Development of the Method for Quantitative Determination of Neomycin Sulphate in Veterinary Preparations With Precolumn Derivatization and Fluorescent Detection

Abstract

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.