Abstract
A simple, rapid and reliable reversedβphase ionβpair chromatography method by HPLC coupled to an evaporative light scattering detector (ELSD) has been developed to simultaneously determine chloride, spectinomycin and its related substances in a sample. The column was a TSKgel ODSβ100V. The mobile phase was ACN/aqueous solution of 15βmM ammonium acetate adjusted with TFA to pH 3.0 (2:98 v/v), in an isocratic mode. The drift tube temperature was set at 50Β°C and the nebulizing gas flow rate of air was 3.5βL/min for ELSD detection. Almost all of the reported degradation compounds of spectinomycin such as actinamine, actinospectinoic acid and biosynthesis intermediates such as dihydrospectinomycin diastereoisomers were baseline separated. MS was utilized for the identification of spectinomycin and its seven related substances. The method for the assay of spectinomycin was successfully validated with respect to accuracy, precision (RSD less than 2%), linearity (throughout the linear range 0.025β3βmg/mL, r=0.9993), sensitivity (LOD: 100βng on column) and robustness. The experimental results demonstrated that the simultaneous determination of chloride, spectinomycin and related substances is feasible in a single run, which suggests applicability in routine assays.