Cephalexin

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 . H₂O   365.40 

C₁₆H₁₇N₃O₄S   347.40 

5-Thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid, 7-[(aminophenylacetyl)amino]-3-methyl-8-oxo-, monohydrate, [6R-[6α,7β (R*)]]-; (6R,7R)-7-[(R)-2-Amino-2-phenyl acetamido]-3-methyl-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid monohydrate CAS RN®:  23325-78-2; UNII: OBN7UDS42Y. 

Anhydrous CAS RN®: 15686-71-2; UNII: 5SFF1W6677. 

1 DEFINITION 

Cephalexin has a potency of NLT 950 µg/mg and NMT 1030 µg/mg of C₁₆H₁₇N₃O₄S, calculated on the anhydrous basis. 

2 IDENTIFICATION 

Change to read: 

A. Spectroscopic Identification Tests 〈197〉, Infrared Spectroscopy: 197K

B. The retention time of the major peak of the Sample solution corresponds to that of the Standard solution, as obtained in the Assay. 

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 mg/mL of Cephalexin 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) per mg of the Cephalexin 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 solution (mg/mL) 

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

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

Acceptance criteria: 950–1030 µg/mg on the anhydrous basis 

4 IMPURITIES 

Organic Impurities 

Procedure 1 

Solution A: Dissolve 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: Dissolve 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: 5 mg/mL of Cephalexin 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 from 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 of the Sample solution other than the cephalexin peak. 

Calculate the percentage of each cephalexin-related substance: 

Result = I/C × 100 

I = concentration of each cephalexin-related substance in the Sample solution as determined from the calibration curve (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 

Total impurities: NMT 5.0% 

Procedure 2: Dimethylaniline 〈223〉: Meets the requirement 

5 SPECIFIC TESTS 

Optical Rotation, Specific Rotation〈781S〉: +149° to +158° 

Sample solution: 5 mg/mL, in pH 4.4 neutralized phthalate buffer (see Reagents, Indicators, and Solutions—Buffer Solutions) Crystallinity 〈695〉: Meets the requirements 

pH 〈791〉: 3.0–5.5, in an aqueous suspension containing 50 mg/mL 

Water Determination, Method I〈921〉: 4.0%–8.0% 

6 ADDITIONAL REQUIREMENTS 

Packaging and Storage: Preserve in tight containers. 

USP Reference Standards 〈11〉 

USP Cephalexin RS