We herein present for the first time the phosphylated (*) tetrapeptide (TP)-adduct GlyGluSer^198*Ala generated from butyrylcholinesterase (BChE) with proteinase K excellently suited for the verification of exposure to toxic organophosphorus nerve agents (OPNA). Verification requires bioanalytical methods mandatory for toxicological and legal reasons. OPNA react with BChE by phosphonylation of the active site serine residue (Ser¹⁹⁸) forming one of the major target protein adducts for verification. After its enzymatic cleavage with pepsin, the nonapeptide (NP) PheGlyGluSer^*AlaGlyAlaAlaSer is typically produced as biomarker. Usually OPNA occur as racemic mixtures of phosphonic acid derivatives with the stereocenter at the phosphorus atom, e.g. (±)-VX. Both enantiomers react with BChE, but the adducted NP does not allow their chromatographic distinction. In contrast, the herein introduced TP-adducts appeared as two peaks when using a stationary reversed phase (1.8 µm) in micro-liquid chromatography–electrospray ionisation tandem-mass spectrometry (µLC–ESI MS/MS) analysis. These two peaks represent diastereomers of the (+)- and (−)-OPNA adducted to the peptide that comprises chiral L-amino acids exclusively. Concentration- and time-dependent effects of adduct formation with (±)-VX and its pure enantiomers (+)- and (−)-VX as well as with (±)-cyclosarin (GF) were investigated in detail characterising enantioselective adduct formation, stability, ageing and spontaneous reactivation. The method was also successfully applied to samples from a real case of pesticide poisoning as well as to samples of biomedical proficiency tests provided by the Organisation for the Prohibition of Chemical Weapons.

我们在此首次提出由丁酰胆碱酯酶 (BChE) 和蛋白酶 K 产生的磷酸化 (*) 四肽 (TP) 加合物 GlyGluSer ^198* Ala，非常适合验证有毒有机磷神经毒剂 (OPNA) 的暴露。出于毒理学和法律原因，验证需要强制使用生物分析方法。^{OPNA 通过活性位点丝氨酸残基 (Ser 198} )的磷酸化与 BChE 反应，形成用于验证的主要目标蛋白加合物之一。用胃蛋白酶酶切后，通常会产生九肽 (NP) PheGlyGluSer ^{* AlaGlyAlaAlaSer 作为生物标志物。}通常OPNA作为立体中心位于磷原子的膦酸衍生物的外消旋混合物存在，例如(±)-VX。两种对映体均与 BChE 反应，但加合的 NP 不允许进行色谱区分。相比之下，在微液相色谱-电喷雾电离串联质谱 (μLC-ESI MS/MS) 分析中使用固定反相 (1.8 µm) 时，本文引入的 TP 加合物出现两个峰。这两个峰代表与仅包含手性L-氨基酸的肽加成的(+)-和(-)-OPNA的非对映异构体。详细研究了 (±)-VX 及其纯对映体 (+)- 和 (−)-VX 以及 (±)-环沙林 (GF) 加合物形成的浓度和时间依赖性效应，以表征对映选择性加合物形成、稳定性、老化和自发再激活。该方法还成功应用于农药中毒真实案例的样本以及禁止化学武器组织提供的生物医学能力测试样本。