Micellar liquid chromatography in clinical chemistry: application to the monitorization of B6 vitamins

Abstract

Background: A micellar reversed-phase liquid chromatographic procedure was developed for the determination of B6 group vitamins, i.e. pyridoxine, pyridoxal and pyridoxamine, in human serum. Methods: Chromatographic conditions used were a C18 column, isocratic mode, flow-rate of 1 ml/min and UV-detection at 290 nm. Optimization of the composition of the mobile phase was performed using an interpretative strategy. Results: After modeling, the composition of the selected mobile phase was 150 mM sodium dodecyl sulphate (SDS)–2% (v/v) pentanol–dihydrogenphosphate buffer 10 mM at pH 3. In this mobile phase, serum samples were injected without pretreatment and analysis time was below 14 min. Calibrations for the three vitamins were linear, with coefficient regression better than 0.999, and intra- and inter-day precision, achieved according to ICH, offering values below 4.3% and 3.2%, respectively. The method was applied to the determination of the B6 vitamins in spiked serum samples, with recoveries around 100%, and in the pharmacokinetic determination of pyridoxine half-life in serum, which was found to be 47.5±3.2 min ( n=5). The procedure was also applied for the analysis of pyridoxine in human serum spiked with several pharmaceutical preparations that contain other drugs which do not produce any kind of interference. Finally, solutions of B6 vitamins kept at −201 °C are stable for up to 3 months. Conclusions: Using the method proposed here, with an SDS–pentanol mobile phase, it is possible to carry out the fast sensitive determination of B6 vitamins in serum following direct injection, without sample pretreatment.