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Open Access 01-11-2011 | Original Article

A novel method for quantification of gemcitabine and its metabolites 2′,2′-difluorodeoxyuridine and gemcitabine triphosphate in tumour tissue by LC–MS/MS: comparison with ¹⁹F NMR spectroscopy

Authors: Tashinga E. Bapiro, Frances M. Richards, Mae A. Goldgraben, Kenneth P. Olive, Basetti Madhu, Kristopher K. Frese, Natalie Cook, Michael A. Jacobetz, Donna-Michelle Smith, David A. Tuveson, John R. Griffiths, Duncan I. Jodrell

Published in: Cancer Chemotherapy and Pharmacology | Issue 5/2011

Abstract

Purpose

To develop a sensitive analytical method to quantify gemcitabine (2′,2′-difluorodeoxycytidine, dFdC) and its metabolites 2′,2′-difluorodeoxyuridine (dFdU) and 2′,2′-difluorodeoxycytidine-5′-triphosphate (dFdCTP) simultaneously from tumour tissue.

Methods

Pancreatic ductal adenocarcinoma tumour tissue from genetically engineered mouse models of pancreatic cancer (KP ^FL/FL C and KP ^R172H/+ C) was collected after dosing the mice with gemcitabine. ¹⁹F NMR spectroscopy and LC–MS/MS protocols were optimised to detect gemcitabine and its metabolites in homogenates of the tumour tissue.

Results

A ¹⁹F NMR protocol was developed, which was capable of distinguishing the three analytes in tumour homogenates. However, it required at least 100 mg of the tissue in question and a long acquisition time per sample, making it impractical for use in large PK/PD studies or clinical trials. The LC–MS/MS protocol was developed using porous graphitic carbon to separate the analytes, enabling simultaneous detection of all three analytes from as little as 10 mg of tissue, with a sensitivity for dFdCTP of 0.2 ng/mg tissue. Multiple pieces of tissue from single tumours were analysed, showing little intra-tumour variation in the concentrations of dFdC or dFdU (both intra- and extra-cellular). Intra-tumoural variation was observed in the concentration of dFdCTP, an intra-cellular metabolite, which may reflect regions of different cellularity within a tumour.

Conclusion

We have developed a sensitive LC–MS/MS method capable of quantifying gemcitabine, dFdU and dFdCTP in pancreatic tumour tissue. The requirement for only 10 mg of tissue enables this protocol to be used to analyse multiple areas from a single tumour and to spare tissue for additional pharmacodynamic assays.

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Title: A novel method for quantification of gemcitabine and its metabolites 2′,2′-difluorodeoxyuridine and gemcitabine triphosphate in tumour tissue by LC–MS/MS: comparison with 19F NMR spectroscopy
Authors: Tashinga E. Bapiro
Frances M. Richards
Mae A. Goldgraben
Kenneth P. Olive
Basetti Madhu
Kristopher K. Frese
Natalie Cook
Michael A. Jacobetz
Donna-Michelle Smith
David A. Tuveson
John R. Griffiths
Duncan I. Jodrell
Publication date: 01-11-2011
Publisher: Springer-Verlag
Published in: Cancer Chemotherapy and Pharmacology / Issue 5/2011
Print ISSN: 0344-5704
Electronic ISSN: 1432-0843
DOI: https://doi.org/10.1007/s00280-011-1613-0

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