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Stable-isotope dilution LC–MS/MS measurement of nitrite in human plasma after its conversion to S-nitrosoglutathione
- Hanff, Erik, Böhmer, Anke, Jordan, Jens, Tsikas, Dimitrios
- Journal of Chromatography B 2014 v.970 pp. 44-52
- acetylcysteine, blood, formates, gas chromatography-mass spectrometry, glutathione, high performance liquid chromatography, humans, hydrochloric acid, ionization, monitoring, nitrites, nitroprusside, pH, quantitative analysis, saliva, stable isotopes, ultrafiltration, urine
- A specific, sensitive and fast LC–MS/MS method with positive electrospray ionization for the quantitative determination of nitrite in human plasma is reported. Added [15N]nitrite served as the internal standard (IS). Endogenous nitrite and IS were converted to their S-nitrosoglutathione (GSNO) derivatives, i.e., GS14NO and GS15NO, respectively, by using excess glutathione (GSH) and HCl. For plasmatic nitrite, fresh plasma (0.5mL) was spiked with the IS (1000nM) and ultrafiltered (cut-off 10kDa). Ultrafiltrate aliquots (100μL) were treated with aqueous GSH at a final concentration of 1mM and 1μL of 5M HCl for 5min. After final sample dilution (1:1, v/v) with acetonitrile–water (70:30, v/v), 2μL aliquots were injected via a thermostated (4°C) autosampler. The mobile phase was acetonitrile–water (70:30, v/v), contained 20mM ammonium formate, had a pH value of 7, and was pumped isocratically at 0.5mL/min. A Nucleoshell column was used for LC separation. The retention time of GSNO was about 0.8min and the total analysis time 5min. Quantification was performed by selected-reaction monitoring the specific mass transition m/z337([M+H]+)→m/z 307([M+H−14NO]+) for GS14NO (i.e., for endogenous nitrite) and m/z338([M+H]+)→m/z307([M+H−15NO]+) for GS15NO (i.e., for the IS). The method was thoroughly validated in human plasma (range, 0–2000nM). The LOD and LOQ values of the LC–MS/MS method were determined to be 1fmol and 5nM [15N]nitrite, respectively. The relative matrix-effect of about 21% was outweighed entirely by the IS. In freshly prepared plasma samples from heparinized blood donated by three healthy subjects, nitrite concentration was determined by LC–MS/MS to be 516, 199 and 369nM. These concentrations were confirmed by using a previously reported GC–MS method and agree with those measured previously by HPLC–UV (334nm) after nitrite conversion to S-nitroso-N-acetylcysteine (SNAC) by N-acetylcysteine (NAC). Measurement of nitrite by LC–MS/MS as GSNO is about 1000 times more sensitive than by HPLC–UV as SNAC. The applicability of the method to microdialysate, urine, and saliva samples from humans was demonstrated. The agreement of two orthogonal MS-based methods indicates that the concentration of nitrite in freshly prepared, non-frozen plasma from heparinized blood of fasted healthy humans is of the order of 400nM.