Tiacumicin B (Fig.1) is a broad-spectrum 18-membered macrolides antibiotic especially against diarrhea-associated Clostridium difficile.Currently, it is undergoing phase Ⅲ clinical trials for the treatment of C.difficile infection (CDI).In our study, C 18 SPE and HPLC-ESI-MS/MS were used for simultaneous determination of tiacumicin B and its analogues in the fermentation broth.Optimum conditions for C 18 SPE were achieved using water/methanol (50∶50, v/v) as the washing solution and water/methanol (20∶80, v/v) for eluting the target compounds.