Gas chromatography-tandem mass spectrometry-based detection of half nitrogen mustards in plasma as a new biomarker of nitrogen mustard exposure

Abstract

The development and optimization of an analytical method for the detection and identification of reactive metabolite of organochlorine chemical warfare agent nitrogen mustards (NMs), 2-[(2-chloroethyl)(alkyl)amino]ethanol (CEAAE), known as half nitrogen mustard, in blood samples is presented, herein. In this study, half nitrogen mustards in plasma are presented as a new and unambiguous biomarker of NM exposure since the fully hydrolyzed product, i.e., amino alcohols, are common industrial chemicals that can be present as such without getting exposed to NMs. Thus, the detection of half nitrogen mustard as a biomarker holds great significance for verification by the Chemical Weapon Convention (CWC) and will also be helpful in understanding the pharmacokinetics of NM-based chemotherapeutic pro-drugs. To the best of our knowledge, this is the first report on the detection of half nitrogen mustards in any matrice, including plasma. A very simple sample preparation protocol was developed for its extraction from plasma samples. Heptafluorobutyrylation and gas chromatography-tandem mass spectrometry in the positive chemical ionization mode were developed for the detection and identification of halfNMs. The developed method has shown excellent analytical figures of merits such as a wide range of linearity (1.0–50 ng mL⁻¹), low limit of detection (0.3–0.5 ng mL⁻¹), and low limit of quantification (1.0 ng mL⁻¹). The interday and intraday reproducibilities were also less than 15%. The developed method was successfully applied to real-world samples; in vitro human plasma was spiked with ∼1 ng mL⁻¹ of all the NMs and in vivo studies were done with rats intravenously exposed to 1 × LD₅₀ of bis(2-chloroethyl)methylamine (HN2).