USP 36 Official Monographs / Hydrocortisone 3843

Pipet 10 mL of this solution into a separator, wash the solu- . tion with two 25-mL portions of methylene chloride, and Hydrocortisone Sodium Phosphate discard the washings. Transfer the aqueous layer to a Injection 100-mL volumetric flask, dilute with water to volume, and mix. » Hydrocortisone Sodium Phosphate Injection is a Procedure—Pipet 2 mL each of the Standard preparation sterile, buffered solution of Hydrocortisone So- and the Assay preparation into separate glass-stoppered, dium Phosphate in Water for Injection. It con- 50-mL conical flasks. To each flask, and to a similar flask containing 2.0 mL of water to provide a blank, add 10.0 mL tains the equivalent of not less than 90.0 percent of Phenylhydrazine hydrochloride solution, and mix. Place the and not more than 115.0 percent of the labeled flasks in a water bath maintained at a temperature of 60° amount of hydrocortisone (C21H30O5). for 2 hours, then cool the solutions to room temperature. Concomitantly determine the absorbances of the solutions Packaging and storage—Preserve in single-dose or multi- from the Assay preparation and the Standard preparation at ple-dose containers, preferably of Type I glass. the wavelength of maximum absorbance at about 410 nm, USP Reference standards 〈11〉— with a suitable spectrophotometer, using the blank to set USP Endotoxin RS the instrument. Calculate the quantity, in mg, of C21H30O5 USP Hydrocortisone RS in each mL of the Injection taken by the formula: USP Hydrocortisone Phosphate Triethylamine RS 0.667(C / V)(AU / AS) C21H31O8P · C6H15N 543.64 Identification— in which 0.667 is the ratio of the molecular weight of hy- A: Ultraviolet Absorption 〈197U〉— drocortisone to that of hydrocortisone phosphate triethyl- Solution: 20 µg per mL, USP Hydrocortisone Phosphate amine; C is the concentration, in µg per mL, of USP Hydro- Triethylamine RS being used to prepare the Standard solu- cortisone Phosphate Triethylamine RS in the Standard tion. preparation; V is the volume, in mL, of Injection taken; and Medium: water. AU and AS are the absorbances of the solutions from the B: Place 5 mL of the Assay preparation, obtained as di- Assay preparation and the Standard preparation, respectively. rected in the Assay, in a glass-stoppered, 50-mL tube, and add 5 mL of a solution prepared by dissolving 50 mg of

alkaline phosphatase enzyme in 50 mL of pH 9 buffer with . magnesium prepared as directed in the Assay under Hydro- cortisone Sodium Phosphate. Allow to stand at room temper- Hydrocortisone Sodium Succinate ature for 2 hours, with occasional mixing, and extract with 25 mL of methylene chloride. Evaporate 15 mL of the meth- C25H33NaO8 484.51 ylene chloride extract on a steam bath to dryness, and dis- Pregn-4-ene-3,20-dione, 21-(3-carboxy-1-oxopropoxy)- solve the residue in 0.5 mL of methylene chloride. Apply 11,17-dihydroxy-, monosodium salt, (11β)-; 5 µL of this solution and 5 µL of a solution of USP Hydrocor- Cortisol 21-(sodium succinate) [125-04-2]. tisone RS in methylene chloride containing 300 µg per mL DEFINITION to a thin-layer chromatographic plate (see Chromatography Hydrocortisone Sodium Succinate contains NLT 97.0% and 〈621〉) coated with a 0.25-mm layer of chromatographic sil- NMT 102.0% of total steroids, calculated as C25H33NaO8, ica gel mixture. Allow the spots to dry, and develop the on the dried basis. chromatogram in a tank completely lined with filter paper, using a solvent system consisting of a mixture of 50 parts of IDENTIFICATION chloroform, 50 parts of acetone, and 1 part of water, until • A. INFRARED ABSORPTION the solvent front has moved about three-fourths of the Sample: Transfer 100 mg of Hydrocortisone Sodium Suc- length of the plate. Remove the plate, mark the solvent cinate to a suitable container, and dissolve in 10 mL of front, and dry. Spray the plate with dilute sulfuric acid (1 in water. In rapid succession, add 1 mL of 3 N hydrochlo- 2), and heat at 105° until brown or black spots appear: the ric acid, shake briefly, immediately decant the aqueous RF value of the principal spot obtained from the solution layer, and wash the precipitate with two additional under test corresponds to that obtained from the Standard 10-mL portions of water, each time removing the water solution. by decanting. Remove as much of the water as possi- Bacterial endotoxins 〈85〉—It contains not more than ble, spread the precipitate in a suitable container, and 1.25 USP Endotoxin Units per mg of hydrocortisone. dry under vacuum at 60° for 3 h. pH 〈791〉: between 7.5 and 8.5. Acceptance criteria: The IR spectrum of a mineral oil Particulate matter 〈788〉: meets the requirements for dispersion of the precipitate so obtained exhibits max- small-volume injections. ima only at the same wavelengths as that of a similar preparation of USP Hydrocortisone Hemisuccinate RS. Other requirements—It meets the requirements under In- • B. ULTRAVIOLET ABSORPTION 〈197U〉 jections 〈1〉. Sample solution: 20 µg/mL in methanol Assay— Analytical wavelength: 242 nm Phenylhydrazine hydrochloride solution—Dissolve 65 mg of Acceptance criteria: Absorptivities, calculated on the phenylhydrazine hydrochloride in 100 mL of dilute sulfuric dried basis, do not differ by more than 3.0%. acid (3 in 5), add 50 mL of isopropyl alcohol, and mix. Pre- • C. IDENTIFICATION TESTS—GENERAL, Sodium 〈191〉: It meets pare this solution fresh daily. the requirements of the flame test. Standard preparation—Dissolve a suitable quantity of USP ASSAY Hydrocortisone Phosphate Triethylamine RS, accurately • ASSAY FOR STEROIDS 〈351〉 weighed, in water, and dilute quantitatively and stepwise Blue tetrazolium solution: 5 mg/mL of blue tetrazolium with water to obtain a solution having a known concentra- in alcohol tion of about 110 µg per mL. Tetramethylammonium hydroxide solu- Assay preparation—Pipet a volume of Injection, equivalent tion: Tetramethylammonium hydroxide TS in alcohol (1 to about 100 mg of hydrocortisone sodium phosphate, into in 10) a 100-mL volumetric flask, and dilute with water to volume. 3844 Hydrocortisone / Official Monographs USP 36

Standard preparation: Prepare as directed in the chap- gle-compartment containers, or in the volume of solution ter for the Standard Preparation, using USP Hydrocorti- designated on the label of containers that are constructed sone Hemisuccinate RS, but dilute the solution with al- to hold, in separate compartments, the Hydrocortisone cohol to a concentration of 12.5 µg/mL. Sodium Succinate for Injection and a solvent. Assay preparation: 12.5 µg/mL of Hydrocortisone So- dium Succinate in alcohol IDENTIFICATION Blank solution: Alcohol • A. INFRARED ABSORPTION Analysis Sample: Transfer a quantity of Hydrocortisone Sodium Samples: Standard preparation, Assay preparation, and Succinate for Injection, equivalent to 100 mg of hydro- Blank solution cortisone sodium succinate, to a suitable container, and Transfer 20.0-mL aliquots of the Samples to separate dissolve in 10 mL of water. In rapid succession, add glass-stoppered, 50-mL conical flasks. To each flask 1 mL of 3 N hydrochloric acid, shake briefly, immedi- add 2.0 mL of Blue tetrazolium solution, mix, and add ately decant the aqueous layer, and wash the precipi- 4.0 mL of Tetramethylammonium hydroxide solution. Al- tate with two additional 10-mL portions of water, each low to stand in the dark for 90 min, add 1.0 mL of time removing the water by decanting. Remove as glacial acetic acid, and proceed as directed in the Pro- much of the water as possible, spread the precipitate in cedure, beginning with “Concomitantly determine the a suitable container, and dry under vacuum at 60° for absorbances…”. 3 h. Calculate the percentage of total steroids, as C25H33NaO8, Acceptance criteria: The IR spectrum of a mineral oil in the portion of Hydrocortisone Sodium Succinate dispersion of the precipitate so obtained exhibits max- taken: ima only at the same wavelengths as that of a similar preparation of USP Hydrocortisone Hemisuccinate RS. Result = (AU/AS) × (CS/CU) × (Mr1/Mr2) × 100 ASSAY

AU = absorbance of the Assay preparation • PROCEDURE AS = absorbance of the Standard preparation Mobile phase: Butyl chloride, water-saturated butyl CS = concentration of USP Hydrocortisone chloride, tetrahydrofuran, methanol, and glacial acetic Hemisuccinate RS in the Standard preparation acid (95:95:14:7:6) (µg/mL) Internal standard solution: 3 mg/mL of USP CU = concentration of hydrocortisone sodium Fluorometholone RS in tetrahydrofuran succinate in the Assay preparation (µg/mL) Diluent: Glacial acetic acid in chloroform (3 in 100) Mr1 = molecular weight of hydrocortisone sodium Solution A: 0.30 mg/mL of USP Hydrocortisone RS in succinate, 484.51 Diluent Mr2 = molecular weight of hydrocortisone Standard solution: 0.65 mg/mL of USP Hydrocortisone hemisuccinate, 462.53 Hemisuccinate RS, prepared as follows. Transfer Acceptance criteria: 97.0%–102.0% on the dried basis 32.5 mg of USP Hydrocortisone Hemisuccinate RS to a 50-mL volumetric flask. Add by pipet 5.0 mL of Internal SPECIFIC TESTS standard solution and 5.0 mL of Solution A. Dilute with • SODIUM CONTENT Diluent to volume. Sample: 1 g Sample solution: Mix the constituted solutions prepared Analysis: Dissolve the Sample, with gentle heating, in from the contents of 10 vials of Hydrocortisone Sodium 75 mL of glacial acetic acid. Add 20 mL of dioxane, Succinate for Injection. Transfer a volume, equivalent to then add crystal violet TS, and titrate with 0.1 N per- 50 mg of hydrocortisone from the resulting constituted chloric acid VS. Each mL of 0.1 N perchloric acid is solution, to a suitable flask containing 10.0 mL of Inter- equivalent to 2.299 mg of sodium (Na). nal standard solution, and dilute with Diluent to Acceptance criteria: 4.60%–4.84% on the dried basis 100.0 mL. Shake thoroughly for 5 min, then allow the • OPTICAL ROTATION, Specific Rotation 〈781S〉 phases to separate, discarding the upper phase. Sample solution: 10 mg/mL in alcohol Chromatographic system Acceptance criteria: +140° to +150° (See Chromatography 〈621〉, System Suitability.) • LOSS ON DRYING 〈731〉 Mode: LC Sample: Dry at 105° for 3 h. Detector: UV 254 nm Acceptance criteria: NMT 2.0% Column: 3.9-mm × 30-cm; packing L3 Flow rate: 1.0 mL/min ADDITIONAL REQUIREMENTS Injection volume: 6 µL • PACKAGING AND STORAGE: Preserve in tight, light-resistant System suitability containers. Sample: Standard solution • USP REFERENCE STANDARDS 〈11〉 Suitability requirements USP Hydrocortisone Hemisuccinate RS Resolution: NLT 2.0 between hydrocortisone hemisuc- cinate and the internal standard Relative standard deviation: NMT 2.0% Analysis . Samples: Standard solution and Sample solution Hydrocortisone Sodium Succinate for [NOTE—The order of elution of peaks is that from the Injection internal standard, hydrocortisone hemisuccinate, and successive smaller peaks representing free hydrocorti- DEFINITION sone and hydrocortisone 17-hemisuccinate, the rela- Hydrocortisone Sodium Succinate for Injection is a sterile tive retention times of which are about 1.0, 1.5, 2.0, mixture of Hydrocortisone Sodium Succinate and suitable and 2.5, respectively.] buffers. It may be prepared from Hydrocortisone Sodium Calculate the percentage of the labeled amount of hy- Succinate, or from Hydrocortisone Hemisuccinate with the drocortisone (C21H30O5) in the portion of Hydrocorti- aid of Sodium Hydroxide or Sodium Carbonate. It con- sone Sodium Succinate for Injection taken: tains the equivalent of NLT 90.0% and NMT 110.0% of Result = (RU/RS) (CS/CU) (Mr1/Mr2) 100 the labeled amount of hydrocortisone (C21H30O5) in sin- × × ×