we suggest that exposure to these toxins are likely to be important factors driving the harbour seal decline in some regions of Scotland.

Detection of human exposure to saxitoxin and neosaxitoxin in urine by online-solid phase extraction-liquid chromatography-tandem mass spectrometry.

Saxitoxin (STX) and neosaxitoxin (NEO) are potent neurotoxins that cause paralytic shellfish poisoning (PSP). PSP typically occurs through the ingestion of bivalve shellfish that have consumed toxin producing dinoflagellates. Due to initial presentation of symptoms being nonspecific, a clinical measurement is needed to confirm exposure to these toxins. Our group has developed an online solid phase extraction hydrophilic interaction liquid chromatography (HILIC) method for the analysis of STX and NEO in human urine with tandem mass spectrometry. A unique feature of this online method is the incorporation of a new synthetic (15)N4-STX labeled internal standard used for quantitation. Manual sample preparation time was reduced by approximately 70% for 98 urine samples as compared to a previously reported method. The lowest reportable limit for STX was improved from 5.0 ng/mL to 1.01 ng/mL and from 10.0 ng/mL to 2.62 ng/mL for NEO. Three analysts validated the method with 20 calibration curves total over 30 days with precision and accuracy within ±15% for all QCs. This new online method rapidly identifies STX and NEO exposure with improved sensitivity, which can facilitate the work of public health authorities to confirm the cases of PSP, complementing the many shellfish monitoring programs worldwide.

Urinary elimination of saxitoxin after intravenous injection.

Paralytic shellfish poisoning is a serious public health concern throughout the world. An analytical method with diagnostic potential was used to isolate and measure saxitoxin, the most potent and studied paralytic shellfish poisoning toxin, in the urine of rats injected i.v. with sublethal doses (2 micrograms/kg) of saxitoxin. Urine was collected at intervals between 4 and 144 hr after injection. Saxitoxin was isolated from urine with an ion-exchange procedure, identified, and measured with a precolumn-oxidation-HPLC procedure coupled with fluorescence detection. The identity of oxidized saxitoxin was confirmed with electrospray ionization mass spectrometry. Four hours after injection, approximately 19% of the injected saxitoxin dose was excreted. By 24 hr, approximately 58% of the administered dose was excreted. Average total urinary excretion of administered saxitoxin was approximately 68% for the full study period. These results demonstrate that small quantities of unmetabolized saxitoxin can be detected in rat urine up to 144 hr after i.v. administration, and that the analytical method may have diagnostic potential for saxitoxin intoxication and paralytic shellfish poisoning.

[3H]-saxitoxinol metabolism and elimination in the rat.

Tritiated saxitoxinol was used to obtain preliminary information on saxitoxin metabolism in the rat. Sublethal doses of tritiated saxitoxinol (18.9-microCi/kg; 3.8 micrograms/kg) were injected i.v. into each of six rats. Urine and fecal samples were collected up to 144 hr post-injection. Within 4 hr, 60% of injected radioactivity was excreted in urine. No radioactivity was found in feces. High performance liquid chromatography analyses of urine showed that saxitoxinol was not metabolized by the rats.

Analysis of saxitoxin in urine by continuous-flow fast-atom bombardment mass spectrometry.

An improved method of saxitoxin analysis in urine using continuous-flow fast-atom bombardment mass spectrometry was developed. Parameters studied were matrix composition, matrix flow, temperature of probe tip, probe-tip design and sample extraction. Optimal detection was obtained using the following matrix composition: 5% glycerol, 0.5% acetic acid, 0.025% sodium dodecylsulfate, 0.1% polyethylene glycol (PEG) 400 and 0.5% PEG 300; probe-tip temperature: (approximately 55 degrees C); flow rate: 5 or 8 microL per min.; probe tip: Olson-Hogge design. The STX standard was detected at 200 pg with signal-to-noise ratio of 11. The percent recovery of saxitoxin from human urine after clean-up on a weak cation exchange column was 75%.

Method for the identification of saxitoxin in rat urine.

Saxitoxin (STX) is one of several related toxins that cause paralytic shellfish poisoning. We used solid-phase extraction (SPE) and prechromatographic oxidation/HPLC with fluorescence detection to isolate, identify, and quantify STX in rat urine. STX recovery from urine with the SPE procedure was approximately 76 +/- 6.5%. The standard curve was linear between 2 and 50 ng/ml. The lower limit of quantification with the method was 2 ng STX/ml of rat urine. Preliminary results with i.v. administration of STX to rats demonstrated that this method can detect and quantify STX in urine.

Toxicokinetics of [3H]saxitoxinol in peripheral and central nervous system of rats.

This study evaluated the toxicokinetics of a saxitoxin (STX) analog, [3H]saxitoxinol (STXOL), in rats. [3H]saxitoxinol (18.9 microCi/kg body weight) was administered iv to male Wistar rats via the penile vein. After injection, [3H]STXOL disappeared rapidly from plasma (t 1/2 = 29.3 min), and 75% of the radiolabel was cleared from plasma within 2 hr. Radioactivity associated with red blood cell membranes was inversely related with the radioactivity associated with hemoglobin, suggesting internalization of STXOL. Distribution of [3H]STXOL, 48 hr after iv exposure, showed that muscle tissues retained 91.2 +/- 7.1%, liver 63.7 +/- 3.8%, heart 17.4 +/- 1.6%, and lung 9.2 +/- 0.8% of the residual dose. High-performance liquid chromatography (HPLC) analysis for saxitoxinol showed three to four major radiolabeled peaks for each of these tissues. By 48 hr, radiolabel associated with the saxitoxinol peak decreased 95% in lungs, heart, and kidneys, with a concomitant increase in unidentified, more polar peaks. No STXOL metabolites were detected in the urine from these animals. Radioactivity accumulation in the brain increased to a maximum of 162% at 8-hr postexposure, compared to values obtained at 10 min, then gradually declined to 148% by 48 hr. HPLC analysis of brain extracts showed that the relative percentage of radioactivity associated with parent toxin gradually decreased, with a concomitant rise in the levels of more polar peaks. Similar results were obtained for spinal cord. The data suggest that saxitoxinol was rapidly cleared from most of the peripheral organs and was little, if any metabolized by muscle cells.