Cephalexin Hydrochloride

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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C₁₆H₁₇N₃O₄S.HCl. H₂O 401.87 

5-Thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid, 7-[(aminophenylacetyl)amino]-3-methyl-8-oxo-, monohydrochloride, monohydrate, [6R- [6α,7β (R*)]]-; 

(6R,7R)-7-[(2R)-2-Amino-2-phenylacetamido]-3-methyl-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid, monohydrochloride, monohydrate; 

7-(d-2-Amino-2-phenylacetamido)-3-methyl-3-cephem-4-carboxylic acid hydrochloride monohydrate CAS RN®: 105879-42-3; UNII:  6VJE5G3D98. 

1 DEFINITION 

Cephalexin Hydrochloride contains the equivalent of NLT 800 µg/mg and NMT 880 µg/mg of cephalexin (C₁₆H₁₇N₃O₄S).

2 IDENTIFICATION 

A. The retention time of the major peak of the Sample solution corresponds to that of the Standard solution, as obtained in the Assay. B. Identification Tests—General, Chloride 〈191〉: 10 mg/mL meets the requirements 

3 ASSAY

Procedure 

Mobile phase: 0.985 g/L of sodium 1-pentanesulfonate in a mixture of acetonitrile, methanol, triethylamine, and water (20:10:3:170), adjusted with phosphoric acid to a pH of 3.0 ± 0.1 

Standard stock solution: 1 mg/mL of USP Cephalexin RS in water 

Standard solution: 0.4 mg/mL of cephalexin in Mobile phase from Standard stock solution 

Sample stock solution: 1.15 mg/mL of Cephalexin Hydrochloride in water 

Sample solution: 0.4 mg/mL of cephalexin in Mobile phase from Sample stock solution 

Chromatographic system 

(See Chromatography 〈621〉, System Suitability.) 

Mode: LC 

Detector: UV 254 nm 

Column: 4.6-mm × 25-cm; packing L1 of low acidity 

Flow rate: 1.5 mL/min 

Injection size: 20 µL 

System suitability 

Sample: Standard solution 

Suitability requirements 

Relative standard deviation: NMT 2.0% 

Analysis 

Samples: Standard solution and Sample solution 

Calculate the quantity, in µg, of cephalexin (C₁₆H₁₇N₃O₄S) in each mg of Cephalexin Hydrochloride taken: 

Result = (r_U/r_S) × (C_S/C_U) × P 

r_U = peak response from the Sample solution 

r_S = peak response from the Standard solution  

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

C_U = concentration of Cephalexin Hydrochloride from the Sample stock solution (mg/mL) 

P = potency of cephalexin in USP Cephalexin RS (µg/mg) 

Acceptance criteria: 800–880 µg/mg 

4 IMPURITIES 

Organic Impurities 

Procedure 1 

Solution A: 1 g of sodium 1-pentanesulfonate in a mixture of 1000 mL of water and 15 mL of triethylamine. Adjust with phosphoric acid to a pH of 2.5 ± 0.1. 

Solution B: 1 g of sodium 1-pentanesulfonate in a mixture of 300 mL of water and 15 mL of triethylamine. Adjust with phosphoric acid to a pH of 2.5 ± 0.1, and add 350 mL of acetonitrile and 350 mL of methanol. 

Mobile phase: See the gradient table below. 

Diluent: 18 mg/mL of monobasic potassium phosphate in water 

Standard solutions: 0.08 mg/mL and 0.16 mg/mL of cephalexin (C₁₆H₁₇N₃O₄S) from USP Cephalexin RS in Diluent, taking into account the  stated potency of the USP Cephalexin RS 

Sample solution: 6 mg/mL of Cephalexin Hydrochloride in Diluent 

Chromatographic system 

(See Chromatography 〈621〉, System Suitability.) 

Mode: LC 

Detector: UV 254 nm 

Column: 4.6-mm × 25-cm; packing L1 of low acidity 

Flow rate: 1 mL/min 

Injection size: 20 µL 

Analysis 

Samples: Standard solutions and Sample solution 

Plot the responses of the cephalexin peaks of the Standard solutions versus their concentrations, calculated on the anhydrous basis, in mg/mL, and draw a straight line through the two points and zero. From the line and the peak responses of the Sample solution, determine the concentration, I, in mg/mL, of each cephalexin-related substance from the Sample solution other than the cephalexin peak. 

Calculate the percentage of each cephalexin-related substance represented by each peak of the Sample solution, other than the cephalexin peak. 

Result = (I/C) × 100 

I = concentration of each cephalexin-related substance other than cephalexin in the Sample solution (mg/mL) C = concentration of cephalexin from the Sample solution (mg/mL) 

Acceptance criteria 

Individual impurities: NMT 1.0% of any individual cephalexin-related substance is found. 

Total impurities: NMT 5.0% 

Procedure 2: Dimethylaniline 〈223〉: Meets the requirement 

5 SPECIFIC TESTS 

Crystallinity 〈695〉: Meets the requirements 

pH 〈791〉: 1.5–3.0, in a solution containing 10 mg/mL 

Water Determination, Method I〈921〉: 3.0%–6.5% 

6 ADDITIONAL REQUIREMENTS 

Packaging and Storage: Preserve in tight containers. 

USP Reference Standards 〈11〉 

USP Cephalexin RS