Smart Eco-Friendly Spectrophotometric Methods Resolving Highly Overlapping Spectra: Application to Veterinary...

Over the last few years, mathematical manipulation has proved to be a very powerful means of successfully resolving severely overlapped spectra for various multicomponent mixtures.

Smart and environmentally friendly spectrophotometric determination approaches were used for two binary mixtures of fixed dose veterinary injections containing flunixin meglumine (FLU) combined with either florfenicol (FLR), or oxytetracycline HCl (OXY).

Regarding the first mixture, both FLU and FLR were determined by three successive resolution techniques, which were; constant multiplication coupled with spectrum subtraction (CM-SS), derivative ratio (DD1), and ratio difference (RD), and two progressive resolution techniques which were absorbance subtraction (AS) and amplitude modulation (AM). Also, graphical representation of concentration of the two drugs through concentration value (CNV) method was also applied. Concerning the second mixture, both FLU and OXY showed severely overlapped spectra and a comparative study was conducted for the determination of each drug by constant center (CC), ratio subtraction via amplitude difference coupled with spectrum subtraction (RS/AD-SS), constant value via amplitude difference (CV-AD), and advanced concentration value (ACV) methods.

Calibration graphs of the first mixture were linear over the range 5-40 µg/mL for FLU, and 3-40 µg/mL for FLR. The proposed methods overcame the problem of the overlapped spectra and the presence of a minor component in the mixture. Regarding the second mixture, calibration graphs were linear over the range 2.5-24 µg/mL for FLU and 4-28 µg/mL for OXY.

The proposed methods were successfully validated as per International Council for Harmonization (ICH) guidelines. The obtained results were statistically compared with the official or reported methods, showing no significant difference concerning accuracy and precision. The methods were evaluated for greenness by three different assessment tools: NEMI, analytical ecoscale, and GAPI.

The methods were successfully applied for the simultaneous determination of the two combinations in synthetic mixtures and their marketed antibiotic veterinary injections: Megluflor® and Floxon®.