by | Related Substance Determine by liquid chromatography; Use the normalization procedure. Carry out of the test as rapidly as possible, avoiding exposure to actinic light and air. Test Solution: Dissolve 1 mg of the substance to be examined without heating in 10ml of the mobile phase. Reference Solution (a): Dissolve 1 mg of Calcitriol CRS without heating in 10ml of the mobile phase. Reference Solution (b): Dilute 1 ml of reference solution (a) to 100 ml of the mobile phase. Dilute 1 ml of this solution to 10 ml with the mobile phase. Reference Solution (c): Heat 2ml of reference solution (a) at 800C for 30 minute. Column: Size: l = 0.25 m, Ø = 4.6 mmStationary phase: octylsilyl silica gel for chrmatograpyl R1 (5 µm),Temperature: 400C Mobile Phase mix 450 volumes of a 1 g/L solution of tris(hydroxymethyl)aminomethane R adjusted to pH 7.0 – 7.5 with phosphoric acid, and 550 volumes of acetonitrile. Flow Rate: 1.0 ml/min. Detection spectrophotometer at 230 nm. Injection 50 µl Run time: Twice the retention time of Calcitriol. Relative Retention: With reference to Calcitriol (retention time = about 14 min): impurity C = about 0.4; pre-calcitriol = about 0.88; impurity A = about 0.95; impurity B = about 1.1. |
| System Suitability: Resolution: minimum 3.5 between the peaks due to pre-calcitriol and Calcitriol in the chromatogram obtained with reference solution (c).Number of theoretical plates: minimum 10,000, calculated for the peak due to Calcitriol in the chromatogram obtained with reference solution (a). Limits: Impurity A, B, C for each impurity, maximum 0.5%.Unspecified impurity: for each impurity, maximum 0.1%.Total impurity: maximum 1.0%. Disregard limit: 0.5 times the area of the principle peak in the chromatogram obtained with reference solution (b) (0.05%); disregard the peak due to pre-calcitriol. |
