Microplate assay with trienzyme extraction for total folate analysis in foods
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In order to verify the trienzyme digestion coupled with a 96-well microplate assay is suitable for folate analysis in a variety of foods, single laboratory method evaluation was conducted using reference materials representing a broad selection of food matrices. A four parameter logistic equation of the calibration curve was generated using nonlinear regression. Accuracy, recovery and precision of the method met acceptable criteria as presented by AOAC International guidelines. Limit of detection and limit of quantitation were 0.3 and 0.6 1/4g/100g, respectively. The results showed that trienzyme digestion coupled with a 96-well microplate assay provided a highly accurate, reproducible and sensitive method for the determination of folate in a variety of foods. The trienzyme digestion was optimized for food folate extraction from vegetables using response surface methodology (RSM). The predicted second-order polynomial model was 2 R adequate (R= 0.947), without significant lack of fit (p > 0.1). Both Pronaseand R.-amylase have significant effects on the extraction. Ridge analysis gave an optimum trienzyme digestion R times: Pronase,1.5 h; [alpha].-amylase, 1.5 h; conjugase, 3 h. The experimental value for CRM 485 (mixed vegetables) under this optimum digestion was close to the predicted value from the model, confirming the validity and adequacy of the model. The optimized trienzyme digestion condition was applied to 5 vegetables and yielded higher folate levels than AOAC Official Method 2004.05. In order to provide reliable folate data for fruits, vegetables and their commercial products, 250 fruit samples and 171 vegetables samples were collected through the National Food and Nutrient Analysis Program (NFNAP) or other United States Department of Agriculture (USDA) food sampling programs. The assay procedure followed AOAC Official Method 2004.05. Folate in most of the fruits and fruit products ranged from 10~80 m g/100g. Most tropical fruits (30-77) are high or concentrated natural sources of folate. Folate contents in vegetables and their products varied from 3 to 307 m g/100g. Edamame showed the highest folate content (307, m g/100g), followed by leafy vegetables (137-249), green beans (28), broccoli (67), corn (38), green peas (31), carrots (26), orange juice (25).