Determination of monoamine oxidase B activity by high-performance liquid chromatography with electrochemical detection |
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| Institution: | 1. Department of Oncology, University of Cambridge, Cambridge, UK;2. Department of Gynecology and Obstetrics, Ribeirao Preto School of Medicine, University of Sao Paulo, Ribeirao Preto, Brazil;3. Department of Physics (Astrophysics), University of Oxford, Oxford, UK;4. Cancer Research UK, Cambridge Institute, Cambridge, UK;5. University of Southampton, Southampton, UK;6. University of Leeds, Leeds, UK;7. Addenbrookes Hospital NHS Trust, Cambridge, UK;8. Institute of Cancer Research, London, UK;9. Cancer Research UK, London, UK;10. Netherlands Cancer Institute, Antoni van Leeuwenhoek Hospital, Amsterdam, The Netherlands;11. Department of Public Health and Primary Care, University of Cambridge, Cambridge, UK;12. Human Genotyping (CEGEN) Unit, Human Cancer Genetics Program, Spanish National Cancer Research Centre (CNIO), Madrid, Spain;13. Biomedical Network on Rare Diseases (CIBERER), Madrid, Spain;14. Cancer Epidemiology Centre, Cancer Council Victoria, Melbourne, Australia;15. Centre for Epidemiology and Biostatistics, Melbourne School of Population and Global Health, The University of Melbourne, Australia;p. Institute of Clinical Medicine, Pathology and Forensic Medicine, University of Eastern Finland, Finland;q. Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, MN, USA;r. Department of Human Genetics & Department of Pathology, Leiden University Medical Center, Leiden, The Netherlands;s. Department of Surgical Oncology, Leiden University Medical Center, Leiden, The Netherlands;t. Sheffield Cancer Research, Department of Oncology, University of Sheffield, Sheffield, UK;u. Academic Unit of Pathology, Department of Neuroscience, University of Sheffield, Sheffield, UK;v. National Cancer Institute, USA;w. Department of Medical Oncology, Family Cancer Clinic, Erasmus MC Cancer Institute, Rotterdam, The Netherlands;x. Division of Clinical Epidemiology and Aging Research, German Cancer Research Center (DKFZ), Heidelberg, Germany;y. Division of Preventive Oncology, German Cancer Research Center (DKFZ), Heidelberg, Germany;z. German Cancer Consortium (DKTK), German Cancer Research Center (DKFZ), Heidelberg, Germany;11. Saarland Cancer Registry, Saarbrücken, Germany |
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| Abstract: | A sensitive high-performance liquid chromatography method with electrochemical detection for measuring monoamine oxidase B activity in blood platelets is described. Dopamine is used as substrate and is incubated with isolated platelets and aldehyde dehydrogenase to convert dihydroxyphenylacetaldehyde to dihydroxyphenylacetic acid (DOPAC). The acid and the added internal standard hydrocaffeic acid are separated from dopamine and the incubation mixture by extraction with 5 ml of ethyl acetate-toluene (5:1, v/v). The organic phase is evaporated under nitrogen stream and the residue dissolved in 0.1 M critic acid. Dihydroxyphenylacetic acid and the internal standard dihydrocaffeic acid are then separated on the Eurosphere 100-C18 5 μm column. The mobile phase used was a mixture of sodium acetate, citric acid, and acetonitrile at pH 2.5. The standard curve was linear from 125 pg to 10 ng. Absolute recovery of DOPAC was 85±3.8% and of hydrocaffeic acid 87±4.1%. The method presented is sensitive (detection limit 8.0 pg of DOPAC injected) and reproducible (coefficient of variation 0.4-1%) with good accuracy (94–98%). |
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