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
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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Keywords: aminothiols, amino acids, plasma, HPLC, hepatitis C
Citation:
Pozdeev V.K., Pozdeyev N.V. (2010) Determination of total aminothiols and neuroactive amino acids in plasma by high performance liquid chromatography with fluorescence detection. Biomeditsinskaya Khimiya, 56(6), 726-738.
Pozdeev V.K. et al. Determination of total aminothiols and neuroactive amino acids in plasma by high performance liquid chromatography with fluorescence detection // Biomeditsinskaya Khimiya. - 2010. - V. 56. -N 6. - P. 726-738.
Pozdeev V.K. et al., "Determination of total aminothiols and neuroactive amino acids in plasma by high performance liquid chromatography with fluorescence detection." Biomeditsinskaya Khimiya 56.6 (2010): 726-738.
Pozdeev, V. K., Pozdeyev, N. V. (2010). Determination of total aminothiols and neuroactive amino acids in plasma by high performance liquid chromatography with fluorescence detection. Biomeditsinskaya Khimiya, 56(6), 726-738.
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