title: Liquid chromatography-mass spectrometry for measuring deoxythioguanosine in DNA from thiopurine-treated patients.
abstract: Adverse reactions and non-response are common in patients treated with thiopurine drugs. Current monitoring of drug metabolite levels for guiding treatment are limited to analysis of thioguanine nucleotides (TGNs) in erythrocytes after chemical derivatisation. Erythrocytes are not the target tissue and TGN levels show poor correlations with clinical response. We have developed a sensitive assay to quantify deoxythioguanosine (dTG) without derivatisation in the DNA of nucleated blood cells. Using liquid chromatography and detection by tandem mass spectrometry, an intra- and inter-assay variability below 7.8% and 17.0% respectively were achieved. The assay had a detection limit of 0.0003125ng (1.1 femtomoles) dTG and was quantified in DNA samples relative to endogenous deoxyadenosine (dA) in a small group of 20 patients with inflammatory bowel disease, all of whom had been established on azathioprine (AZA) therapy for more than 25 weeks. These patients had dTG levels of 20-1360mol dTG/10(6)mol dA; three patients who had not started therapy had no detectable dTG. This method, comparable to previous methods in sensitivity, enables the direct detection of a cytotoxic thiopurine metabolite without derivatisation in an easily obtainable, stable sample and will facilitate a better understanding of the mechanisms of action of these inexpensive yet effective drugs.
authors.affiliation: Institute of Cellular Medicine, Newcastle University, Newcastle upon Tyne, UK. Electronic address: sally.coulthard@newcastle.ac.uk.
authors.forename: Sally A
authors.initials: SA
authors.lastname: Coulthard
authors.affiliation: Northern Institute of Cancer Research, Newcastle University, Newcastle upon Tyne, UK.
authors.forename: Phil
authors.initials: P
authors.lastname: Berry
authors.affiliation: Northern Institute of Cancer Research, Newcastle University, Newcastle upon Tyne, UK.
authors.forename: Sarah
authors.initials: S
authors.lastname: McGarrity
authors.affiliation: Gastroenterology Dept., East Surrey Hospital, Redhill, East Surrey, UK.
authors.forename: Azhar
authors.initials: A
authors.lastname: Ansari
authors.affiliation: Northern Institute of Cancer Research, Newcastle University, Newcastle upon Tyne, UK.
authors.forename: Christopher P F
authors.initials: CPF
authors.lastname: Redfern
mesh_terms: D001773:Blood Cells / Q000737:chemistry / Q000187:drug effects; D002853:Chromatography, Liquid / Q000379:methods; D004247:DNA / Q000097:blood / Q000737:chemistry*; D003849:Deoxyguanosine / Q000031:analogs & derivatives* / Q000032:analysis / Q000097:blood; D006801:Humans; D007166:Immunosuppressive Agents / Q000627:therapeutic use*; D015212:Inflammatory Bowel Diseases / Q000097:blood / Q000188:drug therapy*; D015122:Mercaptopurine / Q000627:therapeutic use*; D053719:Tandem Mass Spectrometry / Q000379:methods; D013872:Thionucleosides / Q000032:analysis* / Q000097:blood
chemical_list: D007166:Immunosuppressive Agents; D013872:Thionucleosides; C002062:alpha-2'-deoxythioguanosine; C010438:beta-2'-deoxythioguanosine; D004247:DNA; D015122:Mercaptopurine; D003849:Deoxyguanosine
keywords: Azathioprine; DNA; Drug monitoring; LC–MS/MS; Mercaptopurine thiopurine; Tandem mass spectrometry; Thioguanine nucleotides