Analytical challenges: determination of tetrodotoxin in human urine and plasma by LC-MS/MS.

Tetrodotoxin (TTX) is a powerful sodium channel blocker found in puffer fish and some marine animals. Cases of TTX poisoning most often result from puffer fish ingestion. Diagnosis is mainly from patient's signs and symptoms or the detection of TTX in the leftover food. If leftover food is unavailable, the determination of TTX in the patient's urine and/or plasma is essential to confirm the diagnosis. Although various methods for the determination of TTX have been published, most of them are for food tissue samples. Dealing with human urine and blood samples is much more challenging. Unlike in food, the amount of toxin in the urine and blood of a patient is generally extremely low; therefore a very sensitive method is required to detect it. In this regard, mass spectrometry (MS) methods are the best choice. Since TTX is a very polar compound, there will be lack of retention on conventional reverse-phase columns; use of ion pair reagent or hydrophilic interaction liquid chromatography (HILIC) can help solve this problem. The problem of ion suppression is another challenge in analyzing polar compound in biological samples. This review will discuss different MS methods and their pros and cons.

Transcriptomic and Proteomic Analyses Reveal the Diversity of Venom Components from the Vaejovid Scorpion Serradigitus gertschi.

To understand the diversity of scorpion venom, RNA from venomous glands from a sawfinger scorpion, Serradigitus gertschi, of the family Vaejovidae, was extracted and used for transcriptomic analysis. A total of 84,835 transcripts were assembled after Illumina sequencing. From those, 119 transcripts were annotated and found to putatively code for peptides or proteins that share sequence similarities with the previously reported venom components of other species. In accordance with sequence similarity, the transcripts were classified as potentially coding for 37 ion channel toxins; 17 host defense peptides; 28 enzymes, including phospholipases, hyaluronidases, metalloproteases, and serine proteases; nine protease inhibitor-like peptides; 10 peptides of the cysteine-rich secretory proteins, antigen 5, and pathogenesis-related 1 protein superfamily; seven La1-like peptides; and 11 sequences classified as "other venom components". A mass fingerprint performed by mass spectrometry identified 204 components with molecular masses varying from 444.26 Da to 12,432.80 Da, plus several higher molecular weight proteins whose precise masses were not determined. The LC-MS/MS analysis of a tryptic digestion of the soluble venom resulted in the de novo determination of 16,840 peptide sequences, 24 of which matched sequences predicted from the translated transcriptome. The database presented here increases our general knowledge of the biodiversity of venom components from neglected non-buthid scorpions.

Sample preparation and RPHPLC determination of diuretics in human body fluids.

This article describes reverse phase high-performance liquid chromatography (RPHPLC) methods for determination of diuretics in different human body fluids (whole blood, plasma, serum or urine). Sample preparation procedures, including solid-phase extraction, liquid-liquid extraction, dilution, precipitation as well as automated RPHPLC procedures, are discussed in order to present the advantages and disadvantages of each type of sample preparation. Also, values of analytical recovery of each procedure used for sample preparation are summarized. The most important RPHPLC parameters (detection mode, stationary phase, mobile phase, sensitivity, etc.) are also summarized and discussed.

Investigation of Metabolite Profile of YM758, a Novel If Channel Inhibitor.

BACKGROUND: YM758 monophosphate is a novel If channel inhibitor that has an inhibitory action for If current and shows a strong and specific activity, selectively lowering the heart rate and decreasing oxygen consumption by heart muscle. OBJECTIVES: The objectives of the current study were to investigate the in vivo metabolic profiles of YM758 in mice, rats, rabbits, dogs, and monkeys and to elucidate the structures of YM758 metabolites. METHODS: Biological samples were analyzed by liquid chromatography hyphenated with a radiometric detection system and liquid chromatography coupled with a mass spectrometer to clarify their metabolic patterns. To elucidate their structures, metabolites were isolated and analyzed by mass spectrometry and nuclear magnetic resonance spectroscopy. RESULTS: Our results from in vivo metabolic profiling in humans and animals indicated there is no significant species difference in the metabolism of YM758, and the metabolic pathways of YM758 are considered to be oxidation, hydration, and demethylation followed by sulfate or glucuronide conjugation.

Tetrodotoxin: chemistry, toxicity, source, distribution and detection.

Tetrodotoxin (TTX) is a naturally occurring toxin that has been responsible for human intoxications and fatalities. Its usual route of toxicity is via the ingestion of contaminated puffer fish which are a culinary delicacy, especially in Japan. TTX was believed to be confined to regions of South East Asia, but recent studies have demonstrated that the toxin has spread to regions in the Pacific and the Mediterranean. There is no known antidote to TTX which is a powerful sodium channel inhibitor. This review aims to collect pertinent information available to date on TTX and its analogues with a special emphasis on the structure, aetiology, distribution, effects and the analytical methods employed for its detection.

Isolation and identification of a sodium channel-inhibiting protein from eggs of black widow spiders.

The eggs of black widow spider (L. tredecimguttatus) have been demonstrated to be rich in biologically active components that exhibit great research value and application foreground. In the present study, a protein toxin, named Latroeggtoxin-II, was isolated from the eggs using the combination of gel filtration, ion exchange chromatography and reversed-phase high performance liquid chromatography. Electrospray mass spectrometric analysis indicated that the molecular weight of the protein was 28.69 kDa, and Edman degradation revealed that its N-terminal sequence was ESIQT STYVP NTPNQ KFDYE VGKDY-. After being abdominally injected into mice and P. americana, the protein could make the animals especially P. americana display a series of poisoning symptoms. Electrophysiological experiments demonstrated that the protein could selectively inhibit tetrodotoxin-resistant Na(+) channel currents in rat dorsal root ganglion neurons, without significant effect on the tetrodotoxin-sensitive Na(+) channel currents. Using multiple proteomic strategies, the purified protein was shown to have only a few similarities to the existing proteins in the databases, suggesting that it was a novel protein isolated from the eggs of black widow spiders.

Modeling of action potential generation in NG108-15 cells.

In order to explore the possibility of identifying toxins based on their effect on the shape of action potentials, we created a computer model of the action potential generation in NG108-15 cells (a neuroblastoma/glioma hybrid cell line). To generate the experimental data for model validation, voltage-dependent sodium, potassium and high-threshold calcium currents, as well as action potentials, were recorded from NG108-15 cells with conventional whole-cell patch-clamp methods. Based on the classic Hodgkin-Huxley formalism and the linear thermodynamic description of the rate constants, ion-channel parameters were estimated using an automatic fitting method. Utilizing the established parameters, action potentials were generated using the Hodgkin-Huxley formalism and were fitted to the recorded action potentials. To demonstrate the applicability of the method for toxin detection and discrimination, the effect of tetrodotoxin (a sodium channel blocker) and tefluthrin (a pyrethroid that is a sodium channel opener) were studied. The two toxins affected the shape of the action potentials differently, and their respective effects were identified based on the predicted changes in the fitted parameters.

Development and validation of a high-throughput double solid phase extraction-liquid chromatography-tandem mass spectrometry method for the determination of tetrodotoxin in human urine and plasma.

A sensitive analytical method for the determination of tetrodotoxin (TTX) in urine and plasma matrices was developed using double solid phase extraction (C18 and hydrophilic interaction liquid chromatography) and subsequent analysis by HPLC coupled with tandem mass spectrometry. The double SPE sample cleanup efficiently reduced matrix and ion suppression effects. Together with the use of ion pair reagent in the mobile phase, isocratic elution became possible which enabled a shorter analysis time of 5.5 min per sample. The assay results were linear up to 500 ng mL(-1) for urine and 20 ng mL(-1) for plasma. The limit of detection and limit of quantification were 0.13 ng mL(-1) and 2.5 ng mL(-1), respectively, for both biological matrices. Recoveries were in the range of 75-81%. To eliminate the effect of dehydration and variations in urinary output, urinary creatinine-adjustment was made. TTX was quantified in eight urine samples and seven plasma samples from eight patients suspected of having TTX poisoning. TTX was detected in all urine samples, with concentrations ranging from 17.6 to 460.5 ng mL(-1), but was not detected in any of the plasma samples. The creatinine-adjusted TTX concentration in urine (ranging from 7.4 to 41.1 ng µmol(-1) creatinine) correlated well with the degree of poisoning as observed from clinical symptoms.

Acute fatal poisoning with pilsicainide and atenolol.

A fatal case of intentional poisoning with two antiarrhythmic agents, pilsicainide, a pure sodium channel blocker, and atenolol, a selective beta1 blocker, is presented. A woman in her twenties was found dead at home and empty pill packages of pilsicainide, atenolol, and aspirin were found near by. Hesitation marks were found on the wrist, and strong fibrous degeneration was observed in the cardiomyocytes of the sinoatrial node. The blood concentrations of pilsicainide and atenolol were 7.83 and 4.94 microg/ml, respectively, both far above the reported therapeutic levels. According to these results, we concluded that death was due to cardiac arrhythmia caused by poisoning with pilsicainide and atenolol. This is the first report of fatal poisoning attributable to an overdose of the combination of these two antiarrhythmic drugs.