AOACSPIFANMethods-2017Awards

llingson

et al

ournal of

AOAC I

nternational

99, N

1, 2016

205

(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.

C. Chemicals and Reagents

(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

.—

-Carnitine, USP (Rockville, MD)

or equivalent.

(i)

Reference standard

.—Choline bitartrate, TCI (Tokyo,

Japan) or equivalent.

(j)

Reference internal standard

.—

-Carnitine-d

HCl, CDN

Isotopes (Pointe Claire, Québec, Canada or equivalent).

(k)

Reference

internal

standard

.—Choline-1,1,2,2-d

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 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.

E. Preparation of Standard Solutions

The carnitine stock standard was prepared at a concentration

of 25 mg/mL by weighing 0.25 g

-carnitine into a 20 mL

polypropylene tube followed by 10 mL water to dissolve. The

purity of

-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

-carnitine-d

and 35 mg choline-1,1,2,2-d

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.

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.

100