A high-performance liquid chromatographic (HPLC) method has been developed to separate sinapine and sinapic acid from other phenolics in canola seed and meal in a single run. The separation was achieved with a reverse-phase C18 column. Owing to the higher recovery of phenolics and ease of use, refluxing with 100% methanol for 20 min was selected as the extraction method for HPLC analysis and determination of total phenolics using Folin-Ciocalteu reagent. A 10-min isocratic/linear/concave gradient and a 15-min isocratic/ linear gradient were selected as the best gradients for the separation of these phenolic compounds. Peak identities for sinapine and sinapic acid were verified with ion exchange separation followed by HPLC analysis. The method was calibrated using sinapine bisulfate and sinapic acid standards; correlation coefficients (R 2 ) for the calibration curves were 0.997 and 0.999 for sinapine bisulfate and sinapic acid, respectively. The extinction coefficient of sinapine was determined to be 1.16 times that of sinapic acid at the detector wavelength (330 nm). Applying this method to routine canola phenolic analyses can greatly reduce the cost by simplifying the procedures and reducing the time required for each determination.Paper no. J9526 in JAOCS 78, 903-910 (September 2001).Sinapine is a major phenolic compound in canola and rapeseed (1,2). Although sinapine may have physiological functions during germination and maturation stages of canola and rapeseed (3,4), sinapine and related phenolic compounds are responsible for the tainting of brown-shelled eggs (5-7) when the meal is used as a poultry feed and may also contribute to the dark color of the meal (8-10). The presence of sinapine and the related phenolic compounds such as sinapic acid, the hydrolyzed product of sinapine, has been a concern for oilseed breeders and processors (11,12). Rapid and sensitive methods for determining sinapine and related phenolics will facilitate canola/rapeseed breeding and processing. Thinlayer chromatography (13), which has been used extensively to separate phenolics, is difficult to quantitate. Colorimetric methods (1,14), when used to determine individual phenolics, require specific reagents for color development. Furthermore, it is difficult to avoid the interference from other compounds in these systems. Some colorimetric methods and ultraviolet (UV) spectrophotometric methods require purification procedures (15,16). Gas chromatography (2,17) requires the hydrolysis of phenolic esters and the derivatization of the resulting phenolic acids before determination. Therefore, it is an indirect method and not directly applicable to nonvolatile phenolic esters. High-performance liquid chromatography (HPLC) is a rapid and sensitive method for qualitative and quantitative analysis of plant phenolics (18). Several HLPC methods for the determination of phenolic compounds have been developed. Clausen et al. (19) used a reverse-phase Nucleosil 5 C8 column and a Nucleosil 5 C18 column with a 30-min linear gradient composed of ...

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Seventeen varieties of cowpeas grown in Arkansas were analyzed for their phenolic constituents using high-performance liquid chromatography (HPLC). Protocatechuic acid was identified as the major phenolic acid present in esterified forms. The amount of protocatechuic acid increased from trace-3.6 to 9.3-92.7 mg/100 g of flour in the 17 varieties of cowpeas after hydrolysis. Six other phenolic acids, including, p-hydroxybenzoic acid, caffeic acid, p-coumaric acid, ferulic acid, 2,4-dimethoxybenzoic acid, and cinnamic acid, were also identified. These phenolic acids were evenly distributed mainly in free acid forms at <7 mg/100 g of flour. Total phenolic contents determined using Folin-Ciocalteu's reagent were largely different among the 17 varieties, ranging from 34.6 to 376.6 mg/100 g of flour. A comparison of the HPLC chromatograms of the 17 cowpea phenolics before and after alkali hydrolysis indicated the conversion of a pattern with evenly distributed peaks to one with a single major peak for protocatechuic acid, suggesting that the chromatograms before hydrolysis better represent the identities of the cowpea varieties.

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