EXAMINATION OF VARIOUS GOSSYPOL DETECTION METHODS FOR IMPROVEMENT OF SENSITIVITY (GLANDED COTTONSEED, HPLC ANALYSIS, PIGMENT, FLUORESCENCE).

摘要

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 (mu)-Bondapak C(,18) column with 0.001 M phosphate buffer pH 3.5 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 (LESSTHEQ) 0.01). With the injection volume varied from 10 to 50 (mu)l, the detection limit ranged from 0.5 to (mu)g/ml.;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 increasing the absorbance, whereas gossypol alone was separated and detected in the HPLC method.;The formation of gossypol-acetal, from the gossypol standard and methanol, was investigated as a mean of analyzing gossypol by fluorometry. This derivative exhibited the maximum emission intensity at 385 nm when it was excited at 280 nm. The relationship between gossypol concentration and the relative intensity of emission radiation was linear from 0.01 to 10 (mu)g/ml, which is 100 times more sensitive than the spectrophotometric method. Unfortunately, the cottonseed extract also contained flavonoids having fluorescent properties which interfered with detection the gossypol-acetal derivative. Additional in-depth study should be conducted to improve this technique as an alternative method for quantifying very low concentration of gossypol in cottonseed products.;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 (mu)g of gossypol with a correlation coefficient for linear regression = 0.992 (p (LESSTHEQ) 0.01).