title: Simultaneous detection of diagnostic biomarkers of alkaptonuria, ornithine carbamoyltransferase deficiency, and neuroblastoma disease by high-performance liquid chromatography/tandem mass spectrometry.
abstract: BACKGROUND
  Urinary homovanillic acid (HVA)/vanillylmandelic acid (VMA), orotic acid (OA), and homogentisic acid (HGA) are diagnostic biomarkers of neuroblastoma, ornithine carbamoyl transferase deficiency (OCTD), and alkaptonuria (AKU), respectively. In this study, a high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for simultaneous quantification of HVA, VMA, OA, and HGA in urine.
  
  
  METHODS
  After sample preparation, which involved only the dilution procedure, samples were quantified by LC-MS/MS. Full-scan MS/MS mode enabled the urinary markers to be quantified with a high degree of specificity and sensitivity. Rather than using a separate enzymatic method to normalize the concentration of creatinine in urine, we quantified the level of creatinine in urine in one LC-MS run.
  
  
  RESULTS
  The limits of detection were 10 μg/l for HGA, 25 μg/l for HVA/VMA, and 50 μg/l for OA with a single-to-noise ratio of 3; the limits of quantification were 50 μg/l for HVA and HGA, 100 μg/l for VMA, and 250 μg/l for OA. The linear dynamic range for quantification of the analytes covered 2 to 3 orders of magnitude, depending on the analyte. The relative standard deviation of the developed LC-MS/MS method was less than 4% for the intra-day validation and 10% for the inter-day validation.
  
  
  CONCLUSIONS
  The results show that our LC-MS/MS technique is a highly sensitive and rapid method for screening for biomarkers that are diagnostic of three metabolic diseases.
authors.affiliation: Department of Medical Research, China Medical University Hospital, Taichung, Taiwan.
authors.forename: Wei-Yi
authors.initials: WY
authors.lastname: Hsu
authors.forename: Ching-Ming
authors.initials: CM
authors.lastname: Chen
authors.forename: Fuu-Jen
authors.initials: FJ
authors.lastname: Tsai
authors.forename: Chien-Chen
authors.initials: CC
authors.lastname: Lai
mesh_terms: D000474:Alkaptonuria / Q000175:diagnosis*; D015415:Biomarkers / Q000032:analysis* / Q000652:urine; D002853:Chromatography, Liquid; D006801:Humans; D057230:Limit of Detection; D009447:Neuroblastoma / Q000175:diagnosis*; D020163:Ornithine Carbamoyltransferase Deficiency Disease / Q000175:diagnosis*; D053719:Tandem Mass Spectrometry; D013997:Time Factors
chemical_list: D015415:Biomarkers