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Comparative Study
. 2011 Nov;68(5):1243-53.
doi: 10.1007/s00280-011-1613-0. Epub 2011 Mar 23.

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 (19)F NMR spectroscopy

Tashinga E Bapiro  1 , 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
Affiliations
Comparative Study

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 (19)F NMR spectroscopy

Tashinga E Bapiro et al. Cancer Chemother Pharmacol. 2011 Nov.

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. (19)F NMR spectroscopy and LC-MS/MS protocols were optimised to detect gemcitabine and its metabolites in homogenates of the tumour tissue.

Results: A (19)F 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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Figures

Fig. 1
Fig. 1
Overlaid spectra of 19F NMR peaks for 30 nmoles of dFdC (green), dFdU (blue), dFdCTP (red), spiked and quantified separately into an untreated KP FL/FL C tumour sample
Fig. 2
Fig. 2
Typical LC–MS/MS chromatogram for tumour tissue homogenate spiked with concentrations of analytes at the lower limit of quantification, a gemcitabine (0.2 ng/mg), b dFdU (0.4 ng/mg) and d dFdCTP (0.2 ng/mg). Internal standards: c dFUR and e 13C, N15CTP
See this image and copyright information in PMC

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