Free and Total Carnitine/Acylcarnitines Quantitative LC-MS/MS Analysis Kit

Acylcarnitines are acetylated forms of L-carnitine derived from the breakdown of amino and fatty acids. Acylcarnitine analysis is performed for the biochemical detection of disorders of mitochondrial fatty acid β-oxidation and organic acid metabolism. The conditions revealed by acylcarnitine analysis have in common the accumulation of short, medium, and/or long-chain acyl-CoA species, which are substrates for one of several carnitine acyl-CoA transferases expressed in different intracellular compartments. Laboratories providing this analysis as a clinical service most often perform flow- injection analysis–tandem mass spectrometry (FIA-MS/MS) as the analytical platform. Recently MS/ MS methods which involve a liquid chromatographic (LC) separation step to quantitate acylcarnitine forms.

Highlights

Quantitative analysis of free carnitine/acylcarnitines in 10 min. for serum/plasma specimens

Just a few pipetting steps for sample preparation; protein crash and shoot.

Additionally, a simple hydrolysis step for the determination of total carnitine

Safeguarded by its own stable labeled isotopes as internal standards

Consuming small volume of patient’s sample

Long life span of HPLC column
Parameters

Carnitine (free)-C0, Acetylcarnitine-C2, Propionylcarnitine-C3, Butyrylcarnitine-C4,
Isovalerylcarnitine-C5, Hexanoylcarnitine-C6, Octanoylcarnitine-C8, Decanoylcarnitine-C10,
Dodecanoylcarnitine-C12, Tetradecanoylcarnitine-C14, Hexadecanoylcarnitine-C16,
Octadecanoylcarnitine-C18 and Octadecenoylcarnitine-C18:1
Matrix

Serum/Plasma

Sample Preparation

Step 1

Sample Preparation For Free Carnitine/Acylcarnitines
Pipette 50 μL of serum/plasma sample into a glass centrifuge tube
Step 2

Add 25 μL of internal standard solution and vortex for 5 sec
Step 3

Add 425 μL of reagent-1 (for protein precipitation), vortex for 5 sec. then, centrifuge at 4000 rpm for 5 min
Step 4

Decant the clear supernatant into a HPLC vial prior to injection

Sample Preparation

Step 1

Sample Preparation For Total Carnitine
Pipette 50 μL of serum/plasma sample into a eppendorf tube
Step 2

Add 50 μL of reagent-2 (for hydrolysis) and vortex for 5 sec. then, incubate for 15 min. at 65 oC.
Step 3

Add 50 μL of reagent-3, vortex for 5 sec. and centrifuge at 4000 rpm for 5 min.
Step 4

Pipette 50 μL of hydrolysate into a glass centrifuge tube
Step 5

Add 25 μL of internal standard solution and vortex for 5 sec.
Step 6

Add 425 μL of reagent-1 (for protein precipitation), vortex for 5 sec. then, centrifuge at 4000 rpm for 5 min
Step 7

Decant the clear supernatant into a HPLC vial prior to injection

Chromatogram
METHOD PERFORMANCE