Thin Layer Chromatography Convulsant Screen Extended by Gas Chromatography-Mass Spectrometry

Abstract

Abstract. Acute onset convulsive disorders in the canine may result from exposure to a variety of toxicants including strychnine, insecticides, metaldehyde, zinc phosphide, methylxanthines, drugs of abuse, bromethalin, and the tremorgenic mycotoxins (roquefortine and penitrem A). Although several of the above can be identified in a single gas chromatography-mass spectrometry (GC-MS) screen most have to be determined by separate tests. This report describes a modification of the strychnine extraction procedure, which allows thin layer chromatographic (TLC) identification of strychnine, bromethalin, roquefortine, and penitrem A in suspect baits, stomach contents or vomitus, and extends the identification to a wide variety of drugs, pesticides, and environmental contaminants by GC-MS. Samples were mixed with base, extracted into CH 2 Cl 2 and the organic fraction back-extracted with acid. The organic fraction (neutrals) was purified by gel permeation chromatography (GPC) and analyzed by TLC to determine penitrem A and bromethalin. The acidic aqueous fraction was adjusted to pH Ͼ 9 and extracted into CH 2 Cl 2 . The resulting CH 2 Cl 2 layer (bases) was then analyzed by TLC to determine strychnine and roquefortine. The organic basic and neutral fractions were recombined with a late eluting GPC fraction and analyzed by GC-MS. Of 312 samples analyzed by TLC from 1995 to 2001, 35 were positive for strychnine alone, 58 were positive for both roquefortine and penitrem A, 4 were positive for roquefortine alone, and 1 was positive for bromethalin. None of the samples were positive for penitrem A alone. Samples negative by TLC were analyzed by the GC-MS extended procedure since mid-1999, and 14 have shown positive for a wide variety of compounds with convulsant activity.

Fatal Bromethalin Poisoning

“…Chemical confirmation of bromethalin poisoning can prove challenging. Chemical analysis of necropsy specimens taken from domestic animals poisoned with bromethalin has demonstrated measurable pesticide residues in fat, liver, kidney, and brain tissues (10,14). Bromethalin readily undergoes photodegradation (10); thus, samples intended for chemical analysis should be protected from the light.…”

Skunk musk causes methemoglobin and Heinz body formation in vitro

“…Skunk musk diluted 1:100 in 100% ethanol was analyzed with an electron‐impact gas chromatograph/mass spectrometer (GC/MS) according to previously reported methods . Briefly, 1 μL of diluted musk was injected into the 210°C split/splitless injector of an Agilent 5890/5972 GC/MS (Santa Clara, CA, USA) held in split mode with an approximate 10:1 split ratio and a helium flow rate of 1.0 mL/min.…”

Mycotoxins

“…25,26 Analysis of bile may be rewarding if vomitus or stomach contents are not available postmortem. Exposure to either penitrem A or roquefortine can be confirmed by analysis of suspect feed or compost, vomitus, gastric lavage washings, or stomach contents; these analytes have also been detected in very low levels in urine and serum.…”