Development and Validation of HPLC-Method Determining Quantitative Content of Ceftiofur Hydrochloride in Suspensions for Injection

Abstract

Ceftiofur is a third-generation cephalosporin used in veterinary medicine to treat infections caused by gram-negative and gram-positive bacteria. It is most often used in the form of suspensions for injections based on a lipid matrix, since the active substance is poorly soluble in water. The analysis of such a drug by direct spectrophotometry is difficult due to the components of the matrix, therefore it is proposed to develop a high-performance liquid chromatography method with UV detection.
 The aim of the work was to develop a method of identification and quantitative determination of ceftiofur hydrochloride in suspensions for injections. The method was developed and validated according to the indicators of selectivity, robustness, linearity and suitability parameters of the chromatographic system. Suspension for injections containing ceftiofur 50 mg/ml was used as a sample-object for method development. The standard sample and the test sample were dissolved in the mobile phase to a concentration of 50 μg/ml. The total uncertainty of the analysis was 1.62%, which is within the limits recommended in DFU 2.0. The samples were separated on a Dionex Ultimate 3000 chromatograph equipped with a Kinetex C18-XB 150×4.6, 5 μm chromatographic column. The mobile phase was a mixture of acetonitrile and 0.05 M ammonium acetate, 0.01 M tetrabutylammonium bromide with a pH of 6.8, titrated with acetic acid, in a volume ratio of 3:7. Ceftiofur hydrochloride was detected spectrophotometrically at a wavelength of 290 nm.
 Under the above conditions, it was possible to completely separate ceftiofur (retention time of the chromatographic peak – 4.4 min) and other components of the studied drug. At the same time, the suitability parameters of the chromatographic system did not exceed the limits specified in the recommendations of the USA Food and Drug Association. For the ceftiofur hydrochloride peak, the efficiency of the chromatographic system was 13,900 theoretical plates. The relative standard deviation (RSD) for the peak areas of the active substance was ±0.11 %, and the peak separation coefficient (RS) of ceftiofur hydrochloride from other components of the drug was 17.3. The symmetry coefficient of the peak was 1.02. The calibration curves were linear in the recommended DFU 2.0 range (80–120% of the nominal concentration of the corresponding active substance). The coefficient of linearity (R2) for the peak area of ceftiofur hydrochloride was 0.9992.