W. Wegscheider
Validation: an Example
Validation: an Example
Learning Objectives: Student
 is able to inquire in sufficient depth on the purpose of analytical work
 is able to translate this information into stepwise strategy keeping in
mind suitable performance characteristics at each step
 can devise experiments to test against these criteria
 performs statistical analysis of obtained data with the goal to make a
valid judgement regarding the suitability of the method
B. Neidhart, W. Wegscheider (Eds.): Quality in Chemical Measurements © Springer-Verlag Berlin Heidelberg 2000
1
W. Wegscheider
Validation: an Example
Validation Must Be Performance Based
 Definition of the need of the client translated to analytical
quality criteria including cost and time
 Compilation of candidate methods
 Critical appraisal of candidate methods with respect to expected
method performance
 Design and execution of validation study
 Evaluation of results, go-ahead or modifications or alternative
method
 Measure the unknown with due regard to previously established
quality criteria
B. Neidhart, W. Wegscheider (Eds.): Quality in Chemical Measurements © Springer-Verlag Berlin Heidelberg 2000
2
W. Wegscheider
Validation: an Example
Worked Example: Fe2O3 in MgO
For certain industrial applications of MgO the upper limit of Fe
oxide needs to be controlled very tightly
Definition of the need of the customer translated to analytical
quality criteria including cost and time
In this application an upper limit of 0.5 % was guaranteed by the
supplier, the customer found 0.64 %, the supplier 0.41 %.
B. Neidhart, W. Wegscheider (Eds.): Quality in Chemical Measurements © Springer-Verlag Berlin Heidelberg 2000
3
W. Wegscheider
4
Validation: an Example
Deriving Minimum Performance Characteristics
from Requirements
As the differences from one another was about 0.1 % Fe2O3 the
proposed method needs to have the following characteristics:
 overall uncertainty (including sampling):
• uncertainty at required level of concentration
from analysis itself:
 limit of quantitation (with matrix):
 limit of detection (with matrix):
 selectivity/interferences:
 linearity:
B. Neidhart, W. Wegscheider (Eds.): Quality in Chemical Measurements © Springer-Verlag Berlin Heidelberg 2000
<0.03 %
~0.01%
<0.03 %
<0.01 %
CRM
at least 0.3-0.7 %
W. Wegscheider
5
Validation: an Example
Preliminary Examination of
Candidate Methods (1)
Method
uncertainty limit of
(no sampl.) quantitation
required (%) 0.01
0.03
limit of
linearity
detection
0.01
0.3-0.7
selectivity
SRM
gravimetry

?
?


titrimetry
?
?


?
?
graphite
furnace AAS

flame AAS



?




WD-XRF





ICP-Q-MS
?



?
B. Neidhart, W. Wegscheider (Eds.): Quality in Chemical Measurements © Springer-Verlag Berlin Heidelberg 2000
W. Wegscheider
6
Validation: an Example
Preliminary Examination of
Candidate Methods (2)
Method
cost
gravimetry
expertise in currently equipment time to
laboratory practiced ready
deliver

?
?
no
titrimetry

?



graphite
furnace AAS
flame AAS




?





WD-XRF

?

?

ICP-Q-MS
?


?
?
B. Neidhart, W. Wegscheider (Eds.): Quality in Chemical Measurements © Springer-Verlag Berlin Heidelberg 2000
?
W. Wegscheider
Validation: an Example
7
Validation Plan
 Produce calibration curves for Fe by flame AA under standard
conditions with and without MgO
 Compare calibration functions
 Evaluate limit of detection and limit of quantitation
 Estimate precision for meeting previously established
requirements (+ margin): allowance for day to day variability, etc.
 Produce recovery data by mixing various aliquots of CRM MgO
with high purity MgO
 Compare with alternative method ?
 Estimate (laboratory) sampling error for sample as received
B. Neidhart, W. Wegscheider (Eds.): Quality in Chemical Measurements © Springer-Verlag Berlin Heidelberg 2000
W. Wegscheider
8
Validation: an Example
Calibration without MgO
26.12.99
Description
Procedure
SOP new
calibration without MgO
flame AA
Christof
Calibration curve
#measurements
#replicates
#conc. levels
25
5
5
x
y1
y2
µg/mL
abs.
0
0.5
1
1.5
2
0.0004
0.0175
0.0348
0.0503
0.063
0.0002
0.0172
0.0351
0.0497
0.0646
Working range
Profile
y3
0
0.0174
0.035
0.0505
0.0649
y4
0.0003
0.0177
0.0348
0.0506
0.0647
B. Neidhart, W. Wegscheider (Eds.): Quality in Chemical Measurements © Springer-Verlag Berlin Heidelberg 2000
0.5
2
y5
y6
0.0004
0.0174
0.0355
0.0502
0.0639
W. Wegscheider
9
Validation: an Example
Calibration with MgO
29.12.99
SOP new
calibration with MgO (200 mg/L)
flame AA
Christof
Calibration curve
#measurements
0
0.5
1
1.5
2
#replicates
0.0002
0.0167
0.0334
0,0502
0.0659
#conc. levels
0.0002
0.0163
0.0339
0,0492
0.066
Working range
Profile
0.0000
0.0163
0.0331
0,0491
0.0661
0.0002
0.0161
0.0331
0,0493
0.0658
B. Neidhart, W. Wegscheider (Eds.): Quality in Chemical Measurements © Springer-Verlag Berlin Heidelberg 2000
0.0001
0.016
0.0333
0.0493
0.0655
W. Wegscheider
10
Validation: an Example
Graphical Comparison
without matrix
with matrix
calibration curve 1. order
calibration curve 2. order
0,07
0,06
0,06
0,05
0,05
0,04
0,04
abs.
abs.
0,07
0,03
0,03
0,02
0,02
0,01
0,01
0
0
-0,5
0
0,5
1
1,5
-0,5
2
0
2,5
0,5
µg/mL
B. Neidhart, W. Wegscheider (Eds.): Quality in Chemical Measurements © Springer-Verlag Berlin Heidelberg 2000
1
mg/mL
mg/l
1,5
2
2,5
W. Wegscheider
Validation: an Example
EXCEL Macro ValiData
Basis of the program is ISO 8466 and DIN 32645
Homogeneity of variances test
Calibration functions
linear OR quadratic following Mandel´s test
weighted linear and weighted quadratic
standard additions
Performance characteristics
Decision limit and detection limit from calibration
Limit of quantitation
Linear range, sensitivity, confidence limits
Recovery studies
orthogonal regression
robust regression
B. Neidhart, W. Wegscheider (Eds.): Quality in Chemical Measurements © Springer-Verlag Berlin Heidelberg 2000
11
W. Wegscheider
12
Validation: an Example
Numerical Comparison
2nd orde r ca libra tion function (y=a +b*x+c*x^2)
without matrix
with matrix
Line a r ca libra tion function (y=a +b*x)
Slope
0.032916
abs./(mg/L)
CI* (slope)
0.032712646
0.033119353 abs./(mg/L)
Ordinate intersect
9.6E-05
abs.
CI (Ordinate intersect)
-0.000153055 0.00034506 abs.
Average(x)
1
mg/L
Average(y)
0.033012
abs.
Residual standard deviation
0.00034755
abs.
Std. deviation of the method
0.01055870805
mg/L
Coeff. of var. of the method
1.055870805
%
t-value (95%)
2.068654794
Qx
12.5
(mg/L)^2
*Confidence interval
B. Neidhart, W. Wegscheider (Eds.): Quality in Chemical Measurements © Springer-Verlag Berlin Heidelberg 2000
W. Wegscheider
13
Validation: an Example
Limits of Detection
Limits of Quantitation
Decision and determination limit
Blank read. meth.
Decision levels (CI)
0.95
Decision limit
0.012155466
Detection limit
0.024310932
Determination limit
Decision and determination limit
Blank read. meth. Calibr. method
Decision levels (CI)
0.95
0.95
Decision limit
0.006345772
0.010236787 mg/l
Detection limit
0.012691545
0.020473573 mg/l
0.036786362 mg/l
Determination limit
B. Neidhart, W. Wegscheider (Eds.): Quality in Chemical Measurements © Springer-Verlag Berlin Heidelberg 2000
without matrix
LOD 0.006 %
with matrix
LOD 0.005 %
LOQ 0.018 %
W. Wegscheider
14
Validation: an Example
Estimation of Recovery
serial dilution of Burned Magnesite SRM 104
calibration curve 1. order
Fe content of 7.07+0.04 % (1 s) with pure MgO
0,7
between 0.1 and
0.6 %
0,6
1.015187226
found/(given)
0,5
0.994663053 1.035711399found/(given)
-0.008726667
found
0,4
-0.016368629-0.001084705found
found
Slope
IC (slope)
Ordinate intersect
IC (Ordinate intersect)
0,3
0,2
0,1
- 0.009 is about
- 1.8 % at 0.5 % level
0
0
0,1
0,2
B. Neidhart, W. Wegscheider (Eds.): Quality in Chemical Measurements © Springer-Verlag Berlin Heidelberg 2000
0,3
0,4
given
0,5
0,6
W. Wegscheider
Validation: an Example
15
Alternative Method: Titrimetry
1. dissolve SRM in HCl, dilute to 100 ml
2. reduce with SnCl2
3. add 20 ml HgCl2 and 50 ml Zimmermann-ReinhartSolution
4. titrate with 0.01 n KMnO4
Result: 7.01 % Fe2O3
Certified: 7.07 + 0.04 %
B. Neidhart, W. Wegscheider (Eds.): Quality in Chemical Measurements © Springer-Verlag Berlin Heidelberg 2000
W. Wegscheider
Validation: an Example
16
Estimation of Uncertainty from Sampling








about 500 g laboratory sample delivered
sampled “according to ISO” by customer
max. concentration about 1 % Fe in dark pieces
min. concentration below LOD in light pieces
many larger grains: ca 1 cm3 corresponds to 3 g
total of 500 g/3 g ~ 160 pieces
80 pieces give a relative uncertainty of 9 %
at the 0.5 % level this is 0.045 % (0.02 % requ. !!!)
CONCLUSION:
the laboratory sample must be about 2000 g
B. Neidhart, W. Wegscheider (Eds.): Quality in Chemical Measurements © Springer-Verlag Berlin Heidelberg 2000
W. Wegscheider
Validation: an Example
Content of Validation Report
Introduction
Test principle
Definition of the type of the analytical test procedure
Definition of validation parameters
Performance of the test
Test outline and acceptance criteria
Equipment qualification
System suitability
Qualification of samples and standards
Summary
References
Results
B. Neidhart, W. Wegscheider (Eds.): Quality in Chemical Measurements © Springer-Verlag Berlin Heidelberg 2000
17