High Throughput LC-MS/MS Method for Simultaneous Estimation of 9-Cis- Retinoic Acid and its Metabolite 4-Oxo-9-Cis-Retinoic Acid in Human Plasma and its Application to a Bioequivalence Study

Abstract

A liquid chromatography mass-spectrometric method is described for the simultaneous estimation of 9-cisretinoic acid and 4-oxo-9-cis-retinoic acid in human plasma using 13-cis-retinoic acid-d5 as an internal standard. Chromatographic separation of these retinoids was achieved on Chromolith Performance RP18 (100 × 4.6 mm) column using binary flow program of two mobile phase. 9-cis-retinoic acid and 4-oxo-9-cis-retinoic acid were extracted from human plasma by liquid-liquid extraction with a mixture of ethyl acetate, n-hexane and iso propyl alcohol (30:65:5, v/v/v). Quantification was achieved by monitoring transitions of m/z 299.4→255.2 for 9-cisretinoic acid, m/z 313.4→269.3 for 4-oxo-9-cis-retinoic acid and 304.4→260.2 for isotretinoin-d5 in multiple reaction monitoring, using turbo ion source in negative polarity. The linearity was established over a dynamic range of 2.85- 450.51 ng/mL and 2.33-180.57 ng/mL for 9-cis-retinoic acid and 4-oxo-9-cis-retinoic acid, respectively. At the lower limit of quantification level, the intra-and inter-day precision values of 9-cis-retinoic acid were within 5.9% and 6.0%, respectively, whereas that of 4-oxo-9-cis-retinoic acid were within 13.8% and 11.3%, respectively. The method was successfully applied for the simultaneous estimation of 9-cis-retinoic acid and 4-oxo-9-cis-retinoic acid in healthy volunteers, after oral administration of 30 mg alitretinoin capsule for bioequivalence study.