A new HPLC method for the simultaneous determination of oxidized and reduced plasma aminothiols using coulometric electrochemical detection.

A new method has been developed that is capable of providing a complete profile of the most common monothiols and disulfides present in plasma or tissue extracts. The method utilizes reversed phase ion-pairing high performance liquid chromatography coupled with coulometric electrochemical detection to simultaneously quantify free oxidized and reduced aminothiols or total aminothiols after chemical reduction. The method is extremely sensitive, with limits of detection in the 5 fmol/mL range for monothiols and 50 fmol/mL for dithiols. The interassay and intraassay coefficients of variation for total and free aminothiols ranged between 1.2 and 5.8%. The mean recoveries for total and plasma aminothiols ranged between 97.1 and 102.8%. The aminothiols are quantified directly, without derivatization, and include methionine, homocysteine, homocystine, cystathionine, cysteine, cystine, cysteinylglycine, and oxidized and reduced glutathione. Because a complete aminothiol profile of metabolites in both the remethylation (anabolic) and transulfuration (catabolic) pathways of homocysteine metabolism can be determined simultaneously, this new method should be useful in determining the metabolic etiology of homocysteinemia and in designing appropriate nutritional intervention strategies. Basic research applications of this method should lead to an increased understanding of the metabolic pathology of aminothiol imbalance.