Analytical Methods ValidationM. Hatta PrabowoDepartment of Pharmaceuticals ChemistryUniversitas Islam Indonesia

Chemical analysisThe 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 objectiveTo demonstrate that the analytical procedure is suitable for its intended purpose

Analytical method validation

Analytical Procedure

Selectivity

Accuracy

Precision- within-run- between-run

Recovery

Limit of Quantitation LOQ

Calibration curve

Robustness

Validation

Stability

Sample Sample preparationpreparation Separation Separation DetectionDetection

Analytical procedure

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?

AccuracyThe 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 acceptedxx xx xx xxxx

xx

PrecisionThe 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.nmol/l

Accuracy(%)

Precision

% %

n

0.76 0.6 5.1 6.1 1823 3.6 1.7 1.8 18122 3.9 1.3 1.3 18

Within-run Between-run

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

Sample No. nmol/l Accuracy LLOQ 1 1.58 5.3% LLOQ 2 1.61 7.3% LLOQ 3 1.46 -2.6% LLOQ 4 1.44 -4.0% LLOQ 5 1.50 0.0% LLOQ 6 1.49 -0.7% Mean: 1.51 0.9%

SD: 0.067 CV%: 4.5

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

RobustnessHow many samples can be analysed in

one run?

85

90

95

100

105

110

115

0 10 20 30 40 50 60 70 80 90 100 110

Sample No.

Foun

d co

ncen

trat

ion

%

Robustness

Stability of your substance

In the automatic injector

In plasma during storage

In room temperature (4 h)

In Freeze/Thaw tests

In stock solutions

Analytical method validation

Analytical Procedure

Selectivity

Accuracy

Precision- within-run- between-run

Recovery

Limit of Quantitation LOQ

Calibration curve

Robustness

Validation

Stability

References1. Guidance for Industry

Bioanalytical Method Validation FDA, May 2001

2. Workshop Report: Shah, V.P. Et al., Pharmaceutical Research: 1992; 9:588-592.

3. 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

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 lowest calibration sample is injected before each run.

The system is accepted if:

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