Evaluation of liquid chromatography with electrochemical detection for determination of 6-thioguanine in clinical samples
Ver Vers, Leonard M.
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A new detection system for the analysis of 6-thioguanine (6-TG) and 6-thioguanine riboside (6-TGR) in plasma samples has been investigated. The investigation began with a study of the electrochemistry of 6-TG and 6-TGR. First, cyclic voltammograms, were obtained for each compound. Second, hydrodynamic voltammograms were obtained. From these two voltammograms, the optimal working voltage of 1.00 volt vs. Ag/AgCl reference for oxidative thin-layer amperometry was noted. Another set of hydrodynamic voltammograms using a mercury electrode were obtained for 6-TG and 6-TGR. From these two voltammograms the detector potential of 0.20 volts vs. Ag/AgCl reference was noted as the working potential. After the optimal working potentials were obtained, linearity experiments were performed using three modes of detection: U.V. absorption, oxidative amperometry and mercury electrode detection of the sulfhydryl group. From a standard solution range of 1.0-200 ng, calibration curves for 6-TG and 6-TGR using all three modes of detection were obtained. Using the data from the calibration curves, regression analysis for both compounds was performed. Then using the Student's t-test at the 95% level, detection limits for both compounds using each detection mode were estimated. The U.V. absorption detection mode had the highest estimated detection limits: 0.91 ng for 6-TG and 0.94 ng for 6-TGR. The Au/Hg electrode had estimated detection limits of 49 pg for 6-TG and 73 pg for 6-TGR. Finally, oxidative amperometry produced estimated detection limits of 14 pg for 6-TG and 31 pg for 6-TGR. The two electrochemical detection modes produced estimated detection limits lower than any previously published. The feasibility of using electrochemical detection for the determination of 6-TG and 6-TGR in clinical samples was shown by comparing plasma chromatograms observed with U.V. absorption, oxidative electrochemical and Au/Hg detection.