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.
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.
Assay—
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.
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—
[NOTE—Use 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 (C
62H
111N
11O
12) 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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-aminobutyryl-N-methylglycyl-N-methyl-L-leucyl-L-valyl-N-methyl-L-leucyl)