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

AOAC Official Method 2015.10 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

( 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 pipetting to avoid contamination and for accuracy with organic solvents.) C. Chemicals and Reagents (a)  Water .—Optima MS 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. (h)  Reference standard .—l-Carnitine (USP, Rockville, MD, USA or equivalent). (i)  Reference standard .—Choline bitartrate (TCI, Tokyo, Japan or equivalent). (j)  Reference internal standard .—l-Carnitine-d 3 HCl (CDN Isotopes, Pointe Claire, Québec, Canada or equivalent). (k)  Reference internal standard .—Choline-1,1,2,2-d 4 chloride (CDN Isotopes or equivalent). ( Note : All use of water in this method must be high-purity MS-grade water.) D. Mobile Phase Preparation Mobile phase A [5 mM ammonium formate in 50 + 50 (v/v) water–acetonitrile with 0.2% formic acid] was prepared by weighing 0.63 g ammonium formate into a 1 L graduated cylinder. Water was added along with a stir bar and mixed to dissolve before diluting to volume with water. The solution was transferred to a 2 Lmobile phase container along with 1 L acetonitrile, 4 mL formic acid, a stir bar, and then thoroughly mixed. Mobile phase B [30 mM ammonium formate in 50 + 50 (v/v) water–acetonitrile with 0.2% formic acid] was

[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, USA) 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, 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 hydrophilic polypropylene (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. (u)  Bottle top dispenser .—5 mL acid resistant (Brand, Essex, CT, USA or equivalent). (v)  Desiccator .—Glass, VWR or equivalent.

Table 2015.10A. MS/MS settings Ionization

Positive ion electrospray (ESI+)

Mode

MRM

Ion spray voltage

1000 V 550°C

Turbo ion spray temp. Declustering potential

120 V

Dwell time

100 msec

Entrance potential

10 V 25 V

Collision cell exit potential

Collision gas Curtain gas

Nitrogen, 5 psig Nitrogen, 20 psig Nitrogen, 60 psig Nitrogen, 60 psig

Nebulizing gas (gas 1) Auxiliary gas (gas 2)

Needle position

Y = 5 mm, X = 5 mm

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