6. AOACSPIFANMethods-2018Awards

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(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 hydrophilic polypropylene (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 × 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. (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. (a)  Water .—Optima MS grade, Thermo Fisher Scientific (Waltham, MA) 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) 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) 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). C. Chemicals and Reagents

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 L mobile 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 prepared by weighing 3.78 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 was also used for the rinse solutions in the autosampler. The carnitine stock standard was prepared at a concentration of 25 mg/mL by weighing 0.25 g l -carnitine into a 20 mL polypropylene tube followed by 10 mL water to dissolve. The purity of l -carnitine from the Certificate of Analysis (CoA) and moisture determined by Karl Fischer titration immediately at the time of weighing was used to calculate the final concentration of carnitine. The choline stock standard was prepared at a concentration of 25 mg/mL choline by weighing 0.62 g choline bitartrate into a 20 mL polypropylene tube followed by 10 mL water to dissolve. The purity of choline bitartrate from the CoA along with a molecular weight conversion from choline bitartrate to choline of 0.41133, was used to calculate the final concentration of choline. Intermediate working standards were prepared at concentrations of 10, 20, 500, 2000, 4000, and 5000 μg/mL for each analyte using both the stock and higher concentration intermediate working standard solutions using appropriate volumes into 20 mL polypropylene tubes with water as the diluent. All stock and intermediate standard solutions were stable for 2 months when stored at 5 ± 3°C and protected from light. Aliquots of the intermediate working standards were treated through the sample analysis, so the concentrations used for the calibration curves for both free and total analyses were the same numerical values as the intermediate working standards but in ng/mL. Internal stock standards were prepared at a concentration of 2 mg/mL by weighing 25 mg l -carnitine-d 3 and 35 mg choline-1,1,2,2-d 4 into separate 20 mL polypropylene tubes. A volume of 10 mL water was added to each to dissolve, and then both solutions quantitatively transferred to a 100 mL polypropylene tube and diluted to volume with water to prepare an intermediate solution at 200 μg/mL. The purity from the CoA was used to calculate the final concentration of each internal standard. Stability of these solutions was monitored while being stored at 5 ± 3°C and protected from light. E. Preparation of Standard Solutions

F. Sample Preparation

Powder IF and adult nutritionals were reconstituted by weighing 25 g and diluting with water to a final weight of 225 g.