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Analysis of spectinomycin in fermentation broth by reversed-phase chromatography

Hong De Yan, Pei Ya Xu, Hai Chan Huang, and Juan Ping Qiu

College of Biological and Environmental Engineering, Zhejiang University of Technology, 310014 Hangzhou, China



Received: 18 January 2009  Revised: 16 March 2009  Accepted: 17 March 2009

Abstract: 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.

Keywords: spectinomycin - derivatization - analysis - separation - HPLC - fermentation broth

Full paper is available at

DOI: 10.2478/s11696-009-0064-0


Chemical Papers 63 (6) 635–640 (2009)

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