Screening and Optimization of the Reaction of Polymyxin B Sulphate with NBD-Cl for the Synchronous Spectrofluorimetric Determination of Polymyxin B Sulphate in Human Plasma.

An accurate and sensitive synchronous spectrofluorimetric method has been developed for the determination of Polymyxin B sulphate (Poly B) in human plasma. The method is based on the reaction of non-fluorescent Poly B with 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) in borate buffer of pH 7 producing a yellow color with maximum relative fluorescence at 440 nm using a constant wavelength difference Δλ = 80 nm. Reaction conditions and other analytical parameters were studied and optimized using factorial design. Three level factorial designs have been employed for the screening, optimization of all experimental variables and determination of their interactions on the final product formation. The variables under investigation were: pH of borate buffer, volume of buffer, volume of NBD-Cl, temperature, time of heating and volume of sulfuric acid. A linear plot between relative fluorescence and concentration was obtained over the concentration range 100.00-1200.00 ng mL(-1). The limit of detection (LOD) and limit of quantification (LOQ) were found to be 10.31 and 31.24 ng mL(-1), respectively. The proposed method was validated according to ICH guidelines and successfully applied for the determination of Poly B in human plasma, where satisfactory results were obtained. The results obtained were statistically compared with those of a published method, where no significant difference was observed.