Quantitative analysis of valienamine in the microbial degradation of validamycin A after derivatization with p-nitrofluorobenzene by reversed-phase high-performance liquid chromatography.
A reversed-phase high-performance liquid chromatography method for the quantitative analysis of valienamine in the microbial degradation of validamycin A, using a procedure for pre-column derivatization of valienamine with p-nitrofluorobenzene is described. Valienamine in the broth was first isolated with the ion-exchange method. The optimized conditions for the derivatization were the reaction time 30 min and reaction temperature 100 degrees C. With the mobile phases consisting of acetonitrile-water (12:88) (eluent A) and methanol (eluent B), the gradient was carried out with 100% of A for 15 min and then 100% of B for another 10 min. The parameters in the process were the flow rate of the mobile phase 1.0 ml/min, the injection volume 20 microl, the column temperature 40 degrees C and wavelength of ultraviolet detection 398 nm in all runs. A good linearity was found in the range of 0.5-150.0 microg/ml. Both intra- and inter-day precisions of valienamine, expressed as the relative standard deviation, were less than 9.4%. Accuracy, expressed as the relative error, range from -0.5 to 2.7%. The mean absolute recovery of valienamine at three different concentrations was 94.2%. The method was proved suitable for the study on the process of microbial degradation of validamycin A to produce valienamine.
Chen
Xiaolong
X
Institute of Bioengineering, Zhejiang University of Technology, Hangzhou 310032, Zhejiang, PR China. Richard_chen@zjut.edu.cn
Zheng
Yuguo
Y
Shen
Yinchu
Y
D001419:Bacteria / Q000378:metabolism; D001711:Biotransformation; D002138:Calibration; D002851:Chromatography, High Pressure Liquid / Q000295:instrumentation / Q000379:methods*; D017077:Culture Media, Conditioned / Q000032:analysis / Q000737:chemistry; D053138:Cyclohexenes; D005464:Fluorobenzenes / Q000737:chemistry; D006595:Hexosamines / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D007294:Inositol / Q000031:analogs & derivatives / Q000378:metabolism; D009578:Nitrobenzenes / Q000737:chemistry; D015203:Reproducibility of Results; D012988:Soil Microbiology; D013696:Temperature; D013997:Time Factors
D017077:Culture Media, Conditioned; D053138:Cyclohexenes; D005464:Fluorobenzenes; D006595:Hexosamines; D009578:Nitrobenzenes; C030790:validamycin A; C030791:valienamine; D007294:Inositol