Development of a reversed-phase liquid chromatographic assay for the quantification of total persipeptides in fermentation broth

Fatemeh Mohammadipanah*, Hamed Kazemi Shariat Panahi, Fatemeh Imanparast, Javad Hamedi

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

12 Citations (Scopus)

Abstract

The emergence and prevalence of multi-drug-resistant bacterial strains increase the potential for outbreaks of incurable infections. The discovery of novel antibiotics and pharmacological preparations requires the identification of novel bioactive small molecules. A specific, sensitive, and reliable quantification method using high-performance liquid chromatography (HPLC) with UV detection was developed for the determination of total persipeptides (A and B), which are cyclic pentapeptides found in the fermentation broth of Streptomyces zagrosensis UTMC 1154 that exhibit bioactivity against methicillin-resistant Staphylococcus aureus (MRSA). A simple liquid–liquid extraction (LLE) method using butanol was employed to extract persipeptides from the fermentation broth prior to HPLC analysis. The chromatographic separation of persipeptides and the internal standard, virginiamycin, was achieved with a gradient of acetonitrile and water on a C18 reversed-phase analytical column in a 25-min analytical run utilizing a flow rate of 0.8 mL min−1 and detection at 210 nm. The whole assay was validated, and the method presented a linear response range with a regression coefficient of determination R2 of 0.9996 for the quantification of persipeptides in the concentration range of 3.9–250.0 µg mL−1, as well as extraction recoveries ranging from 54.78 ± 9.83 % to 56.45 ± 16.33 %. The bias and the precision of the proposed method were <10 %. The detection and quantification limits for the persipeptides were 27 and 83 µg L−1, respectively.

Original languageEnglish
Pages (from-to)1325-1332
Number of pages8
JournalChromatographia
Volume79
Issue number19-20
DOIs
Publication statusPublished - 1 Oct 2016
Externally publishedYes

Keywords

  • Assay development
  • HPLC
  • Liquid-liquid extraction
  • Peptide antibiotics
  • Persipeptides
  • Drug discovery
  • Method validation
  • Method development
  • Stability analysis
  • Robustness testing
  • Fermentation

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