Abstract: A HPLC with mass spectrum derivatization method was developed for the determination of (-) -and (+) -gossypol in cottonseed.After degreased by petroleum ether, the samples were extracted with ultrasoninc by 10% glacial acetic acid of N, N-dimethyl formamide formamide and (R) - (-) -2-amino-1-propanol was selected as the chiral separation reagent.Then the chromatographic separation was carried out on an Agilent XDB-C 18 column (250mm ×4.6mm, 5μm) with a mobile phase consisted of 85% acetonitrile and 15% water within 1.0% formic acid at a flow rate of 1mL /min.The condition of mass spectrometer was operated at m /z 633.2 in positive elctrospray ionization mode.It showed that a good linear relationship between HPLC peak area and gossypol concentration was obtained over the range of 1.93³8.6μg /mL, mean spike recoveries of (-) -and (+) -gossypol at three levels were 99.4% ¹03.0%, with RSD ranged from 1.27% ³.86%.The limits of detection were 2.52ng /mL and 3.41ng /mL, while the limits of quantification were 8.39ng /mL and 11.4ng /mL for (-) -and (+) -gossypol respectively.The method provided simplicity, rapidity, sensitivity and accuracy, and therefore was applicable to the determination of (-) and (+) -gossypol in cottonseed.