Cefazolin

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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1 DEFINITION

Cefazolin contains NLT 95.0% and NMT 103.0% of cefazolin (C₁₄H₁₄N₈O₄S₃), calculated on the anhydrous basis.

2 IDENTIFICATION

Change to read:

A. SPECTROSCOPIC IDENTIFICATION TESTS 〈197〉 , Infrared Spectroscopy : 197K (CN 1-M -2020)

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

3.1 PROCEDURE

Protect all solutions containing cefazolin from light.

Buffer A: 0.9 g/L of anhydrous dibasic sodium phosphate and 1.3 g/L of citric acid monohydrate in water

Buffer B: 5.7 g/L of anhydrous dibasic sodium phosphate and 3.6 g/L of monobasic potassium phosphate in water Mobile phase: Acetonitrile and Buffer A (10:90)

Standard solution: 50 µg/mL of USP Cefazolin RS in Buffer B

Sample solution: 50 µg/mL of Cefazolin in Buffer B

3.1.1 Chromatographic system

(See Chromatography 〈621〉, System Suitability.)

Mode: LC

Detector: UV 254 nm

Column: 3.9-mm × 30-cm; 10-µm packing L1 Flow rate: 2 mL/min

Injection volume: 10 µL

3.1.2 System suitability

Sample: Standard solution Suitability requirements

Relative standard deviation: NMT 2.0% Tailing factor: NMT 1.5

3.1.3 Analysis

Samples: Standard solution and Sample solution

Calculate the percentage of cefazolin (C H N O S ) in the portion of Cefazolin taken:

Result = (r_u/r_s) × (C_s/C_u) × P × 100

r_u = peak response of cefazolin from the Sample solution

r_s = peak response of cefazolin from the Standard solution

C_s = concentration of USP Cefazolin RS in the Standard solution (mg/mL)

C_u = concentration of Cefazolin in the Sample solution (mg/mL)

P = potency of cefazolin in USP Cefazolin RS (mg/mg) Acceptance criteria: 95.0%–103.0% on the anhydrous basis

4 IMPURITIES

4.1 ORGANIC IMPURITIES

Protect all solutions containing cefazolin from light.

Solution A: 6.8 g/L of monobasic potassium phosphate in water

Solution B: 6.8 g/L of monobasic potassium phosphate adjusted with 10% sodium hydroxide to a pH of 6.8 before final dilution with water Solution C: Acetonitrile and Solution A (1:1)

Mobile phase: See Table 1.

Table 1

Blank: Use Solution B.

System suitability stock solution: 2 mg/mL of USP Cefazolin RS in 0.05 M sodium hydroxide. Set the solution aside at room temperature for 5 min. [NOTE—The cefazolin epimer is formed upon treatment of cefazolin with sodium hydroxide.]

System suitability solution: System suitability stock solution and Solution B (1:24)

Standard solution: 25 µg/mL of USP Cefazolin RS in Solution B. Use this solution immediately after preparation. Sample solution: 2.5 mg/mL of Cefazolin in Solution B. Use this solution immediately after preparation.

4.1.1 Chromatographic system

(See Chromatography 〈621〉, System Suitability.) Mode: LC

Detector: UV 210 and 254 nm

Column: 4.6-mm × 25-cm; 5-µm packing L1 Column temperature: 30°

Flow rate: 1.5 mL/min

Injection volume: 20 µL System suitability

Sample: System suitability solution

4.1.2 Suitability requirements

Resolution: NLT 8.0 between cefazolin and cefazolin epimer, 254 nm Analysis

Samples: Blank, Standard solution, and Sample solution

Calculate the percentage of tetrazolylacetic acid and tetrazolylacetamide acetal in the portion of Cefazolin taken:

Result = (r_U(210)/r_S(254)) × (C_s/C_u) × (1/F) × 100

r_U(210) = peak response of tetrazolylacetic acid or tetrazolylacetamide acetal at 210 nm from the Sample solution

r_S(254) = peak response of cefazolin at 254 nm from the Standard solution

C_s = concentration of USP Cefazolin RS in the Standard solution (mg/mL)

C_u = concentration of the Cefazolin in the Sample solution (mg/mL)

F = relative response factor (see Table 2)

Calculate the percentage of each impurity other than tetrazolylacetic acid and tetrazolylacetamide acetal in the portion of Cefazolin taken:

Result = (r_U(254)/r_S(254)) × (C_s/C_u) × (1/F) × 100

r_U(254) = peak response of each impurity other than tetrazolylacetic acid or tetrazolylacetamide acetal at 254 nm from the Sample solution

r_S(254) = peak response of cefazolin at 254 nm from the Standard solution

C_s = concentration of USP Cefazolin RS in the Standard solution (mg/mL)

C_u = concentration of the Cefazolin in the Sample solution (mg/mL)

F = relative response factor (see Table 2)

Acceptance criteria: See Table 2. Disregard peaks corresponding to those in the Blank.

Table 2

^_a    2-(1H-Tetrazol-1-yl)acetic acid.

^b    N-(2,2-Dihydroxyethyl)-2-(1H-tetrazol-1-yl)acetamide.

^c    The identification of this impurity is tentative. The names of the most likely compounds are listed in footnotes d and e.

^d    (R)-2-[2-(1H-Tetrazol-1-yl)acetamido]-2-[(R)-7-oxo-2,4,5,7-tetrahydro-1H-furo[3,4-d][1,3]thiazin-2-yl]acetic  acid.

^e    (6R,7R)-7-[2-(1H-Tetrazol-1-yl)acetamido]-3-(hydroxymethyl)-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic  acid.

^f    5-Methyl-1,3,4-thiadiazole-2-thiol (MMTD).

^g    (6R,7R)-3-(Acetoxymethyl)-7-amino-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (7-ACA).

^h    (6R,7R)-7-[2-(1H-Tetrazol-1-yl)acetamido]-3-methyl-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic  acid.

ⁱ    N-{(5aR,6R)-1,7-Dioxo-1,3,4,5a,6,7-hexahydroazeto[2,1-b]furo[3,4-d][1,3]thiazin-6-yl}-2-(1H-tetrazol-1-yl)acetamide.

^j    (6R,7R)-7-[2-(1H-Tetrazol-1-yl)acetamido]-3-(acetoxymethyl)-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic  acid.

^k    (6R,7R)-7-Amino-3-[(5-methyl-1,3,4-thiadiazol-2-ylthio)methyl]-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic  acid.

^l    Three isomers of this impurity may not be fully resolved by this method. The limit applies to the sum of the isomers, which are as follows: Cefazolin open-ring delta-3: (2R)-2-{(R)-[2-(1H-Tetrazol-1-yl)acetamido](carboxy)methyl}-5-[(5-methyl-1,3,4-thiadiazol-2-ylthio)methyl]-3,6- dihydro-2H-1,3-thiazine-4-carboxylic acid. Cefazolin open-ring delta-2: (2R)-2-{(R)-[2-(1H-Tetrazol-1-yl)acetamido](carboxy)methyl}-5-[(5- methyl-1,3,4-thiadiazol-2-ylthio)methyl]-3,4-dihydro-2H-1,3-thiazine-4-carboxylic acid. Cefazolin open-ring delta-4: (2R)-2-{(R)-[2-(1H-Tetrazol- 1-yl)acetamido](carboxy)methyl}-5-[(5-methyl-1,3,4-thiadiazol-2-ylthio)methyl]-5,6-dihydro-2H-1,3-thiazine-4-carboxylic acid.

^m    (6R,7S)-7-[2-(1H-Tetrazol-1-yl)acetamido]-3-[(5-methyl-1,3,4-thiadiazol-2-ylthio)methyl]-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-   carboxylic acid.

ⁿ    (6R,7R)-3-((5-Methyl-1,3,4-thiadiazol-2-ylthio)methyl)-8-oxo-7-pivalamido-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid.

5 SPECIFIC TESTS

WATER DETERMINATION, Method I 〈921〉: NMT 2.0%

6 ADDITIONAL REQUIREMENTS

PACKAGING AND STORAGE: Preserve in tight containers.

USP REFERENCE STANDARDS 〈11〉

USP Cefazolin RS