Determination of vertilmicin sulfate and its related substances by HPLC-ELSD and HPLC-MS2

A new and simple high-performance liquid chromatography (HPLC)-evaporative light scattering detection (ELSD) method for the determination of vertilmicin sulfate and its related substances is developed. The column is an Agilent SB-C(18) (250 x 4.6 mm, 5 microm). The mobile phase is 0.05 mol/L trifluoroacetic acid-methanol (85:15). Good separation of vertilmicin from the main related substances is achieved. The standard curve is rectilinear in the range of 270-1350 microg/mL (r = 0.9998). The average recovery is 99.8%. The limit of detection is 10 microg/mL. The HPLC-mass spectrometry-mass spectrometry (MS(2)) method is used to characterize the structures of vertilmicin sulfate and its related substances. In positive mode, vertilmicin sulfate and its related substances are elucidated by use of electrospray ion trap MS in the multi-stage MS full scan mode. The possible structure of an unknown impurity in vertilmicin is deduced based on the HPLC-MS(2) data.