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

2015.10 (Jan. 2019) Carn-07 MLT Report FOR ERP USE ONLY DO NOT DISTRIBUTE

Appendix III. Analysis method.

Method Determination of Free and Total Choline and Carnitine in Infant Formula and Adult/Pediatric Nutritional Formula by Liquid Chromatography/Tandem Mass Spectrometry (LCMSMS): (Applicable to the measurement of total or free carnitine and choline by UHPLC MS/ MS in infant formula and adult/pediatric nutritional formula.) Caution: Refer to Material Safety Data Sheets (MSDS) for safety precautions when using chemicals. Use personal protective equipment recommended in MSDS. A. Principle Samples are extracted in water for free carnitine and choline. For total carnitine and choline, samples are digested with nitric acid by microwave. Free and total extracts are both diluted to volume with water, mixed with acetonitrile, and analyzed using liquid chromatography (LC) with tandem mass spectrometric (MS/MS) detection. B. Apparatus (a) LC system . – Prominence (Shimadzu, Kyoto, Japan) or equivalent. (b) MS/MS system . – API 4000 with Electrospray Ionization (ESI) (ABSciex, Framingham, MA) or equivalent. (c) Mass Spectrometry Software . – Analyst (ABSciex) or equivalent. (d) Analytical column . – Zorbax 300-SCX, 3.0 x 50 mm, 5 µm (Agilent, Santa Clara, CA) or equivalent. (e) Microwave . – MARS6 (CEM, Mathews, NC) or equivalent. (f) Microwave turntable, liner, and cap . – MARSXpress, 55 mL PFA Teflon®, 40 position (CEM or equivalent). (g) Vortex mixer. – (VWR, West Chester, PA) or equivalent. (h) Analytical balances. – Model CPA225D (Sartorius, Goettingen, Germany) or equivalent. (i) Horizontal shaker. – Model 6010 (Eberbach, Ann Arbor, MI) or equivalent. (j) Magnetic stir plate. – Model PC-420D (Corning, Corning, NY) or equivalent. (k) Positive displacement pipets. – Microman, various sizes (Gilson, Middleton, WI) or equivalent. (l) Repeater positive displacement pipet . – Repeater Plus (Eppendorf, Hamburg, Germany) or equivalent. (m) Polypropylene tubes . – Digitube, assorted sizes (SCP Science, Montreal, Canada) or equivalent. (n) Mobile phase containers . – 2 L glass (VWR) or equivalent. (o) Syringe filters . – 0.45 µm PTFE and GHP (Pall, Plano, TX) or equivalent. (p) Disposable syringes . – 3 mL (BD Biosciences, Franklin Lakes, NJ) or equivalent. (q) Graduated cylinders . – Assorted sizes (VWR) or equivalent. (r) Magnetic stir bars . – 7.9 x 50 mm (VWR) or equivalent. (s) Autosampler vials/caps . – 1.5 mL silanized crimp top (VWR) or equivalent. (t) Microcentrifuge tubes . – 1.5 mL polypropylene (VWR) or equivalent. (u) Bottle top dispenser . – 5 mL acid resistant (Brand, Essex, CT) or equivalent.

- 20 -