Ceftiofur 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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C₁₉H₁₇N₅O₇S₃ .HCl 560.02 

5-Thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid, 7-[[(2-amino-4-thiazolyl)(methoxyimino)acetyl]amino]-3-[[(2- furanylcarbonyl)thio]methyl]-8-oxo-, monohydrochloride, [6R-[6α,7β(Z)]]-; 

(6R,7R)-7-[2-(2-Amino-4-thiazolyl)glyoxylamido]-3-(mercaptomethyl)-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid, 72-(Z)-(O methyloxime), 2-furoate (ester), monohydrochloride CAS RN®: 103980-44-5. 

1 DEFINITION 

Ceftiofur Hydrochloride contains NLT 844 µg/mg and NMT 956 µg/mg of ceftiofur (C₁₉H₁₇N₅O₇S₃), calculated on the anhydrous and solvent free basis. 

2 IDENTIFICATION 

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

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 

Change to read: 

Procedure 

Solution A: Tetrabutylammonium hydroxide, 40% in water 

Solution B: Dissolve 3.85 g of ammonium acetate and 13.5 mL of Solution A in 700 mL of water. Adjust with glacial acetic acid to a pH of 6.7. Mobile phase: Mix 700 mL of Solution B with 200 mL of methanol and 110 mL of tetrahydrofuran. 

Diluent: 0.05 M ammonium acetate 

Standard solution: 0.16 mg/mL of USP Ceftiofur Hydrochloride RS prepared as follows. Dissolve USP Ceftiofur Hydrochloride RS in methanol using about 4% of the nal volume and dilute with Diluent to volume. 

Sample solution: 0.16 mg/mL of Ceftiofur Hydrochloride prepared as follows. Dissolve Ceftiofur Hydrochloride in methanol using about 4% of the final volume and dilute with Diluent to volume. 

Chromatographic system 

(See Chromatography 〈621〉, System Suitability.) 

Mode: LC 

Detector: UV 254 nm 

Column: 4.6-mm × 25-cm; 5-µm packing L7 

Column temperature: 40° 

Flow rate: 1.5 mL/min 

Injection volume: 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/mg,  of ceftiofur (CC₁₉H₁₇N₅O₇S₃) in the portion of Ceftiofur 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 Ceftiofur Hydrochloride RS in the Standard solution (mg/mL) 

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

P = potency of ceftiofur in USP Ceftiofur Hydrochloride RS (µg/mg) 

Acceptance criteria: 844–956 µg/mg on the anhydrous and solvent-free basis 

4 IMPURITIES 

Low Molecular Weight Impurities 

Solution A: Acetonitrile, trifluoroacetic acid, and water (50:1:950) 

Solution B: Acetonitrile, trifluoroacetic acid, and water (800:1:200) 

Mobile phase: See Table 1. 

Table 1 

Diluent: Acetonitrile and water (1:1) 

System suitability solution: 0.1 mg/mL of USP Ceftiofur System Suitability Mixture RS in Diluent. Sonicate as needed to dissolve. Inject within 20 min of preparation. 

Peak identification solution: 15 µg/mL of USP Cefotaxime Sodium RS in Diluent 

Sample stock solution: 3 mg/mL of Ceftiofur Hydrochloride in Diluent 

Sample solution: 0.3 mg/mL of Ceftiofur Hydrochloride from Sample stock solution in water. Inject the Sample solution within 20 min of preparation. 

Chromatographic system 

(See Chromatography 〈621〉, System Suitability.) 

Mode: LC 

Detector: UV 254 nm 

Column: 4.6-mm × 15-cm; 3-µm packing L1 

Flow rate: 1 mL/min 

Injection volume: 10 µL 

System suitability 

Sample: System suitability solution 

[Note—The relative retention times for ceftiofur delta-3 isomer and ceftiofur are 0.98 and 1.0, respectively.] 

Suitability requirements 

Resolution: NLT 1.5 between ceftiofur delta-3 isomer and ceftiofur 

Analysis 

Samples: Sample solution and Peak identification solution 

[Note—The elution order of the N-deacyl ceftiofur and cefotaxime peaks may be reversed depending on the column used. Determine the location of the cefotaxime peak by using the Peak identification solution.] 

Calculate the percentage of each impurity in the portion of Ceftiofur Hydrochloride taken: 

Result = {r_U/[r_S+ Σ(r_U/F)]} × (1/F) × 100 

r_U = peak response of each impurity from the Sample solution 

r_S = peak response of ceftiofur from the Sample solution 

F = relative response factor (see Table 2) 

Acceptance criteria: See Table 2. The reporting threshold is 0.05% of the total corrected peak area. 

Table 2 

^a(Z)-Ethyl 2-(2-aminothiazol-4-yl)-2-(methoxyimino)acetate. 

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

^c Furan-2-carboxylic acid. 

^d(6R,7R)-7-Amino-3-((furan-2-carbonylthio)methyl)-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid. 

^e(6R,7R)-3-(Acetoxymethyl)-7-((Z)-2-(2-aminothiazol-4-yl)-2-(methoxyimino)acetamido)-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2- carboxylic acid. 

^f (6R,7R)-7-((Z)-2-(2-Aminothiazol-4-yl)-2-(methoxyimino)acetamido)-3-((furan-2-carbonylthio)methyl)-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-3- ene-2-carboxylic acid. 

^g(6R,7R)-7-((E)-2-(2-Aminothiazol-4-yl)-2-(methoxyimino)acetamido)-3-((furan-2-carbonylthio)methyl)-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2- ene-2-carboxylic acid. 

^h S-(4-Hydroxy-5-oxo-2,5-dihydrothiophen-3-yl)methyl furan-2-carbothioate. 

ⁱ(6R,7S)-7-{(6R,7R)-7-[(Z)-2-(2-Aminothiazol-4-yl)-2-(methoxyimino)acetamido]-3-[(furan-2-carbonylthio)methyl]-8-oxo-5-thia-1- azabicyclo[4.2.0]oct-2-enecarboxamido}-3-[(furan-2-carbonylthio)methyl]-8-oxo-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid. 

^j (Z)-2-(Methoxyimino)-2-(2-(tritylamino)thiazol-4-yl)acetic acid. 

High Molecular Weight Impurities (Ceftiofur Polymers) 

To minimize leaching of plastic components, avoid contact between the Mobile phase and plastics during all steps including during the preparation of the Mobile phase, the System suitability solution, and the Sample solution, and when filling the injection vial. Solution A: 45% of potassium hydroxide in water 

Solution B: 0.68 g/L of monobasic potassium phosphate. Adjust with Solution A to a pH of 7.5. 

Mobile phase: 10 g/L of electrophoresis grade sodium dodecyl sulfate in Solution B. Stir or slightly heat to dissolve. Blank: Use the Mobile phase. 

System suitability solution: 0.15 mg/mL of USP Ceftiofur System Suitability Mixture RS in Mobile phase. Sonicate if necessary to dissolve. Inject within 20 min of preparation. 

Sample solution: 0.15 mg/mL of Ceftiofur Hydrochloride in Mobile phase. Inject within 20 min of preparation. 

Chromatographic system 

(See Chromatography 〈621〉, System Suitability.) 

Mode: LC 

Detector: UV 254 nm 

Column: 4.0-mm × 25-cm; 5-µm packing L20 

Flow rate: 1 mL/min 

Injection volume: 20 µL 

Run time: NLT 2 times the retention time of the ceftiofur peak 

System suitability 

Sample: System suitability solution 

[Note—Adjust the chromatographic system so that the retention time of ceftiofur is NLT 2.5 min. The relative retention times of ceftiofur E isomer and ceftiofur are 0.9 and 1.0, respectively.] 

Suitability requirements 

Resolution: NLT 1.0 between ceftiofur and ceftiofur E-isomer 

Analysis 

Samples: Blank and Sample solution 

Calculate the percentage of high molecular weight impurities in the portion of Ceftiofur Hydrochloride taken: 

Result = {100 × (r_U/F)/[(r_U/F) + r_C + r_A ]} − T 

r_U = sum of the responses of all peaks that elute prior to ceftiofur from the Sample solution, corrected for the Blank if necessary 

F = relative response factor, 0.8 

r_C = peak response of ceftiofur from the Sample solution 

r_A = sum of the responses of all peaks that elute after ceftiofur from the Sample solution  

T = total impurities from Table 2 

Acceptance criteria 

Total high molecular weight impurities: NMT 3.9% 

5 SPECIFIC TESTS 

Optical Rotation 〈781S〉, Procedures, Specific Rotation 

Sample solution: 5 mg/mL in dimethylformamide 

Acceptance criteria: −115° to −127° on the anhydrous and solvent-free basis 

Bacterial Endotoxins Test 〈85〉: Where the label states that Ceftiofur Hydrochloride is sterile or that it must be subjected to further processing during the preparation of injectable dosage forms, it contains NMT 1.0 USP Endotoxin Unit/mg of ceftiofur hydrochloride. Water Determination 〈921〉, Method I: NMT 6.0% 

6 ADDITIONAL REQUIREMENTS 

Packaging and Storage: Preserve in tight, light-resistant containers. Store in a freezer. 

Labeling: Label it to indicate that it is intended for veterinary use only. Where it is intended for use in preparing injectable dosage forms, the label states that it is sterile or must be subjected to further processing during the preparation of injectable dosage forms. USP Reference Standards 〈11〉 

USP Cefotaxime Sodium RS 

USP Ceftiofur Hydrochloride RS 

USP Ceftiofur System Suitability Mixture RS 

This is a mixture of ceftiofur, ceftiofur delta-3 isomer, ceftiofur E-isomer, and other impurities. 

Ceftiofur delta-3 isomer 

(6R,7R)-7-((Z)-2-(2-Aminothiazol-4-yl)-2-(methoxyimino)acetamido)-3-((furan-2-carbonylthio)methyl)-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-3-ene-2- carboxylic acid. 

C₁₉H₁₇N₅O₇S₃ 523.56 

 Ceftiofur E-isomer 

(6R,7R)-7-((E)-2-(2-Aminothiazol-4-yl)-2-(methoxyimino)acetamido)-3-((furan-2-carbonylthio)methyl)-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2- carboxylic acid. 

C₁₉H₁₇N₅O₇S₃ 523.56 

USP Endotoxin RS