AOAC OMA# 2011.06 Final Action Review-OMB

1888  B handari et al . : J ournal of AOAC I nternational V ol . 101, N o . 6, 2018

Figure 2011.06. Chromatogram of folate analysis providing elution pattern of folate vitamers. 5-methyltetrahydrofolic acid peak is marked as 5-CH3-THF, 5-formyltetrahydrofolic acid peak is marked as 5-CHO-THF, and folic acid peak is marked as FA. Color images are available online at http://aoac.publisher.ingentaconnect.com/content/aoac/jaoac.

assay = 0.03 mL × 20 mcg/mL (in conjugase activity substrate solution) = about 600 ng. Pte-Glu3 (nanomoles) in conjugase assay = Pte-Glu3 (nanograms) in the assay / 699.6 (Pte-Glu3 molecular weight). Each molecule of Pte-Glu3 on hydrolysis yields 1 molecule of folic acid. The percent conversion of Pte-Glu3 to folic acid = [folic acid (nanomoles) released in the assay / Pte-Glu3 (nanomoles) in the assay] × 100. H. Example Chromatogram See Figure 2011.06 . Formal invitation was sent to more than 40 laboratories around the world by the study director. Initially, 18 laboratories indicated their interest to participate in the study. One of the laboratories could not continue because the samples could not reach them because of Customs clearance issues. Five laboratories could not continue because of a lack of time and changes in their internal priorities. Twelve laboratories participated in the study. One of the laboratories could not qualify for analysis of the MLT samples because their results for the practice samples were far from the expected range. It was noticed that their results of the folic acid method blank were quite high because of interference caused by chromatographic issues. Eleven laboratories qualified to run the MLT samples, and they all completed the study and analyzed all of the MLT samples. They were located in five countries and represented a wide range of food-testing laboratories, including contract analytical services, infant formula manufacturers, research institutes, and food and supplement manufacturers. MLT Samples The study took place using the SPIFAN samples, representing the matrices in the scope of the applicability of the method. I. MLT Protocol Participating Laboratories

The samples included infant formulas and adult nutritionals made from any combination of milk, soy, whey, hydrolyzed proteins, starch, and amino acids, with and without intact proteins. The MLT study included a total of 11 samples that were analyzed in blind duplicates to evaluate the method precision. A Standard Reference Material (SRM 1869, Infant/ Adult Nutritional Formula) from the National Institute of Standards and Technology (Gaithersburg, MD) with the known consensus value for folic acid was included in the set. The potential laboratories who agreed to participate in the study were requested to analyze two practice samples using the method described in the protocol to qualify to test the MLT samples. All of the samples were blinded and codified before sending to the participants. Each collaborator received a detailed study protocol that was agreed in advance by the SPIFAN review panel. The participants also separately received two sets of the samples. One set of the samples consisted of two practice samples (blind coded), and the other set contained 11 MLT samples in blind duplicates. The study was divided in two parts. In the first part of the study, the participants analyzed two practice samples using the method described in the protocol. Any deviation from the method provided was to be recorded and reported. The results were communicated to the study director using an electronic template provided with the protocol. The provided data were reviewed by the study director. The participants whose results were within the expected range were allowed to proceed to the second part of the study to test the MLT samples. The 11 MLT samples were tested over 2 separate days as blind-coded duplicate pairs (11 samples per day). Each participating laboratory provided the results to the study director after the analysis of the MLT samples by using an electronic template similar to the one used in part I for practice samples. The results reported total folate expressed as folic acid μg/100 g of sample. All laboratories also reported additional information, such as the weight of sample aliquot used in the analysis, relevant volumes, and peak areas. Study Design