Skip to Content
Merck
  • Stable-isotope dilution LC-MS/MS measurement of nitrite in human plasma after its conversion to S-nitrosoglutathione.

Stable-isotope dilution LC-MS/MS measurement of nitrite in human plasma after its conversion to S-nitrosoglutathione.

Journal of chromatography. B, Analytical technologies in the biomedical and life sciences (2014-09-23)
Erik Hanff, Anke Böhmer, Jens Jordan, Dimitrios Tsikas
ABSTRACT

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 [(15)N]nitrite served as the internal standard (IS). Endogenous nitrite and IS were converted to their S-nitrosoglutathione (GSNO) derivatives, i.e., GS(14)NO and GS(15)NO, respectively, by using excess glutathione (GSH) and HCl. For plasmatic nitrite, fresh plasma (0.5 mL) was spiked with the IS (1000 nM) and ultrafiltered (cut-off 10 kDa). Ultrafiltrate aliquots (100 μL) were treated with aqueous GSH at a final concentration of 1 mM and 1 μL of 5M HCl for 5 min. After final sample dilution (1:1, v/v) with acetonitrile-water (70:30, v/v), 2 μL 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 0.5 mL/min. A Nucleoshell column was used for LC separation. The retention time of GSNO was about 0.8 min and the total analysis time 5 min. Quantification was performed by selected-reaction monitoring the specific mass transition m/z337([M+H](+))→m/z 307([M+H-(14)NO](+·)) for GS(14)NO (i.e., for endogenous nitrite) and m/z338([M+H](+))→m/z307([M+H-(15)NO](+·)) for GS(15)NO (i.e., for the IS). The method was thoroughly validated in human plasma (range, 0-2000 nM). The LOD and LOQ values of the LC-MS/MS method were determined to be 1 fmol and 5 nM [(15)N]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 369 nM. These concentrations were confirmed by using a previously reported GC-MS method and agree with those measured previously by HPLC-UV (334 nm) 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 400 nM.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Methanol, NMR reference standard
Sigma-Aldrich
Ammonia solution, 0.4 M in THF
Sigma-Aldrich
Ammonia solution, 4 M in methanol
Sigma-Aldrich
Ammonia, puriss., anhydrous, ≥99.95%
Supelco
Methanol, analytical standard
Sigma-Aldrich
Ammonium formate, ≥99.995% trace metals basis
Sigma-Aldrich
Ammonium formate, BioUltra, ≥99.0% (calc. based on dry substance, NT)
Sigma-Aldrich
Ammonia-14N, 99.99 atom % 14N
Supelco
Ammonium formate, eluent additive for LC-MS, LiChropur, ≥99.0%
Sigma-Aldrich
Ammonia solution, 0.4 M in dioxane
Sigma-Aldrich
Methanol, suitable for HPLC, ≥99.9%
Supelco
Methanol, Pharmaceutical Secondary Standard; Certified Reference Material
Sigma-Aldrich
Methanol, ACS reagent, ≥99.8%
Sigma-Aldrich
Methanol, Absolute - Acetone free
Sigma-Aldrich
Methanol, ACS reagent, ≥99.8%
Sigma-Aldrich
Methanol, ACS spectrophotometric grade, ≥99.9%
Sigma-Aldrich
Ammonium formate, reagent grade, 97%
Sigma-Aldrich
Methanol, Laboratory Reagent, ≥99.6%
Supelco
Residual Solvent - Acetonitrile, Pharmaceutical Secondary Standard; Certified Reference Material
Sigma-Aldrich
Methanol, BioReagent, ≥99.93%
Sigma-Aldrich
Ammonia solution, 2.0 M in ethanol
Sigma-Aldrich
Ammonia solution, 7 N in methanol
Sigma-Aldrich
Methanol, suitable for HPLC, gradient grade, suitable as ACS-grade LC reagent, ≥99.9%
Sigma-Aldrich
Ammonia solution, 2.0 M in isopropanol
Sigma-Aldrich
Methanol, ACS reagent, ≥99.8%
Sigma-Aldrich
Methanol, HPLC Plus, ≥99.9%
Sigma-Aldrich
Methanol-12C, 99.95 atom % 12C
Sigma-Aldrich
Methanol solution, NMR reference standard, 4% in methanol-d4 (99.8 atom % D), NMR tube size 3 mm × 8 in.
Sigma-Aldrich
Formic acid solution, BioUltra, 1.0 M in H2O
Sigma-Aldrich
Formic acid, ≥95%, FCC, FG