Bioavailability Bioavailability means the rate and extent to which the active substance is adsorbed from a pharmaceutical product and become available at the site of action

Bioequivalence Two medical products are bioequivalent if they are pharmaceutical equivalent or pharmaceutical alternatives and if their bioavailabilities after administration in the same molar dose are similar to such degree that their effects, with respect to both efficicy and safety, will be essentially the same

Design and conduct of studies The study should be designed in such a way that the formulation effect can be distinguished from other effects. Most common is a two-period, two-sequence crossover design, if the formulations to be compared is two Single dose studies Steady-state studies

Design and conduct of studies Adequate wash out periods between treatments Sampling schedule to provide an adequate estimation of Cmax to cover the plasma concentration time curve long enough, 80% of AUC 24 hours cycle at steady state? drugs with long half-life?

Subjects Healthy volunteers The inclusion/exclusion criteria should be clearly stated in the protocol Both sex 18-55 years old Normal weight Screened for laboratory test medical history medical examination preferable non-smokers and without a history of alcohol or drug abuse

Chemical analysis The bioanalytical part of bioequivalence trials should be conducted according to the applicable principle of Good Laboratory Practice (GLP)

Good Laboratory Practice (GLP) Test plan (Analytical protocol) Sample traceability Documentation, possible to reconstruct the study Analytical method validation report Analytical report signed by responsible investigator

Pre-study phase The method used must be well characterised Stability Specificity Accuracy Precision Limit of quantitation Response function

Validation objective To demonstrate that the analytical procedure is suitable for its intended purpose

Analytical method validation Analytical Procedure Stability Selectivity Robustness Validation Accuracy Calibration curve Precision - within-run - between-run Limit of Quantitation LOQ Recovery

Analytical procedure Sample preparation Separation Detection

Specificity (selectivity) Ability of an analytical method to measure only what it is intended to measure Blank samples from six different subjects Will other drugs, metabolites or endogenous components interfere in the measurements?

Accuracy The closeness of mean test results obtained by the analytical method to the true value (concentration) of the analyte.

Accuracy Accuracy should be measured at minimum 3 levels At least 5 determinations per concentration The mean value should be within 15% of the actual value At the lower limit of quantitation level within 20% is accepted x x x x x x

Precision The closeness of individual measurements of an analyte when the procedure is applied repeatedly to multiple aliquotes of a single homogenous volume of biological matrix

Precision Precision should be measured at minimum 3 levels At least 5 determinations per concentration The calculated CV should not exceed 15% At the lower limit of quantitation level, CV should not exceed 20% Subdivided into within-run and between-run

Precision and Accuracy Conc. Accuracy Precision n nmol/l (%) Within-run Between-run % % 0.76 0.6 5.1 6.1 18 23 3.6 1.7 1.8 18 122 3.9 1.3 1.3 18

Recovery The extraction efficiency of an analytical method Recovery of an analyte need not be 100%

Lower limit of quantitation The lowest standard on the calibration curve should be accepted as the lower limit of quantitation (LLOQ) if

Lower limit of quantification The analyte responce at LLOQ is at least 5 times the blank response The peak should be identifiable and discrete Precision within 20% CV Accuracy of 80-120%

LLOQ (1.50 nmol/l) for morphine

Calibration/Standard curve A calibration curve is the relation between instrument response and known concentrations of the analyte Should be prepared in the same biological matrix as the samples Should consist of 6-8 samples covering the expected range Should include LLOQ and a blank sample Should include a zero sample (with internal standard) Same curve fitting, weighting in prestudy and study Any changes should be documented

Calibration curve

Sample dilution Any required sample dilutions should use like matrix Dilution QC sample should be used

Robustness How many samples can be analysed in one run?

Robustness 115 110 105 Found concentration % 100 10 20 30 40 50 60 70 10 20 30 40 50 60 70 80 90 100 110 95 90 85 Sample No.

Stability of your substance In Freeze/Thaw tests In room temperature (4 h) In the automatic injector In stock solutions In plasma during storage

Analytical method validation Analytical Procedure Stability Selectivity Robustness Validation Accuracy Calibration curve Precision - within-run - between-run Limit of Quantitation LOQ Recovery

References Guidance for Industry Bioanalytical Method Validation FDA, May 2001 Workshop Report: Shah, V.P. Et al., Pharmaceutical Research: 1992; 9:588-592. Workshop Report: Shah, V.P. et al., Pharmaceutical Research: 2000; 17:1551-1557

Costs Validation = 130-180.000 SEK Stability = 15-20.000 SEK for each time point QA = 11.000 SEK/study

The study phase (1) ...in which the validated bioanalytical method is applied to the actual analysis of samples from the biostudy mainly in order to confirm the stability, accuracy and precision.

The study phase (2) Calibration curve in each run Six Quality Control samples in each run Pre-stablished SOPs for procedures (method) Acceptance criteria for a run - accuracy and precision of the calibration curve - accuracy and precision of the QC samples - repeat analysis It is preferable to analyse all study samples from a subject in a single run

The study phase (3) The QC samples should be used to accept or reject the run (2 samples at 3 levels) Four QC samples out of six should be within 15% of their nominal value Two QC samples can be outside ±15% but not both at the same concentration

System suitability test The lowest calibration sample is injected before each run. The system is accepted if: Signal to noise ratio is above 5 for the substance. The peak shape is acceptable after visual inspection of the chromatogram The retention times are within 10% of the previous run.

The analytical report should include Results for all calibration curves Results for all quality control samples Representative number of chromatograms Should include data from subjects who eventually dropped-out Reanalysed samples and the reason for reanalyses The analytical validation report The responsible investigator(s) should sign for their respective section of the report

Chiral active substances The bio-analytical method should be enantiomeric Unless Both products contain the same stable singel enantiomer Both products contain the racemate and both enantiomers show linear pharmacokinetics

Also guidance for Reference and test product Data analysis In vitro dissolution comlementary to a bioequivalence study Reporting of results Application for products containing new active substances Application for products containing approved active substances is given