6 mm, 5 ��m) column with mobile phase containing a mixture of sol

6 mm, 5 ��m) column with mobile phase containing a mixture of solvent A (water) and Solvent B (Methanol) selleckbio in the ratio of 50:950 v/v respectively. Isocratic method was used with runtime of 25 minute for sample and 12 minute for standard Solution. The mobile phases were filtered through nylon 0.45-��m membrane filters and degassed in sonicator. The flow rate of the mobile phase was 0.8 mL/min. The column temperature was maintained at 45��C and the eluted compounds were monitored at the wavelength of 277 nm. The injection volume was 500 ��l. Preparation of standard solution A standard stock solution (Stock A) of BHT were prepared in diluent-1 (water:Acetonitrile, 1:9) with a concentration of 0.2 mg/mL. Working standard solution was prepared from above stock solution (stock A) by further dilution with diluent-2 (water: Acetonitrile, 3:7) to get final concentration of 0.

32 ��g/mL of BHT. Preparation of sample solution Five capsules of paricalcitol was taken and cut the each capsule shell and transferred accurately the whole capsules with content in to a 50-mL volumetric flask. Added 25 mL of diluent-1 (water:Acetonitrile,1:9) and sonicate for about 20 minutes in the sonicator with vigorous intermediate shaking. Allow the flask to stand at room temperature and make the volume up to the mark with diluent-1(water:Acetonitrile, 1:9) and mix well. Further diluted with diluent-2 (water: Acetonitrile, 3:7) to get the concentration of BHT as 0.32 ppm.

RESULTS AND DISCUSSION Method development and optimization As BHT concentration is very low, the main objective was to develop a method for detection of BHT and the chromatographic method should able to separate critical closely eluting compounds from BHT, with a shorter run time. The blend containing 0.32 mcg/mL of BHT was used for method optimization. An isocratic method employed using Milli-Q water and methanol in the ratio of 20:80 as mobile phase, Alltima C18 (250 �� 4.6 mm) 5-��m column with flow rate of 2.0 mL/min on HPLC equipped with photo diode array detector.60% acetonitrile was used as diluent. BHT peak was merged with excipient peak. To resolve the peak an attempt were made with different ratio of water and methanol in mobile phase by changing Ace C18, 250 �� 4.6 mm, 5-��m column. BHT peak was very well resolved but the recovery found in lower side.

Therefore to achieve satisfactory recovery, different experiments were conducted in various diluents. Recovery was increased by changing the diluent and sonication time but peak shape was not found symmetrical. Further experiment were conducted by different GSK-3 diluent ratio of water and acetonitrile and decided to keep two diluent, first diluent for extraction of BHT from paricalcitol capsule where organic solvent ratio is more and second diluent to get symmetrical peak shape. Based on these experiments, the conditions were further optimized as described below.

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