Examination of various gossypol detection methods for improvement of sensitivity

Typescript (photocopy).A high performance liquid chromatographic (HPLC) technique was developed to analyze gossypol in cottonseed products with higher sensitivity and specificity than the official spectrophotometric method. Seventy percent aqueous acetone extract of cottonseed products was analyzed for free gossypol content on a μ-Bondapak C18 column with a mobile phase consisting of Tetrahydrofuran(UV):0.001 M phosphate buffer pH 3.5 (60:40 v:v) at a flow rate of 1.7 ml/min. The chromatogram was detected by a UV detector at 254 nm, and peak height was measured for quantification by an external standard method. The relationship between amount of gossypol injected and peak height was linear over at least 1000 folds (10 to 1000 ng) with a correlation coefficient for linear regression = 0.998 (p [less than or equal to] 0.01). With the injection volume varied from 10 to 50 μl, the detection limit ranged from 0.5 to 1 μg/ml. Aminopropanol digestion of bound gossypol and the HPLC method shortened the time for analysis of total gossypol to only 1 hr, compared with 1 day in the spectrophotometric method. The standard curve was linear, from 0.1 to 0.8 Mg of gossypol with a correlation coefficient for linear regression = 0.992 (p [less than or equal to] 0.01). Gossypol in cottonseed oil was selectively separated by extraction of cottonseed oil with a hexane and N,N,dimethylformamide:water (2:1 v:v) solvent mixture. After filtration, the solvent mixture was injected into the HPLC and eluted in less than 15 min. The spectrophotometric method showed a gossypol content in glanded cottonseed oil 5 to 6 times higher than did the HPLC method. This probably resulted from the gossypol derivatives and other interfering compounds possibly reacting with p-anisidine to develop color and increasind the absorbance, whereas gossypol alone was separated and detected in the HPLC method..