Determination of S-nitrosoglutathione in plasma: Comparison of two methods

In this work we compared the results of the GSNO determination in human plasma by two independent methods. The first method is based on the pre-column derivatization of GSNO thiolic part by p-hydroxymercury benzoate (PHMB) and followed by the determination of GS-PHMB product by reversed phase chromatography coupled to chemical vapour generation atomic fluorescence spectrometry (RPC-CVGAFS). The second method is based on RPC separation of GSNO from interfering compounds and the post-column, on-line enzymatic hydrolysis of GSNO by commercial γ-glutamyl transferase (GGT) and fluorescence detection.Endogenous GSNO was determined only in plasma from blood sampled by syringe (not by Vacutainers®) and ranged between 157 and 257 nM on the basis of RPC-CVGAFS method, and between 90 and 225 nM by RPC-FD method. There was a good correlation between the two methods (slope = 1.06 ± 0.09, R2 = 0.9543). RPC-CVGAFS method based on PHMB derivatization determined a GSNO concentration 60 ± 20 nM in excess with respect to RPC-FD method. Sampling issues connected with common blood sampling procedures like venipuncture and sampling in syringe or Vacutainers® still introduce in GSNO analysis unknown factors, which require further investigations.