6. AOACSPIFANMethods-2018Awards

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204 E llingson et al .: J ournal of AOAC I nternational V ol . 99, N o . 1, 2016

Infant Formula and Adult Nutritionals

Determination of Free and Total Choline and Carnitine in Infant Formula and Adult/Pediatric Nutritional Formula by Liquid Chromatography/Tandem Mass Spectrometry (LC/MS/MS): Single-Laboratory Validation, First Action 2015.10 D avid J. E llingson , J effrey J. S hippar , and J ustin M. G ilmore Covance Laboratories, Nutritional Chemistry and Food Safety, 3301 Kinsman Blvd, Madison, WI 53704-2523

Analytical methods for the analysis of both L-carnitine and choline are needed for reliable and accurate determination in infant formula and adult/pediatric nutritional formula. These compounds are different in how they are utilized by the human body, but are structurally similar. L-carnitine and choline are quaternary ammonium compounds, enabling both to be retained under acidic conditions with strong cation exchange (SCX)chromatography.Thismethodanalyzesbothcompounds simultaneously as either the free forms or as a total amount that includes bound sources such as phosphatidylcholine or acetylcarnitine. The free analysis consists of water extraction and analysis by LC/MS/MS, while the total analysis consists of extraction by acid assisted microwave hydrolysis and analysis by LC/MS/MS. Calibration standards used for calculations are extracted with all samples in the batch. Asingle laboratory validation (SLV) was performed following the guidelines of the AOAC Stakeholder Panel on Infant Formula and Adult Nutritionals (SPIFAN) utilizing the kit of materials provided. The results achievedmeet the requirements of SMPR 2012.010 and 2012.013 for L-carnitine and total choline, respectively. A n analytical method for the analysis of l -carnitine and total choline in infant formula (IF) and adult/pediatric nutritional formula is needed tomeet the StandardMethod Performance Requirements (SMPRs ® ) of the AOAC Stakeholder Panel on Infant Formula and Adult Nutritionals (SPIFAN; 1, 2). There are currently other methods that can either quantify carnitine or total choline in IFs (3–5). We developed a method that can analyze these compounds simultaneously using strong cation-exchange (SCX) chromatography because both are quaternary ammonium compounds and can be retained under acidic conditions. This method can be used to quantify the free or total choline and carnitine content of a sample. The free portion is analyzed using water extraction, whereas the total extraction uses Received June 1, 2015. Accepted by AK July 31, 2015. This method was approved by the Expert Review Panel for Infant Formula and Adult Nutritionals as First Action. The Expert Review Panel for Infant Formula and Adult Nutritionals invites method users to provide feedback on the First Action methods. Feedback from method users will help verify that the methods are fit-for-purpose and are critical for gaining global recognition and acceptance of the methods. Comments can be sent directly to the corresponding author or methodfeedback@aoac.org. Corresponding author’s e-mail: david.ellingson@covance.com DOI: 10.5740/jaoacint.15-0144

acid-assisted microwave hydrolysis. The total amount can include contribution from bound sources such as phosphatidylcholine or acetylcarnitine. Both extraction methods use LC tandem mass spectrometry (MS/MS) analysis with electrospray ionization (ESI). Calibration standards are included through each extraction procedure for greater assurance with quantification, and the acidic conditions of the total extraction allowdirect injection after dilution with acetonitrile for fast analysis. A single-laboratory validation (SLV) was performed with this method using the SPIFAN SLV kit of materials. All requirements of SMPRs were met, except the ability to differentiate l - and d -carnitine. Verification for recovery of the bound forms of choline and carnitine commonly found in these matrixes was also completed. AOAC Official Method 2015.10 Determination of Free and Total Choline and Carnitine in Infant Formula and Adult/Pediatric Nutritional Formula Liquid Chromatography/Tandem Mass Spectrometry (LC/MS/MS) First Action 2015 [Applicable to the determination of free and total choline and carnitine in infant formula and adult/pediatric nutritional formula.] Caution : It is recommended that all preparation steps with

nitric acid be performed within a fume hood, and the necessary personal protective equipment used when handling.

A. Principle

The method uses a water extraction for free analysis and acid-assisted microwave hydrolysis for total analysis. Both compounds are simultaneously analyzed and quantified by LC/MS/MS with ESI.

B. Apparatus

(a)  LC system .—Prominence, Shimadzu (Kyoto, Japan) or equivalent. (b)  MS/MS system .—API 4000 with ESI, ABSciex (Framingham, MA) or equivalent. (c)  Mass spectrometry software .—Analyst (ABSciex) or equivalent. (d)  Analytical column .—Zorbax 300-SCX, 3.0 × 50 mm, 5 μm, Agilent (Santa Clara, CA) or equivalent.