Single laboratory method performance evaluation for the analysis of total food folate by trienzyme extraction and microplate assay.

Single laboratory method performance parameters, including the calibration curve, accuracy, recovery, precision, limit of detection (LOD), and limit of quantification (LOQ), were evaluated for the analysis of total food folate by the trienzyme extraction and microplate assay with Lactobacillus casei subsp. rhamnosus. Standard Reference Material (SRM) 1546 (meat homogenate), SRM 2383 (baby food composite), SRM 1846 (infant formula), Certified Reference Material (CRM) 121 (wholemeal flour), and CRM 485 (mixed vegetables), representing a broad selection of food matrices, were used to evaluate the performance of the method. A generated 4-parameter logistic equation of the calibration curve was y= (0.0705 - 1.0396)/(1 + (x/0.0165) (1.3072)) + 1.0396 (P < 0.0001). The test of parallelism demonstrated that matrix components in the food extracts did not affect the accuracy. Measured values of the SRMs and CRMs were within their certified or reference values. Recoveries for all reference materials met the requirements of the AOAC guidelines for single laboratory validation. Precision measured as repeatability, including simultaneous and consecutive replicates for each SRM and CRM, met the Horwitz criterion. LOD and LOQ values were 0.3 and 0.6 mug/100 g, respectively. The results showed that trienzyme digestion using alpha-amylase, Pronase(R), and conjugase from chicken pancreas 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.