Post-Mortem Changes of Methomyl in Blood with Hemoglobin

Abstract

Methomyl, (E,Z)-methyl N-{[(methylamino)carbonyl]oxy}ethanimidothioate, is a widely used pesticide that has been detected in many fatal cases of accidental exposure or suicide. Forensic toxicologists have been baffled that the blood methomyl concentration in persons who have died of methomyl poisoning is much lower than the expected concentration in blood. In this study, we speculated two mechanisms underlying the insufficient recovery of methomyl in blood. First, methomyl is decomposed by serum albumin as esterase. Second, methomyl is bound to a specific blood protein, resulting in insufficient recovery in the free form. However, human serum albumin does not show esterase activity for the decomposition of methomyl. On the contrary, specific methomyl hemoglobin adducts have been detected by liquid chromatography quadrupole time-of-flight mass spectrometry (LC-Q/TOF-MS). The mass spectra indicated that methomyl was specifically bound to tryptophan (W), tyrosine (Y), and valine (V) residues in hemoglobin. The amounts of W-and V-adducts dose-dependently increased in vitro when the methomyl concentration was lower than the lethal concentration. In addition, the W-adduct was detected in blood sampled from an autopsied subject who died of intentional methomyl ingestion, suggesting that the W-adduct could be used as a biomarker of methomyl poisoning. We were able to estimate the amount of methomyl ingested on the basis of the amount of the W-adduct.

Effects of human serum albumin on post-mortem changes of malathion

“…X500R (AB Sciex, Foster City, CA, USA) and 5600 + QTOF (AB Sciex) were equipped with Prominence UFLC (Shimadzu, Kyoto, Japan) and Nexera X2 (Shimadzu) as their liquid chromatographs, respectively. The conditions for liquid chromatography and mass spectrometry were according to our previous reports 11 , and summarized in Table 1 .…”

“…A 0.1 mL of a reaction mixture including blood proteins (final concentrations, HSA 40 mg/mL or Hb 100 mg/mL) and malathion (final concentration, 1 μg/mL) was incubated at 37 °C for 0 and 24 h. We used HSA and Hb at the concentrations reported in the previous literatures 8 , 11 . After the incubation, diazepam-d5 (50 ng/mL) as the internal standard in 1.9 mL of ACN was added to the reaction mixture.…”

“…A 0.11 mL aliquot of a reaction mixture containing 40 mg/mL HSA and 10 mg/mL malathion in 0.5 M Tris–HCl buffer was incubated at 37 °C for 24 h. The enzyme digestion by pronase was performed by the methods reported in our previous literature 11 . Briefly, a 6 mg portion of pronase was added to the reaction mixture, and an overnight incubation was carried out at 37 °C.…”

“…The aliquot was washed three times with 0.3 mL of 0.5 M Tris–HCl buffer and centrifuged again at 9984× g for 10 min. The enzyme digestion by pronase was performed by the methods reported in our previous literature 11 . Briefly, 30 mg portion of pronase and 0.3 mL of 0.5 M Tris–HCl buffer were added to the aliquot of the blood sample, and the blood sample was incubated at 37 °C overnight.…”

“…In this study, liquid chromatography quadrupole time-of-flight mass spectrometry (LC-Q/TOF–MS) was used to meet the analytical requirements for these evaluations. LC-Q/TOF–MS is an effective technique to evaluate unidentified metabolites and to identify protein adducts because it can provide the accurate molecular mass by TOF–MS and molecular information by tandem MS 11 .…”