Analysis of antineoplastic polysaccharides from Mycobacterium bovis BCG vaccine by high performance anion exchange chromatography with pulsed amperometric detection

Abstract Aqueous extracts of Mycobacterium bovis BCG vaccine, Tice® substrain, showed potent antineoplastic activity against a murine S180 sarcoma cell line in vivo. Following Sephadex LH-20 chromatography, one fraction (PSIA) was found to have antineoplastic activity, and contains complex polysaccharides. PS1A was further separated into four subfractions by Sephadex G-75 column chromatography. An HPLC method has been established for the analyses of polysaccharides in PS1A and its subfractions. Using a Dionex DX500 HPLC system, separation was achieved on a CarboPac PA1 anion exchange column by gradient elution with 0.1 M NaOH and 0.9 M NaOH/1 M NaOAc. Pulsed amperometry with three potential waveforms (E1 =0.1 v, E2=0.7v and E3=-0.1v) was used for detection. The HPLC chromatogram of PS1A showed three major peaks with retention times of 8.5, 15.5 and 19.25 min., respectively, and these three peaks have been identified in the subfractions of PS1A.