Fatal strychnine poisoning: application of gas chromatography and tandem mass spectrometry.

The history and toxicological findings in a case of suicidal fatal strychnine poisoning are presented along with a description of the analytical methods. Detection and quantitation of strychnine in body fluids and tissues was performed by gas chromatography (GC) with nitrogen-phosphorus detection, using organic extraction and calibration by a standard addition method. Strychnine concentrations in subclavian blood (1.82 mg/mL), inferior vena cava blood (3.32 mg/mL), urine (3.35 mg/mL), bile (11.4 mg/mL), liver (98.6 mg/kg), lung (12.3 mg/kg), spleen (11.8 mg/kg), brain (2.42 mg/kg), and skeletal muscle (2.32 mg/kg) were determined. Confirmation of strychnine in blood and tissue was performed by GC with detection by tandem ion-trap mass spectrometry (MS). GC-MS-MS analysis, employing electron ionization followed by unit mass resolution and collision-induced dissociation of strychnine, resulted in confirmatory ions with mass-to-charge ratios of 334 (parent ion), 319, 306, 277, 261, 246, 233, and 220. Additional confirmation was provided by GC-MS-MS-MS analysis of each confirmatory ion, revealing an ion fragmentation pathway consistent with the molecular structure of strychnine. The case demonstrates body tissue and fluid distribution of strychnine in a fatal poisoning and the application of tandem MS in medical examiner casework.

Benztropine identification and quantitation in a suicidal overdose.

For a human fatality involving suspected overdose with the anticholinergic agent benztropine, GC-MS analysis was utilized for identification, quantitation, and investigation of metabolism. Organic extracts of blood and urine were analyzed by a capillary-column gas chromatograph interfaced with an ion-trap mass spectrometer, which was programmed for wide-spectrum data acquisition. Electron impact and chemical ionization were used for benztropine detection. The chemical structures of the ion fragments are proposed. Benztropine-d3 was synthesized and used as an internal standard. Quantitative determinations of benztropine revealed 0.183 mg/L in blood and 7.12 mg/L in urine from the decedent. Drug concentrations were interpreted relative to the case findings, published data, and a limited evaluation of the therapeutic concentrations in psychiatric patients. In addition, the possible metabolic conversion to norbenztropine was investigated by the synthesis of the norbenztropine derivative. Chromatographic evaluation of samples from the case study did not reveal significant bioconversion via the N-desmethylation pathway.

Liquid-chromatographic determination of urinary 5-hydroxy-3-indoleacetic acid, with fluorescence detection.

We describe and evaluate a procedure for measuring urinary 5-hydroxy-3-indoleacetic acid by "high-performance" liquid chromatography. After a simple organic extraction, the analyte and internal standard are chromatographed on a reversed-phase column and are detected by native fluorescence. The detection limit (3 ng per injection), between-day precision (CV 5.2%), absolute recovery (70%), analytical recovery (99%), and working linear range (up to 15 mg/L) have been determined. Compared with colorimetric results with nitrosonaphthol, values obtained with the chromatographic method are significantly lower. Reference values and clinical experience with the method are reported. The method is simple, free from interferences, and suitable for use in routine analysis in the clinical laboratory.

Thin-layer chromatography, with fluorescence detection, of benzodiazepines and tricyclic antidepressants in serum from emergency-room patients.

We describe a rapid, sensitive procedure for detecting benzodiazepines and tricyclic antidepressants in serum. After the compounds are adsorbed on charcoal and eluted with an organic solvent mixture, the drugs are thin-layer chromatographed and detected by their fluorescence. Because it is so fast and easy (turn-around time of about 1 h), the method can be used for emergency toxicology screening. An analysis requires 1.0 mL of serum, and the limit of detection for each of the drugs is about 0.75 mg/L. The procedure is sensitive enough to allow the use of serum instead of urine for the detection of these drugs, because serum has certain advantages over urine as the sample and better reflects the state of the patient's toxicity.

Liquid-chromatographic assay for urinary homovanillic acid, with fluorescent detection.

We describe a procedure for measuring urinary homovanillic acid by "high-performance" liquid chromatography. The pH of urine samples is adjusted, and they are chromatographed directly on an octyl silica column. The effluent is reacted with a ferricyanide reagent, to convert homovanillic acid to a product that is fluorescent at 420 nm on excitation at 320 nm. The standard curve is linear up to a homovanillic acid concentration of 20 mg/L; the detection limit is 0.3 mg/L. The method is evaluated for precision, recovery, potential interference, and reference values, and compared (r = 0.99) with a published spectrophotometric method. The proposed method is suitable for the routine analysis for homovanillic acid in urine.

Fluorometry of ethylene glycol in serum.

We describe an assay for ethylene glycol in serum, which is based on the Hantzsch condensation reaction. Ethylene glycol is dehydrogenated by sodium periodate to form two molecules of formaldehyde, which is then combined with ammonium ion and acetylacetone to form a fluorescent diacetyllutidine derivative. The detection limit of this method is 50 mg/L. The procedure is sensitive, requires only 100 micro L of serum, and the standard curve is linear over a working range of 50 to 250 mg/L. Average analytical recovery ranged from 99 to 102% and within-run precision studies showed CVs of < 5%. Reagents are easily prepared and available.

Fluorometric measurement of theophylline.

We have developed a simple, sensitive procedure in which theophylline in a solution of cerium(IV) acts as an oxidizable compound to produce fluorescent cerium(III) when combined with a microsolvent for extraction at pH 7.9. This method provides excellent sensitivity and specificity because other xanthines and phenobarbital, secobarbital, amobarbital, and phenytoin do not interfere. Day-to-day reproducibility (coefficient of variation, 6%) is attained for theophylline concentrations of 10 and 20 mg/L. Results correlate well with liquid chromatography and enzyme immunoassay procedures. Total analysis time for a single sample is 10 min.

Emergency determination of acetaminophen.

I describe a senstive colorimetric micromethod in which a pH 11.0 carbonate buffer is used to induce acetaminophen to react with Folin-Ciocalteau reagent at room temperature. These reagents and a simple solvent extraction favor formation of a stable indophenol dye with acetaminophen, giving this procedure a good degree of specificity. Results correlate well with those by liquid-chromatographic procedures, and day-to-day precision is less than 8%.

Evaluation of EMIT methods for the determination of the five major antiepileptic drugs on an automated kinetic analyser.

We have evaluated the performance of enzyme-multipled immunoassay methods for the five major antiepileptic drugs on an automated system, the Perkin-Elmer Model KA-150 Kinetic Analyzer. The precision in the normal duplicate mode was found to be in the range of 6% to 10% for all five tests over a typical working day. All EMIT methods were compared to gas-liquid chromatographic procedures and, in addition, the phenytoin and phenobarbital assays were compared to a liquid-chromatographic method. The phenytoin assay was also compared to RIA and to a manual spectroscopic method. In general, most of the comparison studies resulted in acceptable correlation, although one gas chromatographic method did not correlate very well with the phenytoin and phenobarbital immunoassays.

Simple procedure for measuring total protein in urine.

We describe a procedure for measuring total protein in urine. The method is simple, sensitive, and free of interference from drugs that are known to affect other commonly used methods. The CV for a 1.1 g/liter urine control in daily, routine use is 4.6%. The procedure involves adsorbing the protein onto cellulose powder, binding of Ponceau S dye by the protein, washing away excess dye, and eluting the bound dye into dilute NaOH for colorimetry. Comparison of results by this method with those by the biuret and turbidimetric procedures showed good agreement in those cases where the specimens were suitable for assay by the comparison methods.