AOAC SPIFAN Nutrients ERP-Final Review (Jan. 2019)

Chol-08/Carn-07 (February 2016) FOR ERP USE ONLY DO NOT DISTRIBUTE

Accuracy Accuracy was tested by spiking (fortifying) with varying concentrations of choline or carnitine in a variety of SPIFAN materials that would best encompass the different types of products and cover the analytical range of the method. Recovery was tested in both the free and total analyses. Recovery towards the SMPR LOQ can be found in Tables 2 and 3. A high fat RTF adult nutritional was spiked with choline to achieve the upper SMPR range for total choline at 250 mg/100g (Table 16). The other products chosen for spike recovery were the SPIFAN Blank milk based RTF infant formula, soy based powder infant formula, and a high protein RTF adult nutritional (Tables 17-22). Each product had endogenous levels of choline and carnitine, so 50 and 100% overspikes were utilized to assess recovery. Each of the products were spiked in duplicate across three days using multiple analysts. The marginal recovery (recovery of spiked amount) plus total recovery (recovery of endogenous + spiked amount) are provided in these tables due to the variability that can be encountered when spiking much less than the endogenous amount. Outliers were not removed from the recovery analysis and calculations used all recovery results. The mean recoveries obtained meet the SMPR requirements over the analytical ranges required. Another assessment of the total analysis was performed to determine the efficiency of hydrolyzing bound forms of carnitine and choline. Raw materials of acetylcarnitine, acetylcholine, and phosphatidylcholine were tested through the total analysis at levels that would be at the upper range of the SMPR (Table 23). Acetylcholine and acetylcarnitine showed recoveries over 100% that indicated complete hydrolysis of these forms. Nominal purities for the raw material provided by the manufacturer were used to calculate the recoveries. Although the recovery analysis of these materials may not be the most accurate, it still is good indication that these forms are being completely hydrolyzed through the method. The phosphatidylcholine showed more variable recoveries, but the overall mean recovery verifies an almost complete hydrolysis of this bound form. NIST SRM 1849a was also analyzed over four days in triplicate and results obtained meet the certified ranges or fall close to the informational mass fraction value given. The carnitine certified range is understood as free carnitine and not total carnitine. The SRM showed an increase in the total analysis from the free analysis, which indicates the presence of bound forms of carnitine in the SRM.