Isolation of Peptide Components of Bacitracin by Preparative HPLC and Solid Phase Extraction (SPE)

Abstract

This paper describes isolation procedure of antimicrobially active components of bacitracin (Bc) and its main oxidative degradation products needed for further stability investigations. The procedure of isolation consisted of chromatographic separation of components and degradation products, purification of isolates using solid phase extraction (SPE), and lyophilisation of pure samples. A gradient preparative HPLC separation on Kromasil 5C8 (250 × 16 mm I.D.) column was based on our previously developed analytical method, increasing the ratio of methanol against acetonitrile in the mobile phase and adjusting the flow rate, gradient profile, Bc concentration, and injection volume to achieve an efficient separation of compounds of interest under scale‐up conditions. Eluates collected after chromatographic separations were concentrated and purified using SPE procedure, simultaneously removing buffer salts derived from the mobile phase. Pure substances eluted from the SPE cartridges were then dried by lyophilisation and used for identification purposes and for further stability experiments. Identity and purity of all substances were checked and confirmed chromatographically, using diode array detection on fast monolithic silica gel Chromolith RP‐18e (100 × 4.6 mm I.D.) column after each step of the isolation procedure. Finally, the lyophilisates of active Bc components (A, B1, B2, and B3) and their corresponding oxidative degradation products (F, H1, H2, and H3) were analysed, and their identity confirmed by fast atom bombardment (FAB) mass spectrometry.

Keywords:

• Bacitracin
• Preparative HPLC separation
• Scale up
• Solid phase extraction
• Monolithic silica gel RP‐18e (endcapped) column

Acknowledgment

The authors are grateful to Dr. Bogdan Kralj for FAB measurements from the Jozef Stefan Institute, Ljubljana, Slovenia.