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.

The aim of work was to develop a method for the identification and quantification of neomycin sulfate in intracisternal veterinary preparations. The method was developed and validated by: selectivity, linearity and suitability parameters of the chromatographic system. A four-component intracisternal drug with a 25 mg/g neomycin content was used as an object sample for method development. The standard sample and the test sample were dissolved in purified water to a concentration of 100 μg/ml, followed by derivatization with 9-fluorenylmethoxycarbonyl chloride at room temperature. The total uncertainty of the analysis was 1.62 %, which is within the limits recommended in SFU 2.0. The samples were separated on a Dionex Ultimate 3000 chromatograph equipped with an Acclaim C18 150 × 4.6.5 μm chromatographic column. The mobile phase was a mixture of acetonitrile and water in a volume ratio of 95:5. Neomycin sulfate was detected fluorescently at wavelengths λex - 254 nm, λem - 303 nm. Under the above conditions, it was possible to completely separate the two isoforms of neomycin (retention time of chromatographic peaks - 6.57 min and 6.98 min), 9-fluorenylmethoxycarbonyl chloride and other components of the study drug (retention time of the corresponding chromatographic peaks - 2-5 min). In this case, the parameters of the suitability of the chromatographic system did not exceed the limits specified in the recommendations of the USA Food and Drug Association. For peaks of neomycin sulfate, the efficiency of the chromatographic system was 7700–7950 theoretical plates. The relative standard deviation (RSD) for the peak areas of the active substance was ± 0.33%, and the coefficient of separation of the peaks (RS) of neomycin sulfate from the peaks of the derivatization reagent and other components of the drug was 11.9. The coefficient of peak symmetry was 1.18. The calibration curves were linear in the recommended SFU 2.0 range (80–120% of the nominal concentration of the corresponding active substance). The coefficient of linearity (R2) for the sum of neomycin sulfate peak areas was – 0.9991.