AOAC Final Action Methods in 2019

( u )  Bottle top dispenser .—5 mL acid resistant (Brand, Essex, CT, USA or equivalent). ( v )  Desiccator .—Glass (VWR or equivalent.) Note : Nonspecific binding can occur with these analytes when using glassware, so plasticware should be used at all times for standard/sample preparation. All laboratory plasticware should be single-use whenever possible. Positive displacement pipets are also mandatory for pipeting to avoid contamination and for accuracy with organic solvents. C. Chemicals and Reagents ( a )  Water/ultra-pure water (UPW) .—OptimaMS grade (Thermo Fisher Scientific, Waltham, MA, USA or equivalent). ( b )  Acetonitrile .—Optima MS grade (Thermo Fisher Scientific or equivalent). ( c )  Ammonium formate .—Optima MS grade (Thermo Fisher Scientific or equivalent). ( d )  Formic acid .—Optima MS grade (Thermo Fisher Scientific or equivalent). ( e )  Nitric acid .—70% (w/w), ACS grade (Avantor, Center Valley, PA, USA or equivalent). ( f )  Isopropanol .—Optima MS grade (Thermo Fisher Scientific or equivalent). ( g )  Desiccant .—VWR or equivalent. Note : All use of water in this method must be high-purity MS grade water. Perform all preparation steps with nitric acid in a fume hood, and wear necessary personal protective equipment when handling. D. Reference Standards ( a )  Reference standards .—( 1 )  L-Carnitine.— U.S. Pharmacopeia (Rockville, MD, USA) or equivalent). ( 2 )  Choline bitartrate.— TCI (Tokyo, Japan) or equivalent. ( b )  Reference internal standards .—( 1 )  L-Carnitine-d3 HCl.— CDN Isotopes (Pointe Claire, Québec, Canada) or equivalent. ( 2 )  Choline-1,1,2,2-d4 chloride.— CDN Isotopes or equivalent. E. Procedure ( a )  Reagent preparation .—( 1 )  Preparation of mobile phase.— ( a )  Mobile phase A.— 5 mM ammonium formate in water:ACN (50 + 50, v/v) with 0.2% formic acid. Weigh 0.32 ± 0.01 g ammonium formate into a 1 L mobile phase bottle containing 500 mL UPW. Add a stir bar and then mix on a stir plate until dissolved. Add 500  L acetonitrile to the mobile phase container, along with 2.00 mL formic acid. Add a stir bar and mix on a stir plate until thoroughly mixed, typically for about 2 min. Solution is stable for 2 weeks when stored at room temperature. ( b )  Mobile phase B.— 30 mM ammonium formate in water:ACN (50 + 50, v/v) with 0.2% formic acid. Weigh 1.89 ± 0.01 g ammonium formate into a 1 L mobile phase bottle containing 500 mL UPW. Add a stir bar and then mix on a stir plate until dissolved. Add 500 mL acetonitrile to the mobile phase container, along with 2.00 mL formic acid. Add a stir bar and mix on a stir plate until thoroughly mixed, typically for about 2 min. Solution is stable for 2 weeks when stored at room temperature. Note : Rinse all mobile phase bottles thoroughly prior to use with UPW and isopropanol. Use additional rinse with concentrated acid if high background signal is present. ( c )  Injector wash .—Mobile phase B is used as HPLC injector wash.

AOAC Official Method 2015.10 Free and Total Choline and Carnitine in Infant Formula and Adult/Pediatric Nutritional Formula Liquid Chromatography/Tandem Mass Spectrometry First Action 2015 Revised First Action 2016 Final Action 2019

[Applicable to the measurement of total or free carnitine and choline by ultra-high-performance liquid chromatography with tandem mass spectrometry (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 LC-MS/ MS detection. B. Apparatus ( a )  LC system .—Prominence (Shimadzu, Kyoto, Japan) or equivalent. ( b )  MS/MS system .—API 4000 with electrospray ionization (ESI; AB Sciex, Framingham, MA, USA) or equivalent. ( c )  MS software .—Analyst (AB Sciex) or equivalent. ( d )  Analytical column .—Zorbax 300-SCX, 3.0 × 50 mm, 5 µm (Agilent, Santa Clara, CA, USA) or equivalent. ( e )  Microwave .—MARS6 (CEM, Mathews, NC, USA) 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, USA) or equivalent. ( h )  Analytical balances .—Model CPA225D (Sartorius, Goettingen, Germany) or equivalent. ( i )  Horizontal shaker .—Model 6010 (Eberbach, Ann Arbor, MI, USA) or equivalent. ( j )  Magnetic stir plate .—Model PC-420D (Corning, Corning, NY, USA) or equivalent. ( k )  Positive displacement pipets .—Microman, various sizes (Gilson, Middleton, WI, USA) 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, USA or equivalent). ( p )  Disposable syringes .—3 mL (BD Biosciences, Franklin Lakes, NJ, USA or equivalent). ( q )  Graduated cylinders .—Assorted sizes (VWR or equivalent). ( r )  Magnetic stir bars .—7.9 × 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).

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