440 / 670
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.
Chol-08/Carn-07 (February 2016)
FOR ERP USE ONLY
DO NOT DISTRIBUTE