Very Long Chain Fatty Acids (VLCFA)

VERY LONG CHAIN FATTY ACIDS, PRISTANIC AND PHYTANIC ACIDS ANALYSIS USING GC-MS

Peroxisomes are organelles surrounded by membrane in the form of spheres. Peroxisomes play an important role in beta oxidation of very long chain fatty acids (C22:0, C24:0, C26:0) and alpha oxidation of 3-methyl fatty acids (phytanic acid). The Zellweger syndrome is best known peroxisomal disorders. Zellweger syndrome is a rare disease characterized by decreased or absent peroxisomes in the cells of the liver, kidney and brain. Increased levels of very long chain fatty acids (VLCFA) in blood and decreased plasmalogen levels in erythrocytes are the most important parameters to aid in diagnosis.

VERY LONG CHAIN FATTY ACIDS, PRISTANIC AND PHYTANIC ACIDS

The diagnosis of X-ALD may be raised by the above clinical signs or symptoms, including isolated adrenal insufficiency and is commonly achieved by laboratory evaluation of increased concentrations of VLCFA in plasma. Testing typically includes three VLCFA parameters: the level of hexacosanoic acid (C26:0), and the ratio of hexacosanoic acid to tetracosanoic acid (C26:0/ C24:0), and to docosanoic acid (C26:0/C22:0).

  • Features

    • Very long-chain fatty acids, (VLCFA) pristanic and phytanic acid analysis to aid in diagnosis of peroxisomal biogenesis disorders (Zellweger Syndrome Spectrum), X-linked adrenoleukodystrophy (X- ALD).

    • Detection of multiple target analytes include pristanic and phytanic acids in a single run (14 min).

  • Matrix

    • Serum
  • PARAMETERS

    • Pristanic Acid, phytanic Acid, docosanoic acid, tetracosanoic acid and hexacosanoic acid.

Sample Preparation


  • Step 1
    • Pipet 200 μl serum sample into a glass vial
  • Step 2
    • Reagent mix prepare into the volumetric flask on ice in fume hood. Reagent 3 is added drop-wise while swirling on R1 and R2, vortex for 10
  • Step 3
    • 600 μl of Reagent-mix, vortex for 15 seconds.
  • Step 4
    • Put the vial into the empty glass tube. İncubate in water bath at 90 min 70°C.
  • Step 5
    • After cooling to room temperature, the methyl derivatives are transferred in eppendorf, extracted 800 µl Reagent 4 and centrifuge at 3500rpm 2 min.
  • Step 6
    • Pipet 600 µl extracted serum samples are dried under stream of nitrogen, dissolved 150 µl Reagent 4, and vortex 10 sec.
  • Step 7
    • Transfer the final solution into the insert of HPLC vials.
  • EXAMPLE CHROMATOGRAMS

  • ANALYTICAL PERFORMANCE

    Compound

    R2

    LOQ

    %Recovery

    Repeatability

    %RSD

    Pristanic Acid Methyl Ester

    0,9991

    0,18

    100,615

    1,365

    Phytanic Acid  Methyl Ester

    0,9999

    1,635

    93,522

    1,919

    Behenic Acid  Methyl Ester  (C22:0)

    0,9946

    3,046

    83,163

    1,087

    Lignoceric Acid  Methyl Ester  (C24:0)

    0,9949

    2,161

    99,706

    1,406

    Cerotic Acid  Methyl Ester  (C26:0)

    0,9933

    0,159

    119,49

    3,031

     

  • Calibration Curves

    Calibration Curve of C22:0 methyl ester

     

    Calibration Curve of C24:0 methyl ester

    Calibration Curve of C26:0 methyl ester

    Calibration Curve of Pristanic Acid Methyl Ester

    Calibration Curve of Phytanic Acid Methyl Ester

ADAPTING DIAGNOSTICS IN CHROMATOGRAPHY COUPLED MASS SPECTROMETRY..

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