GC–MS analysis of S-nitrosothiols after conversion to S-nitroso- N-acetyl cysteine ethyl ester and in-injector nitrosation of ethyl acetate

Abstract

S-Nitrosothiols from low-molecular-mass and high-molecular-mass thiols, including glutathione, albumin and hemoglobin, are endogenous potent vasodilators and inhibitors of platelet aggregation. By utilizing the S-transnitrosation reaction and by using the lipophilic (p K L 0.78) and strong nucleophilic synthetic thiol N-acetyl cysteine ethyl ester (NACET) we have developed a GC–MS method for the analysis of S-nitrosothiols and their 15N- or 2H– 15N-labelled analogs as S-nitroso- N-acetyl cysteine ethyl ester (SNACET) and S 15NACET or d 3-S 15NACET derivatives, respectively, after their extraction with ethyl acetate. Injection of ethyl acetate solutions of S-nitrosothiols produced two main reaction products, compound X and compound Y, within the injector in dependence on its temperature. Quantification was performed by selected-ion monitoring of m/ z 46 (i.e., [NO 2] −) for SNACET and m/ z 47 (i.e., [ 15NO 2] −) for S 15NACET/d 3-S 15NACET for compound X, and m/ z 157 for SNACET and m/ z 160 for d 3-S 15NACET for compound Y. In this article we describe the development, validation and in vitro and in vivo applications of the method to aqueous buffered solutions, human and rabbit plasma. Given the ester functionality of SNACET/S 15NACET/d 3-S 15NACET, stability studies were performed using metal chelators and esterase inhibitors. The method was found to be suitable for the quantitative determination of various S-nitrosothiols including SNACET externally added to human plasma (0–10 μM). Nitrite contamination in ethyl acetate was found to interfere. Our results suggest that the concentration of endogenous S-nitrosothiols in human plasma does not exceed about 200 nM in total. Oral administration of S 15NACET to rabbits (40–63 μmol/kg body weight) resulted in formation of ALB-S 15NO, [ 15N]nitrite and [ 15N]nitrate in plasma.