We read with great interest the work by RodrÃguez-Ortigosa et al.,1 who reported that the biliary secretion of S-nitrosoglutathione (GSNO) is involved in rat hypercholeresis. Biliary GSNO was identified and quantified by the direct infusion of the supernatant of deproteinized bile into a quadrupole time-of-flight mass spectrometry (MS) instrument. The presence of GSNO in the bile suggests a role of S-nitrosothiols in bile flow regulation. In our opinion, the identity and quantity of GSNO in the rat bile, as reported by RodrÃguez-Ortigosa et al., lack solid proof. The analysis of GSNO and other endogenous S-nitrosothiols in biological fluids is highly challenging.2 Beyond cautious analytical handling, consideration and rationalization of previous observations are required. Major analytical problems include very low concentrations and poor chemical stability. The concentration of GSNO in biological fluids is on the threshold of the picomolar/nanomolar range.2, 3 Tandem mass spectrometry (MS/MS) coupled to liquid chromatography (LC) is the most reliable methodology for the unequivocal identification and accurate quantification of GSNO.3, 4 The renunciation of the LC step and the use of simple MS instead of MS/MS are fraught with danger. In the positive electrospray ionization mode, GSNO ionizes to produce the most characteristic ions, [M+H]+ [mass-to-charge ratio (m/z) = 337] and [M+H−NO]+• (m/z = 307), which results from the loss of •NO (30 Da) from [M+H]+ (Fig. 1). Thus far, collision-induced dissociation (CID) of m/z = 337 has not been reported to yield m/z = 319 (dehydrated GSNO). Moreover, CID-induced loss of H2O (18 Da) seems to occur only after the loss of the labile NO moiety of GSNO (producing m/z = 2894; Fig. 1) and in stable glutathione conjugates of bile acids.5 (A,B) LC-MS and (C,D) LC-MS/MS analyses of synthetic GSNO (molecular mass = 336) and GS15NO (molecular mass, 337) by positive electrospray ionization with the Xevo tandem quadrupole MS instrument from Waters (Eschborn, Germany). Product ion mass spectra were obtained by the subjection of the precursor ions at m/z = 337 for GSNO and at m/z = 338 for GS15NO to CID with argon (collision energy = 12 eV). Additional experimental conditions were as follows: capillary, cone, and extractor voltages of 1.02 kV, 26 V, and 3 V, respectively; source and desolvation temperatures of 150°C and 650°C, respectively; desolvation gas (nitrogen) and collision gas flow rates of 600 L/hour and 0.15 mL/minute, respectively; a high-performance liquid chromatography column (held at 30°C) with dimensions of 50 × 2.10 mm (Kinetex 2.6-μm, 100-Ã… hydrophilic interaction liquid chromatography column, Phenomenex); and gradient elution between solvent A [90%; 5 mM ammonium acetate and 0.1 vol % HCOOH in acetonitrile (pH 5.8)] and solvent B [50%; 5 mM ammonium acetate and 0.1 vol % HCOOH (pH 5.8)] within 5 minutes at a flow rate of 0.3 mL/minute. The retention time for GSNO and GS15NO was 3.45 minutes. The identification of GSNO in biological fluids is best performed by LC-MS/MS through the generation of product ions from m/z = 337 of the LC peak eluting with the retention time of GSNO. The product ion mass spectrum must include the most characteristic ion at m/z = 307 (Fig. 1).4 Quantification is best performed through the monitoring of m/z = 307 produced by CID of m/z = 337 after chromatographic separation.3, 4 15N-labeled S-nitrosoglutathione (GS15NO) is best suited as an internal standard4 (Fig. 1). S-Nitroso-N-acetylcysteine, the mercapturic acid of NO, has not been found in humans.6 Our groups were not able to detect GSNO in the bile of normal rats at concentrations greater than 200 nM, the detection limit of the spectrophotometric method. The presence of GSNO in bile and its role in bile flow regulation still need to be demonstrated. Dimitrios Tsikas Ph.D.*, Alexander A. Zoerner Ph.D.*, Frank-Mathias Gutzki Ph.D.*, Ranieri Rossi Ph.D. , * Institute of Clinical Pharmacology Hannover Medical School Hannover, Germany, Department of Evolutionary Biology Laboratory of Pharmacology and Toxicology University of Siena Siena, Italy.