Development and validation of a high-performance thin-layer chromatography assay for the analysis of tacrolimus ointments

A simple and rapid high-performance thin-layer chromatography (HPTLC) assay was developed and validated for the quantification of tacrolimus ointments on silica gel 60 F₂₅₄ HPTLC plates, using toluene–acetonitrile–ethyl acetate–glacial acetic acid (6:2:2:0.1, V/V) as the mobile phase and p-anisaldehyde‒sulphuric acid as the derivatising reagent. The analysis was performed at 366 nm. The method was fully validated according to the International Council for Harmonisation guidelines and found to be sensitive, precise, accurate and reproducible with tacrolimus presenting as a sharp band at R_F 0.20, good linear regression between 20 and 140 ng/band (r² = 0.9989) and sensitivity levels in terms of limit of detection and limit of quantification of 5.68 ng and 17.20 ng, respectively. The method is therefore deemed suitable for the routine quality control analysis of tacrolimus ointments.