Very Long Chain Fatty Acids (VLCFA)

VERY LONG CHAIN FATTY ACIDS, PRISTANIC AND PHYTANIC ACIDS IN SERUM GC-MS KIT

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, (VLCFA) pristanic and phytanic acid analysis to aid in diagnosis of peroxisomal biogenesis disorders (Zellweger Syndrome Spectrum), X-linked adrenoleukodystrophy (X- ALD). 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 eight VLCFA parameters: the level of C22:0, C24:0, C26:0, pristanic and phytanic acid and the ratio of C26:0/ C22:0, C24:0/C22:0, pristanic acid/phytanic acid.

  • Highlights

    • Total run time is 14 min.
    • All analytes, including pristanic phytanic acid, aredetected in a single sample preparation
    • Consuming small volume of patient’s sample
    • Long life span of GC column
  • Matrix

    • Serum
  • PARAMETERS

    • Pristanic acid, Phytanic acid, Docosanoic acid (C22:0), Tetracosanoic acid (C24:0) and
      Hexacosanoic acid (C26:0).

Sample Preparation


  • Step 1
    • Pipette 200 μl serum sample into a glass via
  • Step 2
    • Reagent mix prepare into the volumetric flask on ice in fume hood. Reagent 3 is added dropwise while swirling on R1 and R2, vortex for 10 sec.
  • Step 3
    • 600 μl of Reagent-mix, vortex for 15 sec.
  • Step 4
    • Locate the vial into the empty glass tube. Incubate 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
    • Pipette 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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