CPSA Digest 2003

Day 3

WeOB1

A Method Development Paradigm

Common Misconceptions

• Assay validation does not equal assay optimization.
• A validated method is not necessarily a tight method (A method only needs to answer the question being asked; if batch size is 50 then why invest additional time to develop an assay for thousands of samples).
• Repeating an assay a number of times does not constitute validation.

Stages

• Stage I - Initial method development; Spend the bulk of time here.
     - Sample preparation and method development
     - LC method development
     - LC-MS/MS method development
• Stage II - Pre-validation; Does the method have sufficient quality to complete the study?
• Stage III - Validation; This stage should occupy about one week.
• Stage IV - Application.

Sample Extraction

Interested in:

• Recovery
• Interference
• Precision, accuracy & ruggedness

The "ideal" extraction method:

• Works for all pharmaceutical compounds
• 100% recovery
• No ion suppression
• Utilize 96-well format

Common Extraction Protocols

• "Dilute and Shoot" - Protein Precipitation    - Fast method development
     - Most susceptible to ion suppression
     - Matrix effects seen when assaying incurred samples
     - Most likely to foul the mass spectrometer source
• Solid-Phase Extraction (SPE) and Liquid-Liquid Extraction (LLE) Methods    - Robust and scalable
     - Method development can be time-consuming
     - Clean injections presented to mass spec source

• When the extraction is very selective, less effort needs to be spent on the chromatography.
• Chromatography is inherently more difficult to optimize than the extraction protocol.
• When a clean sample is presented to the analytical system, more flexibility exists with the chromatography.

First choice - SCX solid-phase extraction

• Molecule is protonated at low pH and is attracted by ion exchange functional group.
• Remove lipids and neutrals with an aggressive organic wash.
• Ensure release of drug during elution step by making the analyte neutral; the eluant is volatile for a subsequent evaporation step if necessary.

• Tighter particle packing reduces eddy diffusion to maximize interaction of analyte with sorbent pores.
• Silica Quality; run of the mill 40-60 μm does not have as tight control over quality of material.
• 10 μm particles are prepared using HPLC processes and result in higher quality, fewer impurities and a more uniform particle size.

Extraction Fine Tuning
Perform a "2D" experiment; Load/Elute at different pHs

• Load/wash under acidic or basic conditions
     - Analyte retention
     - Removal of polar interferences
• Elute under acidic or basic conditions
     - Minimum organic required for recovery of analyte
     - Retain hydrophobic interferences

% Organic vs. pH
Increase % organic from 0 to 100% Optimum load/wash is up to 40% methanol since analyte is retained on column On elution, 80% organic and greater yields analyte = cleanliness Leave polar materials on column; not looking for 100% recovery

HPLC Method Development

• Mobile phase solvent
     - Volatility, amount of water, ionization issues
• Mobile phase additives
     - olatility, concentration, ion suppression, buffering capacity
• Column
     - Retention time, peak shape, resolution

LC-MS/MS Method Development

• Optimize MS/MS conditions
     - APCI versus Electrospray
     - +/- ion mode
• Reproducibility
• Carryover
• Determine limit of detection (LOD) and limit of quantitation (LOQ)

Method Development & Validation

• Develop extraction protocol
  1 to 2 weeks
• Develop chromatography
  1 to 2 weeks
• Validate analytical assay (per species & matrix)
  2 to 4 weeks

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