Simplified Method for Quantifying Sulfur Mustard Adducts to Blood Proteins by Ultrahigh Pressure Liquid Chromatography–Isotope Dilution Tandem Mass Spectrometry

Abstract

Sulfur mustard binds to reactive cysteine residues, forming a stable sulfur-hydroxyethylthioethyl [S-HETE] adduct that can be used as a long-term biomarker of sulfur mustard exposure in humans. The digestion of sulfur mustard-exposed blood samples with proteinase K following total protein precipitation with acetone produces the tripeptide biomarker [S-HETE]-Cys-Pro-Phe. The adducted tripeptide is purified by solid phase extraction, separated by ultra-high pressure liquid chromatography, and detected by isotope dilution tandem mass spectrometry. This approach was thoroughly validated and characterized in our laboratory. The average interday relative standard deviation was ≤ 9.49%, and the range of accuracy was between 96.1-109% over a concentration range of 3.00 to 250. ng/mL with a calculated limit of detection of 1.74 ng/mL. A full 96-well plate can be processed and analyzed in 8 h which is five times faster than our previous 96-well plate method and only requires 50 µL of serum, plasma, or whole blood. Extensive ruggedness and stability studies and matrix comparisons were conducted to create a robust, easily transferrable method. As a result, a simple and high-throughput method has been developed and validated for the quantitation of sulfur mustard blood protein adducts in low volume blood specimens which should be readily adaptable for quantifying human exposures to other alkylating agents.

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

“…The results indicated that pH value of enzymatic buffer had significant influence on the yield of adducted tripeptide with variation of over 50 %, and the highest signal response was from pH 8.0 of 20 mM Tris HCl, which had not previously been reported. The investigated digestion reaction at 40, 50, and 60°C showed that temperature was a critical factor, and there were higher signals at 50 and 60°C, which is consistent with that of previous study [17]. Conditions in enzyme concentration (2.00, 4.00, 6.00 mg•mL -1 ) and digestion time (1.0, 2.0, 4.0, 8.0, 12.0 h) were also optimized and investigated.…”

“…However, the level of 4.00 mg•mL -1 enzyme and digestion time ≥4.0 h had relatively high and stable yields of the tripeptide adduct. Obviously, longer digestion time was needed in our study than 1.5 h of the previous study [17], which could be well explained by the fact that the digestion mixture was stirred in the previous study, while it was not stirred during digestion in our study. Interestingly, longer time for digestion (16,24, and 48 h) was also investigated with the result that the yields of the tripeptide adduct were still stable to 48 h, suggesting that proteinase K digestion of ALB can form stable tripeptide adduct.…”

“…Proteinase K isolated from Tritirachium album has been confirmed to be more reliable and effective than currently available pronase for digestion of total proteins into the [S-HETE]-CPF tripeptide adduct [17]. Therefore, in this study, proteinase K was chosen to produce the tripeptide.…”

“…In addition to MS method, LC conditions were also optimized for good separation of [S-HETE]-CPF and [S-ETE]-CPF tripeptides from the matrix, and for further decreasing matrix interference in MS analysis. In previous studies, the short run-time for analysis of [S-HETE]-CPF tripeptides was the focus instead of good separation, resulting in the necessity of an additional SPE procedure after digestion [9,16,17]. Here, effective separation was the main consideration, and was achieved by combined isocratic with gradient elution program utilizing a shorter C 18 reversed-phase column (50 mm length).…”

“…It appears that these problems can be addressed by a recently developed and validated method for quantification of HDalbumin adduct, in which there is a simplified procedure for decreased analysis time, low usage amount of blood specimens, and reproducible tripeptide yields produced by novel selected proteinase K [17]. In spite of thie, the method could only make quantification of [S-HETE]-CPF tripeptide instead of HD concentration exposed, and the need for an isotope labeling [S-HETE]-CPF as IS was not readily available for many ordinary laboratories.…”