title: Quantification of carnitine and acylcarnitines in biological matrices by HPLC electrospray ionization-mass spectrometry.
abstract: BACKGROUND
  Analysis of carnitine and acylcarnitines by tandem mass spectrometry (MS/MS) has limitations. First, preparation of butyl esters partially hydrolyzes acylcarnitines. Second, isobaric nonacylcarnitine compounds yield false-positive results in acylcarnitine tests. Third, acylcarnitine constitutional isomers cannot be distinguished.
  
  
  METHODS
  Carnitine and acylcarnitines were isolated by ion-exchange solid-phase extraction, derivatized with pentafluorophenacyl trifluoromethanesulfonate, separated by HPLC, and detected with an ion trap mass spectrometer. Carnitine was quantified with d(3)-carnitine as the internal standard. Acylcarnitines were quantified with 42 synthesized calibrators. The internal standards used were d(6)-acetyl-, d(3)-propionyl-, undecanoyl-, undecanedioyl-, and heptadecanoylcarnitine.
  
  
  RESULTS
  Example recoveries [mean (SD)] were 69.4% (3.9%) for total carnitine, 83.1% (5.9%) for free carnitine, 102.2% (9.8%) for acetylcarnitine, and 107.2% (8.9%) for palmitoylcarnitine. Example imprecision results [mean (SD)] within runs (n = 6) and between runs (n = 18) were, respectively: total carnitine, 58.0 (0.9) and 57.4 (1.7) micromol/L; free carnitine, 44.6 (1.5) and 44.3 (1.2) micromol/L; acetylcarnitine, 7.74 (0.51) and 7.85 (0.69) micromol/L; and palmitoylcarnitine, 0.12 (0.01) and 0.11 (0.02) micromol/L. Standard-addition slopes and linear regression coefficients were 1.00 and 0.9998, respectively, for total carnitine added to plasma, 0.99 and 0.9997 for free carnitine added to plasma, 1.04 and 0.9972 for octanoylcarnitine added to skeletal muscle, and 1.05 and 0.9913 for palmitoylcarnitine added to skeletal muscle. Reference intervals for plasma, urine, and skeletal muscle are provided.
  
  
  CONCLUSIONS
  This method for analysis of carnitine and acylcarnitines overcomes the observed limitations of MS/MS methods.
authors.affiliation: Department of Pharmacology, Case Western Reserve University School of Medicine, Cleveland, OH 44106, USA.
authors.forename: Paul E
authors.initials: PE
authors.lastname: Minkler
authors.forename: Maria S K
authors.initials: MS
authors.lastname: Stoll
authors.forename: Stephen T
authors.initials: ST
authors.lastname: Ingalls
authors.forename: Shuming
authors.initials: S
authors.lastname: Yang
authors.forename: Janos
authors.initials: J
authors.lastname: Kerner
authors.forename: Charles L
authors.initials: CL
authors.lastname: Hoppel
mesh_terms: D000107:Acetylation; D002138:Calibration; D002331:Carnitine / Q000032:analysis* / Q000737:chemistry* / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D006801:Humans; D015394:Molecular Structure; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D002331:Carnitine