Experiences with monolithic LC phases in quantitative bioanalysis.

Abstract

The applicability of monolithic liquid chromatographic (LC) phases in the field of quantitative bioanalysis has been evaluated. Two existing methods with fluorescence detection (the determination of bexarotene in plasma and the determination of dextromethorphan plus metabolites in urine) were successfully transferred from a conventional reversed-phase column to a 10 cm x 4.6 mm i.d. monolith. By simply increasing the mobile phase flow-rate, run times were about 3-fold reduced, while the chromatographic resolution of the analytes remained unaffected. In both cases, a very good correlation was found between the results of clinical samples obtained with the original method and those obtained with the adapted method. Two methods with tandem mass spectrometric detection were set up. For one of these methods (nifedipine in plasma), the separation of the analyte from interfering matrix components did not need a high plate number; the resolution found on a 10-cm monolith at 6 ml/min and that on a 3-cm conventional column at 2 ml/min were comparable and achieved in the same period of time. As the validation results on both column types were similar and considering the limited compatibility of mass spectrometric detection with high solvent flow rates, the conventional column was concluded to be the best choice for this application. For the determination of estradiol in plasma, however, there was so much interfering material that needed to be separated from the analyte, that the best results were obtained with three 10-cm monolithic columns coupled in series and because of the possibility to apply a relatively high flow-rate, a reasonable run time was still achieved.