DETERMINATION OF ENDOGENOUS LEVELS OF 13-CIS-RETINOIC ACID (ISOTRETINOIN), ALL-TRANS-RETINOIC ACID (TRETINOIN) AND THEIR 4-OXO METABOLITES IN HUMAN AND ANIMAL PLASMA BY HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY WITH AUTOMATED COLUMN-SWITCHING AND ULTRAVIOLET DETECTION

A highly sensitive HPLC method with automated column switching was dev eloped for the simultaneous determination of endogenous levels of 13-c is-retinoic acid (isotretinoin), all-trans-retinoic acid (tretinoin) a nd their 4-oxo metabolites in plasma samples from man, Cynomolgus monk ey, rabbit, rat and mouse. Plasma (0.4 mi) was deproteinated by adding ethanol (1.5 mi) containing the internal standard acitretin. After ce ntrifugation, 1.4 mi of the supernatant were directly injected onto th e precolumn packed with LiChrospher 100 RP-18 (5 mu m) 1.25% ammonium acetate and acetic acid-ethanol (8:2, v/v) was used as mobile phase du ring injection and 1% ammonium acetate and 2% acetic acid-ethanol(102: 4, v/v) was added, on-line, to decrease the elution strength of the in jection solution. After backflush purging of the precolumn, the retain ed components were transferred to the analytical column in the backflu sh mode, separated by gradient elution and detected at 360 nm. Two cou pled Superspher 100 RP-18 endcapped columns (both 250X4 mm) were used for the separation, together with a mobile phase consisting of acetoni trile-water-10% ammonium acetate-acetic acid: (A) 600:300:60:10 (v/v/v /v), (B) 950:20:5:20 (v/v/v/v), and (C) 990:5:0:5 (v/v/v/v). The metho d was linear in the range 0.3-100 ng/ml, at least, with a quantificati on limit of 0.3 ng/ml. The mean recoveries from human plasma were 93.2 %-94.4% and the mean inter-assay precision was 2.8%-3.2% (range 0.3-10 0 ng/ml). Similar results were obtained for animal plasma. The analyte s were found to be stable in the plasma of all investigated species st ored at -20 degrees C for 4.3 months and at -80 degrees C for 9 months , at least. At this temperature, human plasma samples were even stable for 2 years. The method was successfully applied to more than 6000 hu man and 1000 animal plasma samples from clinical and toxicokinetic stu dies. Endogenous levels determined in control patients and pregnant wo men were similar to published data from volunteers. (C) 1997 Elsevier Science B.V.