Determine by liquid chromatography;
NOTE-Carry out the assay as rapidly as possible, avoiding exposure to actinic light and air.
Test solution. Dissolve 1.0 mg of the substance under examination in 10.0 ml of the mobile phase.
Reference solution (a). A 0.01 per cent w/v solution of calcitriol RS in the mobile phase.
Reference solution (b). Dilute 1.0 ml of reference solution (a) to 100 ml with the mobile phase.
Reference solution (c). Keep 2 ml of reference solution (a) at 80° for 30 minutes.
-a stainless steel column 25 cm x 4.6 mm, packed with octadecylsilane bonded to porous silica (5 µm),
-column temperature: 40°,
-mobile phase: a mixture of 45 volumes of a solution containing 0.1 per cent w/v of tris(hydroxymethyl) aminomethane with the pH adjusted to 7.3 with orthophosphoric acid and 55 volumes of acetonitrile,
-flow rate: 1 ml per minute,
-spectrophotometer set at 230 nm,
-injection volume: 50 µl.
Inject reference solution (c). The relative retention times with reference to calcitriol for precalcitriol is about 0.9 and the resolution between the peaks due to calcitriol and pre-calcitriol is not less than 3.5. Inject reference solution (a) in replicate. The relative standard deviation is not more than 2.0 per cent and the column efficiency for calcitriol peak is not less than 10000 theoretical plates. Inject reference solution (a) and the test solution. Run the chromatogram for twice the retention time of calcitriol. Calculate the content of C27H4403.