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Jing et al.:
J
ournal of
AOAC I
nternational
V
ol.
98, N
o
. 5, 2015
1397
in a vacuum oven at 65°C (±3°C) for 4 h. (
b
) Weigh 170 (±17) mg
into an appropriate weighing boat. (
c
)
Quantitatively transfer
into a 50 mL volumetric flask. (
d
) Dissolve and bring to volume
with laboratory water. (
Note
: choline chloride is the form
weighed, but many laboratories prefer working in units of
choline hydroxide, which is how the method is presented here.)
(
2
)
L-Carnitine (approximately 750 µg/mL).—
(
a
)
Weigh
150.0 ± 8 mg into an appropriate weighing boat as quickly
as possible. (
b
)
Transfer into a 200 mL volumetric flask.
(
c
)
Dissolve and bring to volume with laboratory water.
(
d
)
Correct weight for moisture content. (
e
) Aliquot the stock
solution into 2 mL plastic vials, with each vial receiving at least
1 mL and store ≤–15°C. Expiration: 1 year.
(
c
)
Internal standard (IS) stock solutions.—
(
1
)
Choline
chloride-d
9
(approximately 20000 µg/mL).—
(
a
) Transfer 1 g
choline chloride-d
9
into a 50 mL volumetric flask. (
b
) Dissolve
and bring to volume with laboratory water. (
c
) Aliquot the stock
solution into 2 mL plastic vials with each vial receiving about
1 mL and store at ≤–15°C. Expiration: 1 year.
(
2
)
L-Carnitine
chloride-d
3
HCl
(approximately
10000 µg/mL).—
(
a
) Transfer 0.5 g L-carnitine chloride-d
3
to a
50 mL flask. (
b
) Dissolve and bring to volume with laboratory
water. (
c
) Aliquot the stock solution into 2 mL plastic vials with
each vial receiving about 1 mL and store at ≤–15°C. Expiration:
1 year.
(
d
)
Mixed intermediate internal standard solution
(MIX-IS)
.—Prepare fresh on day of analysis. Must use same
MIX-IS solution for both calibration working standards and
samples.
(
1
)
Transfer 400 µL of each choline chloride-d
9
and 400 µL
carnitine chloride-d
3
HCl stock solution by a calibrated pipet
into a 10 mL volumetric flask.
(
2
)
Bring to volume with laboratory water and mix well.
(
e
)
Intermediate working standards (IWS).
—Prepare fresh
on day of analysis.
(
1
)
Pipet 100 µL MIX-IS into three individual 50 mL
volumetric flasks.
(
2
)
Using Table
2014.04A
, add the required volumes of the
stock choline and carnitine solution by proper sized calibrated
pipets.
(
3
)
Bring to volume with laboratory water and mix well.
(
f
)
Working standard solution (WS).
–Prepare fresh on day of
analysis. Use 90% acetonitrile in final working solution.
(
1
)
Pipet 200 µL of each IWS (IWS 1–3) into three 10 mL
volumetric flasks.
(
2
)
Add 800 µL laboratory water to each volumetric flask.
(
3
)
Bring to volume with acetonitrile and mix well.
(
4
)
Transfer to autosampler vials for analysis.
(
5
)
Table
2014.04B
summarizes the concentrations of
choline and carnitine and their associated ISs in the three IWS
and in the three WS.
F. Procedure
(
a
)
Liquids
.—For liquids and ready-to-feed products,
select sample weights between 1 and 5 g based on expected
concentration of the two nutrients in each sample.
(
b
)
Powders.
—Powder products are reconstituted with water
prior to analysis, typically 11.1% (w/w; 25 g powder added to
200 g water). Typically a 5 g sample aliquot is taken from the
reconstituted material for analysis, but adjust as needed.
(
c
)
Sample analysis.—
(
1
) Weigh sample into a tarred
microwave reaction vessel.
(
2
) Add 125 µL MIX-IS.
(
3
) Add water to sample to achieve a total sample volume of
approximately 8 mL.
(
4
) Add 2.2 mL of approximately 70% nitric acid and seal
vessels. The final volume should be close to 10 mL (sample +
acid + water + MIX-IS = 10 mL) prior to microwave digestion
which makes acid concentration approximately 3.5 M.
(
5
) Vortex vessels for 30 s, and then place vessels into
turntable and insert into microwave system.
(
6
) Complete microwave digestion using the conditions
defined in Table
2014.04C
. Operate with microwave venting to
a fume hood.
(
7
) After cool down (by air), uncap each vessel, and add
3.5 mL concentrated ammonium hydroxide to each vessel.
Operate in a fume hood.
(
8
) Vortex each sample for 30 s.
(
9
) Allow samples to stand in a hood for 30 min to react any
acylcarnitines under basic conditions.
(
10
) Vortex samples for 30 s.
(
11
) Transfer 1 mL digested sample into a disposable
centrifuge tube containing 25 mL laboratory water.
(
12
) Vortex for 30 s.
(
13
) Filter about 1 mL diluted sample using a 0.45 µm nylon
syringe filter.
(
14
) Transfer 0.1 mL filtered solution into autosampler vial
containing 0.9 mL acetonitrile. Mix well. Sample is ready for
analysis.
(
15
) Samples diluted with acetonitrile are stable for 24 h
Table 2014.04C. Microwave digestion parameters
Power
100% (1600 W)
Ramp to temperature, min
10
Hold time, min
40
Temperature,
°
C
120
Table 2014.04D. Mass analysis parameters (Xevo TQ-S)
Standard
Retention time typical, min
Molecular ion
(precursor), amu Product ion, amu Dwell, s
Cone voltage, V Collision energy, V
Choline
2.5
104.2
60.3
0.025
40
32
Choline chloride-d
9
2.5
113.4
69.3
0.025
40
30
Carnitine
4.4
162
103
0.025
30
28
Carnitine chloride-d
3
4.4
165
103
0.025
30
26
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