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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