Liquid chromatography–mass spectrometry for measuring deoxythioguanosine in DNA from thiopurine-treated patients

Sally A. Coulthard*, Phil Berry, Sarah McGarrity, Azhar Ansari, Christopher P.F. Redfern

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

7 Citations (Scopus)

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 0.0003125 ng (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 20–1360 mol dTG/106 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.

Original languageEnglish
Pages (from-to)175-180
Number of pages6
JournalJournal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences
Volume1028
DOIs
Publication statusPublished - 15 Aug 2016

Bibliographical note

Funding Information:
This study was funded by The BROAD Foundation (IBD-0355R2) and MRC Confidence in Concept funds awarded to Newcastle University. The funding bodies had no role in study design, collection, analysis and interpretation of data, in the writing of the report or in the decision to submit the article for publication. We are grateful to the clinical staff at the East Surrey Hospital, Royal Bournemouth and Christchurch Hospitals, North Tees Hospital, South Tyneside Hospital, City Hospital Sunderland and James Cook Hospital South Tees who contributed patient samples to the study.

Publisher Copyright:
© 2016 The Authors

Other keywords

  • Azathioprine
  • DNA
  • Drug monitoring
  • LC–MS/MS
  • Mercaptopurine thiopurine
  • Tandem mass spectrometry
  • Thioguanine nucleotides

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