Determination of total aminothiols and neuroactive amino acids in plasma by high performance liquid chromatography with fluorescence detection

• Published in: Biomeditsinskaia khimiia Vol. 56; no. 6; p. 726
• Main Authors: Pozdeev, V K, Pozdeev, N V
• Format: Journal Article
• Language: Russian
• Published: Russia (Federation) 01.11.2010
• Subjects: Adult; Amino Acids - blood; Chromatography, High Pressure Liquid; Female; Hepatitis C, Chronic - blood; Humans; Male; Middle Aged; Neurotransmitter Agents - blood; Sensitivity and Specificity; Spectrometry, Fluorescence
• ISSN: 2310-6972
• DOI: 10.18097/pbmc20105606726

This paper describes a simple and sensitive reversed-phase HPLC method for the determination of total homocysteine, total cysteine, total glutathione (GSH+GSSG), and neuroactive amino acids (Asp, Glu, Tau, GABA) using precolumn derivatization with ortho-phtaldialdehyde and fluorimetric detection at 360 and 470 nm for emission and excitation, respectively. Derivatization was performed with ortho-phthaldialdehyde in the presence of 2-mercaptoethanol after alkylation of the free sulfhydryl groups with iodoacetic acid. For determination of total aminothiols, the disulfide bonds were reduced and protein-bound thiols were released by addition of dithiothreitol to the plasma sample. The advantage of this method is the simultaneous determination of both homocysteine/cysteine/glutathione and neuroactive amino acids in the sample. The plasma levels of studied compounds were determined in 14 healthy volunteers (20-45 years old) and 55 patients with chronic hepatitis C (20-49 years old) and the resulting numbers were in a good agreement the studies published earlier. The calibration curves were linear over a concentration range of 5-100 microM in plasma (r2 = 0.985-0.996). The intraday and interday coefficients of variation were 3-6% and 4-7%, respectively. The recovery of the standards added to the plasma samples ranged from 94 to 102%. The limits of detection (LOD) were 0.2-0.5 ng per 10 microl injection volume (signal-to-noise ratio of 3).