Acylcarnitine Analysis by Tandem Mass Spectrometry

Emily H. Smith1, Dietrich Matern1

1 Mayo Clinic College of Medicine, Rochester, Minnesota
Publication Name:  Current Protocols in Human Genetics
Unit Number:  Unit 17.8
DOI:  10.1002/0471142905.hg1708s64
Online Posting Date:  January, 2010
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Carnitine plays an essential role in fatty acid metabolism, as well as modulation of intracellular concentrations of free coenzyme A by esterification of acyl residues. Acylcarnitine analysis of various biological fluids is a sensitive method to detect >20 inborn errors of metabolism that result in abnormal accumulation of acylcarnitine species due to several organic acidemias and most fatty acid β‐oxidation disorders. In addition, acylcarnitine analysis may aid in monitoring treatment of known patients affected with these inborn errors of metabolism. This unit describes protocols that can be used to measure acylcarnitine species of various carbon chain lengths in several biological specimen types including plasma, dried blood and bile spots, and urine, by derivatization to butylesters and flow‐injection electrospray ionization tandem mass spectrometry (ESI‐MS/MS). Curr. Protoc. Hum. Genet. 64:17.8.1‐17.8.20. © 2010 by John Wiley & Sons, Inc.

Keywords: acylcarnitines; carnitine; fatty acid oxidation; inborn errors of metabolism; organic acids; tandem mass spectrometry

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Table of Contents

  • Introduction
  • Basic Protocol 1: Plasma Acylcarnitine Analysis by ESI‐MS/MS
  • Alternate Protocol 1: Urine Acylcarnitine Analysis by ESI‐MS/MS
  • Alternate Protocol 2: Acylcarnitine Analysis in Dried Blood or Bile Spots by ESI‐MS/MS
  • Support Protocol 1: Preparation of Controls for Acylcarnitine Analysis in All Specimen Types
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: Plasma Acylcarnitine Analysis by ESI‐MS/MS

  • Labeled and unlabeled internal standards (see protocol 4)
  • 98% to 100% formic acid
  • Normal control plasma in 1.5‐ml microcentrifuge tubes (see protocol 4)
  • Abnormal control plasma in 1.5‐ml microcentrifuge tubes (see protocol 4)
  • Patient samples: plasma in tube containing anticoagulant (e.g., heparin)
  • Quality control standard mixture (see protocol 4)
  • Nitrogen gas, in‐house, scrubbed with Big Hydrocarbon Trap BHT‐1 (Chrom Tech)
  • 3 N HCl in n‐butanol (butanolic‐HCl; Regis Chemical, cat. no. 201009)
  • 80% acetonitrile in water
  • 1‐ml screw‐cap micro vials (Chrom Tech)
  • Thermolyne MaxiMix 2 type 37600 vortex mixer
  • Centrifuge (Abbott, cat. no. 3531)
  • Reacti‐Vials (Chrom Tech, cat. no. 402001)
  • Pierce Reacti‐Vap evaporating unit
  • Pierce Reacti‐Therm heating module
  • Autosampler vials (Fisher, cat. no. 0334064)
  • Autosampler caps (Fisher, cat. no. 0337749)
  • Tandem mass spectrometry system (Applied Biosystems/MDS SCIEX API 3000)
  • PE Series 200 LC autosampler
  • MS/MS TurboIon spray source
  • PE Series 200 isocratic LC pump
  • 0.5‐µm filters (Chrom Tech, model no. A‐431)
  • 0.5‐µm Peek filter‐end fitting (Chrom Tech, model no. A‐428X)
  • Back pressure LC column (BDS Hypersil C18, 3‐µm, 50 × 1–cm; Keystone Scientific, cat. no. 053‐46‐1)

Alternate Protocol 1: Urine Acylcarnitine Analysis by ESI‐MS/MS

  • Normal control (see protocol 4)
  • Abnormal control (see protocol 4)
  • Quality control standard mixture (see protocol 4)
  • Blood or bile sample
  • Internal standard/extraction solution prepared in methanol
  • Filter paper (Whatman 903 Protein Saver Card)
  • Automatic puncher (e.g., Wallac DBS Puncher, Perkin‐Elmer Life and Analytical Sciences) or manual punchers (e.g., M.C. Mieth Manufacturing)
  • Plate rotator

Alternate Protocol 2: Acylcarnitine Analysis in Dried Blood or Bile Spots by ESI‐MS/MS

  • Normal range pooled plasma, urine, blood, or bite
  • Unlabeled stock standards for controls dissolved in methanol (final working standard solution is prepared in acetonitrile—for plasma or urine acylcarnitine analysis—or methanol—for blood or bile on filter paper—at concentrations based on the upper limit of the reference range for the respective acylcarnitine species)
    • Acetylcarnitine (C 2)
    • Propionylcarnitine (C 3)
    • Butyrylcarnitine (C 4)
    • Isovalerylcarnitine (C 5)
    • Hexanoylcarnitine (C 6)
    • Octanoylcarnitine (C 8)
    • Decanoylcarnitine (C 10)
    • Dodecanoylcarnitine (C 12)
    • Tetradecanoylcarnitine (C 14)
    • Hexadecanoylcarnitine (C 16)
    • Octadecanoylcarnitine (C 18)
  • Isotopically labeled stock standards (internal standards) dissolved in methanol (final working internal standard solution is prepared in acetonitrile—for plasma or urine acylcarnitine analysis—or methanol—for blood or bile on filter paper—at the following concentrations:
    • 1.330 µmol/liter acetyl‐D 3‐carnitine (C 2)
    • 0.266 µmol/liter propionyl‐D 3‐carnitine (C 3)
    • 0.266 µmol/liter butyryl‐D 3‐carnitine (C 4)
    • 0.266 µmol/liter octanoyl‐D 3‐carnitine (C 8)
    • 0.266 µmol/liter dodecanoyl‐D 3‐carnitine (C 12)
    • 0.543 µmol/liter palmitoyl‐D 3‐carnitine (C 16)
  • 2‐ml screw‐cap vials
  • Additional or different internal standards can be used. Labeled and unlabeled acylcarnitine standards are available from several sources, including Dr. Herman J. ten Brink, VU University Medical Center, Amsterdam, The Netherlands ( or Cambridge Isotope Laboratories, (
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Literature Cited

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