title: Quantitative measurement of porphobilinogen in urine by stable-isotope dilution liquid chromatography-tandem mass spectrometry.
abstract: BACKGROUND
  Measurement of porphobilinogen (PBG) is useful in the diagnosis of the acute neurologic porphyrias. Currently used colorimetric assays lack analytical and clinical sensitivity and specificity.
  
  
  METHODS
  We developed a liquid chromatography-electrospray tandem mass spectrometry (LC-MS/MS) method for the measurement of PBG in 1 mL of urine, using 5-(aminoethyl)-4-(carboxymethyl) 1H-2,4-[(13)C]pyrolle-3-propanoic acid ([2,4-(13)C]PBG; 2.75 microg) as internal standard. After solid-phase extraction, LC-MS/MS analysis was performed in the selected-reaction monitoring (SRM) mode. PBG and [2,4-(13)C]PBG were monitored through their own precursor and product ion settings (m/z 227 to 210 and m/z 229 to 212, respectively). The retention time of PBG and [2,4-(13)C]PBG was 1.0 min in a 2.3-min analysis.
  
  
  RESULTS
  Daily calibrations (n = 6) between 0.1 and 2.0 mg/L were linear and reproducible. Inter- and intraassay CVs were 3.2-3.5% and 2.6-3.1%, respectively, at mean concentrations of 0.24, 1.18, and 2.15 mg/L. The regression equation for the comparison between an anion-exchange column method (y) and the LC-MS/MS method (x) was: y = 0.84x + 0.74 (S(y/x) = 5.8 mg/24 h; r = 0.85; n = 100). In 47 volunteers, PBG excretion was 0.02-0.42 mg/24 h, lower than reported reference intervals (up to 2.0 mg/24 h) based on colorimetric methods. In 85 samples with PBG < or =0.5 by LC-MS/MS, 8 (9.4%) had values > or =2.0 mg/24 h by the anion-exchange method (mean +/- SD, 4.3 +/- 1.8 mg/24 h). In 11 patients with confirmed diagnoses of acute porphyria and increased PBG by LC-MS/MS, 2 had values within the reported reference intervals by a quantitative anion-exchange method.
  
  
  CONCLUSIONS
  The quantitative LC-MS/MS method for PBG measurement exhibits greater analytical specificity and improved clinical sensitivity and specificity than currently available methods.
authors.affiliation: Biochemical Genetics Laboratory, Department of Laboratory Medicine and Pathology, Mayo Clinic, 200 First St. SW, Rochester, MN 55905, USA.
authors.forename: R E
authors.initials: RE
authors.lastname: Ford
authors.forename: M J
authors.initials: MJ
authors.lastname: Magera
authors.forename: K M
authors.initials: KM
authors.lastname: Kloke
authors.forename: P A
authors.initials: PA
authors.lastname: Chezick
authors.forename: A
authors.initials: A
authors.lastname: Fauq
authors.forename: J P
authors.initials: JP
authors.lastname: McConnell
mesh_terms: D002853:Chromatography, Liquid; D006801:Humans; D007201:Indicator Dilution Techniques; D011162:Porphobilinogen / Q000652:urine*; D012016:Reference Values; D021241:Spectrometry, Mass, Electrospray Ionization
chemical_list: D011162:Porphobilinogen