2. 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

3. 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

4. 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

5. 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?

6. 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

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

8. 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

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

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

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

12. Analytical procedure
Sample preparation
Separation
Detection

13. 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?

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

15. 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

16. 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

17. 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

18. 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

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

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

21. 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 %

22. LLOQ (1.50 nmol/l) for morphine

23. 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

24. Calibration curve

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

26. Robustness
How many samples can be analysed in one run?

27. Robustness 115 110 105 Found concentration % 100 10 20 30 40 50 60 7010 20 30 40 50 60 70 80 90
100
110 95 90 85
Sample No.

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

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

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

31. Costs Validation = 130-180.000 SEK
Stability = SEK for each time point
QA = SEK/study

32. 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.

33. 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

34. 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

35. 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.

36. 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

37. 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

38. 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