Change to read:

Add the following:

A: Infrared Absorption 197K.

B: The retention time of the major peak in the chromatogram of the Assay preparation corresponds to that in the chromatogram of the Standard preparation, as obtained in the Assay.

Add the following:

Change to read:

Tris buffer solution, Mobile phase, System suitability solution, and Chromatographic system— Proceed as directed in the Assay.

USP31

Test solution— Prepare as directed for Assay preparation.

Procedure— Inject a volume (about 50 µL) ofUSP31 the Test solution into the chromatograph, record the chromatograms for at least two times the retention time of the calcitriol peak, identify the impurities listed in Table 1, and measure the peak responses. Calculate the percentage of any individual impurity in the portion of Calcitriol taken by the formula: in which ri is the peak response of any individual peak other than the main calcitriol peak and the pre-calcitriol peak; and rs is the sum of the responses of all the peaks: in addition to not exceeding the limits in Table 1, not more than 1.0% of total impurities is found. Disregard any peak less than 0.1%.

Change to read:

Tris buffer solution— Dissolve 1.0 g of tris(hydroxymethyl)aminomethane in 900 mL of water, adjust with phosphoric acid to a pH of 7.0 to 7.5, dilute with water to make 1000 mL, and mix.

Mobile phase— Prepare a filtered and degassed mixture of acetonitrile and Tris buffer solution (55:45). Make adjustments if necessary (see System Suitability under Chromatography 621).

Standard preparation— Transfer an accurately weighed quantity of USP Calcitriol RS to a suitable volumetric flask, dissolve first in acetonitrile (without heating), using 55% of the final volume, then dilute with Tris buffer solution to volume, and mix to obtain a solution having a known concentration of about 100 µg of calcitriol per mL. [note—Allow the solution to warm up to room temperature before diluting with Tris buffer solution to final volume.]USP31

System suitability solution— Heat 2.0 mL of the Standard preparation at 80 for 30 minutes.

Assay preparation— Transfer an accurately weighed quantity of Calcitriol to a suitable volumetric flask, dissolve first in acetonitrile (without heating), using 55% of the final volume, then dilute with Tris buffer solution to volume, and mix to obtain a solution having a known concentration of about 100 µg of calcitriol per mL. [note—Allow the solution to warm up to room temperature before diluting with Tris buffer solution to final volume.]USP31

Chromatographic system (see Chromatography 621)— The liquid chromatograph is equipped with a 230-nm detector and a 4.6-mm × 25-cm column that contains 5-µm packing L7. The flow rate is about 1 mL per minute. The column temperature is maintained at 40. Chromatograph the System suitability solution, and record the peak responses as directed for Procedure: the relative retention times are about 0.9 for pre-calcitriol and 1.0 for calcitriol; and the resolution, R, between pre-calcitriol and calcitriol is not less than 3.5. Chromatograph the Standard preparation, and record the peak responses as directed for Procedure: the column efficiency is not less than 10,000 theoretical plates; and the relative standard deviation for replicate injections is not more than 1.0%.

Procedure— Separately inject equal volumes (about 50 µL) of the Standard preparation and the Assay preparation into the chromatograph, record the chromatograms, and measure the responses for the calcitriol and pre-calcitriol peaks. Calculate the percentage of C27H44O3 in the portion of Calcitriol taken by the formula: in which CS and CU are the concentrations, in µg per mL, of calcitriol in the Standard preparation and the Assay preparation, respectively; and rU and rS are the sums of the calcitriol and pre-calcitriol peak responses obtained from the Assay preparation and the Standard preparation, respectively.