Introduction: 1,5-AG can be used as a sensitive marker for short- term glycemic control. A commercial chemical method from a single vendor is available for measurement of 1,5-AG. Few LC-MS/MS methods exist with almost none in clinical application. Method: 50 μL of plasma was added to 100 μL of 10 mg/L aqueous 13 C-1,5-AG internal standard containing 10% trichloroacetic acid in a 1 mL microcentrifuge tube. After vortexing, this mixture was centrifuged for 5 min at 14,800 g. 20 μL of supernatant was added to 1 mL of methanol before injecting into a Waters UPLC-MS/MS fitted with an Acquity UPLC BEH Amide column, set at 30 °C, with a isocratic flow of 0.3 mL/min (10% aqueous ammonium hydroxide and 90% methanolic ammonium hydroxide). Detection (in negative ion mode) was made by multiple reaction monitoring of 1,5-AG (163>101.1) and 13 C-1,5-AG (169.1>105.1). Total analytical run time was 2.5 minutes. Results: Both 1,5-AG and 13 C-1,5-AG eluted at 1.64 min. The method was linear up to 100 mg/L. Precision at LOD and LOQ was 16.9% and 5.7%, respectively. Recovery throughout the measurement averaged 105%. Limits of detection and quantification were 0.2 mg/L and 0.4 mg/L. No interference from haemoglobin (419 g/L), turbidity (lipemic index of 60), and bilirubin (600 μmol/L) was observed. Conclusion: A simple, fast, reliable and, highly specific UPLC- MSMS method for measurement of 1,5-AG was developed. These attributes make this method suitable for routine clinical application. doi:10.1016/j.clinbiochem.2011.06.017 P507 Serum biomarkers in white matter disease (Wmd) — A preliminary study P.C. Chan a,b , D. Navaranjan f , A.M. Philips a , J. Pettersen d , A. Ganda c , S. Black c,e a Department of Clinical Pathology, Sunnybrook Health Sciences Centre, Toronto, Canada b Department of Laboratory Medicine and Pathobiology, University of Toronto, Canada c Linda C. Campbell Cognitive Neurology Research Unit, Sunnybrook Health Sciences Centre, Toronto, Canada d Division of Neurology, University of British Columbia & The Northern Medical Program, University of Northern British Columbia, Canada e Department of Medicine, University of Toronto, Canada f Department of Biological Sciences, University of Toronto, Canada Introduction: White matter lesions as detected by MRI hyper- intensities (WMH) have been associated with increased risk of dementia and cognitive deficits. Objective: In this exploratory study, we hypothesize that certain serum biomarkers contribute to the development of WMH and predict cognitive performance. Methods: Serum samples were collected from 14 normal control (NC) subjects, 17 Alzheimer disease (AD) (7 also had Cerebrovascular Disease, CVD), and 25 WMD (16 had mild cognitive impairment) patients from the Stroke Prevention and Cognitive Neurology Clinics at Sunnybrook Health Sciences Centre. Serum levels of matrix metalloproteinase-9, matrix metalloproteinase-2, brain derived neurotrophic factor, fibrinogen, homo- cysteine, vitamin B12 and C-reactive protein were measured by ELISA or standard clinical laboratory methods. WMH was measured by MRI and global cognitive status was assessed by Mini-Mental State Examination (MMSE). Multiple linear regression was used to identify contributing biomarkers to WMH. Biomarker levels among different patient groups were compared using one-way ANOVA. All analyses were made using GraphPad Instat and a P-value <0.05 was considered significant. Results: None of the serum biomarker levels differed among NC, AD and WMD patients. Vitamin B12 was the only significant con- tributing factor in the prediction of WMH (r = 0.32). WMH correlated with MMSE only among AD patients (r = 0.50), and differed significantly between AD and AD + CVD patients. Discussion: The reason for the positive correlation between B12 and WMH is not clear and deserves further investigation. WMH appears to be useful in confirming vascular aetiology and predicting cognitive decline particularly among AD patients. doi:10.1016/j.clinbiochem.2011.06.018 P508 Quantification of urinary 5-Methyl-2′-Deoxycytidine by isotope-dilution liquid chromatography-tandem mass spectrometry with on-line solid-phase extraction M.R. Chao a,b , Y.J. Li b , C.W. Hu c a Department of Occupational Safety and Health, Chung Shan Medical University. Taichung 402, Taiwan b Institute of Medical and Molecular Toxicology, Chung Shan Medical University, Taichung 402, Taiwan c Department of Public Health, Chung Shan Medical University, Taichung 402, Taiwan Objectives: Alterations in global DNA methylation has been implicated in several pathobiological processes. Quantification of 5- methyl-2′-deoxycytidine (5mdC) in urine or blood could be used to assess and monitor global DNA methylation status in patients. We firstly describe the use of on-line solid-phase extraction (SPE) and isotope-dilution liquid chromatography-tandem mass spectrometry (LC-MS/MS) for automated measurement of urinary 5mdC. Methods: Automated purification of urine was accomplished with a switching valve and an Inertsil ODS-3 column. After the addition of 5- (methyl-d 3 )-2′-deoxycytidine (d 3 -5mdC) as an internal standard, urine samples were analyzed without further sample purification. The samples were analyzed in the positive ion multiple reaction monitoring (MRM) mode. Optimal MRM conditions were obtained for three channels: m/z 242 →126 (quantifier ion) and 242 →109 (qualifier ion) for 5mdC, and m/z 245 →129 for d 3 -5mdC. This method was applied to measure urinary 5mdC in 45 healthy male subjects. Results: The limit of detection was 0.024 ng/mL (4.98 fmol in an injection volume of 50 μL). Intra- and inter-day imprecision (CV) was <15%. Mean recovery of 5mdC in urine was 97–110%. Mean urinary concentrations of 5mdC was firstly demonstrated to be 5.24 ± 3.54 ng/mg creatinine. Conclusions: This on-line SPE LC-MS/MS method is sufficiently sensitive, precise, and rapid to provide high-throughput direct analysis of urinary 5mdC. This method may help to assess the global DNA methylation status of patients in daily clinical practice and may facilitate the research into the epigenetic diseases. doi:10.1016/j.clinbiochem.2011.06.019 P509 Mathematical estimation of iatrogenic anemia onset due to repetitive diagnostic blood loss in critical care patients A.C. Chin a , M.E. Lyon a,c , G.A. Slotsve b , A.W. Lyon a a Department of Pathology and Laboratory Medicine, University of Calgary and Calgary Laboratory Services, Calgary, Alberta, Canada b Department of Economics, Northern Illinois University, DeKalb, IL, USA c Department of Pharmacology and Therapeutics, and Paediatrics, University of Calgary and Calgary Laboratory Services, Calgary, Alberta, Canada Anemia in critical care patients is multifactorial involving rates of diagnostic blood loss, erythropoiesis, and red cell senescence. Transfusions to alleviate anemia initiated by unnecessary repetitive phlebotomy, confer risks of adverse events. Past studies have Abstracts 1168