(100C)(SU / SS)

Internal standard solution and Mobile phase—Prepare as directed in the Assay under Hydrocortisone Hemisuccinate.

Standard preparation— Weigh accurately about 32.5 mg of USP Hydrocortisone 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 Hydrocortisone 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 Hydrocortisone Sodium Succinate for Injection. Transfer an accurately measured volume of the resulting constituted solution, equivalent to about 50 mg of hydrocortisone, 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.

Procedure— Proceed as directed for Procedure in the Assay under Hydrocortisone Hemisuccinate. The order of elution of peaks is that from the internal standard, hydrocortisone hemisuccinate, and successive smaller peaks representing free hydrocortisone and hydrocortisone 17-hemisuccinate, whose relative retention times are about 1.0, 1.5, 2.0, and 2.5, respectively. Measure the areas of the peaks from the internal standard, hydrocortisone hemisuccinate, and hydrocortisone 17-hemisuccinate. Calculate the ratio of the summation of the areas of the hydrocortisone hemisuccinate and hydrocortisone 17-hemisuccinate peaks to that of the internal standard in the chromatogram obtained from the Standard preparation, RS, and the same ratio in the chromatogram obtained from the Assay preparation, RU. Calculate the quantity, in mg, of hydrocortisone (C21H30O5) in the portion of constituted solution taken by the formula: in which 0.784 is the ratio of the molecular weight of hydrocortisone to that of hydrocortisone hemisuccinate; C is the concentration, in mg per mL, of USP Hydrocortisone Hemisuccinate RS in the Standard preparation; and RU and RS are the ratios as defined above, and to this quantity add the amount, in mg, of free hydrocortisone found in the test for Free hydrocortisone.