Compound
Concentration (nmol/mL)
D9-l-Carnitine (C0)
0.760
D3-Acetycarnitine (C2)
0.190
D3-Propionylcanitine (C3)
0.038
D3-Butyrylcarnitine (C4)
0.038
D9-Isovalerylcarnitine (C5)
0.038
D3-Octanoylcarnitine (C8)
0.038
D9-Tetradecanoylcarnitine (C14)
0.038
D3-Hexadecanoylcarnitine (C16)
0.076
2.
Quality controls:
(a)
Prepare stock of carnitine/acylcarnitine compounds (Cambridge Isotopes) according to Table 2. Stable for 2 years at −20 °C.
Table 2
Preparation of stock carnitine/acylcarnitine solutions
|
Compound |
M.W. |
Amount in vial (μg) |
Reconstitute vial with this amount of methanol (mL) |
Resulting concentration (nmol/mL) |
|---|---|---|---|---|
|
C0 |
198 |
5000 |
2.52 |
10,000 |
|
C2 |
240 |
5000 |
2.1 |
10,000 |
|
C3 |
254 |
5000 |
1.97 |
10,000 |
|
C4 |
268 |
5000 |
1.86 |
10,000 |
|
C5 |
282 |
5000 |
1.77 |
10,000 |
|
C8 |
324 |
5000 |
1.54 |
10,000 |
|
C14 |
408 |
5000 |
1.22 |
10,000 |
|
C16 |
436 |
5000 |
1.15 |
10,000 |
(b)
Use stock carnitine/acylcarnitine compounds to prepare primary combo carnitine/acylcarnitine solution (Table 3). Stable for 2 years at −20 °C.
Table 3
Preparation of primary combo carnitine/acylcarnitine solution
|
Compound |
Stock solution (μL) |
Resulting concentration (nmol/mL) |
|---|---|---|
|
C0 |
800 |
4000 |
|
C2 |
400 |
2000 |
|
C3 |
20 |
100 |
|
C4 |
10 |
50 |
|
C5 |
10 |
50 |
|
C8 |
10 |
50 |
|
C14 |
10 |
50 |
|
C16 |
10 |
50 |
(c)
Use primary combo carnitine/acylcarnitine solution to prepare working controls as shown in Table 4. Stable for 1 year at −20 °C.
Table 4
Preparation of working quality controls
|
QC1 |
QC2 |
QC3 |
QC4 | |
|---|---|---|---|---|
|
UTAK serum (μL) |
200 |
995 |
980 |
950 |
|
Primary combo (μL) |
0 |
5 |
20 |
50 |
|
0.9 % Saline (μL) |
800 |
0 |
0 |
0 |
|
Resulting concentrations |
Below normal limit |
Approximately the upper limit of normal values |
Approximately 4× upper limit of normal |
Approximately >9× upper limit of normal |
2.4 Analytical Equipment and Supplies
1.
MS/MS 4000Q TRAP (AB Sciex).
2.
Prominence HPLC (Shimadzu).
3.
Dry block at 60 °C.
4.
Sample evaporator, Turbovap (Zymark).
5.
Fume Hood.
3 Methods
3.1 Stepwise Procedure
1.
To a 1.5 mL microcentrifuge tube, add 20 μL sample or control.
2.
Add 400 μL of working internal standard.
3.
Close cap, vortex, and allow to stand for 5 min.
4.
Centrifuge for 5 min at 12,000 × g for 10 min.
5.
Transfer 300 μL of supernatant into a 13 × 100 mm tube.
7.
To the resulting residue, add 100 μL of 3 N HCl in butanol.
8.
Cap tubes and incubate in dry block for 20 min at 60 °C.
10.
Reconstitute with 900 μL of 80 % acetonitrile/water.
11.
Transfer sample to autosampler vial.
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