Dual-electrode amperometric detection for the determination of SR4233 and its metabolites with microbore liquid...

3-Amino-1,2,4-benzotriazine-2,4-di-N-oxide (SR4233) is a promising new antineoplastic agent based on reductive activation. SR4233 and its major metabolites (SR4317 and SR4330) are all easily reduced at a carbon electrode. Reductive amperometric detection can therefore provide high selectivity and low detection limits with chromatographic analysis and is an ideal approach to detection of SR4233 in microdialysis samples. However, in order to use amperometric detection in the reductive mode, sample deoxygenation is necessary. This is typically done by purging the sample with either argon or nitrogen prior to injection. This approach is not feasible for microdialysis samples because only 5-10 microliters is usually available. In this report, a microbore liquid chromatographic method with dual-electrode amperometric detection is described for the determination of SR4233 and its metabolites without predeoxygenation. A dual-electrode amperometric detector was used in the series configuration with an upstream potential of -450 mV to reduce SR4233 and its metabolites to a common product and a downstream potential of +400 mV to oxidize this product. Oxygen is only electroactive at the upstream electrode because of its irreversible behavior. This method is compatible with the small sample volumes provided by microdialysis sampling. Linear calibration graphs were obtained up to 55 microM for SR4233, and 140 microM for both SR4317 and SR4330. The detection limits were 70 nM for SR4233, and 50 nM for SR4317 and SR4330. The average intra-day variation over 5 days was 1.8% (SR4233), 1.4% (SR4330), and 1.8% (SR4317), whereas the inter-day variation over 5 days was 14.1% (SR4233), 8.6% (SR4317), and 2.6% (SR4330).