International Journal of Applied Science and Engineering
Published by Chaoyang University of Technology

Yerra Koteswara Raoa, Hung-Yuh Linb, Wen-Shi Wu c, Yew-Min Tzengb1

a Institute of Biotechnology, National Dong Hwa University, Hualien, Taiwan, R.O.C.
b Institute of Biochemical Sciences and Technology, Chaoyang University of Technology, Wufeng, Taiwan, R.O.C.
c Department of Horticulture, Chinese Culture University, Taipei, Taiwan, R.O.C.


 

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ABSTRACT


Two methods, high-performance liquid chromatography (HPLC) and micellar electrokinetic chromatography (MEKC), for analysis of multicomponent lipopeptide antibiotic iturin A are described. After conducting a series of optimizations, the HPLC separation of iturin A2, A3, A4, A5, A6, A7, and A8 isomers were achieved by a NPS RP-C18 column with an isocratic elution of acetonitrile/10 mM ammonium acetate (40:60, v/v), and flow rate of 1 mL/min. On the other hand, the MEKC separation achieved at 25 °C using an applied voltage of 20 kV, and background electrolyte consisting of 20 mM boric acid buffer (pH 8.72) with 50 mM sodium dodecyl sulfate (SDS) and 10% (v/v) acetonitrile. The calibration curve was linear to 20-200 µg/mL and 50-500 µg/mL for HPLC and MEKC with R2 = 0.9954 and R2 = 0.9967, respectively. The detection limits of iturin A from the strain B128 cultivation medium were in the 2.5-7.0 µg/mL and 5.0-13.0 µg/mL ranges, and the reproducibility of within-day assay was 98.1 and 98.7%, for HPLC and MEKC, respectively (a UV detector was applied in the both cases). Both methods were selective, robust, and specific, allowing reliable quantification of iturin A, and could be useful for clinical and biomedical investigations of related antibiotics.


Keywords: Bacillus amyloliquefaciens B128; High-performance liquid chromatography; Micellar electrokinetic chromatography; Lipopeptide antibiotic; Iturin A


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ARTICLE INFORMATION




Accepted: 2008-11-12
Available Online: 2008-11-01


Cite this article:

Rao, Y.K., Lin, H.-Y., Wu, W.-S., Tzeng, Y.-M. 2008. Evaluation of HPLC and MEKC methods for the analysis of lipopeptide antibiotic iturin A produced by Bacillus amyloliquefaciens. International Journal of Applied Science and Engineering, 6, 85–96. https://doi.org/10.6703/IJASE.2008.6(2).85