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-C(18) (250 mm x 4.6 mm, 5 microm). The mobile phase was 25 mM trifluoroacetic acid. The drift tube temperature was 40 degrees C. The pressure of nebulizing gas was 3.5 bar. Good separation of spectinomycin from main related substances could be achieved. The standard curve was rectilinear in the range of 0.07-3.8 mg/ml (r = 0.9997). Precision was 1.0% (R.S.D.). The limit of detection was 6 microg/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.