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Analytical considerations. Drs. Jan Welink. Training workshop: Assessment of Interchangeable Multisource Medicines, Kenya, August 2009. Guidance. FDA Guidance for Industry Bioanalytical method validation, May 2001 ICH Guidance for industry

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slide1

Analytical considerations

Drs. Jan Welink

Training workshop: Assessment of Interchangeable Multisource Medicines, Kenya, August 2009

guidance
Guidance
  • FDA Guidance for Industry
    • Bioanalytical method validation, May 2001
  • ICH Guidance for industry
    • Validation of analytical methods: definitions and terminology, June 1995
    • Validation of analytical procedures: methodology, November 1996
gcp glp
GCP/GLP
  • GCP/GLP compliance
    • Clinical studies have to be performed under conditions complying with the principles of Good Clinical Practice, and for analytical methods and sample data handling conditions complying with the principles of Good Laboratory Practice are required.
    • For older studies without statement of compliance with the above mentioned principles, the assessor should rely on the quality of the submitted report.
choice of method
Choice of method
  • Method used for the determination of drugs and/or metabolites should be:

Sensitive

Accurate

Discriminative

Precise

sensitivity
Sensitivity
  • Method should be able to quantify the drug in the sampled specimen at least 10 % of the maximum concentration reached after dosing.

Limit of Quantification (LOQ): 1/10 Cmax

discriminative
Discriminative
  • The method should be able to discriminate between the selected analyte and interfering compounds from the environment or from other compounds administered simultaneously
accuracy
Accuracy
  • The method must be accurate enough to measure the true value (concentration) of the analyte in a relative small sample
precision
Precision
  • The analytical method should be precise enough to reveal identical results when the procedure is applied repeatedly to multiple aliquots of a single homogeneous volume of the biological matrix
validation
Validation

To measure is to know!

validation10
Validation
  • Range
  • Accuracy
  • Precision
  • Robustness
  • Specificity
  • Detection limit (LOD)
  • Quantification limit (LOQ)
  • Linearity
validation specificity
Validation-specificity
  • Investigation of specificity should be conducted during the validation phase of the assay
  • The procedures used to demonstrate specificity should be clearly reported
  • Must be applied with structurally similar materials
  • Choices base on scientific judgements
validation lod
Validation-LOD
  • Various methods possible

visual evaluation

  • minimum level at which the analyte can be detected reliably

signal-to noise

  • 3:1 ratio is acceptable

standard deviation of the slope and response

  • LOD = 3.3 σ / S
    • σ = standard deviation of the response
    • S = slope of the calibration curve
validation loq
Validation-LOQ
  • Based on signal-to noise
    • Reliable quantification is a 10:1 ratio
  • Based on SD of the response and the slope
    • LOQ = 10 σ / S
      • σ = standard deviation of the response
      • S = slope of the calibration curve
validation15
Validation

LOQ, LOD and SNR

  • Limit of Quantitation
  • Limit of Detection
  • Signal to Noise Ratio

Peak BLOQ

Peak ALOD

noise

Baseline

validation lod loq
Validation-LOD/LOQ

Recommended data:

  • The LOD and LOQ and the method used for the LOQ should be presented
  • The limits should be validated by the analyses of a suitable number of samples prepared at the LOD and LOQ limits
validation linearity
Validation-linearity
  • Should be evaluated across the range of concentrations expected during the study
  • A minimum of five concentrations used in the range is recommended
  • The correlation coefficient, y-intercept slope of the regression and residual sum of squares should be submitted
  • Deviations from the regression line should be analysed for evaluating linearity
validation range
Validation-range
  • The specified range is derived from linearity studies and should cover the extremes of the concentrations probably reached during the study
  • The range should be justified in the report based on scientific information
validation accuracy
Validation-accuracy
  • Accuracy should be assessed on samples spiked with known amounts of the analyte
  • Accuracy should be assessed using determinations over a minimum of 3 concentration levels (low, medium and high)
  • Accuracy should be reported as percent recovery from the added amount
validation precision
Validation-precision
  • Repeatability
    • concentrations covering the specified range
  • Intermediate precision
    • Like days, analysts, equipment
  • Reproducibility
    • Determined if analyses take place in separate periods
validation accuracy precision24
Validation-accuracy/precision

Between-day:

Intra-day:

validation accuracy precision25
Validation-accuracy/precision:

Accuracy/precision calibrators:

validation accuracy precision26
Validation-accuracy/precision

FDA

Accuracy

Precision

within-run

between-run

within-run

between-run

normally: <15%

LLOQ: <20%

normally: <15%

LLOQ: <20%

validation robustness
Validation-robustness
  • Robustness should be considered during development phase
  • Shows the reliability of the analytical method with respect to variations in the method parameters
  • In case variations occur they should be suitably controlled and if present adequately tested and documented
validation robustness28
Validation-robustness

Typical examples:

  • Stability of the analytical solutions
    • Influence of variations of pH of the mobile phase
    • Influence of variations of mobile phase composition
    • Influence of temperature and flow rate
  • Extraction conditions
    • pH and extraction time
validation recovery
Validation-recovery

Recovery:

  • Extraction efficiency analytical method
    • consistent
    • precise
    • reproducible

Recovery:

80%

75%

91%

97%

65%

73%

Recovery:

15%

16%

13%

15%

16%

14%

mean: 81.1%

CV: 14.7%

mean: 14.8%

CV: 7.9%

validation stability
Validation-stability

Stability assessed prior subject sample analysis!

  • Required data
    • Freeze and thaw stability
    • Short term temperature stability
    • Long term stability
    • Stock solution stability
    • Post preparation stability
analysis clinical samples
Analysis clinical samples
  • The analytical method should be validated before the start of obtaining clinical samples.
  • Each analytical run should contain sufficient QC samples at the beginning, middle and end at at least 3 levels (LQC, MQC and HQC).

QC

QC

QC

QC

QC

QC

analysis clinical samples33
Analysis clinical samples
  • Acceptation or rejection of a run should be predefined before the actual start of the analysis of the clinical samples.

FDA criteria

QC

QC

QC

QC

QC

QC

analysis clinical samples34
Analysis clinical samples
  • All samples of 1 subject in 1 run
  • Subject sample reanalysis should be predefined before the actual start of the analysis of the clinical samples.

Reasons:

- improper sample injection

- mail function

- concentration > HLOQC

- unexpected value

- PK reason

QC

QC

QC

QC

QC

QC

analysis clinical samples35
Analysis clinical samples

- unexpected value

- PK reason

report
Report
  • All methods should be covered by adequate Standard Operating Procedures (SOP’s) for general and analysis specific procedures
  • Before the start of an analytical procedure an adequate study plan has to be written or be incorporated in the study protocol
report37
Report
  • A specific detailed description of the bioanalytical method should be written
  • All experiments used to make claims or draw conclusions should be presented in the report
  • GLP compliance/inspections/audits