Complete two-dimensional separation for analysis of acidic compounds in plasma using column-switching reversed-phase liquid chromatography

A complete two-dimensional separation technique for the determination of acidic compounds in plasma was developed by using column-switching reversed-phase liquid chromatography. This technique was based on solute peak enrichment at the top of the second column during heart-cutting and an ion-pair chromatographic separation in the second column using tetrabutylammonium ion, where different separation modes in the first and second columns and solute peak enrichment at the top of the second column during heart-cutting were achieved coincidentally. Retention behaviors of two solutes, zidovudine-β-d-glucuronide (AZT-β-d-Gluc) and probenecid, in the first and second column and solute peak enrichment at the top of the second column were investigated for establishment of the system. Different retention behaviors of the solutes in the first and second column, which were evaluated by changes in capacity factor versus acetonitrile concentration in the mobile phases, and peak enrichment could be accomplished by using ion-pair chromatography in the second column. System suitability was confirmed by assessing the number of theoretical plates (N) of the second column for the solutes after heart-cutting. The N values in the second column after column switching were almost same as those in the case that the solutes were directly injected onto the second column. These results indicate that complete two-dimensional separation should be achieved by using this system. Furthermore, this technique was applied to method development for the determination of AZT-β-d-Gluc and probenecid in rat plasma. The peaks of each analyte in the plasma extract obtained by deproteinization were well separated from those of endogenous substances, and easy determination of the analytes could be accomplished at the ng/ml level only by changing the acetonitrile concentration in the mobile phases.