122 / 258
1396
Jing
et al.
: J
ournal of
AOAC I
nternational
Vol. 98, No. 5, 2015
(BEH) HILIC 1.7
µ
m, 2.1 × 100 mm (Waters Corp., Milford,
MA) or equivalent.
(
b
)
Liquid chromatograph.
—Waters Acquity UPLC Binary
or equivalent.
(
c
)
Solvent manager.
—Capable of 15 000 psi or equivalent.
(Waters Corp.)
(
d
)
Detector.
—Waters Acquity TQD or Xevo TQS triple
quadrupole mass spectrometer (Waters Corp.) or equivalent.
(
e
)
Injector.
—Waters Acquity sample manager with
integrated column oven or equivalent.
(
f
)
Autosampler
.—Waters Acquity with cooler and vials, or
equivalent.
(
g
)
Nitrogen generator
.—Peak Scientific (Billerica, MA)
Model NM30LA or equivalent.
(
h
)
Data system.
—Waters MassLynx, latest revision or
equivalent.
(
i
)
Microwave digestion system.
—MarsXpress (CEM,
Matthews, NC) or equivalent.
(
j
)
Reaction vessel.
—50 mL Teflon
®
(CEM) or equivalent.
(
k
)
Centrifuge tubes.
—Polypropylene, 50 mL capacity,
disposable, or equivalent.
(
l
)
Vortex mixer with flat and cone top.
(
m
)
Balance 1.—
Readable to at least 0.0001 g (Mettler-
Toledo AT200, Columbus, OH)
or equivalent.
(
n
)
Balance 2.—
Readable to at least 0.001 mg (Mettler-
Toledo XP 6) or equivalent.
(
o
)
Syringe.—
1 mL Luer tip.
(
p
)
Filters.
—0.45 µm nylon membrane with syringe tip or
equivalent.
(
q
)
Pipets.—
5–25, 50–250, and 100–1000 μL adjustable.
(
r
)
Pipet tips.
(
s
)
pH meter.
(
t
)
Volumetric flasks.—
10, 50, 100, 250 mL.
C. Chemicals and Solvents
(
a
)
Acetonitrile.
—Fisher Optimal (Fisher Scientific, Fair
Lawn, NJ) LC/MS grade, or equivalent.
(
b
)
Nitric acid.
—69–70% reagent grade, J.T. Baker (Avantor
Center Valley, PA)
or equivalent.
(
c
)
Ammonium acetate.
—Fluka (Sigma-Aldrich, St. Louis,
MO) LC/MS additive grade or equivalent.
(
d
)
Ammonium hydroxide.
—28–30% reagent grade [for
adjusting pH in mobile phase also; check before use; MACRON
(Avantor Center Valley, PA)
or equivalent)].
(
e
)
Deionized laboratory water.—
≥18 Mohm/cm (EMD
Millipore Corp. Billerica, MA, or equivalent).
(
f
)
pH buffer solutions
.—pH 4.0, 7.0, 10.0.
D. Standards
(
a
)
L-Carnitine
.—USP Reference Standard Cat. No.
1359903 or equivalent; store desiccated.
(
b
)
L-Carnitine-d
3
HCl (methyl-d
3
)
.—Cambridge Isotope
Laboratories Inc. (Andover, MA)
DLM-1871-0.1; store
refrigerated.
(
c
)
Choline chloride
.—USP Reference Standard Cat. No.
1133547 or equivalent.
(
d
)
Choline chloride-d
9
(trimethyl-d
9
98%)
.—DLM-549-1
(Cambridge Isotope Laboratories Inc.).
E. Preparation of Reagents/Standard Solutions
(
a
)
Mobile phase.—
(
1
)
Mobile phase A.
—10 mMammonium
acetate [water–acetonitrile (95 + 5, v/v)]. Quantitatively
transfer 770 (±15) mg ammonium acetate into a 1 L glass bottle
and dissolve with 950 mL laboratory water. Adjust pH up to
8.2–8.6 by ammonium hydroxide,
C
(
d
). Record the amount
of ammonium hydroxide added. Add 50 mL acetonitrile to the
bottle and mix well. Store at room temperature. Expiration:
1 week.
(
2
)
Mobile phase B.
—10 mM ammonium acetate
[water–acetonitrile (5 + 95, v/v)]. Weigh 770 (±15) mg
ammonium acetate into a 100 mL beaker. Dissolve with 50 mL
laboratory water. Add same amount of ammonium hydroxide
as added into mobile phase A and then mix well. Quantitatively
transfer the 50 mL solution into a 1 L glass bottle. Add
950 mL acetonitrile into the bottle and mix well. Store at room
temperature. Expiration: 2 weeks.
(
b
)
Native stock solutions.—
(
1
)
Choline hydroxide
(approximately 3000 µg/mL).—
Store at 2–8°C. Expiration:
1 month (currently trying to extend to 1 year). (
a
)
Dry
approximately 200 mg choline chloride USPReference Standard
Table 2014.04A. Volumes of each stock solution for IWS
IWS
Stock vol., µL
MIX-IS, µL
Final vol., mL
1
25
100
50
2
125
100
50
3
250
100
50
Table 2014.04B. Concentration of standards
µg/mL
Standard
Choline
Choline chloride-d
9
Carnitine
Carnitine chloride-d
3
Diluent
Final vol., mL
IWS 1
1.5
1.6
0.375
0.8
Water
50
IWS 2
7.5
1.6
1.875
0.8
Water
50
IWS 3
15
1.6
3.750
0.8
Water
50
ng/mL
90%
WS 1
30
32
7.5
16
MeCN
a
10
WS 2
150
32
37.5
16
MeCN
10
WS 3
300
32
75
16
MeCN
10
a
MeCN = Acetonitrile.
122