ISSN print edition: 0366-6352
ISSN electronic edition: 1336-9075
Registr. No.: MK SR 9/7
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 www.springerlink.com.
Chemical Papers 63 (6) 635–640 (2009)