An improved method for retrospective quantification of sulfur mustard exposure by detection of its albumin adduct using ultra-high pressure liquid chromatography-tandem mass spectrometry

Abstract

Sulfur mustard (HD) adduct to human serum albumin (ALB) at Cys-34 residue has become an important and long-term retrospective biomarker of HD exposure. Here, a novel, sensitive, and convenient approach for retrospective quantification of HD concentration exposed to plasma was established by detection of the HD-ALB adduct using ultra-high pressure liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) with a novel non-isotope internal standard (IS). The HD-ALB adduct was isolated from HD-exposed plasma with blue Sepharose. The adduct was digested with proteinase K to form sulfur-hydroxyethylthioethyl ([S-HETE])-Cys-Pro-Phe tripeptide biomarker. The tripeptide adduct could be directly analyzed by UHPLC-MS/MS without an additional solid phase extraction (SPE), which was considered as a critical procedure in previous methods. The easily available 2-chloroethyl ethylsulfide (2-CEES) as HD surrogate was first reported to be used as IS in place of traditional d8-HD for quantification of HD exposure. Furthermore, 2-CEES was also confirmed to be a good IS alternative for quantification of HD exposure by investigation of product ion spectra for their corresponding tripeptide adducts which exhibited identical MS/MS fragmentation behaviors. The method was found to be linear between 1.00 and 250 ng•mL(-1) HD exposure (R(2)>0.9989) with precision of <4.50% relative standard deviation (%RSD), accuracy range between 96.5% and 114%, and a calculated limit of detection (LOD) of 0.532 ng•mL(-1). The lowest reportable limit (LRL) is 1.00 ng•mL(-1), over seven times lower than that of the previous method. The entire method required only 0.1 mL of plasma sample and took under 7 h without special sample preparation equipment. It is proven to be a sensitive, simple, and rugged method, which is easily applied in international laboratories to improve the capabilities for the analysis of biomedical samples related to verification of the Chemical Weapon Convention (CWC).

A mass spectrometric platform for the quantitation of sulfur mustard-induced nucleic acid adducts as mechanistically relevant biomarkers of exposure

“…In particular, mass spectrometry provides full chemical specificity, high analytical sensitivity, accuracy, and reproducibility, and is, therefore, considered highly suited for exposure verification in court procedures (Remane et al 2016). One approach to verify SM exposure by mass spectrometry represents the measurement of SM-protein adducts in blood, e.g., serum albumin and hemoglobin, which enables relatively long time windows (Gandor et al 2015;John et al 2016a;Liu et al 2015;Newmark et al 2007;Noort et al 2008;. Therefore, blood-derived SM-protein adducts represent well-suited forensic 'biomarkers of exposure'.…”

Quo vadis blood protein adductomics?

“…t (Liu et al 2015), LOQ was calculated from the lowest reportable limit obtained from plasma incubated with 1ng sulfur mustard /mL plasma, 1 ml of plasma 40 mg of albumin were assumed; t1) (Andacht et al 2014); t2) (John et al 2016); Probably, the LOQ for the albumin adduct of the adducted T3 peptide (ALVLIAFAQYLQQC(-14BQ)PFEDHVK) (Smith et al 2021) with a logD of -9.25 could be lowered significantly using other enzyme combinations yielding 14BQ-CPF or 4BQ-C with a logD of -0.78 and -1.63, respectively, if the same digestion yields are obtained. To evaluate the digestion yields synthetic standards are needed.…”

“…3 , name in Table S1; c (Rynoe et al 2003 ), no CAS number, [4-(2,2,2-trifluoroacetyl)oxy-3-(2,2,2-trifluoroacetyl)sulfanyl-phenyl] 2,2,2-trifluoroacetate; d (Sabbioni et al 2010 ), CAS:1200446-92-9; e CAS:1200446-89-4; f also 1,2-benzoquinone-adducts, (Waidyanatha et al 1998 ); g (Sabbioni et al 2012 ), CAS:1416719-29-3; h CAS:1416719-28-2; i CAS:1416719-26-0; j (Kumar and Sabbioni 2010 ), CAS:1211456-36-8; k CAS:1211456-38-0; l CAS:1609242-21-8; m (Peng and Turesky 2014 ), no CAS number; n (Noort et al 2000 ), CAS:775312-71-5, S -[2-[(2-hydroxyethyl)thio]ethyl]-CPF; o (Yeo et al 2008 ), CAS: 1016983-35-9, S -[2-[ethyl(2-hydroxyethyl)amino]ethyl]-CPF; p CAS:428508-48-9; q CAS:1016983-38-2; r NEM-modified albumin (NES-Alb; expected modification:, S -(1-ethyl-2,5-dioxo-3-pyrrolidinyl)-L-cysteine (Preston et al 2017 ); r1) calculated from the modification level (0.2% = 2.4 pmol) of the synthetic standard NES-Alb in a total of 79 µg Alb. LOQ 2.4 pmol/79 µg Alb = 43.8 pmol/mg; s (Smith et al 2021 ), an on column LOQ of 7 fmol/160 ng was listed and this value was converted for 1 mg albumin, + lysC = lysine endopeptidase; t (Liu et al 2015 ), LOQ was calculated from the lowest reportable limit obtained from plasma incubated with 1ng sulfur mustard /mL plasma, 1 ml of plasma 40 mg of albumin were assumed; t1) (Andacht et al 2014 ); t2) (John et al 2016 ); u CAS:1211456-34-6; v (Barknowitz et al 2014 ), CAS:1536466-52-0; w CAS:1536466-53-1; x to & from cruciferous vegetables: x from gluconasturtin, y glucotropaeolin, z glucoraphanin, § sinigrin, & 1-methoxy-3-indolylmethyl glucosinolate; $ in vitro synthesized standards, that were just characterized by MS…”

“…t (Liu et al 2015 ), LOQ was calculated from the lowest reportable limit obtained from plasma incubated with 1ng sulfur mustard /mL plasma, 1 ml of plasma 40 mg of albumin were assumed; t1) (Andacht et al 2014 ); t2) (John et al 2016 );…”

Sulfur Mustard: Persisting Threat

“…Jedním z nejvíce studovaných analytů jsou adukty s N-terminálním valinem (hydroxyethylthioethyl-valin) nebo histidinem (hydroxyethylthioethylhistidin). Tyto analyty lze stanovit metodou GC-MS až 120 dní po expozici, tedy po dobu životnosti erytrocytu (31,32). V krevní plazmě lze též stanovit alkylační adukt s albuminem.…”