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

Objective

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

Abbreviations

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
â†‘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.

Conclusions

â†‘ analytes â†‘ 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.

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