Free methylprednisolone—
Using the chromatograms obtained in the
Assay, measure the areas of the peaks from the internal standard and free methylprednisolone. Calculate the ratio of the area of the free methylprednisolone peak to that of the internal standard in the chromatogram obtained from the
Standard preparation,
SS, and the same ratio in the chromatogram obtained from the
Assay preparation,
SU. Calculate the quantity, in mg, of free methylprednisolone in the
Assay preparation taken by the formula:
100C(SU / SS),
in which
C is the concentration, in mg per mL, of
USP Methylprednisolone RS in the
Standard preparation; and
SU and
SS are the ratios as defined above. The amount of free methylprednisolone is not more than 6.6% of the labeled amount of methylprednisolone.
Assay—
Internal standard solution—
Prepare a solution of
USP Fluorometholone RS in tetrahydrofuran containing about 3 mg per mL.
Mobile phase—
Prepare a filtered mixture of butyl chloride, water-saturated butyl chloride, tetrahydrofuran, methanol, and glacial acetic acid (95:95:14:7:6). Make adjustments if necessary (see
System Suitability under
Chromatography 
621
).
Standard preparation—
Weigh accurately about 32.5 mg of
USP Methylprednisolone Hemisuccinate RS, and transfer it to a 50-mL volumetric flask. Add by pipet 5.0 mL of
Internal standard solution and 5.0 mL of a solution of glacial acetic acid in chloroform (3 in 100) containing in each mL an accurately known quantity of about 0.30 mg of
USP Methylprednisolone RS. Dilute with glacial acetic acid in chloroform (3 in 100) to volume, and mix.
Assay preparation—
Mix the constituted solutions prepared from the contents of 10 vials of Methylprednisolone Sodium Succinate for Injection. Transfer an accurately measured volume of the resulting constituted solution, equivalent to about 50 mg of methylprednisolone, to a suitable flask containing 10.0 mL of Internal standard solution, and dilute with glacial acetic acid in chloroform (3 in 100) to 100.0 mL. Shake thoroughly for 5 minutes, then allow the phases to separate, discarding the upper phase.
Chromatographic system
(see
Chromatography 621)—The liquid chromatograph is equipped with a 254-nm detector and a 3.9-mm × 30-cm column that contains packing L3. The flow rate is about 1.0 mL per minute. Chromatograph the
Standard preparation, and record the peak responses as directed for
Procedure: the order of elution of peaks is the internal standard peak, methylprednisolone hemisuccinate peak, and successive smaller peaks of free methylprednisolone and methylprednisolone 17-hemisuccinate.
Procedure—
Separately inject equal volumes (about 6 µL) of the
Standard preparation and the
Assay preparation into the chromatograph, record the chromatograms, and measure the peak areas for the internal standard, methylprednisolone hemisuccinate, and methylprednisolone 17-hemisuccinate. Calculate the quantity, in mg, of methylprednisolone (C
22H
30O
5) in the portion of constituted solution taken by the formula:
0.789(100C)(RU / RS),
in which 0.789 is the ratio of the molecular weight of methylprednisolone to that of methylprednisolone hemisuccinate;
C is the concentration, in mg per mL, of
USP Methylprednisolone Hemisuccinate RS in the
Standard preparation; and
RU and
RS are the ratios of the sum of the peak areas for methylprednisolone hemisuccinate and methylprednisolone 17-hemisuccinate to the peak area of the internal standard obtained from the
Standard preparation and the
Assay preparation, respectively. To this quantity add the amount, in mg, of free methylprednisolone found in the test for
Free methylprednisolone.
for 3 hours: it loses not more than 2.0% of its weight.