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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