Fully automated determination of selective retinoic acid receptor ligands in mouse plasma and tissue by reversed-phase liquid chromatography coupled on-line with solid-phase extraction

A fully automated reversed-phase HPLC method was developed for the quantitative assay of three retinoids (Am-580, CD-2019 and CD-437) which selectively activate the retinoic acid receptors RAR alpha, RAR beta and RAR gamma, respectively. Mouse plasma, embryo and maternal tissues were prepared for injection by on-line solid-phase extraction (SPE) and valve-switching techniques. Following automatic injection, the sample was loaded on preconditioned disposable cartridges, cleaned-up and then transferred onto the analytical column to be eluted in the backflush mode, separated by gradient elution and detected by UV, while a new cartridge was concomitantly conditioned. The overall recovery was quantitative allowing for external standardization. The calibration curves were linear in all biological samples tested so far, with a correlation coefficient (r) >0.99. The intra-day precision was less than or equal to 7.8% (n=5-6) and the inter-day variability was less than or equal to 9.4% (n=3). The lower limit of detection was 2.5 ng/ml or ng/g for CD-2019 and CD-437, and 5 ng/ml for Am-580 with a SIN ratio of 5 using a sample weight of 25 mu l or mg. The method is now in routine use in our laboratory for the assessment of the pharmacokinetic profiles of these retinoids. The small sample size required, the simple sample prepration and the rapid analysis with high degree of automation make this method convenient for microanalysis of biological samples both in animal and human studies.