Journal of Chromatography B, 879 (2011) 1485–1495 Contents lists available at ScienceDirect Journal of Chromatography B journal homepage: www.elsevier.com/locate/chromb 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  Alexandra Schwarz a , Darko Modun b , Karsten Heusser a , Jens Tank a , Frank-Mathias Gutzki a , Anja Mitschke a , Jens Jordan a , Dimitrios Tsikas a,∗ a Institute of Clinical Pharmacology, Hannover Medical School, Carl-Neuberg-Str. 1, D-30625 Hannover, Germany b Department of Pharmacology, University of Split School of Medicine, Soltanska 2, 21000 Split, Croatia article info Article history: Received 15 February 2010 Accepted 5 May 2010 Available online 12 May 2010 Keywords: Circulation Derivatization Ferricyanide Hemoglobin Kinetics Quantification abstract Previously, we reported on the usefulness of pentafluorobenzyl bromide (PFB-Br) for the simultane- ous 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 meth- ods 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 arti- cle 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 abso- lutely 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 ery- throcytes (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. © 2010 Elsevier B.V. All rights reserved. 1. Introduction The major fraction of endogenously produced nitric oxide (NO) is oxidized to nitrate (NO 3 - ) within the erythrocytes by oxyhe- moglobin (HbO 2 ) [1]. NO partly autoxidizes to nitrite (NO 2 - ) which is also oxidized to nitrate by HbO 2 in erythrocytes. However, in human blood the half-life of nitrite is about 6000 times longer than that of NO, i.e., about 12 min versus <0.1 s (reviewed in Ref. [1]).  This paper is part of the special issue “Enhancement of Analysis by Analytical Derivatization”, Jack Rosenfeld (Guest Editor). ∗ Corresponding author. Tel.: +49 511 532 3959; fax: +49 511 532 2750. E-mail address: tsikas.dimitros@mh-hannover.de (D. Tsikas). Nitrite and nitrate circulate in blood, both in plasma and in red blood cells, and they are excreted in the urine. Under certain con- ditions nitrate may be an indicator of systemic NO production [2]. Recently, circulating nitrite has been suggested to reflect endothe- lial NO synthesis (reviewed in Ref. [3]). Pioneer work showed that nitrate and nitrite are evenly distributed between plasma and blood cells [4]. However, recent studies reported that nitrite is present in red blood cells at higher concentrations than in plasma, i.e., that nitrite accumulates within the erythrocytes, as measured by chemi- luminescence [5]. Others reported that nitrite distribution in blood compartments is dependent upon the bicarbonate/CO 2 concentra- tion in the blood [6], but this was not confirmed [7]. Distribution of nitrite in blood may be of particular importance, as erythro- cytes may not only oxidize nitrite to nitrate, but they may also reduce nitrite to NO in certain conditions such as hypoxia [8,9]. 1570-0232/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.jchromb.2010.05.011