High performance liquid chromatography-tandem mass spectrometry was used to investigate a nonapeptide H2N-Tyr-Val-Asn-Val-Asn-Met-Gly-Leu-Lys-CONH2(Mr1035.6) and its main by-products from solid phase synthesis. The target sequence was assembled in stepwise fashion from the C-terminal to N-terminal using Fmoc chemistry on a Fmoc-PAL-PEG•PS resin manually. The product was deprotected and cleaved from the resin by treatment with reagent R(90%TFA,5%thioanisole, 3%ethanedithiol, 2%anisole,V/V/V/V) for 2.5 hours at room temperature(20～25). Reverse-phase HPLC showed that the crude product contained a major component, two minor components (one early eluting and the other late eluting) and several trace-level components. The major component and the two minor components were analyzed by high performance liquid chromatography-tandem mass spectrometry, respectively. The major component was confirmed as the desired nonapeptide. The molecular weight of the early-eluting component was 16 more than that of the desired nonapeptide, and the by-product was proven to consist of two kinds of nonapeptide derivatives, one with Tyr1 oxidized and the other with Met6 oxidized. The late-eluting by-product was confirmed as resulting from 12 increase in mass of Tyr1 residue, which has not been reported early. The possible origins of those by-products were discussed.