Development and validation of efficient stable isotope dilution LC–HESI–MS/MS method for the verification of β-lyase metabolites in human urine after sulfur mustard exposure

As a part of the project for screening unequivocal biomarkers after sulfur mustard exposure, a quantitative method for the determination of β-lyase metabolites 1,1′-sulfonylbis-[2-(methylsulfinyl)ethane] (SBMSE) and 1-methylsulfinyl-2-[2-(methylthio)ethylsulfonyl]ethane (MSMTESE) was validated. Full validation was conducted according to the FDA guidelines for method validation using pooled human urine as a sample matrix. The metabolites were extracted from urine with an optimized sample preparation procedure using ENV+ solid phase extraction cartridge with reduced volume of sample and solvents. Metabolites were detected by improved and faster liquid chromatography–heated electrospray ionization–tandem mass spectrometry (LC–HESI–MS/MS) method with two transitions of each chemical using non-buffered eluents, post-column splitter and higher flow-rate. These provided over five times faster analysis than previously published method providing 4.5 min/sample cycle time, to achieve up to 200 samples per day (24 h). Quantification was performed using deuterium labelled internal standard of SBMSE. The method was linear over the concentration range of 5–200 ng/ml (average correlation coefficients were R2 = 0.997 and R2 = 0.989) for both β-lyase metabolites, SBMSE and MSMTESE, respectively. The average retention times for SBMSE and MSMTESE were 1.96 ± 0.01 min and 3.24 ± 0.03 min (n = 54). Calculated limits of detection were 4 ng/ml for both SBMSE and MSMTESE, respectively. Lower limits of quantification were 10 ng/ml and 11 ng/ml for SBMSE and MSMTESE, respectively. This validated method was successfully used in the First Confidence Building Exercise on Biomedical Samples Analysis organized by the Organisation for the Prohibition of Chemical Weapons (OPCW). Identification criteria for analysing unequivocal biomarkers of sulfur mustard with LC–MS/MS after alleged use is discussed and proposed based on the validation and exercise results.