Abstract: This study aimed to establish a high performance liquid chromatography (HPLC) method for the determination of free gossypol and its degradation product tetramethoxy gossypol in commercially available cottonseed oil in order to provide a scientific basis for quality control of cottonseed oil. The chromatographic separation was accomplished on an Inertsil? ODS-3 column (250 mm × 4.6 mm, 5 mm) using a mobile phase comprised of methanol and 2% phosphoric acid (85:15, V/V) at a flow rate of 1.0 mL/min, and the column temperature was set at 32 ℃. The detection wavelength was 235 nm, and the injection volume was 10 mL. Good linear relationship was achieved for gossypol in the concentration range of 0.245–1.226 mg/mL (r = 0.999 5). The relative standard deviation (RSD) of precision and repeatability was 0.88% and 1.73% (n = 6), respectively. The average recovery was 101.2%, with RSD of 1.75% (n = 3). The method also showed a good linear relationship for tetramethoxy gossypol in the concentration range of 0.211–1.056 mg/mL (r = 0.999 6). The RSD of precision and repeatability was 1.56% and 1.11% (n = 6), respectively. The average recovery was 100.3%, with RSD of 1.12% (n = 3). Free gossypol was detected in 25 out of 90 batches of commercially available cottonseed oils in the range of 0.159– 1.661 mg/mL, and tetramethoxy gossypol in 56 in the range of 0.093–1.661 mg/mL. This method displayed good accuracy, high precision and easy operation and could be widely used to monitor the detoxification of cottonseed oil.

Key words: high performance liquid chromatography, free gossypol, tetramethoxy gossypol, determination