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» Cyclosporine contains not less than 98.5 percent and not more than 101.5 percent of cyclosporine A (C62H111N11O12), calculated on the dried basis.
Packaging and storage Preserve in tight, light-resistant containers.
USP Reference standards 11
USP Cyclosporine Resolution Mixture RS
This material is a 100:1 mixture of cyclosporine and cyclosporine U.
Identification The chromatogram of the Assay preparation obtained as directed in the Assay exhibits a major peak for cyclosporine, the retention time of which corresponds to that exhibited in the chromatogram of the Standard preparation, as obtained in the Assay.
Loss on drying 731 Dry about 100 mg, accurately weighed, in a capillary-stoppered bottle in vacuum at a pressure not exceeding 5 mm of mercury at 60 for 3 hours: it loses not more than 2.0% of its weight.
Heavy metals, Method II 231: 0.002%.
Related compounds Using the chromatograms obtained from Standard preparation 2 and the Assay preparation in the Assay, calculate the percentage of each impurity by the formula:
2000(C / W)(ri / rS2)in which C is the concentration, in mg per mL, of USP Cyclosporine RS in Standard preparation 2; W is the weight, in mg, of Cyclosporine taken to prepare the Assay preparation; ri is the response of an individual impurity observed in the chromatogram of the Assay preparation; and rS2 is the response of the main cyclosporine peak in the chromatogram obtained from Standard preparation 2: not more than 0.7% of any individual impurity is found, and the sum of all such impurities is not more than 1.5%, any impurities corresponding to less than 0.05% being disregarded.
Mobile phase Prepare a mixture of water, acetonitrile, tert-butyl methyl ether, and phosphoric acid (520:430:50:1). Make adjustments if necessary (see System Suitability under Chromatography 621).
Diluent Prepare a mixture of acetonitrile and water (1:1).
Standard preparation 1 Dissolve an accurately weighed quantity of USP Cyclosporine RS in Diluent to obtain a solution having a known concentration of about 1.25 mg per mL.
Standard preparation 2 Transfer 2.0 mL of Standard preparation 1 to a 250-mL volumetric flask, dilute with Diluent to volume, and mix. This solution contains about 0.01 mg of USP Cyclosporine RS per mL.
Assay preparation Dissolve about 25 mg of Cyclosporine, accurately weighed, in Diluent, dilute with Diluent to 20.0 mL, and mix.
Resolution solution Prepare a solution of USP Cyclosporine Resolution Mixture RS in Diluent having a concentration of about 1.25 mg per mL.
Chromatographic system (see Chromatography 621) The liquid chromatograph is equipped with a 210-nm detector, a 0.25-mm × 1-m stainless steel tube connected to a 4-mm × 25-cm column that contains 3- to 5-µm packing L1. The tube and column are maintained at 80. The flow rate is about 1.2 mL per minute. Chromatograph the Resolution solution, and record the responses as directed for Procedure: the cyclosporine U peak and the main cyclosporine peak are resolved from each other. Chromatograph Standard preparation 1, and record the responses as directed for Procedure: the relative standard deviation for replicate injections is not more than 1.0%. Chromatograph Standard preparation 2, and record the responses as directed for Procedure: the relative standard deviation for replicate injections is not more than 10%.
Procedure [noteUse peak areas where peak responses are indicated. ] Separately inject equal volumes (about 20 µL) of Standard preparation 1, Standard preparation 2, and the Assay preparation into the chromatograph, record the chromatograms, and measure the peak responses. Calculate the percentage of cyclosporine A (C62H111N11O12) in the Cyclosporine taken by the formula:
(CP/10U)(rU / rS)in which C is the concentration, in mg per mL, of USP Cyclosporine RS in Standard preparation 1; P is the specified purity, in µg per mg, of USP Cyclosporine RS; U is the concentration, in mg per mL, of specimen in the Assay preparation; and rU and rS are the main cyclosporine peak responses obtained from the Assay preparation and Standard preparation 1, respectively.
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USP35NF30 Page 2793Pharmacopeial Forum: Volume No. 27(2) Page 2135