Journal of Chromatography B, 927 (2013) 147–157 Contents lists available at SciVerse ScienceDirect Journal of Chromatography B j ourna l h om epage: www.elsevier.com/locate/chromb UPLC–MS/MS measurement of S-nitrosoglutathione (GSNO) in human plasma solves the S-nitrosothiol concentration enigma  Dimitrios Tsikas ∗ , Mario Schmidt, Anke Böhmer, Alexander A. Zoerner, Frank-Mathias Gutzki, Jens Jordan Institute of Clinical Pharmacology, Hannover Medical School, Carl-Neuberg-Strasse 1, D-30625 Hannover, Germany a r t i c l e i n f o Article history: Received 2 November 2012 Accepted 25 January 2013 Available online 4 February 2013 Keywords: Artefacts Fast-liquid chromatography Nitric oxide Quantification S-Nitrosothiols Tandem mass spectrometry Validation a b s t r a c t We developed and validated a fast UPLC–MS/MS method with positive electrospray ionization (ESI+) for the quantitative determination of S-nitrosoglutathione (GSNO) in human plasma. We used a published protocol for the inactivation of plasma -glutamyltransferase (GT) activity by using the GT transi- tion inhibitor serine/borate and the chelator EDTA for the stabilization of GSNO, and N-ethylmaleimide (NEM) to block SH groups and to avoid S-transnitrosylation reactions which may diminish GSNO concen- tration. S-[ 15 N]Nitrosoglutathione (GS 15 NO) served as internal standard. Fresh blood was treated with NEM/serine/borate/EDTA, plasma spiked with GS 15 NO (50 nM) was ultrafiltered (cut-off 10 kDa) and 10 L aliquots of the ultrafiltrate were analyzed by UPLC–MS/MS. Five HILIC columns and an Acquity UPLC BH amide column were tested. The mobile phase was acetonitrile–water (70:30, v/v), contained 20 mM ammonium formate, had a pH value of 7, and was pumped isocratically (0.5 mL/min). The Nucle- oshell column allowed better LC performance and higher MS sensitivity. The retention time of GSNO was about 1.1 min. Quantification was performed by selected-reaction monitoring the mass transition m/z 337 ([M+H] + ) → m/z 307 ([M+H 14 NO] •+ ) for GSNO (i.e., GS 14 NO) and m/z 338 ([M+H] + ) → m/z 307 ([M+H 15 NO] •+ ) for GS 15 NO. NEM/serine/borate/EDTA was found to stabilize GSNO in human plasma. The method was validated in human plasma (range, 0–300 nM) using 50 nM GS 15 NO. Accuracy and precision were in generally acceptable ranges. A considerable matrix effect was observed, which was however out- weighed by the internal standard GS 15 NO. In freshly prepared plasma from heparinized blood donated by 10 healthy subjects, no endogenous GSNO was determined above 2.8 nM, the limit of quantitation (LOQ) of the method. This study challenges previously reported GSNO plasma concentrations being far above the present method LOQ value and predicts that the concentration of low-molecular-mass and high-molecular-mass S-nitrosothiols are in the upper pM- and lower nM-range, respectively. © 2013 Elsevier B.V. All rights reserved. 1. Introduction S-Nitrosothiols or organic thionitrites with the general formula R S N O (RSNO) are reaction products of organic thiols (R S H; RSH), notably cysteinyl thiols, and higher oxides of nitric oxide (NO) such as dinitrogen trioxide (N 2 O 3 ). Despite its radical nature, NO is relatively stable in heme-free aqueous solutions. Three of the most reactive NO-species towards RSH are nitrous acid (H O N O; HONO, pK a 3.3), the nitrosyl cation ( + NO), and N 2 O 3 . Under acidic conditions, HONO is readily formed from nitrite, the autoxidation product of NO. From an analytical standpoint, RSNO are “prob- lem children” of the NO family [1–3]. RSNO’s S-nitroso group is  This paper belongs to the “Fast Liquid Chromatography” by P.D. Tzanavaras and C.K. Zacharis (Guest Editors). ∗ Corresponding author. Tel.: +49 511 532 3984; fax: +49 511 532 2750. E-mail address: tsikas.dimitros@mh-hannover.de (D. Tsikas). thermally labile and chemically very reactive towards thiols, tran- sition metal ions such as Cu 2+ and reducing agents such as ascorbic acid. On the other hand, the S-nitroso group is readily formed under acidic conditions from HONO and RSH. Both nitrite and RSH are ubiquitous in biological systems. Together, these mechanisms give rise to abundant artefactual formation during sample preparation and analysis. RSNO lack self-fluorescence. The molar absorptivity coefficient of the S-nitroso group, the most characteristic func- tional group of RSNO, is quite low (ε ≈ 0.8 mM -1 × cm -1 around 334 nm) and does not allow sensitive quantification below about 1 M by HPLC-UV [4]. Hence, physiological RSNO are commonly measured indirectly, for instance by converting the S-nitroso group to NO or nitrite for detection (for instance Refs. [5–16]). For recent reviews on RSNO analysis see Refs. [17–19]). Twenty years ago, RSNO from low-molecular-mass (LMM) and high-molecular-mass (HMM) RSH have been reported to occur in various biological samples and to exert a variety of phys- iological functions [20]. Best investigated HMM RSNO include 1570-0232/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jchromb.2013.01.023