Fatal poisoning with Taxus baccata: quantification of paclitaxel (taxol A), 10-deacetyltaxol, baccatin III, 10-deacetylbaccatin III, cephalomannine (taxol B), and 3,5-dimethoxyphenol in body fluids by liquid chromatography-tandem mass spectrometry.

This method development was to confirm the fatal ingestion of toxic yew plant material in postmortem samples (stomach content, urine, femoral blood, cardiac blood, bile, and brain tissue) collected from a 22-year-old man who committed suicide by ingesting yew leaves. The analytical method was based on a liquid-liquid extraction under alkaline conditions followed by LC-MS-MS analysis. Chromatographic separation was achieved by HPLC on a Kinetex C18 2.6u (100 × 3 mm) coupled to a QTRAP 5500 system. The method allows the simultaneous identification and quantification of the yew alkaloids taxoids paclitaxel (taxol A), 10-deacetyltaxol, baccatin III, 10-deacetylbaccatin III, cephalomannine (taxol B), and 3,5-dimethoxyphenol; the alkaloidal diterpenoids monoacetyltaxine, taxine B, monohydroxydiacetyltaxine, triacetyltaxine, and monohydroxytriacetyltaxine were also identified. The initial hypothesis of yew tree (Taxus baccata) poisoning was confirmed. The quantitative evaluation revealed taxoid concentrations ranging from 4.5 to 132 µg/L (stomach content), 1 to 200 µg/L (urine), <0.5 to 12 µg/L (cardiac blood), <0.5 to 7.3 µg/L (femoral blood), and 4.9 to 290 µg/L (bile). In brain tissue, none of these taxoids could be detected (<0.5 µg/L). In urine, after enzymatic hydrolysis, the concentration of 3,5-dimethoxyphenol (3,5-DMP) was 23,000 µg/L. The alkaloidal diterpenoids were found in all postmortem samples. The newly developed LC-MS-MS method enables the identification of alkaloidal and non-alkaloidal diterpenoids and 3,5-dimethoxyphenol in human body fluids and tissues for the confirmation of accidental or intentional poisonings with yew plant material.

Accidental intoxication with Veratrum album.

A 49-year-old man consumed two glasses (approximately 2 x 20 mL) of a beverage containing yellow gentian (Gentiana lutea). Shortly after ingestion, he developed nausea, vomiting, and oral paraesthesia. On admission to the hospital he suffered from severe bradycardia (35 beats/min) and hypotension (50/30 mm Hg), and he was treated with activated charcoal, antiemetics (metoclopramide, ondansetron), atropine, and intravenous electrolytic solution. The initial suspicion of Veratrum poisoning could be confirmed by identifying protoveratrines A (ProA) and protoveratrine B (ProB) in a sample from the beverage as well as in the patients serum by liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS-MS). The yellow-colored beverage contained 25% ethanol (by headspace gas chromatography), 20.4 mg/L ProA, and 13.7 mg/L ProB. The serum concentration of ProA was 1162 ng/L and ProB was 402 ng/L. Veratridine, cevadine, and jervine were not detected, neither in the beverage nor in the serum sample. The lower limits of quantitation for all compounds is 10 microg/L (S/N > 10, beverage) and 100 ng/L (S/N > 10, serum). After treatment, the patient completely recovered from the symptoms within 24 h and was discharged from the hospital. The analytical method described was developed for the simultaneous identification and quantitation of five Veratrum alkaloids. The method is based on a liquid-liquid extraction followed by LC-MS-MS analysis. The time needed for analysis was 6 min.

High-performance liquid chromatographic assay for the measurement of azathioprine in human serum samples.

A quantitative, highly sensitive HPLC-based method for the direct measurement of azathioprine is described, introducing a newly synthesized 9-methyl derivative of this immunosuppressant as internal standard in combination with isocratic HPLC and UV-absorbance measurement at 285 nm. Analysis was performed on a RP18 select B column with acetonitrile-0.01 M potassium phosphate buffer (12:88, v/v) at pH 2.3 as mobile phase. Results of precision analysis from serum samples spiked with 3.125, 12.5, and 25 ng azathioprine, respectively, were (mean +/- S.D.): 3.148 +/- 0.259 ng (8.22%), 12.594 +/- 0.571 ng (4.53%), and 25.016 +/- 0.658 ng (2.63%) with C.V. values in parentheses for n = 5. The accuracy of the assay ranged from -7.6 to 0.7% (expressed as % bias) tested on five consecutive days. The limit of quantification was at 2.5 +/- 0.256 ng (C.V. 10.25%), thus allowing drug monitoring in long-term patients. The method can also be used to evaluate individual pharmacokinetic parameters of a single patient, as well as for drug monitoring of a cohort of patients who suffer from azathioprine-induced symptoms of toxicity. An example of the pharmacokinetic behaviour in an individual is given in this paper.

Processing of tetanus and botulinum A neurotoxins in isolated chromaffin cells.

Tetanus and botulinum A neurotoxins were introduced into the cytosol of chromaffin cells by means of an electric field in which the plasma membrane is forced to form pores of approximately 1 micron at the sites facing the electrodes. As demonstrated by electron microscopy, both [125I] and gold-labelled tetanus toxin (TeTx) diffuse through these transient openings. Dichain-TeTx, with its light chain linked to the heavy chain by means of a disulfide bond, causes the block of exocytosis to develop more slowly than does the purified light chain. The disulfide bonds, which in both toxins hold the subunits together, were cleaved by the intrinsic thioredoxin-reductase system. Single chain TeTx, in which the heavy and light chains are interconnected by an additional peptide bond, was far less effective than dichain-TeTx at blocking exocytosis, which indicates that proteolysis is the rate-limiting step. The toxins were degraded further to low-molecular weight fragments which, together with intact toxins and subunits, were released by the cells. The intracellular half-life of [125I] dichain-TeTx was approximately three days. The number of light-chain molecules required to maintain exocytosis block in a single cell, as calculated by two different methods, was less than 10. The long duration of tetanus poisoning may result from the persistence of intracellular toxin due to scarcity of free cytosolic proteases. This may also hold for the slow recovery from botulism.

IgA protease from Neisseria gonorrhoeae inhibits exocytosis in bovine chromaffin cells like tetanus toxin.

When tetanus toxin from Clostridium tetani or IgA protease from Neisseria gonorrhoeae is translocated artificially into the cytosol of chromaffin cells, both enzymes inhibit calcium-induced exocytosis, which can be measured by changes in membrane capacitance. The block of exocytosis caused by both proteases cannot be reversed by enforced stimulation with increased calcium concentration. This effect differs from the botulinum A neurotoxin-induced block of exocytosis that can be overcome by elevation of the intracellular calcium concentration. Tetanus toxin is about 50-fold more potent than IgA protease in cells stimulated by carbachol. In this case, the release of [3H]noradrenaline was determined. Trypsin and endoprotease Glu-C are hardly effective and only at concentrations that disturb the integrity of the cells. Like tetanus toxin, IgA protease also splits synaptobrevin II, though at a different site of the molecule. However, unlike tetanus toxin, it does not cleave cellubrevin. It is concluded that the membranes of chromaffin vesicles contain synaptobrevin II, which, as in neurons, appears to play a crucial part in exocytosis.