Quantification of Free Carnitine and Acylcarnitines in Plasma or Serum Using HPLC/MS/MS

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
QS this combo carnitine/acylcarnitine solution to 2 mL using methanol (730 μL)
 
(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.
 
6.
Evaporate to dryness under a stream of nitrogen at 40 °C (see Note 1 ).
 
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.
 
9.
Evaporate to dryness under a stream of nitrogen at 40 °C (see Notes 2 and 3 ).
 
10.
Reconstitute with 900 μL of 80 % acetonitrile/water.
 
Oct 21, 2016 | Posted by in BIOCHEMISTRY | Comments Off on Quantification of Free Carnitine and Acylcarnitines in Plasma or Serum Using HPLC/MS/MS

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