Determination of spectinomycin hydrochloride and its related substances by HPLC–ELSD and HPLC–MS n

Abstract: A new and simple high-performance liquid chromatography–evaporative light scattering detection (HPLC–ELSD) method for the determination of spectinomycin hydrochloride and its related substances was developed. The column was Agilent SB-C18 (250mm×4.6mm, 5μm).The mobile phase was 25mM trifluoroacetic acid. The drift tube temperature was 40°C. The pressure of nebulizing gas was 3.5bar. Good separation of spectinomycin from main related substances could be achieved. The standard curve was rectilinear in the range of 0.07–3.8mg/ml (r =0.9997). Precision was 1.0% (R.S.D.). The limit of detection was 6μg/ml. The method is simple and rapid, and the results are accurate and reproducible. The HPLC–MS n method was used to characterize the structures of impurities contained in the spectinomycin. In positive mode, impurities were elucidated by use of electrospray ion trap mass spectrometry in the multi-stage MS full scan mode. The possible structures of impurities C and D in spectinomycin were deduced based on the HPLC–MS n data. [Copyright &y& Elsevier]