Determination of Nitrogen Mustard Metabolites in Urine Using High-Performance Liquid Chromatography-High-Resolution Mass Spectrometry

Liquid chromatography-high-resolution mass spectrometry that combines the capabilities of highly selective separation of mixtures under study, valid detection of unknown substances, and high sensitivity is widely used for the detection of biologically active components in mixtures with a complex composition, to which biological fluids (blood, urine, etc.) belong. A method for the simultaneous extraction of highly polar biomarkers of nitrogen mustards such as N-triethanolamine (TEA), N-ethyldiethanolamine (EDEA), and N-methyldiethanolamine (MDEA) from urine followed by their determination by high-performance liquid chromatography (HPLC) combined with high-resolution tandem mass spectrometry is proposed. The mass spectra of fragmentation of protonated molecular ions of TEA, EDEA, and MDEA are studied, and possible structural formulas of the fragment ions are given. The conditions of the sample preparation of urine and mass spectrometric detection in the multiple reaction monitoring mode are optimized. The option of fivefold dilution with deionized water is chosen as a method of sample preparation of urine for the analysis. The separation of the components is performed in the reversed-phase chromatography mode with the retention times for TEA, EDEA, and MDEA of 2.00, 2.05, and 1.92 min, respectively. The time required to complete all the steps of the analysis of urine samples does not exceed 25 min. The detection limits of the biomarkers in urine are 1 ng/mL for TEA and 2 ng/mL for EDEA and MDEA. The developed approach makes it possible to determine the fact of application of specific nitrogen mustards in inquiry of possible exposure of a living organism to blister agents.