ZOOM Online: HPLC Analytical Method Development and Validation

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Good HPLC methods must satisfy both technical requirements (sensitivity, specificity, linearity, accuracy and precision) as well as business needs (reliability in routine use and a run time appropriate to the number of samples to be tested). These requirements are equally important in both a development and routine QC context: decisions during drug development must be based on reliable data, and routine QC testing, including stability studies, must control risks to product quality and patient safety.

This course presents a logical, step-wise approach to the development of HPLC methods (Day 1) and then explains how to validate chromatographic methods in line with regulatory expectations and best practice (Day 2).

The course is intended for analytical scientists who have experience of operating HPLC instrumentation.

Who Should Attend?

Scientists working with HPLC who need to further their understanding of the technique in order to develop better methods faster. Scientists who have to validate HPLC methods in accordance with current internationally-accepted guidance. HPLC technicians working in R&D laboratories, quality control laboratories and stability testing laboratories. Managers with a responsibility for generating regulatory submissions.

Course Programme

Day 1 - HPLC Method Development

Revision of chromatographic theory

  • Separation modes
  • Factors affecting resolution
  • Peak symmetry
  • Band broadening
  • The effect of particle size and extra-column volume on efficiency

Important chemical concepts

  • Factors affecting analyte/stationary phase interactions (polarity, hydrogen bonding and pKa)
  • Stationary phase endcapping

Analyte properties affecting pKa, solubility and detectability

Workshop: reading solute structures

Matrix properties

  • Effect on extraction
  • Chemical interference
  • Selectivity and detector wavelength
  • Injection solvent strength

Method performance requirements

  • Defining method performance requirements
  • Measurement uncertainty vs. specification limits

Sample preparation

  • Selective sample preparation
  • Choice of filter membrane
  • Chemical and physical stability of samples

Developing the separation

  • Starting conditions for different separation types

Neutral/ionisable organic molecules

Special cases

  • Separation modes: isocratic, gradient, ion pairing/suppression, HILIC, aqueous normal phase, normal phase, ion exchange and size exclusion

Retention mechanisms

  • Choice of stationary phase (including silanol activity considerations)
  • Mobile phase pH and solute pKa
  • Choice of pH buffer
  • Temperature effects
  • Core-shell and UHPLC columns
  • Detector selection

Gradient elution

  • When to use gradient elution
  • Significance of gradient delay volume
  • Retention and resolution models in gradient separations
  • Gradient profile optimisation
  • The effect of column dimensions and temperature
  • Step-wise gradient method development strategy

Case studies

Workshop: selecting starting conditions for method development

Day 2 - HPLC Method Validation

Regulatory guidance (ICH, US, EU, WHO)

Validation terminology

Setting meaningful acceptance criteria

  • Acceptance criteria based on specification limits
  • Measurement uncertainty and sources of error
  • Typical acceptance criteria
  • Lifecycle approach to method validation: Analytical Target Profile (ATP) and analytical control strategy

Experimental approaches to method validation

  • Specificity: with and without impurity standards
  • Linearity: best practice
  • Use of spiking experiments
  • Options for evaluating sensitivity
  • Recommended robustness experiments

Phase-appropriate method validation

Workshop " setting method validation acceptance criteria

Writing effective analytical methods, validation protocols and reports

  • Pre-validation check-list
  • Contents of method and validation documentation
  • Mistake-proofing analytical methods

Dealing with validation failures

Setting system suitability criteria

  • Regulatory guidance
  • Statistically-based methods

Verifying compendial procedures

  • Regulatory guidance
  • Approaches for different method types

Workshop " planning a method validation exercise

What people said about ZOOM Online: HPLC Analytical Method Development and Validation

"I had high expectation, and it was delivered"

"Slides were clearly laid out and not too heavy or full of jargon"

"A lot of information, very good to follow"

"Dynamic and interactive"

"Excellent comprehensive course materials and well delivered"

" Roland is very knowledgeable and shares loads of personal experience that gives us different ways of looking at things "

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