Quantitative high-performance liquid chromatographic determination of retinoids in human serum using on-line solid-phase extraction and column switching - Determination of 9-cis-retinoic acid, 13-cis-retinoic acid, all-trans-retinoic acid, 4-oxo-all-trans-retinoic acid and 4-oxo-13-cis-retinoic acid

A fully automated isocratic high-performance liquid chromatographic method for the determination of 9-cis-retinoic acid, 13-cis-retinoic acid, all-trans-retinoic acid, 4-oxo-13-cis-retinoic acid and 4-oxo-all-trans-retinoic acid, has been developed using on-line solid-phase extraction and a column switching technique allowing clean-up and pre-concentration in a single step, A 500-mu l sample of serum was diluted with 750 mu l of a solution containing 20% acetonitrile and the internal standard 9,10-dimethylanthracene. About 1000 mu l of this mixture was injected on a 20X4.6 mm I.D. poly ether ether ketone (PEEK) pre-column with titanium frits packed with Bondapak C-18 37-53 mu m, 300 Angstrom particles. Proteins and very polar compounds were washed out to waste, from the pre-column, with 0.05% trifluoroacetic acid (TFA)-acetonitrile (8.5:1.5, v/v). More than 200 aliquots of diluted serum could be injected on this pre-column before elevated back-pressure enforces replacement. Components retained on the pre-column were backflushed to the analytical column for separation and detection at 360 nm. Baseline separation was achieved using a single 250x4.6 mm I.D. Suplex pKb-100 column and a mobile phase containing 69:10:2:16:3 (v/v) of acetonitrile-methanol-n-butanol-2% ammonium acetate-glacial acetic acid. A total time of analysis of less than 30 min, including sample preparation, was achieved. Recoveries were in the range of 79-86%. The limit of detection was 1-7 ng/ml serum and the precision, in the concentration range 20-1000 ng/ml, was between 1.3 and 4.5% for all five compounds, The method was applied for the analysis of human serum after oral administration of 60 mg Roaccutan, The method is well suited for pharmacological studies, while the endogenous levels of some retinoic acid isomers are below the limit of quantitation.