Hydrocortisone

This article is compiled based on the United States Pharmacopeia (USP) – 2025 Edition

Issued and maintained by the United States Pharmacopeial Convention (USP)

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C₂₁H₃₀O₅ 362.46 

Pregn-4-ene-3,20-dione, 11,17,21-trihydroxy-, (11β)-; 

Cortisol CAS RN®: 50-23-7; UNII: WI4X0X7BPJ. 

1 DEFINITION 

Hydrocortisone contains NLT 97.0% and NMT 102.0% of C H O , calculated on the dried basis. 

2 IDENTIFICATION 

Change to read: 

Spectroscopic Identification Tests 〈197〉, Infrared Spectroscopy: 197M

Change to read: 

Spectroscopic Identification Tests 〈197〉, Ultraviolet-Visible Spectroscopy: 197U

Analytical wavelength: 242 nm 

Sample solution: 10 µg/mL in methanol 

Acceptance criteria: Absorptivities, calculated on the dried basis, do not differ by more than 2.5%. 

3 ASSAY 

Procedure 

Mobile phase: Acetonitrile, methanol, and water (25:25:50) 

Diluent: Methanol and water (1:1) 

Internal standard solution: 1 mg/mL of Propylparaben in methanol 

Standard stock solution: 1 mg/mL of USP Hydrocortisone RS in methanol 

Standard solution: 2.0 mL of Standard stock solution and 2.0 mL of Internal standard solution. Dilute with Diluent to 50 mL. Sample stock solution: 1 mg/mL of Hydrocortisone in methanol 

Sample solution: 2.0 mL of Sample stock solution and 2.0 mL of Internal standard solution. Dilute with Diluent to 50 mL. Chromatographic system 

(See Chromatography 〈621〉, System Suitability.) 

Mode: LC 

Detector: UV 254 nm 

Column: 4.6-mm × 15-cm; 5-µm packing L1 

Flow rate: 1 mL/min 

Injection size: 10 µL 

System suitability 

Sample: Standard solution 

[Note—The relative retention times for hydrocortisone and propylparaben are about 1.0 and 1.8, respectively.] Suitability requirements 

Resolution: NLT 9.0 between the hydrocortisone and propylparaben peaks 

Column efficiency: NLT 3000 theoretical plates for hydrocortisone 

Tailing factor: NMT 1.2 

Relative standard deviation: NMT 2.0% 

Analysis 

Samples: Standard solution and Sample solution 

Calculate the percentage of hydrocortisone (C₂₁H₃₀O₅) in the portion of Hydrocortisone taken:  

Result = (R_U/R_S ) × (C_S /C_U) × 100 

R_U = peak response ratio of hydrocortisone to the internal standard from the Sample solution

R_S = peak response ratio of hydrocortisone to the internal standard from the Standard solution

C_S = concentration of USP Hydrocortisone RS in the Standard solution (mg/mL) 

C_U = nominal concentration of hydrocortisone in the Sample solution (mg/mL) 

Acceptance criteria: 97.0%–102.0% on the dried basis 

4 IMPURITIES 

Residue on Ignition 〈281〉 

Sample: 100 mg 

Acceptance criteria: NMT 0.5% 

Organic Impurities 

Mobile phase: Butyl chloride, tetrahydrofuran, methanol, glacial acetic acid, and water (890:56:28:24:0.4) 

Diluent: Butyl chloride, tetrahydrofuran, methanol, and glacial acetic acid (81.5:10:8:0.5) 

Standard solution: 40 µg/mL of USP Hydrocortisone RS in Diluent. [Note—Sonicate for 5 min.] 

Sample solution: 2 mg/mL of Hydrocortisone in Diluent. [Note—Sonicate for 5 min.] 

Chromatographic system 

(See Chromatography 〈621〉, System Suitability.) 

Mode: LC 

Detector: UV 254 nm 

Column: 4.6-mm × 15-cm; 3-µm packing L3 

Flow rate: 1.5 mL/min 

Injection size: 5 µL 

System suitability 

Sample: Standard solution 

Suitability requirements 

Tailing factor: NMT 2.0 

Relative standard deviation: NMT 5% 

Analysis 

Samples: Standard solution, Sample solution, and Diluent. [Note—Ignore artifact peaks.] 

Calculate the percentage of impurities in the portion of Hydrocortisone taken: 

Result = (r_U/r_S ) × (C_S /C_U) × 100 

r_U = = individual impurity peak area from the Sample solution 

r_S = peak area from the Standard solution 

C_S = concentration of USP Hydrocortisone RS in the Standard solution (mg/mL) 

C_U = concentration of hydrocortisone in the Sample solution (mg/mL) 

Acceptance criteria 

Individual impurities: NMT 0.5% 

Total impurities: NMT 2.0% 

5 SPECIFIC TESTS 

Optical Rotation, Specific Rotation〈781S〉 

Sample solution: 10 mg/mL, in dioxane 

Acceptance criteria: +150° to +156° 

Loss on Drying 〈731〉: Dry a sample at 105° for 3 h: it loses NMT 1.0% of its weight. 

6 ADDITIONAL REQUIREMENTS 

Packaging and Storage: Preserve in well-closed containers. Store at controlled room temperature. 

USP Reference Standards 〈11〉 

USP Hydrocortisone RS