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

Pozdeev V.K.1 , Pozdeyev N.V.2

1. Saint-Petersburg Research Institute of Influenza, Russian Academy of Medical Sciences, Northwestern Branch
2. Department of Medicine, Harbor Hospital
Section: Experimental/Clinical Study
DOI: 10.18097/PBMC20105606726      UDK: 577.112.3:543.544:616.36-002.14      PubMed Id: 21395075
Year: 2010  Volume: 56  Issue: 6  Pages: 726-738
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 μM 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 μl injection volume (signal-to-noise ratio of 3).
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Pozdeev V.K., Pozdeyev N.V. (2010) Biomeditsinskaya khimiya, 56(6), 726-738.
This paper is also available as the English translation:10.1134/S199075081003011X
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