A: Dilute with methanol, if necessary, a volume of Injection to obtain a solution containing 500 µg per mL. Apply 20 µL of this solution and 20 µL of a solution of USP Morphine Sulfate RS in a mixture of methanol and water (1:1) containing 500 µg per mL to a suitable thin-layer chromatographic plate (see Chromatography 621) coated with a 250-µm layer of chromatographic silica gel mixture. Allow the spots to dry, and develop the chromatogram in a solvent system consisting of a mixture of acetone, methanol, and ammonium hydroxide (50:50:1) until the solvent front has moved about three-fourths of the length of the plate. Remove the plate from the developing chamber, mark the solvent front, and allow the solvent to evaporate. Locate the spots on the plate by examination under short-wavelength UV light: the RF value of the principal spot obtained from the Injection corresponds to that obtained from the Standard solution.

B: It responds to the barium chloride test for Sulfate 191.

Mobile phase , Standard preparation, System suitability preparation, and Chromatographic system—Prepare as directed in the Assay under Morphine Sulfate.

Assay preparation— Transfer an accurately measured volume of Injection, equivalent to about 24 mg of morphine sulfate, to a 100-mL volumetric flask, dilute with Mobile phase to volume, and mix.

Procedure— Proceed as directed for Procedure in the Assay under Morphine Sulfate. Calculate the quantity, in mg, of morphine sulfate pentahydrate [(C17H19NO3)2·H2SO4·5H2O] in each mL of the Injection taken by the formula: in which 758.85 and 668.77 are the molecular weights of morphine sulfate pentahydrate and anhydrous morphine sulfate, respectively, V is the volume, in mL, of Injection taken, and the other terms are as defined therein.