An enhanced throughput method for quantification of sulfur mustard adducts to human serum albumin via isotope dilution tandem mass spectrometry.

Here, we report an enhanced throughput method for the diagnosis of human exposure to sulfur mustard. A hydroxyethylthioethyl (HETE) ester-adducted tripeptide, produced by pronase digestion of human serum albumin, was selected as the quantitative exposure biomarker. Cibacron Blue enrichment was developed from an established cartridge method into a 96-well plate format, increasing throughput and ruggedness. This new method decreased sample volume 2.5-fold. Addition of a precipitation and solid-phase extraction concentration step increased the sensitivity of the method. With the conversion to a 96-well plate and optimization of chromatography, the method resulted in a 3-fold decrease in analysis time. Inclusion of a confirmation ion has increased specificity. The method was found to be linear between 0.050 and 50 µM sulfur mustard exposure with a precision for both quality control samples of ≤6.5% relative standard deviation and an accuracy of >96%. The limit of detection (3So) was calculated to be ∼0.0048 µM, an exposure value similar to that of the HETE-albumin adduct method first described by Noort and co-workers (Noort et al., 1999; Noort el al., 2004) which used protein precipitation to isolate albumin. A convenience set of 124 plasma samples from healthy unexposed individuals was analyzed using this method to assess background levels of exposure to sulfur mustard; no positive results were detected.