title: Extraction of tamoxifen and its metabolites from formalin-fixed, paraffin-embedded tissues: an innovative quantitation method using liquid chromatography and tandem mass spectrometry.
abstract: OBJECTIVE
  Tamoxifen is a key therapeutic option for breast cancer treatment. Understanding its complex metabolism and pharmacokinetics is important for dose optimization. We examined the possibility of utilizing archival formalin-fixed paraffin-embedded (FFPE) tissue as an alternative sample source for quantification since well-annotated retrospective samples were always limited.
  
  
  METHODS
  Six 15 μm sections of FFPE tissues were deparaffinized with xylene and purified using solid-phase extraction. Tamoxifen and its metabolites were separated and detected by liquid chromatography-tandem mass spectrometry using multiple-reaction monitoring.
  
  
  RESULTS
  This method was linear between 0.4 and 200 ng/g for 4-hydroxy-tamoxifen and endoxifen, and 4-2,000 ng/g for tamoxifen and N-desmethyl-tamoxifen. Inter- and intra-assay precisions were <9 %, and mean accuracies ranged from 81 to 106 %. Extraction recoveries were between 83 and 88 %. The validated method was applied to FFPE tissues from two groups of patients, who received 20 mg/day of tamoxifen for >6 months, and were classified into breast tumor recurrence and non-recurrence. Our preliminary data show that levels of tamoxifen metabolites were significantly lower in patients with recurrent cancer, suggesting that inter-individual variability in tamoxifen metabolism might partly account for the development of cancer recurrence. Nevertheless, other causes such as non-compliance or stopping therapy of tamoxifen could possibly lead to the concentration differences.
  
  
  CONCLUSIONS
  The ability to successfully study tamoxifen metabolism in such tissue samples will rapidly increase our knowledge of how tamoxifen's action, metabolism and tissue distribution contribute to breast cancer control. However, larger population studies are required to understand the underlying mechanism of tamoxifen metabolism for optimization of its treatment.
authors.affiliation: Pharmacokinetic Unit, Tom Baker Cancer Centre, Alberta Health Services, Calgary, AB, Canada.
authors.forename: Ella S M
authors.initials: ES
authors.lastname: Ng
authors.forename: S Bill
authors.initials: SB
authors.lastname: Kangarloo
authors.forename: Mie
authors.initials: M
authors.lastname: Konno
authors.forename: Alexander
authors.initials: A
authors.lastname: Paterson
authors.forename: Anthony M
authors.initials: AM
authors.lastname: Magliocco
mesh_terms: D001943:Breast Neoplasms / Q000737:chemistry* / Q000188:drug therapy / Q000378:metabolism*; D002853:Chromatography, Liquid / Q000379:methods*; D005260:Female; D005557:Formaldehyde; D006801:Humans; D009364:Neoplasm Recurrence, Local / Q000737:chemistry / Q000378:metabolism; D010232:Paraffin; D010865:Pilot Projects; D012189:Retrospective Studies; D013629:Tamoxifen / Q000031:analogs & derivatives / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D013629:Tamoxifen; C016601:afimoxifene; D005557:Formaldehyde; C055492:4-hydroxy-N-desmethyltamoxifen; D010232:Paraffin