Analytical considerations Drs. Jan Welink Training workshop: Assessment of Interchangeable Multisource Medicines, Kenya, August 2009

Guidance FDA Guidance for Industry ICH 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 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 Sensitive Accurate Discriminative Precise Method used for the determination of drugs and/or metabolites should be: Sensitive Accurate Discriminative Precise

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 The method should be able to discriminate between the selected analyte and interfering compounds from the environment or from other compounds administered simultaneously

Accuracy The method must be accurate enough to measure the true value (concentration) of the analyte in a relative small sample

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 To measure is to know!

Validation Specificity Detection limit (LOD) Quantification limit (LOQ) Linearity Range Accuracy Precision Robustness

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-specificity

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 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

World Health Organization Validation World Health Organization 22 April, 2017 LOQ, LOD and SNR Limit of Quantitation Limit of Detection Signal to Noise Ratio Peak B LOQ There are no specific criteria set for the Limit of Quantitation (LOQ) and Limit of Detection (LOD) but guidance is available from specifications and pharmacopeias. The noise is measured by running the instrument at maximum gain with no test being processed. The ripple generated is noise due to the instrument’s electronics, etc. The peak is measured relative to this noise and the ratio is calculated. This is known as the Signal to Noise Ratio (SNR). Generally: LOD SNR should be greater than 2:1. Peak A is acceptable for LOD but not for quantitation; The LOD can be calculated if the standard deviation (SD) of the response (which is standard deviation of the blank) and the slope is determined: LOD = 3.3 x SD slope Similarly LOQ = 10 x SD The LOQ SNR should generally be above 10:1. Peak B is suitable for quantitation; Precision as a percentage relative standard deviation should be 5 - 10% at the limit for LOQ. Peak A LOD noise Baseline

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 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-linearity

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 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-accuracy HQC MQC LQC

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/precision

Validation-accuracy/precision Between-day: Intra-day:

Validation-accuracy/precision: Accuracy/precision calibrators:

Validation-accuracy/precision FDA Accuracy Precision within-run between-run within-run between-run normally: <15% LLOQ: <20% normally: <15% LLOQ: <20%

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-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-robustness

Validation-recovery Recovery: Extraction efficiency analytical method consistent precise reproducible Recovery: 80% 75% 91% 97% 65% 73% Recovery: 15% 16% 13% 14% mean: 81.1% CV: 14.7% mean: 14.8% CV: 7.9%

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 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 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 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 samples - unexpected value - PK reason

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

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

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