ELSEVIER Journal of Chromatography A, 729 (1996) 125-136 JOURNALOF CHROMATOGRAPHY A 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,~ C H.M.M. Arafa , F.M.A. Hamada b, M.M.A. Elmazar, H. Nau a ~'htstitut f iir Toxikologie und Embryopharmakologie, Freie Universitiit Berlin, Garystrasse 5, D-1419.5 Berlin, Germany hPharmacy College, AI-Azhar University, Cairo, Egypt ~Pharmacy College, King Saud Universi~, Riyadh, Saudi Arabia Abstract 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 RARa, RARfl and RART, 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 -<7.8% (n=5-6) and the inter-day variability was -<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 S/N ratio of 5 using a sample weight of 25/.d 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. Keywords: Sample preparation; Retinoids; Retinol 1. Introduction Retinoids, natural and synthetic analogs of vitamin A alcohol (retinol; ROH), are implicated in a wide variety of key biological processes including cell growth and differentiation, embryogenesis and epi- thelial homeostasis [1,2]. Clinically, retinoids have proven efficacy in the treatment of dermatological diseases such as acne, psoriasis and photoaged skin *Corresponding author. [3,4], as well as in certain malignancies [5]. The molecular mechanisms of such compounds are thought to be mediated, for the most part, through regulation of gene expression by two subclasses of nuclear retinoic acid receptors RARs and RXRs [6,7]. Both of these subclasses contain three receptor subtypes arbitarily designated a, fl, and y, which are encoded by separate genes [8[. The clinical usefulness of retinoids is limited by a number of side effects such as bone and lipid toxicities [9], and teratogenicity [10,11]. Therefore, new retinoid congeners with superior therapeutic 0021-9673/96/$15.00 © 1996 Elsevier Science B.V. All rights reserved SSDI 0021-9673(95)00980-9