Statistical analysis for analytical methods validations

Statistical analysis for analytical methods validations
AUTHORS:
Corneo R - Nanni V - Tizzani E
Nguyen Van Danh - Vanasia A
ASTRAZENECA SPA ITALY
A.VANASIA Srl
INTRODUCTION
In analytical chemistry every new analytical method, after the development step, must be
validated before routine use.
The validation process consist to evaluate the method results obtained with some statistical
approaches in order to define the systematic errors and application limits.
SCOPE OF VALIDATION PROCESS
The principal aim of the validation process is to discover the analytical limits, precision
and accuracy grade in order to evaluate the future analytical data obtained using the
method.
A full validation process should permit to verify if an anomalous result should be refused
or accepted. In this way we can maintain a constant analytical quality of results.
METHODOLOGY AND CONTENTS
In analytical chemistry there is a variety of analytical methods classified by analytical
methodology:
1. manual method (every operation is performed manually by analysts
2. automatic method (some or totally operation is performed by instrument).
For both these categories we will perform analytical validation. Obviously for the
automatic method the validation process is more complex and detailed because we have to
consider not only the systematic error of manual operations bur also the systematic errors
of every single part of automatic systems used. Actually in modern laboratories we
increase the automatic analysis therefore we need more efficiency for validation exercise.
The validation process is regulated by an International Guidelines or by the International
Pharmacopoeias (USP or PhEur).

Generally a validation process consist of the following study:
• Linearity
• Method or/and System Repeatability
• Accuracy
• Intermediate Precision
• Quantification Limit
• Detection Limit
• Robustness
All the data series produced with these studies should be evaluated with a specific program
in order to apply the same approach and elaboration. Moreover it is evident that every
calculation approach must be validated before the use.
For these reasons we choose the standard SAS statistical applications. In particular we
used the GLM REG Proc. with relative output files and options statistical evaluation for
various model acceptability limits (e.g. intercept calculation and relative confidence
limits).
Some models are correlate with automated graphics procedures.
This program is divided in:
• easier data entry
• pick list menu
• automated output
• automated graphics

Main Menu
Insert new Test

Select Test
Select existing Test

Modify Test’s Title
Screen of Test
Screen Accuracy of Data Entry/Modify
Accuracy
Title
TEST OF UK1 (FIRST)
Test Date __________ Level (%) 0.05 (0.05 0.10 0.25 0.50
1.00)
Analist
____________________
Content (%) 87.03 Degrado (%) 0.684
Weight mg % Theoric % Practic
1. 6.29 0.04271 0.042
2. 8.15 0.05535 0.052
3. 7.56 0.05134 0.048
4. 8.05 0.05467 0.052
5. 7.93 0.05385 0.051
6. 8.26 0.05609 0.053
7. 7.44 0.05052 0.047
8. 7.26 0.0493 0.046
9. ____________ ____________ ____________
10. ____________ ____________ ____________

Screen Linearity of Data Entry/Modify
Linearity Title TEST OF UK1 (FIRST)
Test Date __________ Level (%) _____ Analist ____________________ Unit MCG/ML
% Theoric Weight Mg X Value Y Area
Items : 1 0.05 10.13 2.12302 11280
1 0.05 9.98 2.09159 11051
2 0.1 19.98 4.18737 22764
2 0.1 20.18 4.22929 23546
3 0.2 40.34 8.45438 47523
3 0.2 40.38 8.46277 47341
4 0.4 80.75 16.92344 96199
4 0.4 80.8 16.93395 96140
5 0.5 101.05 21.17788 120200
5 0.5 100.7 21.10453 118552
6 0.6 121.54 25.47214 145930
6 0.6 121.25 25.41137 145243
7 0.8 161.52 33.85108 196441
7 0.8 161.58 33.86366 193612
8 1 202.08 42.35158 242366
8 1 201.92 42.31805 242741
9 ____________ ____________ ____________ ____________
9 ____________ ____________ ____________ ____________
10 ____________ ____________ ____________ ____________
10 ____________ ____________ ____________ ____________
Screen System Repeatability of Data Entry/Modify
System Repeatability (Precisione del Sistema)
Title
TEST OF UK1 (FIRST)
Test Date 04/08/2000 Level (%) 90 ( B=Before A=After )
Analist ____________________ Weight mg ____________
Area
1 2150570
2 2147458
3 2147478
4 2147382
5 2145879
6 2144850
7 2142292
8 2142521
9 2144085
10 2141689

Screen Intermediate Precision of Data Entry/Modify
Intermediate Precision
Title : TEST OF UK1 (FIRST)
Date 18/11/1999 18/11/1999 19/11/1999 19/11/1999
Name : Analist 1 Analist 2 Analist 3 Analist 4
LOCATELLI COLOMBO LOCATELLI COLOMBO
Order :
1. 0.113 0.213 0.108 0.203
2. 0.107 0.203 0.111 0.204
3. 0.11 0.21 0.109 0.205
4. 0.107 0.202 0.111 0.209
5. 0.107 0.214 0.11 0.206
6. 0.11 0.205 0.11 0.205
7. 0.115 0.214 0.11 0.204
8. 0.109 0.202 0.115 0.212
9. ____________ ____________ ____________ ____________
10 ____________ ____________ ____________ ____________
Screen Quantification Limit of Data Entry/Modify
Quantification Limit
Title
TEST OF UK1 (FIRST)
Test Date __________
Analist ____________________
Injection Level p/p mg Area
1 0.05 7.54 10932
2 0.05 7.15 9996
3 0.05 8.02 11773
4 ____________ ____________ ____________
1 0.025 4.17 5975
2 0.025 3.65 5076
3 0.025 3.96 5813
4 ____________ ____________ ____________
1 0.01 1.8 2168
2 0.01 2.03 2772
3 0.01 3.15 4570
4 ____________ ____________ ____________
1 0.005 2.48 1458
2 0.005 1.86 1374
3 0.005 1.57 973
4 ____________ ____________ ____________
1 ____________ ____________ ____________
2 ____________ ____________ ____________
3 ____________ ____________ ____________
4 ____________ ____________ ____________

Examples : Output of Linearity’s Test
Assay=TEST OF UK1 (FIRST)
Model: MODEL1
Dependent Variable: YAREA
Analysis of Variance
Sum of
Mean
Source DF Squares Square F Value Prob>F
Model 1 95135853350 95135853350 117896.127 0.0001
Error 14 11297249.362 806946.383
C Total 15 95147150599
Root MSE 898.30194 R-square 0.9999
Dep Mean 110058.06250 Adj R-sq 0.9999
C.V. 0.81621
Parameter Estimates
Parameter Standard T for H0:
Variable DF Estimate Error Parameter=0 Prob > |T|
INTERCEP 1 -1305.874836 394.49692146 -3.310 0.0052
XVALUE 1 5767.236825 16.79646991 343.360 0.0001
Linearity
Confidence Limit 95% of Intercept * Y = -1305.874836 +
5767.2368255 X
THEORY
Lower Upper Degrees T_student Sqrt Xvalue
Assay 95th 95th Freedom 97.5(%)DF Number Unit YAREA XVALUE
TEST OF UK1 (FIRST) -2151.99 -459.763 14 2.14479 4 MCG/ML 11280 2.1230 0.05
11051 2.0916 0.05
22764 4.1874 0.10
23546 4.2293 0.10
47523 8.4544 0.20
47341 8.4628 0.20
96199 16.9234 0.40
96140 16.9340 0.40
120200 21.1779 0.50
118552 21.1045 0.50
145930 25.4721 0.60
145243 25.4114 0.60
196441 33.8511 0.80
193612 33.8637 0.80
242366 42.3516 1.00
242741 42.3181 1.00