6. AOACSPIFANMethods-2018Awards

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pediatric, and adult nutritionals, and none of the published myo-inositol methods will only determine both free myo- inositol and myo-inositol bound as phosphatidylinositol. In 2011 and 2012, the AOAC Expert Review Panel (ERP) on SPIFAN Nutrient Methods granted First Action status to two inositol methods, AOAC 2011.18 (4) and AOAC 2012.12  (5). Both of these methods use ion chromatography and pulsed amperometric detection with a gold electrode, but they differ in their sample preparation procedures and chromatographic separations. Single- laboratory validations (SLVs) were completed with both of these methods, and in March 2013AOAC 2011.18 was selected by the ERP for further evaluation with a multilaboratory collaborative study to determine method reproducibility (4, 6). Consistent with the requirements of the SMPRs, the method chosen by the ERP for a multilaboratory collaborative study (AOAC 2011.18 ) determines only free and phosphatidylinositol bound myo-inositol. With this method, free myo-inositol and phosphatidylinositol bound myo- inositol are extracted using two different sample preparation procedures, separated by ion chromatography using a combination of Dionex PA1 and MA1 columns with column switching, and detected with pulsed amperometry using a gold electrode. Free myo-inositol is extracted from samples with dilute hydrochloric acid and water. Phosphatidylinositol is extracted from samples with chloroform and separated from other fats with silica SPE cartridges. Myo-inositol is then released from the glycerol backbone with concentrated acetic and hydrochloric acids at 120°C. Initially 15 laboratories expressed interest in completing the multilaboratory collaborative study, but only 10 laboratories were able to participate. The 10 participating laboratories were located in five different countries. The remaining five laboratories were not able to participate because of time and resource constraints and issues with the importation of samples into their countries. One of the participating laboratories did not receive the liquid products, and one laboratory was only able to complete the free myo-inositol testing. Before actual multilaboratory collaborative study samples were analyzed, each participating laboratory was asked to analyze a practice sample, Standard Reference Material (SRM) 1849a, in duplicate in order to identify and resolve any testing issues that they may have had executing the method. During the analysis of the practice sample for phosphatidylinositol bound myo-inositol, it was discovered that there were significant differences between the ovens used by the study participants. Even though all oven temperatures were set at 110°C, it appeared that in some laboratories samples did not receive enough heat during the hydrolysis step to efficiently release bound myo- inositol. In all cases when laboratories were asked to increase oven temperatures 10–20°C, myo-inositol recoveries improved. After approval of the practice sample results by the Study Directors, laboratories began testing the study samples. Multilaboratory Collaborative Study

Each participating laboratory received blind duplicates of nine SPIFAN matrixes fortified with myo-inositol or with significant levels of inherent myo-inositol. The SPIFAN matrixes analyzed included SRM 1849a, an infant formula partially hydrolyzed milk based powder, an infant formula partially hydrolyzed soy-based powder, a child formula powder, an infant elemental powder, an infant formula milk-based powder, an infant formula soy-based powder, an infant formula milk-based RTF, and an unfortified infant formula milk-based RTF. Per SPIFAN requirements, participants were asked to reconstitute all powders prior to analysis. SRM 1849a was reconstituted by dissolving the entire contents of the sachet (10 g) in 90 mL water. All other powders were reconstituted by dissolving 25 g of powder in 200 mL laboratory water. For free myo-inositol analyses, participants were asked to analyze all 18 samples on one day, and for phosphatidylinositol bound myo-inositol analyses participants were asked to split the 18 samples into two groups of 10 and eight according to the data reporting sheets in the study protocol and to test each group on a separate day. Although the original AOAC First Action method 2011.18 published in the Journal of AOAC INTERNATIONAL  (4) included a step to dilute each sample with water before adding 50% sodium hydroxide after sample hydrolysis, this step was accidentally omitted from the Official First Action method on the AOAC website and the original AOAC 2011.18 multilaboratory study protocol. This error was discovered after one of the participating laboratories reported a more vigorous than expected reaction when 50% sodium hydroxide was added to the hydrolyzed sample. The protocol was corrected, and the revised protocol was sent to all study participants. It should also be noted that the original method validation data published in the First Action 2011.18 manuscript (4) were generated using direct powder weights. Upon completion of the sample analyses, participating laboratories were asked to send all of their data to the Study Directors. This included all standard and sample chromatograms for the instrument check, practice and test sample analyses, standard curve information, calculations, and completed Reporting of Analysis Forms with dilution and sample weights. Participants were also asked to report any deviations to the method and any relevant comments based on their experiences with the method. All data were statistically analyzed according to AOAC INTERNATIONAL guidelines to determine overall mean, repeatability SD (s r ), repeatability RSD (RSD r ), reproducibility SD (s R ), reproducibility RSD (RSD R ), and Horwitz ratio (HorRat; 7). Cochran ( P = 0.025, one-tail) and Grubbs’ (single and double, P = 0.025, two-tail) tests were used to determine statistical outliers. Myo-inositol SPIFAN SMPRs for repeatability were ≤5% RSD at myo-inositol concentrations of 2–68 mg/100 g RTF liquid. Myo-inositol SPIFAN SMPRs for reproducibility were ≤8% RSD in products with myo-inositol concentrations ranging from 2 to 68 mg/100 g of RTF liquid.