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Your guide to method development

When we consider method development as part of the bioanalytical process from discovery, through to pre-clinical and clinical studies, its purpose is to define the method, provide sound scientific evidence for the method design and check its suitability for its intended purpose.

It’s important for method development activities to be adequately documented to support a reproducible method that is ready for validation. If the experiments below are incorporated into the method development plan and properly executed, then the resultant method design should be deemed suitable to proceed with validation:

  • Analyte information – structure, properties, pKa value, solubility, stability in solution and adsorption properties (to plastic or glass)
  • Internal standard – stable labelled, analogue. Should be monitored for consistency of response
  • Anticoagulant and counter ion
  • Instrument ionisation mode, conditions and parameters. Minor parameters may be optimised to account for instrument to instrument variability and improve response (i.e.voltage and gas settings)
  • Chromatography column, mobile phase composition, gradient profile
  • If metabolites are known and standards are available, then an evaluation of metabolite impact on quantitation of analyte is required
  • Sample preparation – simple dilution in diluent, protein precipitation, liquid-liquid extraction, supported-liquid extraction or solid phase extraction, other
  • Method optimisation to determine the achievable LLOQ
  • QC preparation at an appropriate to range
  • Calibration curve weighting
  • Phospholipid and dosing vehicle impact evaluation
  • Recovery
  • Haemolysis
  • Matrix stability (including whole blood and collection stability if appropriate)
  • Extract stability
  • Initial Carryover Evaluation
  • Post-column infusion experiment

Before starting method validation, a pre-validation run should be performed to assess the following:

  • Accuracy and precision
  • Carryover
  • Freeze / thaw stability
  • Bench top stability
  • Long term stability
  • Recovery
  • Matrix effects
  • Selectivity

Length of run should mimic longest expected sample set, padded with blanks.

Once the sample preparation parameters are refined, they must remain fixed during validation. Changes in aliquot volume or sample preparation parameters necessitate at least partial revalidation.