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Analytical considerations
Drs. Jan Welink
Training workshop: Assessment of Interchangeable Multisource Medicines, Kenya, August 2009
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GuidanceGuidance
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
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GCP/GLPGCP/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.
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Choice of methodChoice of method
Method used for the determination of drugs and/or metabolites should be:
SensitiveAccurateDiscriminativePrecise
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SensitivitySensitivity
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
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DiscriminativeDiscriminative
The method should be able to discriminate between the selected analyte and interfering compounds from the environment or from other compounds administered simultaneously
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AccuracyAccuracy
The method must be accurate enough to measure the true value (concentration) of the analyte in a relative small sample
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PrecisionPrecision
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
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ValidationValidation
To measure is to know!
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ValidationValidation
Specificity
Detection limit (LOD)
Quantification limit (LOQ)
Linearity
Range
Accuracy
Precision
Robustness
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Validation-specificityValidation-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
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Validation-specificityValidation-specificity
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Validation-LODValidation-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
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Validation-LOQValidation-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
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LOQ, LOD and SNR
Limit of Quantitation
Limit of Detection
Signal to Noise Ratio
noise
Peak ALOD
Peak BLOQ
Baseline
Validation
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Validation-LOD/LOQValidation-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
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Validation-linearityValidation-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
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Validation-linearityValidation-linearity
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Validation-rangeValidation-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
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Validation-accuracyValidation-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
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Validation-accuracyValidation-accuracy
LQC
MQC
HQC
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Validation-precisionValidation-precision
Repeatability– concentrations covering the specified range
Intermediate precision– Like days, analysts, equipment
Reproducibility– Determined if analyses take place in separate periods
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Validation-accuracy/precisionValidation-accuracy/precision
Accuracy/precision:
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Validation-accuracy/precisionValidation-accuracy/precision
Intra-day:Between-day:
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Validation-accuracy/precision:Validation-accuracy/precision:
Accuracy/precision calibrators:
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Validation-accuracy/precisionValidation-accuracy/precision
FDA
Accuracy
within-run between-run
normally: <15%
LLOQ: <20%
Precision
within-run between-run
normally: <15%
LLOQ: <20%
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Validation-robustnessValidation-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
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Validation-robustnessValidation-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
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Validation-robustnessValidation-robustness
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Validation-recoveryValidation-recovery
Recovery:
Extraction efficiency analytical method– consistent– precise– reproducible
Recovery:80%75%91%97%65%73%
mean: 81.1%
CV: 14.7%
Recovery:15%16%13%15%16%14%
mean: 14.8%
CV: 7.9%
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Validation-stabilityValidation-stability
Required data – Freeze and thaw stability– Short term temperature stability– Long term stability– Stock solution stability– Post preparation stability
Stability assessed prior subject sample analysis!
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Analysis clinical samplesAnalysis 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 QCQC QC QCQC
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Analysis clinical samplesAnalysis clinical samples
Acceptation or rejection of a run should be predefined before the actual start of the analysis of the clinical samples.
QC QCQC QC QCQC
FDA criteria
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Analysis clinical samplesAnalysis 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.
QC QCQC QC QCQC
Reasons: -improper sample injection
-mail function -concentration > HLOQC
-unexpected value -PK reason
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Analysis clinical samplesAnalysis clinical samples
-unexpected value
-PK reason
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ReportReport
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
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ReportReport
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
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EndEnd