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 (19)F NMR spectroscopy.
abstract: OBJECTIVE
  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.
  
  
  CONCLUSIONS
  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.
authors: Cancer Research UK, Cambridge Research Institute, Li Ka Shing Centre, Robinson Way, Box 278, Cambridge CB2 0RE, UK.
authors: Tashinga E
authors: TE
authors: Bapiro
authors: Frances M
authors: FM
authors: Richards
authors: Mae A
authors: MA
authors: Goldgraben
authors: Kenneth P
authors: KP
authors: Olive
authors: Basetti
authors: B
authors: Madhu
authors: Kristopher K
authors: KK
authors: Frese
authors: Natalie
authors: N
authors: Cook
authors: Michael A
authors: MA
authors: Jacobetz
authors: Donna-Michelle
authors: DM
authors: Smith
authors: David A
authors: DA
authors: Tuveson
authors: John R
authors: JR
authors: Griffiths
authors: Duncan I
authors: DI
authors: Jodrell
mesh_terms: D000818:Animals; D000970:Antineoplastic Agents / Q000378:metabolism*; D002138:Calibration; D003570:Cytidine Triphosphate / Q000031:analogs & derivatives* / Q000378:metabolism; D003841:Deoxycytidine / Q000031:analogs & derivatives* / Q000378:metabolism; D004195:Disease Models, Animal; D005467:Floxuridine / Q000031:analogs & derivatives* / Q000378:metabolism; D005461:Fluorine; D006801:Humans; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D051379:Mice; D012015:Reference Standards; D053719:Tandem Mass Spectrometry / Q000379:methods*; D000093542:Gemcitabine
chemical_list: D000970:Antineoplastic Agents; D005467:Floxuridine; D003841:Deoxycytidine; D005461:Fluorine; D003570:Cytidine Triphosphate; C533693:gemcitabine triphosphate; C084565:2',2'-difluoro-2'-deoxyuridine; D000093542:Gemcitabine