A validated high-performance thin-layer chromatographic method for the simultaneous separation of 13-cis-retinoic acid (isotretinoin) and all-trans-retinoic acid (tretinoin)

Abstract

A new, simple, and rapid normal-phase high-performance thin-layer chromatography method was developed for the simultaneous separation of retinoic acid isomers (tretinoin and isotretinoin). The isomers were baseline-resolved on aluminum-backed silica gel 60 F254 layer using toluene-ethyl acetate-methanol (8:1:0.5, v/v) as the mobile phase. Quantification was achieved with ultraviolet (UV) detection at 334 nm. The developed analytical method was validated according to International Conference on Harmonization guidelines in terms of parameters like accuracy, precision, linearity, and specificity. Good linearity was observed in the concentrations ranging from 30 to 70 ng band−1 for both the isomers. Recovery study results ranged from 95 to 102% for both tretinoin and isotretinoin. The final optimized methodology was successfully applied to separate the isomers and was proven to be reproducible and accurate for its quantitative determination.