Analysis of causes of death reveals the difficulties in determining the role of substitution drugs in the death process, as many other factors may be involved, including circumstances surrounding death, past history, differential selection of subjects into either substitution modality and concomitant intake of other drugs (especially benzodiazepines and neuroleptics). The potential for synergistic or additive actions by other isolated molecules-particularly opioids, benzodiazepines, other psychotropes and alcohol-must be also considered.

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Studies in mice and guinea pigs have shown that albumin is a new biomarker of organophosphorus toxicant (OP) and nerve agent exposure. Our goal was to determine whether OP-labeled albumin could be detected in the blood of humans exposed to OP. Blood from four OP-exposed patients was prepared for mass spectrometry analysis by digesting 0.010 ml of serum with pepsin and purifying the labeled albumin peptide by offline high performance liquid chromatography. Dimethoxyphosphate-labeled tyrosine 411 was identified in albumin peptides VRY(411)TKKVPQVSTPTL and LVRY(411)TKKVPQVSTPTL from two patients who had attempted suicide with dichlorvos. The butyrylcholinesterase activity in these serum samples was inhibited 80%. A third patient whose serum BChE activity was inhibited 8% by accidental inhalation of dichlorvos had undetectable levels of adduct on albumin. A fourth patient whose BChE activity was inhibited 60% by exposure to chlorpyrifos had no detectable adduct on albumin. This is the first report to demonstrate the presence of OP-labeled albumin in human patients. It is concluded that tyrosine 411 of human albumin is covalently modified in the serum of humans poisoned by dichlorvos and that the modification is detectable by mass spectrometry. The special reactivity of tyrosine 411 with OP suggests that other proteins may also be modified on tyrosine. Identification of other OP-modified proteins may lead to an understanding of neurotoxic symptoms that appear long after the initial OP exposure.

Carbofuran is a pesticide whose acute toxicity is due to inhibition of acetylcholinesterase. Butyrylcholinesterase (BChE) in plasma is inhibited by carbofuran and serves as a biomarker of poisoning by carbofuran. The goal was to develop a method to positively identify poisoning by carbofuran. Sera from an attempted murder and an attempted suicide were analyzed for the presence of carbofuran adducts on BChE. The BChE from 1 ml of serum was rapidly purified on a 0.2 ml procainamide-Sepharose column. Speed was essential because the carbofuran-BChE adduct decarbamylates with a half-life of about 2 h. The partially purified BChE was boiled to denature the protein, thus stopping decarbamylation and making the protein vulnerable to digestion with trypsin. The labeled peptide was partially purified by HPLC before analysis by LC/MS/MS in the multiple reaction monitoring mode on the QTRAP 2000 mass spectrometer. Carbofuran was found to be covalently bound to Ser 198 of human BChE in serum samples from two poisoning cases. Multiple reaction monitoring triggered MS/MS spectra positively identified the carbofuran-BChE adduct. In conclusion a mass spectrometry method to identify carbofuran poisoning in humans has been developed. The method uses 1 ml of serum and detects low-level exposure associated with as little as 20% inhibition of plasma butyrylcholinesterase.

The goal was to develop a method to detect pesticide adducts in tryptic digests of butyrylcholinesterase in human plasma from patients poisoned by pesticides. Adducts to butyrylcholinesterase in human serum may serve as biomarkers of pesticide exposure because organophosphorus and carbamate pesticides make a covalent bond with the active site serine of butyrylcholinesterase. Serum samples from 5 attempted suicides (with dichlorvos, Aldicarb, Baygon and an unknown pesticide), and from 1 patient who accidentally inhaled dichlorvos were analyzed. Butyrylcholinesterase was purified from 2 ml serum by ion exchange chromatography at pH 4, followed by procainamide affinity chromatography at pH 7. The purified butyrylcholinesterase was denatured, digested with trypsin, and the modified peptide isolated by HPLC. The purified peptide was analyzed by multiple reaction monitoring in the QTRAP 4000 mass spectrometer. This method successfully identified the pesticide-adducted butyrylcholinesterase peptide in 4 patients whose butyrylcholinesterase was inhibited 60-84%, but not in 2 patients whose inhibition levels were 8 and 22%. It is expected that low inhibition levels will require analysis of larger serum plasma volumes. In conclusion, a mass spectrometry method for identification of exposure to live toxic pesticides has been developed, based on identification of pesticide adducts on the active site serine of human butyrylcholinesterase.