title: Plasma allantoin measurement by isocratic liquid chromatography with tandem mass spectrometry: method evaluation and application in oxidative stress biomonitoring.
abstract: BACKGROUND
  Allantoin in human plasma is a specific biomarker of oxidative stress. We describe a sensitive method to measure plasma allantoin using isocratic liquid chromatography and mass spectrometry (LC-MS/MS).
  
  
  METHODS
  Direct injection of deproteinized plasma into the LC-MS/MS system was performed. The method was technically evaluated. Results on 200 healthy and 35 Type 2 diabetic Chinese subjects were compared.
  
  
  RESULTS
  Dose-response of allantoin was linear to at least 21 pmol (20 μmol/l in plasma); LOD was 0.16 pmol; recovery 99.2-100.2% at 1-5 μmol/l; accuracy, 98.5-100.8%; within-day and between-day CVs (n=6), <4.0% (at 5.00-40 μmol/l) and <2.0% (at 1-5 μmol/l), respectively. Plasma allantoin in diabetic patients was ~8-fold higher than in healthy subjects; mean (SD): 8.82 (7.26) and 1.08 (0.86) μmol/l, respectively (p<0.0001). Allantoin was slightly higher in healthy men than in age- and BMI-matched women: 1.21 (0.99) μmol/l, n=88 compared to 0.97 (0.74) μmol/l, n=112; p<0.001. No association with age was seen. Gender difference was also seen in the diabetes patients: men, n=14, 11.57 (8.57) μmol/l; women, n=21, 6.99 (5.75) μmol/l, p<0.05.
  
  
  CONCLUSIONS
  Based on 95th percentiles of the healthy subjects, plasma allantoin of >2.2 μmol/l in women and >3.1 μmol/l in men indicates increased oxidative stress. Allantoin in diabetes subjects is clearly and markedly increased. The method will facilitate future studies of oxidative stress in human biomonitoring studies.
authors.affiliation: Department of Health Technology & Informatics, The Hong Kong Polytechnic University, Kowloon, Hong Kong.
authors.forename: Wai-Yuen
authors.initials: WY
authors.lastname: Chung
authors.forename: Iris F F
authors.initials: IF
authors.lastname: Benzie
mesh_terms: D000328:Adult; D000481:Allantoin / Q000097:blood*; D015415:Biomarkers / Q000097:blood; D016022:Case-Control Studies; D002853:Chromatography, Liquid; D003924:Diabetes Mellitus, Type 2 / Q000097:blood* / Q000175:diagnosis; D005260:Female; D017022:Flow Injection Analysis; D006801:Humans; D057230:Limit of Detection; D008297:Male; D008875:Middle Aged; D018670:Monitoring, Ambulatory; D018384:Oxidative Stress; D012016:Reference Values; D015203:Reproducibility of Results; D053719:Tandem Mass Spectrometry
chemical_list: D015415:Biomarkers; D000481:Allantoin
keywords: Allantoin; Biomarker evaluation; Diabetes; LC–MS/MS; Mass spectrometry; Oxidative stress