Interference with Saville's method in determination of low-molecular weight S-nitrosothiols by ultrafiltration.

To detect low-molecular weight S-nitrosothiols in human plasma, we used a system combining HPLC for separation and Saville's method for colorimetric detection of S-nitrosothiols. The sensitivity and detection limit was 1-2 nM for both S-nitrosocysteine and S-nitrosoglutathione. When plasma was analyzed after ultrafiltration (with units requiring higher g force [5000 g], irrespective to the material of the membrane) to eliminate high molecular substances, a signal corresponding to S-nitrosoglutahione was recognized. This signal behaved as real S-nitrosoglutathione as it was partially Hg(2+)-sensitive and gradually decayed with time. However, the use of pre-washed units or another ultrafiltration unit that required lower g force (1800 g) or direct application of plasma to the HPLC-Saville's method system did not result in such signal. Based on these observations, it is important to be aware of the interference originating from the ultrafiltration unit and its potential effect on the precise quantification of low molecular weight S-nitrosothiols using Saville's method.