Development and validation of HPTLC method for estimation of tacrolimus in formulations

A new, simple, and rapid high-performance thin-layer chromatographic method with a derivatization procedure was developed and validated for quantitative determination of tacrolimus. Tacrolimus was chromatographed on silica gel 60 F(254) TLC plate using toluene-acetonitrile-glacial acetic acid (6:4:0.1, by volume) as mobile phase. Tacrolimus was visualized using a derivatization reagent containing anisaldehyde-sulfuric acid in absolute alcohol and quantified by densitometric analysis in the reflectance mode at 675 nm. The method was found to give compact spots for the drug (R(f) = 0.40 +/- 0.03). The linear regression analysis data for the calibration plots showed good linear relationship with r(2) = .9989 in the concentration range 100-800 ng/spot. The method was validated for precision, recovery, repeatability, and robustness as per the International Conference on Harmonization guidelines. The minimum detectable amount was found to be 28.90 ng, whereas the limit of quantitation was found to be 97.04 ng. Statistical analysis of the data showed that the method is precise, accurate, reproducible, and selective for the analysis of tacrolimus. The method was successfully employed for the estimation of equilibrium solubility and quantification of tacrolimus as a bulk drug and in commercially available capsules and in-house developed self-microemulsifying formulations.