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1.
Transfer 0.5 grams L-Carnitine -d
3
to a 50 mL flask
2.
Dissolve and bring to volume with laboratory water
d.
Mixed Intermediate Internal Standard Solution (MIX-IS)
. Prepare fresh on the day of
analysis - must use same MIX-IS solution for both calibration working standards and
samples.
i.
Transfer 400 µL of each -choline-chloride-d
9
and 400 µL-carnitine-d
3
HCl stock
solution by a calibrated pipette into a 10 mL volumetric flask
ii.
Bring to volume with laboratory water and mix well.
e.
Intermediate Working Standards (IWS)
– Prepare fresh on the day of analysis
i.
Pipette 100 µL of the mixed intermediate Internal Standard solution (MIX-IS)
into three individual 50 mL volumetric flasks.
ii.
Using Table 1, add the required volumes of the stock choline and carnitine
solution by proper sizes calibrated pipets.
iii.
Bring to volume with laboratory water and mix well.
Table 1. Volumes of each Stock Solution for IWS
Stock Volume (µL)
MIX-IS (µL)
Final Volume (mL)
IWS -1
25
100
50
IWS -2
125
100
50
IWS -3
250
100
50
f.
Working Standard Solution (WS)
– Prepare fresh on the day of analysis. Use 90%
acetonitrile in final working solution.
i.
Pipette 200 µL of each Intermediate Working Standards (IWS-1, 2, and 3) into
three10 mL volumetric flasks
ii.
Add 800 µL laboratory water each volumetric flask
iii.
Bring to volume with acetonitrile and mix well
iv.
Transfer to autosampler vials for analysis.
Table 2. Concentration of Standards
Name
Choline
Choline-d
9
Carnitine Carnitine-d
3
Diluent
VF
µg/mL
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%
WS1
30
32
7.5
16
MeCN
10
WS2
150
32
37.5
16
MeCN
10
WS3
300
32
75
16
MeCN
10
Candidates for 2016 Method of the Year
327