An Enhanced Throughput Method for Quantification of Sulfur Mustard Adducts to Human Serum Albumin via Isotope Dilution Tandem Mass Spectrometry

摘要

Here we report an enhanced throughput method for the diagnosis of human exposure to sulfur mustard (HD). 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 by 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 HD exposure with precision for both quality control samples ≤6.5% relative standard deviation (RSD) and accuracy >96%. The limit of detection (3So) was calculated to be ∼0.0048 µM, an exposure value similar to the HETE-albumin adduct method first described by Noort(1-2) 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 HD; no positive results were detected.