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Abstract
A Single Laboratory Validation (SLV) was conducted to determine free and total choline
and carnitine in infant formula and adult/pediatric nutritional formula utilizing Liquid
Chromatography/Tandem Mass Spectrometry (LC/MS/MS). The free method involves
extracting choline and carnitine using water extraction, addition of acetonitrile, filtration,
and injection. The total method involves extraction of choline and carnitine using acid
assisted microwave hydrolysis with nitric acid, followed by filtration, addition of
acetonitrile, and injection. The separation utilizes strong cation exchange (SCX) with
electrospray ionization (ESI) in positive mode.
Introduction
A single laboratory validation was conducted on the Stakeholder Panel of Infant Formula
and Adult Nutritionals (SPIFAN) test kit of materials to determine if this method meets
the Standard Method Performance Requirements (SMPR) of 2012.010 and 2012.013 for
L-Carnitine and Total Choline in Infant Formula and Adult/Pediatric Nutritional
Formula. The SMPR method criteria for Limit of Quantitation (LOQ), Limit of
Detection (LOD), Analytical Range, Repeatability, and Accuracy are all addressed in the
report. Linearity and Specificity are also included. The time of analysis for either free or
total analyses were less than 10 hours from sample preparation to result. All powdered
samples were reconstituted before each analysis by weighing approximately 25 grams of
material and diluting to approximately 225 grams with purified water prior to sampling.
National Institute of Standards and Technology Standard Reference Material (NIST
SRM) 1849a was reconstituted 10X by weighing approximately 1 g of material and
diluting to 10 grams with purified water prior to sampling. All ready to feed (RTF)
products were weighed as received, except both high fat and high protein adult nutritional
RTF products that were diluted 5X before weighing for total choline and carnitine
analysis. The list of matrices used for testing are shown in Table 1. The SMPR for total
choline and L-carnitine are in Tables 2 and 3. The method is provided as a separate
attachment.
Chol-08/Carn-07 (February 2016)
FOR ERP USE ONLY
DO NOT DISTRIBUTE