SIMULTANEOUS DETERMINATION OF ALL-TRANS-RETINOIC, 13-CIS-RETINOIC AND9-CIS-RETINOIC ACID, THEIR 4-OXO METABOLITES AND ALL-TRANS-RETINOL INHUMAN PLASMA BY HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY

All-trans-retinoic acid (all-trans-RA) and 13-cis-retinoic acid (13-ci s-RA), due to their effects on cell differentiation, proliferation and angiogenesis, improved treatment results in some malignancies. Pharma cokinetic studies of all-trans-RA and 13-cis-RA along with monitoring of retinoic acid metabolites may help to optimize retinoic acid therap y and to develop new effective strategies for the use of retinoic acid s in cancer treatment. Therefore, we developed a HPLC method for the s imultaneous determination in human plasma of the physiologically impor tant retinoic acid isomers, all-trans-, 13-cis- and 9-cis-retinoic aci d, their 4-oxo metabolites, 13-cis-4-oxoretinoic acid (13-cis-4-oxo-RA ) and all-trans-4-oxoretinoic acid (all-trans-4-oxo-RA), and vitamin A (all-trans-retinol). Analysis was performed on a silica gel column wi th UV detection at 350 nm using a binary multistep gradient composed o f n-hexane, 2-propanol and glacial acetic acid. For liquid-liquid extr action a mixture of n-hexane, dichloromethane and 2-propanol was used. The limits of detection were 0.5 ng/ml for retinoic acids and 10 ng/m l for all-trans-retinol. The method showed good reproducibility for al l components (within-day C.V.: 3.02-11.70%; day-to-day C.V.: 0.01-11.3 4%). Furthermore, 9-cis-4-oxoretinoic acid (9-cis-4-oxo-RA) is separat ed from all-trans-4-oxo-RA and 13-cis-4-oxo-RA. In case of clinical us e of 9-cis-retinoic acid (9-cis-RA) the pharmacokinetics and metabolis m of this retinoic acid isomer can also be examined.