Analysis of retinoids by high-performance liquid chromatography using programmed gradient separation.

Vitamin A (retinol), its metabolites and analogues have been heavily focused upon due to their clinical value and utility. Retinol can be used as a reflection of dietary or nutritional status, while the retinol analogues (isotretinoin, tretinoin, etretinate) are being proven as potent dermatologic and anti-tumor agents. High-performance liquid chromatography (HPLC) has been demonstrated to be applicable to the measurement of these compounds in blood. HPLC assays have been reported for retinol [l--4], 13&s-retinoic acid [ 5, 61, all-trtrns-retinoic acid [ 7, 81, etretinate [ 9, lo] , plus retinol and retinal isomers [ ll---151. Several assay systems have been reported for the isocratic separation of mixtures of retinoids [ 13-151. Of these, the methods of Frolick et al. [ 141 and McClean et al. [15] allow for the measurement of numerous retinoids in biological specimens through the use of single isocratic systems. Using these methods the required time for separation of compounds can run as long as 36 min [14]. The natural and synthetic retinoids, plus their respective major metabolites, have differences in polarity that make chromatographic separation difficult in a short time period. During the long separation times the later elutmg peaks become broad and require an integrator. To counteract this