Methods to be Reviewed by OMB in 2016

126 / 363

Ellingson et al.: Journal of AOAC International Vol. 99, N o. 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.—l-Carnitine, USP (Rockville, MD)
or equivalent.
(i) Reference standard.—Choline bitartrate, TCI (Tokyo,
Japan) or equivalent.
(j) Reference internal standard.—l-Carnitine-d3 HCl, CDN
Isotopes (Pointe Claire, Québec, Canada or equivalent).
(k) Reference internal standard.—Choline-1,1,2,2-d4
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 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-d3
and 35 mg choline-1,1,2,2-d4 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.
Candidates for 2016 Method of the Year
125