by | Assay Cholecalciferol contains not less than 97.0 per cent and not more than 103.0 per cent of C27H44O. Carry out the following procedure as rapidly as possible in subdued light and protected from air. Determine by liquid chromatography. Test solution. Weigh accurately about 50.0 mg of the substance under examination, dissolve in 10 ml of toluene without heating and dilute to 100.0 ml with the mobile phase; dilute 5.0 ml of this solution to 50.0 ml with the mobile phase; further dilute 5.0 ml of this solution to 50.0 ml with the mobile phase. Reference solution (a). Dissolve 50.0 mg of Cholecalciferol RS in 10 ml of toluene without heating and dilute to 100.0 ml with the mobile phase; dilute 5.0 ml of this solution to 50.0 ml with the mobile phase (Solution A); further dilute 5.0 ml of solution A to 50.0 ml with the mobile phase. Reference solution (b). Reflux 5.0 ml of solution A, under nitrogen, on a water-bath for 60 minutes to obtain a solution of Cholecalciferol, precholecalciferol and trans-Cholecalciferol. Chromatographic system – a stainless steel column 25 cm × 4.6 mm, packed with porous silica particles (5 μm) (such as Nucleosil 50-S 5 μm), – mobile phase: a mixture of 997 volumes of hexane and 3 volumes of 1-pentanol, – flow rate. 2 ml per minute, – spectrophotometer set at 254 nm, – a 20 μl loop injector. Inject reference solution (b) and record the chromatogram adjusting the sensitivity so that the height of the peak due to Cholecalciferol is more than 50 per cent of full-scale deflection. The approximate relative retention times calculated with reference to Cholecalciferol are 0.4 for pre cholecalciferol and 0.5 for trans-Cholecalciferol. The resolution between pre cholecalciferol and trans-Cholecalciferol should be not less than 1.0; if necessary adjust the proportions of the constituents and flow rate of the mobile phase to obtain the required resolution. Inject reference solution (a) and record the chromatogram adjusting the sensitivity so that the height of the peak due to Cholecalciferol is more than 50 per cent of full-scale deflection. |
