title: Determination of choline, betaine, and dimethylglycine in plasma by a high-throughput method based on normal-phase chromatography-tandem mass spectrometry.
abstract: BACKGROUND
  The quaternary ammonium compounds, choline and betaine, and dimethylglycine (DMG) reside along a metabolic pathway linked to the synthesis of neurotransmitters and membrane phospholipids and to homocysteine remethylation and, therefore, folate status. Lack of a convenient, high-throughput method for the determination of these compounds has prevented population-based studies of their possible associations with lifestyle, nutrition, and chronic diseases.
  
  
  METHODS
  Serum or plasma samples were deproteinized by mixing with three volumes of acetonitrile that contained d(9)-choline and d(9)-betaine as internal standards. We used a normal-phase silica column for the separation of choline (retention time, 2.8 min), betaine (1.3 min), DMG (1.15 min), and internal standards, which were detected as positive ions by tandem mass spectroscopy in the multiple-reaction monitoring mode, using the molecular transitions m/z 104-->60 (choline), m/z 113-->69 (d(9)-choline), m/z 118-->59 (betaine), m/z 127-->68 (d(9)-betaine), and m/z 104-->58 (DMG).
  
  
  RESULTS
  For all three metabolites, the assay was linear in the range 0.4-400 micromol/L, and the lower limit of the detection (signal-to-noise ratio = 5) was < or =0.3 micromol/L. The within- and between-day imprecision (CVs) was 2.1-7.2% and 3.5-8.8%, respectively. The analytical recovery was 87-105%. The fasting plasma concentrations (median, 25th-75th percentiles) were 8.0 (7.0-9.3) micromol/L for choline, 31.7 (27.0-41.1) micromol/L for betaine, and 1.66 (1.30-2.02) micromol/L for DMG in 60 healthy blood donors. In individuals who had eaten a light breakfast, plasma concentrations of all three metabolites were significantly (25-30%) higher than in fasting individuals.
  
  
  CONCLUSIONS
  This is the first method for the combined measurement of choline, betaine, and DMG in human plasma or serum. The assay is characterized by simple sample preparation, no derivatization, high throughput, imprecision (CV) <10%, detection limits below the values seen in volunteers, and the high specificity provided by tandem mass spectroscopy.
authors.affiliation: LOCUS for Homocysteine and Related Vitamins, University of Bergen, N-5021 Bergen, Norway.
authors.forename: Pål I
authors.initials: PI
authors.lastname: Holm
authors.forename: Per Magne
authors.initials: PM
authors.lastname: Ueland
authors.forename: Gry
authors.initials: G
authors.lastname: Kvalheim
authors.forename: Ernst A
authors.initials: EA
authors.lastname: Lien
mesh_terms: D000328:Adult; D000368:Aged; D001622:Betaine / Q000097:blood*; D001782:Blood Donors; D002794:Choline / Q000097:blood*; D002851:Chromatography, High Pressure Liquid; D005215:Fasting; D005260:Female; D006801:Humans; D008297:Male; D013058:Mass Spectrometry; D008875:Middle Aged; D012521:Sarcosine / Q000031:analogs & derivatives* / Q000097:blood*
chemical_list: D001622:Betaine; C025138:dimethylglycine; D002794:Choline; D012521:Sarcosine