title: Stable-isotope dilution GC-MS approach for nitrite quantification in human whole blood, erythrocytes, and plasma using pentafluorobenzyl bromide derivatization: nitrite distribution in human blood.
abstract: Previously, we reported on the usefulness of pentafluorobenzyl bromide (PFB-Br) for the simultaneous derivatization and quantitative determination of nitrite and nitrate in various biological fluids by GC-MS using their (15)N-labelled analogues as internal standards. As nitrite may be distributed unevenly in plasma and blood cells, its quantification in whole blood rather than in plasma or serum may be the most appropriate approach to determine nitrite concentration in the circulation. So far, GC-MS methods based on PFB-Br derivatization failed to measure nitrite in whole blood and erythrocytes because of rapid nitrite loss by oxidation and other unknown reactions during derivatization. The present article reports optimized and validated procedures for sample preparation and nitrite derivatization which allow for reliable quantification of nitrite in human whole blood and erythrocytes. Essential measures for stabilizing nitrite in these samples include sample cooling (0-4°C), hemoglobin (Hb) removal by precipitation with acetone and short derivatization of the Hb-free supernatant (5 min, 50°C). Potassium ferricyanide (K(3)Fe(CN)(6)) is useful in preventing Hb-caused nitrite loss, however, this chemical is not absolutely required in the present method. Our results show that accurate GC-MS quantification of nitrite as PFB derivative is feasible virtually in every biological matrix with similar accuracy and precision. In EDTA-anticoagulated venous blood of 10 healthy young volunteers, endogenous nitrite concentration was measured to be 486±280 nM in whole blood, 672±496 nM in plasma (C(P)), and 620±350 nM in erythrocytes (C(E)). The C(E)-to-C(P) ratio was 0.993±0.188 indicating almost even distribution of endogenous nitrite between plasma and erythrocytes. By contrast, the major fraction of nitrite added to whole blood remained in plasma. The present GC-MS method is useful to investigate distribution and metabolism of endogenous and exogenous nitrite in blood compartments under basal conditions and during hyperemia.
authors.affiliation: Institute of Clinical Pharmacology, Hannover Medical School, Carl-Neuberg-Str. 1, D-30625 Hannover, Germany.
authors.forename: Alexandra
authors.initials: A
authors.lastname: Schwarz
authors.forename: Darko
authors.initials: D
authors.lastname: Modun
authors.forename: Karsten
authors.initials: K
authors.lastname: Heusser
authors.forename: Jens
authors.initials: J
authors.lastname: Tank
authors.forename: Frank-Mathias
authors.initials: FM
authors.lastname: Gutzki
authors.forename: Anja
authors.initials: A
authors.lastname: Mitschke
authors.forename: Jens
authors.initials: J
authors.lastname: Jordan
authors.forename: Dimitrios
authors.initials: D
authors.lastname: Tsikas
mesh_terms: D004912:Erythrocytes / Q000737:chemistry*; D005464:Fluorobenzenes / Q000737:chemistry*; D008401:Gas Chromatography-Mass Spectrometry / Q000379:methods*; D006801:Humans; D006940:Hyperemia / Q000097:blood; D007553:Isotope Labeling / Q000379:methods; D009573:Nitrites / Q000097:blood*; D009587:Nitrogen Isotopes / Q000097:blood; D010949:Plasma / Q000737:chemistry*; D005466:Fluorocarbons
chemical_list: D005464:Fluorobenzenes; D009573:Nitrites; D009587:Nitrogen Isotopes; C025063:pentafluorobenzyl bromide; D005466:Fluorocarbons