Analytical Challenges In The Development Of An Assay For Capecitabine And Three Of Its Metabolites

Lori D. Payne* and Ryan Morrison^
*Bioanalytical Systems, Inc., McMinnville, OR, USE
^Pfizer Global Research & Development, Chesterfield, MO, USA


To develop a robust bioanalytical assay for capecitabine and three of its metabolites in human plasma to support clinical oncology studies.


Cape = capecitabine (MW 359)
5DFC/5’-DFCR = 5’-deoxy-5-fluorocytidine (MW 245)
5FUR/5’-DFUR = 5’-deoxy-5-fluorouridine (MW 246)
5FU = 5-fluorouracil (MW 128)

Metabolic Pathway: Capecitabine to 5-FU

Analytical Challenges

  • Mulitple analytes
  • Metabolites are highly water soluble (poorly retained)
  • Low molecular weight for 5FU (interferences - clean up required)
  • Both positive and negative ionization mode required
  • Dimerization seen at high concentrations
  • Stable label internal standard available only for parent
  • Capecitabine unstable in plasma!

Step 1: Determine MS Conditions

Challenge: Positive/Negative Ionization

Step 2: DryLab Experiments/Chromatorgraphy

Challenge: Poor retention.

  • Poor retention on RP-18 except for capecitabine
  • Low organic gradient required for the three analytes
  • Metabolite retention order switch with NH4 formate to formic acid
  • Good peak shape
  • Polarity change required to capture parent and metabolites; minor drifts in retention times could be problematic
  • MPA: formic acid, NH4 formate and NH4 carbonate
  • MeOH and ACN as MPB

Step 3: Internal Standard Candidates based on Structural Similarity

Challenge 1: Positive/Negative Ionization
Challenge 2: No good candidates

Screen more IS candidates

  • Gemcitabine (known to be unstable)
  • 5-chloro-2-deoxy-uridine
  • 2-deoxy-uridine

Step 4: Optimize Chromatography

Challenge 1: Short Column Life
Challenge 2: Optimize Chromatography Conditions for all Analytes
Challenge 3: Internal Standard Variability

  • Better peak shape on new column
  • ↑CV as column aged
  • Continued variability of IS and 5FUR (earliest eluting peak) response with time
  • Isocratic hold at beginning
  • pH of carbonate buffer
  • Concentration of carbonate buffer
  • Gradient (linear, step, final)
  • Re-equilibration time
  • Isocratic hold at end

Introduction of New Concept: Mass Spec Re-equilibration Time

Two isocratic runs (two injections/one extraction), one for cape (4 min) and one for metabolites (6 min) instituted. Overall run time the same.


  • ↑ analytes ↑ chance of failure statistically
  • Concept of “mass spec” re-equilibration beyond column reequilibration for early eluting compounds
  • Sometimes two isocratic runs = the time of a gradient
  • Capecitabine and metabolites assay successfully validated and used for oncology clinical trials.