Analysis of spectinomycin in fermentation broth by reversed-phase chromatography

A pre-column derivatized high-performance liquid chromatographic (HPLC) method with ultraviolet-visible detection was developed to measure the concentrations of spectinomycin in fermentation broth. Derivatization reagents, 2,4-dinitrophenylhydrazine in acetonitrile (5 mg mL⁻¹) and trifluoroacetic acid in acetonitrile (0.8 mol L⁻¹), were added to an aliquot of the fermentation broth, and the mixture was incubated for 60 min at 70°C. The resulting derivative was separated from other compounds by isocratic elution in a reversed-phase column Zorbax SB-C18 (250 mm × 4.6 mm, 5 μm). Mobile phase consisted of acetonitrile, tetrahydrofuran, and water (φ _r = 40: 35: 25) and the flow rate was 1.0 mL min⁻¹. The detection wavelength was 415 nm. The standard curve for spectinomycin sulfate was linear with correlation coefficients of 0.9997 in the range of 25 μg mL⁻¹ to 600 μg mL⁻¹. The relative standard deviation values ranged from 0.43 % to 2.18 % depending on the concentration of samples. The average recovery was 101.5 %. The limit of detection was 50 ng mL⁻¹.