Determination of trimethylpsoralen in blood, ophthalmic fluids, and skin

Trimethylpsoralen (TMP) levels in the blood of vitiligo patients were determined through the use of a high-performance liquid chromatographic method. TMP was extracted from blood buffered at pH 9.0 with 95:5 (V/V) hexane-isopropanol mixture; evaporated to dryness, and reconstituted in 50 microliters of ethanol. A 10-microliters aliquot was injected into a Micropack MCH-10 column (Varian HPLC model #5000). The mobile phase consisted of a mixture of water and acetonitrile with a linear gradient. The retention time of TMP was 16.5 min. The calibration curve of the external standards was linear. Overall recovery of the internal stands was 75-92%, with the lower detection limit of TMP at 2 ng/ml. Peak blood levels as low as 140 ng/ml and as high as 800 ng/ml were obtained in vitiligo patients 1-2 hr following the oral administration of 30 mg of trioxsalen tablets (Paul B. Elder Co.). Blood TMP levels peaked consistently at 2 hr when patients were fasted for 8 hr prior to drug ingestion. These results are consistent with the clinical observation that maximum response due to phototherapy is obtained 1-2 hr after oral administration of the drug. Two hours after oral drug administration, TMP levels in the epidermis, dermis, and whole skin of the guinea pig (in ng per g tissue) were: epidermis, 226 +/- 15; dermis, 25 +/- 6; and whole skin 176 +/- 12. Also detected were TMP levels 244 +/- 17 ng/ml in aqueous humor and 63 +/- 6 mg/ml in vitreous humor. These results point to the fact that the eyes of patients must be protected from overexposure to sunlight after psoralen-ultraviolet treatment.