科研之友
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摘要
Exposure to toxic organophosphorus pesticides (OPP) represents a serious problem in the public healthcare sector and might be forced in terroristic attacks. Therefore, reliable verification procedures for OPP-intoxications are required for forensic, toxicological and clinical reasons. We developed and optimized a toolbox of methods to detect adducts of human serum albumin (HSA) with OPP considered as long-term biomarkers. Human serum was incubated with diethyl-oxono and diethyl-thiono pesticides for adduct formation used as reference. Afterwards serum was subjected to proteolysis using three proteases separately thus yielding phosphorylated tyrosine residues (Y*) detected as single amino acid (pronase), as hexadecapeptide LVRY*(411)TKKVPQVSTPTL (pepsin) and as the tripeptide Y*(TK)-T-411 (trypsin), respectively. Adducts were analyzed via microbore liquid chromatography coupled to electrospray ionization (mu LC-ESI) and tandem-high-resolution mass spectrometry (MS/HR MS). Using paraoxon-ethyl as model OPP for adduct formation, methods were optimized with respect to MS/HR MS-parameters, protease concentrations and incubation time for proteolysis. HSA-adducts were found to be stable in serum in vitro at + 37 degrees C and -30 degrees C for at least 27 days and resulting biomarkers were stable in the autosampler at 15 degrees C for at least 24 h. Limits of identification of adducts varied between 0.25 mu M and 4.0 mu M with respect to the corresponding pesticide concentrations in serum. Applicability of the methods was proven by successful detection of the adducts in samples of OPP-poisoned patients thus demonstrating the methods as a reliable toolbox for forensic and toxicological analysis.
- 出版日期2018-8
