Development and validation of a reverse phase-liquid chromatographic method for the estimation of butylated hydroxytoluene as antioxidant in paricalcitol hard gelatin capsule formulation dosage form

Introduction: A novel and simple isocratic reverse phase liquid chromatographic (RP-LC) method was developed for the quantitative determination of antioxidant-butylated hydroxy toluene (BHT) in paricalcitol hard gelatin capsule. In the paricalcitol capsule BHT concentration is very low. This method is precisely able to estimate BHT at low concentration at about 0.0039 μg/mL and to separate BHT from paricalcitol main compound and other oil-based excipients. Materials and Methods: The method was developed by using ACE-C18 (250 × 4.6 mm) 5-μm column with mobile phase containing a mixture of solvent A (water) and solvent B (methanol) in the ratio of 5:95 v/v, respectively. The flow rate was 0.8 mL/min with column temperature of 45 °C and detection wavelength at 277 nm. The developed method was validated as per ICH guidelines with respect to specificity, linearity, limit of detection, limit of quantification, accuracy, precision and robustness. Results: In the precision study the % RSD for the result of BHT was below 1.5% at target concentration level. The limit of detection, limit of quantification are 0.0013 μg/mL and 0.0039 μg/mL, respectively and précision at TOQ level (0.0039 μg/mL) was with 6.2% RSD. The method was linear with concentration rage of 0.0039-0.64 μg/mL with the correlation coefficient greater than 0.999 and % bias at 100% level are within + 2%. The percentage recoveries for BHT were calculated observed from 98.8 to 104.8%. Conclusion: The developed method was found to be precise, accurate, linear, selective and robust.