6 mm, 5 ��m) column with mobile phase containing a mixture of solvent A (water) and Solvent B (Methanol) in the ratio of 50:950 v/v respectively. The mobile phase was filtered through nylon 0.45-��m membrane filters and degassed in sonicator. Isocratic method was those used with runtime of 25 minute for sample and 12minute for standard. Water and acetonitrile in the ratio of 1:9 was used as diluent-1 and water: Acetonitrile in the ratio of 3:7 was used as diluent-2. The flow rate of the mobile phase was 0.8 mL/min. The column temperature was maintained at 45��C and the eluted compound was monitored at the wavelength of 277 nm. The injection volume was 500 ��L. Method validation The proposed method was validated as per ICH guideline.
[17] The following validation characteristics were addressed: system suitability, specificity, precision, limit of detection and quantification, linearity, range, accuracy, solution stability, mobile phase stability and robustness. System suitability System suitability was checked for the conformance of suitability and reproducibility of chromatographic system for analysis. The system suitability was evaluated on the basis of USP tailing factor and theoretical plates of BHT and relative standard deviation (RSD) of five injections of standard solution. System suitability was determined before sample analysis from five replicate injections of the standard solution containing 0.32 ��g/ mL BHT [Figure 2]. The acceptance criteria were % RSD should not be more than 2.0%, USP tailing factor should less than 2.0 and theoretical plate should be more than 3000 for BHT peak from standard solution.
All critical parameters tested met the acceptance criteria [Table 1]. Figure 2 Typical chromatograms of standard solution Table 1 System suitability test results Specificity A specificity study to establish the interference of placebo was conducted. Study was performed on Placebo (Placebo contains without BHT, with Paricalcitol and other excipients) in duplicate equivalent to about the weight of placebo present in portion of test preparation as per test method. Chromatograms of placebo had shown no peaks at the retention time of BHT, this indicates that the excipients used in the formulation do not have any interference in estimation of BHT in Paricalcitol capsules [Figure 3].
Figure 3 Typical chromatograms of placebo Precision The precision of method was verified by repeatability and intermediate precision at target concentration level (0.32 ��g/mL). Repeatability was checked by injecting six individual preparations of BHT in paricalcitol hard gelatin capsule as per test method [Figure 4]. %RSD of result for BHT was calculated. The intermediate precision of the method was also evaluated using different analyst and different instrument and performing the analysis on different days. Figure 4 Typical chromatograms of sample solution The % RSD for the result GSK-3 of BHT in repeatability study was within 1.