Quantitative Analysis for
Botanical Products
Gabriel Giancaspro Vice President Foods Dietary Supplements Herbal Medicines
Spectrophotometric (cascara sennosides)
HPLC (Ginkgo St Johnrsquos wort Milk Thistle)
NMR (Krill Oil Aloe vera (soon) Chitosan)
Quantitative Methods in Pharmacopeia
2
Validation of quantitative procedures
3
USP
bull Is the process which establishes through laboratory
studies that a procedure meet the requirements for the
intended analytical application
ICH
bull The objective of the validation of an analytical procedure is
to demonstrate that it is appropriate for the intended use
bull lt1225gt Validation of Compendial Procedures Validation will be required when
ndash an analytical procedure is used to test a non-official article
ndash an official article is tested using an alternative procedure (see
USP General Notices 630 )
bull lt1226gt Verification of Compendial Procedures Verification will be required the first time an official article is tested
using a USP procedure
bull lt1224gt Transfer of Analytical Procedures Transfer will applies when a non-compendial procedure is moved
from one lab to another
Validation Verification and Transfer
Analytical Performance Characteristics
bull Accuracy
bull Precision
bull Repeatability
bull Intermediate Precision
bull Reproducibility
bull Specificity
bull Detection Limit Quantification Limit
bull Linearity
bull Range
bull Robustness
lt1225gt Validation of Pharmacopeial Procedures
Performance Characteristics
Category I Category II Category III Category IV
Qty Limit
Accuracy
Yes
Yes
No
Precision Yes Yes No Yes No
Specificity Yes Yes Yes Yes
LOD No No Yes No
LOQ No Yes No No
Linearity Yes Yes No No
Range Yes Yes No
May be required depending on the type of test
Specificity
Definition ndash The methodrsquos ability to specifically measure the
analyte in the presence of the components in the analytical
matrix
bull Impurities degradation products andor excipients are
available
bull When impurities degradation products or excipients are
not available bull Compare the results with another well-characterised analytical
method
bull Perform an artifical degradation under drastic conditions (light
heat acids y bases etc)
bull Peak purity
bull Peak shape analysis
bull Diode array detection
bull HPLC-MS
Specificity
Linearity
Definition ndash The ability of a method to produce results that are directly proportional to the concentration of the sample at a given interval
bull 5 levels of concentration Different dilutions of an stock
solution or separate weights of the component
bull 3 samples per level of concentration
Linearity
bull Analysis and collection of data
bull Graphical evaluation
bull Esimtating the parameters of the model by least
squares
bull Calculation of the confidence intervals for the estimated
parameters
Coefficient of correlation
i
i
i
i
i
ii
yyxx
yyxx
r
)()(
))((
2
Confidence intervals
2
)ˆ( 2
n
yy
S i
ii
xy
i
i
xy
b
xx
SS
2)(
b plusmn t(n-2) Sb
a plusmn t(n-2) Sa
i
i
i
i
xyaxxn
x
SS2
2
)(
-20
-15
-10
-5
0
5
10
15
20
0 5 10 15 20 25 30 35
Resid
uals
Cc (mgml)
Anaacutelisis de residuos
bull Check that the graph has a random pattern
bull A trend indicates a failure in the chosen model
bull An increase or decrease in residue dispersion indicates that the data
is not homoscedastic
Residue Analysis
Residue Analysis
Accuracy
Definition - The difference between results obtained by
the method and the actual value
bull It is calculated by determining the recovery of the method
bull Three replicates at three levels bull 80 -120 for the principal component bull 50 - 150 for impurities
ad
obs
C
CR
100
Recovery test
Amount added (mg) Amound detected (mg) Recovered
2435 2470 1014
2515 2541 1010
2515 2517 1001
3004 3079 1025
2974 3018 1015
3014 3038 1008
3533 3570 1010
3463 3456 998
3673 3701 1008
Mean 1010
SD 07945
Ecuacion de la recta
Encontrado = a + ( b x Antildeadido)
b= 09989
Ecuacion de la recta
Encontrado = a + ( b x Antildeadido)
b= 09989
a= 03272 Valores de t experimentales
y = 09989x + 03272Rsup2 = 09974
20
25
30
35
40
20 25 30 35 40
ob
serv
ed
added
Accuracy
Fraction Unit Range of Recovery ()
100 100 98-102
01 01 95-105
001 100 ppm 90-107
000000001 1 ppb 40-120
Acceptable Recovery depending on the analyte level
Gonzaacutelez et al Trends in Anal Chem 26 (3) 2007
Precision
Definition - The degree of agreement between results
when a procedure is repeatedly applied to a
homogenous sample
bull Repeatability Intermediate Precision Reproducibility
bull Calculation of the SD or RSD
bull Repeatability is precision under the same operating
conditions for a short period of time It is determined
by a minimum of 9 measurements within the given
range of the procedure (3 concentrations 3
replications) or a minimum of 6 replications at 100
bull Intermediate Precision indicates intra-laboratory
variations different days different analysts different
equipment
bull Reproducibility indicates inter-laboratory variations
Precision
Robustness
Definition ndash A measure of a methodrsquos ability to accept
small but deliberate variations in the parameters of
the method
bull Steps to Consider ndash Design and execution of the experiment
ndash Calculating the effects
ndash Statisticalgraphical analysis of the effects
ndash Relevant conclusions and eventual modifications of the
method
bull Changes in pH column mobile phase etc
Experimental design
bull One factor at a time
bull Factorial Designs bull Full Factorial
bull Fractional Factorial
bull Plackett-Burman
Robustness
Full Factorial Design (2k runs)
pH
Flow
TEA
pH
Flow
TEA
pH
Flow
TEA
pH
Flow
TEA
Particle size
Organic
Fractional Factorial Design ndash 5 factors
pH
Flow
TEA
pH
Flow
TEA
pH
Flow
TEA
pH
Flow
TEA
Particle size
Organic
7 factors (A-G) 2 levels
Exp Factors Result
A B C D E F G
1 + + + + + + + y1
2 + + - + - - - y2
3 + - + - + - - y3
4 + - - - - + + y4
5 - + + - - + - y5
6 - + - - + - + y6
7 - - + + - - + y7
8 - - - + + + - y8
Placket - Burman
Eff = ( y+ - y-)4
25
26
Extract according to the procedure and analyze
Repeat extraction
Combine aliquots and analyze correcting by dilution factor
Use the results for the combined corrected extraction as the
denominator for recovery calculation
Perform statistical analysis
Anderson ML amp Burney DP (1998) J AOAC Int 81
1005ndash1010
Accuracy of extraction procedure
27
28
29
30
Specificity
31
Specificity Phyllanthus amarus by peak purity
32
33
34
35
36
37
Ruggedness and robustness
38
Response factor variation with different columns
39
40
41
42
No instrumental separation
Less specific than chromatographic procedures
Specificity (selectivity) achieved by sample preparation
Specific color reactions
No problems to validate linearity accuracy and precision as per
HPLC
However the higher the complexity in sample preparation the
lowest the precision and ruggedness
Spectrometric analytical procedures
43
Identity well controlled Orthogonal methods with multiple
fingerprints
Ubiquitous markers in high concentration provided ID is well
controlled
Sample preparation with multiple extractions washings acidbasic
Color reactions with high specificity
When is OK to use a less specific quantitative procedure
44
Examples Black Cohosh St John wort Milk Thistle
Often times is not possible to obtain the same results
Results may be correlated but not absolute
Use all known peaks in the chromatogram for the class to
measure and calculate the total (use response factors)
Verify correlation between the two methods
Set new limits according to results
Moving from Spectrophotometric to HPLC
45
46
47
48
Silymarins in Milk Thistle
49
Typical chromatogram
50
Needs redefinition of limits
A more specific test will give consistently lower results
Correlation between tests is recommended
In some cases conversion factors have been proposed
Gaedcke F Pharmeuropa Scientific Notes [2009 2009(1)5-10]
Acceptance criteria after transfer
51
Spectrophotometric (cascara sennosides)
HPLC (Ginkgo St Johnrsquos wort Milk Thistle)
NMR (Krill Oil Aloe vera (soon) Chitosan)
Quantitative Methods in Pharmacopeia
2
Validation of quantitative procedures
3
USP
bull Is the process which establishes through laboratory
studies that a procedure meet the requirements for the
intended analytical application
ICH
bull The objective of the validation of an analytical procedure is
to demonstrate that it is appropriate for the intended use
bull lt1225gt Validation of Compendial Procedures Validation will be required when
ndash an analytical procedure is used to test a non-official article
ndash an official article is tested using an alternative procedure (see
USP General Notices 630 )
bull lt1226gt Verification of Compendial Procedures Verification will be required the first time an official article is tested
using a USP procedure
bull lt1224gt Transfer of Analytical Procedures Transfer will applies when a non-compendial procedure is moved
from one lab to another
Validation Verification and Transfer
Analytical Performance Characteristics
bull Accuracy
bull Precision
bull Repeatability
bull Intermediate Precision
bull Reproducibility
bull Specificity
bull Detection Limit Quantification Limit
bull Linearity
bull Range
bull Robustness
lt1225gt Validation of Pharmacopeial Procedures
Performance Characteristics
Category I Category II Category III Category IV
Qty Limit
Accuracy
Yes
Yes
No
Precision Yes Yes No Yes No
Specificity Yes Yes Yes Yes
LOD No No Yes No
LOQ No Yes No No
Linearity Yes Yes No No
Range Yes Yes No
May be required depending on the type of test
Specificity
Definition ndash The methodrsquos ability to specifically measure the
analyte in the presence of the components in the analytical
matrix
bull Impurities degradation products andor excipients are
available
bull When impurities degradation products or excipients are
not available bull Compare the results with another well-characterised analytical
method
bull Perform an artifical degradation under drastic conditions (light
heat acids y bases etc)
bull Peak purity
bull Peak shape analysis
bull Diode array detection
bull HPLC-MS
Specificity
Linearity
Definition ndash The ability of a method to produce results that are directly proportional to the concentration of the sample at a given interval
bull 5 levels of concentration Different dilutions of an stock
solution or separate weights of the component
bull 3 samples per level of concentration
Linearity
bull Analysis and collection of data
bull Graphical evaluation
bull Esimtating the parameters of the model by least
squares
bull Calculation of the confidence intervals for the estimated
parameters
Coefficient of correlation
i
i
i
i
i
ii
yyxx
yyxx
r
)()(
))((
2
Confidence intervals
2
)ˆ( 2
n
yy
S i
ii
xy
i
i
xy
b
xx
SS
2)(
b plusmn t(n-2) Sb
a plusmn t(n-2) Sa
i
i
i
i
xyaxxn
x
SS2
2
)(
-20
-15
-10
-5
0
5
10
15
20
0 5 10 15 20 25 30 35
Resid
uals
Cc (mgml)
Anaacutelisis de residuos
bull Check that the graph has a random pattern
bull A trend indicates a failure in the chosen model
bull An increase or decrease in residue dispersion indicates that the data
is not homoscedastic
Residue Analysis
Residue Analysis
Accuracy
Definition - The difference between results obtained by
the method and the actual value
bull It is calculated by determining the recovery of the method
bull Three replicates at three levels bull 80 -120 for the principal component bull 50 - 150 for impurities
ad
obs
C
CR
100
Recovery test
Amount added (mg) Amound detected (mg) Recovered
2435 2470 1014
2515 2541 1010
2515 2517 1001
3004 3079 1025
2974 3018 1015
3014 3038 1008
3533 3570 1010
3463 3456 998
3673 3701 1008
Mean 1010
SD 07945
Ecuacion de la recta
Encontrado = a + ( b x Antildeadido)
b= 09989
Ecuacion de la recta
Encontrado = a + ( b x Antildeadido)
b= 09989
a= 03272 Valores de t experimentales
y = 09989x + 03272Rsup2 = 09974
20
25
30
35
40
20 25 30 35 40
ob
serv
ed
added
Accuracy
Fraction Unit Range of Recovery ()
100 100 98-102
01 01 95-105
001 100 ppm 90-107
000000001 1 ppb 40-120
Acceptable Recovery depending on the analyte level
Gonzaacutelez et al Trends in Anal Chem 26 (3) 2007
Precision
Definition - The degree of agreement between results
when a procedure is repeatedly applied to a
homogenous sample
bull Repeatability Intermediate Precision Reproducibility
bull Calculation of the SD or RSD
bull Repeatability is precision under the same operating
conditions for a short period of time It is determined
by a minimum of 9 measurements within the given
range of the procedure (3 concentrations 3
replications) or a minimum of 6 replications at 100
bull Intermediate Precision indicates intra-laboratory
variations different days different analysts different
equipment
bull Reproducibility indicates inter-laboratory variations
Precision
Robustness
Definition ndash A measure of a methodrsquos ability to accept
small but deliberate variations in the parameters of
the method
bull Steps to Consider ndash Design and execution of the experiment
ndash Calculating the effects
ndash Statisticalgraphical analysis of the effects
ndash Relevant conclusions and eventual modifications of the
method
bull Changes in pH column mobile phase etc
Experimental design
bull One factor at a time
bull Factorial Designs bull Full Factorial
bull Fractional Factorial
bull Plackett-Burman
Robustness
Full Factorial Design (2k runs)
pH
Flow
TEA
pH
Flow
TEA
pH
Flow
TEA
pH
Flow
TEA
Particle size
Organic
Fractional Factorial Design ndash 5 factors
pH
Flow
TEA
pH
Flow
TEA
pH
Flow
TEA
pH
Flow
TEA
Particle size
Organic
7 factors (A-G) 2 levels
Exp Factors Result
A B C D E F G
1 + + + + + + + y1
2 + + - + - - - y2
3 + - + - + - - y3
4 + - - - - + + y4
5 - + + - - + - y5
6 - + - - + - + y6
7 - - + + - - + y7
8 - - - + + + - y8
Placket - Burman
Eff = ( y+ - y-)4
25
26
Extract according to the procedure and analyze
Repeat extraction
Combine aliquots and analyze correcting by dilution factor
Use the results for the combined corrected extraction as the
denominator for recovery calculation
Perform statistical analysis
Anderson ML amp Burney DP (1998) J AOAC Int 81
1005ndash1010
Accuracy of extraction procedure
27
28
29
30
Specificity
31
Specificity Phyllanthus amarus by peak purity
32
33
34
35
36
37
Ruggedness and robustness
38
Response factor variation with different columns
39
40
41
42
No instrumental separation
Less specific than chromatographic procedures
Specificity (selectivity) achieved by sample preparation
Specific color reactions
No problems to validate linearity accuracy and precision as per
HPLC
However the higher the complexity in sample preparation the
lowest the precision and ruggedness
Spectrometric analytical procedures
43
Identity well controlled Orthogonal methods with multiple
fingerprints
Ubiquitous markers in high concentration provided ID is well
controlled
Sample preparation with multiple extractions washings acidbasic
Color reactions with high specificity
When is OK to use a less specific quantitative procedure
44
Examples Black Cohosh St John wort Milk Thistle
Often times is not possible to obtain the same results
Results may be correlated but not absolute
Use all known peaks in the chromatogram for the class to
measure and calculate the total (use response factors)
Verify correlation between the two methods
Set new limits according to results
Moving from Spectrophotometric to HPLC
45
46
47
48
Silymarins in Milk Thistle
49
Typical chromatogram
50
Needs redefinition of limits
A more specific test will give consistently lower results
Correlation between tests is recommended
In some cases conversion factors have been proposed
Gaedcke F Pharmeuropa Scientific Notes [2009 2009(1)5-10]
Acceptance criteria after transfer
51
Validation of quantitative procedures
3
USP
bull Is the process which establishes through laboratory
studies that a procedure meet the requirements for the
intended analytical application
ICH
bull The objective of the validation of an analytical procedure is
to demonstrate that it is appropriate for the intended use
bull lt1225gt Validation of Compendial Procedures Validation will be required when
ndash an analytical procedure is used to test a non-official article
ndash an official article is tested using an alternative procedure (see
USP General Notices 630 )
bull lt1226gt Verification of Compendial Procedures Verification will be required the first time an official article is tested
using a USP procedure
bull lt1224gt Transfer of Analytical Procedures Transfer will applies when a non-compendial procedure is moved
from one lab to another
Validation Verification and Transfer
Analytical Performance Characteristics
bull Accuracy
bull Precision
bull Repeatability
bull Intermediate Precision
bull Reproducibility
bull Specificity
bull Detection Limit Quantification Limit
bull Linearity
bull Range
bull Robustness
lt1225gt Validation of Pharmacopeial Procedures
Performance Characteristics
Category I Category II Category III Category IV
Qty Limit
Accuracy
Yes
Yes
No
Precision Yes Yes No Yes No
Specificity Yes Yes Yes Yes
LOD No No Yes No
LOQ No Yes No No
Linearity Yes Yes No No
Range Yes Yes No
May be required depending on the type of test
Specificity
Definition ndash The methodrsquos ability to specifically measure the
analyte in the presence of the components in the analytical
matrix
bull Impurities degradation products andor excipients are
available
bull When impurities degradation products or excipients are
not available bull Compare the results with another well-characterised analytical
method
bull Perform an artifical degradation under drastic conditions (light
heat acids y bases etc)
bull Peak purity
bull Peak shape analysis
bull Diode array detection
bull HPLC-MS
Specificity
Linearity
Definition ndash The ability of a method to produce results that are directly proportional to the concentration of the sample at a given interval
bull 5 levels of concentration Different dilutions of an stock
solution or separate weights of the component
bull 3 samples per level of concentration
Linearity
bull Analysis and collection of data
bull Graphical evaluation
bull Esimtating the parameters of the model by least
squares
bull Calculation of the confidence intervals for the estimated
parameters
Coefficient of correlation
i
i
i
i
i
ii
yyxx
yyxx
r
)()(
))((
2
Confidence intervals
2
)ˆ( 2
n
yy
S i
ii
xy
i
i
xy
b
xx
SS
2)(
b plusmn t(n-2) Sb
a plusmn t(n-2) Sa
i
i
i
i
xyaxxn
x
SS2
2
)(
-20
-15
-10
-5
0
5
10
15
20
0 5 10 15 20 25 30 35
Resid
uals
Cc (mgml)
Anaacutelisis de residuos
bull Check that the graph has a random pattern
bull A trend indicates a failure in the chosen model
bull An increase or decrease in residue dispersion indicates that the data
is not homoscedastic
Residue Analysis
Residue Analysis
Accuracy
Definition - The difference between results obtained by
the method and the actual value
bull It is calculated by determining the recovery of the method
bull Three replicates at three levels bull 80 -120 for the principal component bull 50 - 150 for impurities
ad
obs
C
CR
100
Recovery test
Amount added (mg) Amound detected (mg) Recovered
2435 2470 1014
2515 2541 1010
2515 2517 1001
3004 3079 1025
2974 3018 1015
3014 3038 1008
3533 3570 1010
3463 3456 998
3673 3701 1008
Mean 1010
SD 07945
Ecuacion de la recta
Encontrado = a + ( b x Antildeadido)
b= 09989
Ecuacion de la recta
Encontrado = a + ( b x Antildeadido)
b= 09989
a= 03272 Valores de t experimentales
y = 09989x + 03272Rsup2 = 09974
20
25
30
35
40
20 25 30 35 40
ob
serv
ed
added
Accuracy
Fraction Unit Range of Recovery ()
100 100 98-102
01 01 95-105
001 100 ppm 90-107
000000001 1 ppb 40-120
Acceptable Recovery depending on the analyte level
Gonzaacutelez et al Trends in Anal Chem 26 (3) 2007
Precision
Definition - The degree of agreement between results
when a procedure is repeatedly applied to a
homogenous sample
bull Repeatability Intermediate Precision Reproducibility
bull Calculation of the SD or RSD
bull Repeatability is precision under the same operating
conditions for a short period of time It is determined
by a minimum of 9 measurements within the given
range of the procedure (3 concentrations 3
replications) or a minimum of 6 replications at 100
bull Intermediate Precision indicates intra-laboratory
variations different days different analysts different
equipment
bull Reproducibility indicates inter-laboratory variations
Precision
Robustness
Definition ndash A measure of a methodrsquos ability to accept
small but deliberate variations in the parameters of
the method
bull Steps to Consider ndash Design and execution of the experiment
ndash Calculating the effects
ndash Statisticalgraphical analysis of the effects
ndash Relevant conclusions and eventual modifications of the
method
bull Changes in pH column mobile phase etc
Experimental design
bull One factor at a time
bull Factorial Designs bull Full Factorial
bull Fractional Factorial
bull Plackett-Burman
Robustness
Full Factorial Design (2k runs)
pH
Flow
TEA
pH
Flow
TEA
pH
Flow
TEA
pH
Flow
TEA
Particle size
Organic
Fractional Factorial Design ndash 5 factors
pH
Flow
TEA
pH
Flow
TEA
pH
Flow
TEA
pH
Flow
TEA
Particle size
Organic
7 factors (A-G) 2 levels
Exp Factors Result
A B C D E F G
1 + + + + + + + y1
2 + + - + - - - y2
3 + - + - + - - y3
4 + - - - - + + y4
5 - + + - - + - y5
6 - + - - + - + y6
7 - - + + - - + y7
8 - - - + + + - y8
Placket - Burman
Eff = ( y+ - y-)4
25
26
Extract according to the procedure and analyze
Repeat extraction
Combine aliquots and analyze correcting by dilution factor
Use the results for the combined corrected extraction as the
denominator for recovery calculation
Perform statistical analysis
Anderson ML amp Burney DP (1998) J AOAC Int 81
1005ndash1010
Accuracy of extraction procedure
27
28
29
30
Specificity
31
Specificity Phyllanthus amarus by peak purity
32
33
34
35
36
37
Ruggedness and robustness
38
Response factor variation with different columns
39
40
41
42
No instrumental separation
Less specific than chromatographic procedures
Specificity (selectivity) achieved by sample preparation
Specific color reactions
No problems to validate linearity accuracy and precision as per
HPLC
However the higher the complexity in sample preparation the
lowest the precision and ruggedness
Spectrometric analytical procedures
43
Identity well controlled Orthogonal methods with multiple
fingerprints
Ubiquitous markers in high concentration provided ID is well
controlled
Sample preparation with multiple extractions washings acidbasic
Color reactions with high specificity
When is OK to use a less specific quantitative procedure
44
Examples Black Cohosh St John wort Milk Thistle
Often times is not possible to obtain the same results
Results may be correlated but not absolute
Use all known peaks in the chromatogram for the class to
measure and calculate the total (use response factors)
Verify correlation between the two methods
Set new limits according to results
Moving from Spectrophotometric to HPLC
45
46
47
48
Silymarins in Milk Thistle
49
Typical chromatogram
50
Needs redefinition of limits
A more specific test will give consistently lower results
Correlation between tests is recommended
In some cases conversion factors have been proposed
Gaedcke F Pharmeuropa Scientific Notes [2009 2009(1)5-10]
Acceptance criteria after transfer
51
bull lt1225gt Validation of Compendial Procedures Validation will be required when
ndash an analytical procedure is used to test a non-official article
ndash an official article is tested using an alternative procedure (see
USP General Notices 630 )
bull lt1226gt Verification of Compendial Procedures Verification will be required the first time an official article is tested
using a USP procedure
bull lt1224gt Transfer of Analytical Procedures Transfer will applies when a non-compendial procedure is moved
from one lab to another
Validation Verification and Transfer
Analytical Performance Characteristics
bull Accuracy
bull Precision
bull Repeatability
bull Intermediate Precision
bull Reproducibility
bull Specificity
bull Detection Limit Quantification Limit
bull Linearity
bull Range
bull Robustness
lt1225gt Validation of Pharmacopeial Procedures
Performance Characteristics
Category I Category II Category III Category IV
Qty Limit
Accuracy
Yes
Yes
No
Precision Yes Yes No Yes No
Specificity Yes Yes Yes Yes
LOD No No Yes No
LOQ No Yes No No
Linearity Yes Yes No No
Range Yes Yes No
May be required depending on the type of test
Specificity
Definition ndash The methodrsquos ability to specifically measure the
analyte in the presence of the components in the analytical
matrix
bull Impurities degradation products andor excipients are
available
bull When impurities degradation products or excipients are
not available bull Compare the results with another well-characterised analytical
method
bull Perform an artifical degradation under drastic conditions (light
heat acids y bases etc)
bull Peak purity
bull Peak shape analysis
bull Diode array detection
bull HPLC-MS
Specificity
Linearity
Definition ndash The ability of a method to produce results that are directly proportional to the concentration of the sample at a given interval
bull 5 levels of concentration Different dilutions of an stock
solution or separate weights of the component
bull 3 samples per level of concentration
Linearity
bull Analysis and collection of data
bull Graphical evaluation
bull Esimtating the parameters of the model by least
squares
bull Calculation of the confidence intervals for the estimated
parameters
Coefficient of correlation
i
i
i
i
i
ii
yyxx
yyxx
r
)()(
))((
2
Confidence intervals
2
)ˆ( 2
n
yy
S i
ii
xy
i
i
xy
b
xx
SS
2)(
b plusmn t(n-2) Sb
a plusmn t(n-2) Sa
i
i
i
i
xyaxxn
x
SS2
2
)(
-20
-15
-10
-5
0
5
10
15
20
0 5 10 15 20 25 30 35
Resid
uals
Cc (mgml)
Anaacutelisis de residuos
bull Check that the graph has a random pattern
bull A trend indicates a failure in the chosen model
bull An increase or decrease in residue dispersion indicates that the data
is not homoscedastic
Residue Analysis
Residue Analysis
Accuracy
Definition - The difference between results obtained by
the method and the actual value
bull It is calculated by determining the recovery of the method
bull Three replicates at three levels bull 80 -120 for the principal component bull 50 - 150 for impurities
ad
obs
C
CR
100
Recovery test
Amount added (mg) Amound detected (mg) Recovered
2435 2470 1014
2515 2541 1010
2515 2517 1001
3004 3079 1025
2974 3018 1015
3014 3038 1008
3533 3570 1010
3463 3456 998
3673 3701 1008
Mean 1010
SD 07945
Ecuacion de la recta
Encontrado = a + ( b x Antildeadido)
b= 09989
Ecuacion de la recta
Encontrado = a + ( b x Antildeadido)
b= 09989
a= 03272 Valores de t experimentales
y = 09989x + 03272Rsup2 = 09974
20
25
30
35
40
20 25 30 35 40
ob
serv
ed
added
Accuracy
Fraction Unit Range of Recovery ()
100 100 98-102
01 01 95-105
001 100 ppm 90-107
000000001 1 ppb 40-120
Acceptable Recovery depending on the analyte level
Gonzaacutelez et al Trends in Anal Chem 26 (3) 2007
Precision
Definition - The degree of agreement between results
when a procedure is repeatedly applied to a
homogenous sample
bull Repeatability Intermediate Precision Reproducibility
bull Calculation of the SD or RSD
bull Repeatability is precision under the same operating
conditions for a short period of time It is determined
by a minimum of 9 measurements within the given
range of the procedure (3 concentrations 3
replications) or a minimum of 6 replications at 100
bull Intermediate Precision indicates intra-laboratory
variations different days different analysts different
equipment
bull Reproducibility indicates inter-laboratory variations
Precision
Robustness
Definition ndash A measure of a methodrsquos ability to accept
small but deliberate variations in the parameters of
the method
bull Steps to Consider ndash Design and execution of the experiment
ndash Calculating the effects
ndash Statisticalgraphical analysis of the effects
ndash Relevant conclusions and eventual modifications of the
method
bull Changes in pH column mobile phase etc
Experimental design
bull One factor at a time
bull Factorial Designs bull Full Factorial
bull Fractional Factorial
bull Plackett-Burman
Robustness
Full Factorial Design (2k runs)
pH
Flow
TEA
pH
Flow
TEA
pH
Flow
TEA
pH
Flow
TEA
Particle size
Organic
Fractional Factorial Design ndash 5 factors
pH
Flow
TEA
pH
Flow
TEA
pH
Flow
TEA
pH
Flow
TEA
Particle size
Organic
7 factors (A-G) 2 levels
Exp Factors Result
A B C D E F G
1 + + + + + + + y1
2 + + - + - - - y2
3 + - + - + - - y3
4 + - - - - + + y4
5 - + + - - + - y5
6 - + - - + - + y6
7 - - + + - - + y7
8 - - - + + + - y8
Placket - Burman
Eff = ( y+ - y-)4
25
26
Extract according to the procedure and analyze
Repeat extraction
Combine aliquots and analyze correcting by dilution factor
Use the results for the combined corrected extraction as the
denominator for recovery calculation
Perform statistical analysis
Anderson ML amp Burney DP (1998) J AOAC Int 81
1005ndash1010
Accuracy of extraction procedure
27
28
29
30
Specificity
31
Specificity Phyllanthus amarus by peak purity
32
33
34
35
36
37
Ruggedness and robustness
38
Response factor variation with different columns
39
40
41
42
No instrumental separation
Less specific than chromatographic procedures
Specificity (selectivity) achieved by sample preparation
Specific color reactions
No problems to validate linearity accuracy and precision as per
HPLC
However the higher the complexity in sample preparation the
lowest the precision and ruggedness
Spectrometric analytical procedures
43
Identity well controlled Orthogonal methods with multiple
fingerprints
Ubiquitous markers in high concentration provided ID is well
controlled
Sample preparation with multiple extractions washings acidbasic
Color reactions with high specificity
When is OK to use a less specific quantitative procedure
44
Examples Black Cohosh St John wort Milk Thistle
Often times is not possible to obtain the same results
Results may be correlated but not absolute
Use all known peaks in the chromatogram for the class to
measure and calculate the total (use response factors)
Verify correlation between the two methods
Set new limits according to results
Moving from Spectrophotometric to HPLC
45
46
47
48
Silymarins in Milk Thistle
49
Typical chromatogram
50
Needs redefinition of limits
A more specific test will give consistently lower results
Correlation between tests is recommended
In some cases conversion factors have been proposed
Gaedcke F Pharmeuropa Scientific Notes [2009 2009(1)5-10]
Acceptance criteria after transfer
51
Analytical Performance Characteristics
bull Accuracy
bull Precision
bull Repeatability
bull Intermediate Precision
bull Reproducibility
bull Specificity
bull Detection Limit Quantification Limit
bull Linearity
bull Range
bull Robustness
lt1225gt Validation of Pharmacopeial Procedures
Performance Characteristics
Category I Category II Category III Category IV
Qty Limit
Accuracy
Yes
Yes
No
Precision Yes Yes No Yes No
Specificity Yes Yes Yes Yes
LOD No No Yes No
LOQ No Yes No No
Linearity Yes Yes No No
Range Yes Yes No
May be required depending on the type of test
Specificity
Definition ndash The methodrsquos ability to specifically measure the
analyte in the presence of the components in the analytical
matrix
bull Impurities degradation products andor excipients are
available
bull When impurities degradation products or excipients are
not available bull Compare the results with another well-characterised analytical
method
bull Perform an artifical degradation under drastic conditions (light
heat acids y bases etc)
bull Peak purity
bull Peak shape analysis
bull Diode array detection
bull HPLC-MS
Specificity
Linearity
Definition ndash The ability of a method to produce results that are directly proportional to the concentration of the sample at a given interval
bull 5 levels of concentration Different dilutions of an stock
solution or separate weights of the component
bull 3 samples per level of concentration
Linearity
bull Analysis and collection of data
bull Graphical evaluation
bull Esimtating the parameters of the model by least
squares
bull Calculation of the confidence intervals for the estimated
parameters
Coefficient of correlation
i
i
i
i
i
ii
yyxx
yyxx
r
)()(
))((
2
Confidence intervals
2
)ˆ( 2
n
yy
S i
ii
xy
i
i
xy
b
xx
SS
2)(
b plusmn t(n-2) Sb
a plusmn t(n-2) Sa
i
i
i
i
xyaxxn
x
SS2
2
)(
-20
-15
-10
-5
0
5
10
15
20
0 5 10 15 20 25 30 35
Resid
uals
Cc (mgml)
Anaacutelisis de residuos
bull Check that the graph has a random pattern
bull A trend indicates a failure in the chosen model
bull An increase or decrease in residue dispersion indicates that the data
is not homoscedastic
Residue Analysis
Residue Analysis
Accuracy
Definition - The difference between results obtained by
the method and the actual value
bull It is calculated by determining the recovery of the method
bull Three replicates at three levels bull 80 -120 for the principal component bull 50 - 150 for impurities
ad
obs
C
CR
100
Recovery test
Amount added (mg) Amound detected (mg) Recovered
2435 2470 1014
2515 2541 1010
2515 2517 1001
3004 3079 1025
2974 3018 1015
3014 3038 1008
3533 3570 1010
3463 3456 998
3673 3701 1008
Mean 1010
SD 07945
Ecuacion de la recta
Encontrado = a + ( b x Antildeadido)
b= 09989
Ecuacion de la recta
Encontrado = a + ( b x Antildeadido)
b= 09989
a= 03272 Valores de t experimentales
y = 09989x + 03272Rsup2 = 09974
20
25
30
35
40
20 25 30 35 40
ob
serv
ed
added
Accuracy
Fraction Unit Range of Recovery ()
100 100 98-102
01 01 95-105
001 100 ppm 90-107
000000001 1 ppb 40-120
Acceptable Recovery depending on the analyte level
Gonzaacutelez et al Trends in Anal Chem 26 (3) 2007
Precision
Definition - The degree of agreement between results
when a procedure is repeatedly applied to a
homogenous sample
bull Repeatability Intermediate Precision Reproducibility
bull Calculation of the SD or RSD
bull Repeatability is precision under the same operating
conditions for a short period of time It is determined
by a minimum of 9 measurements within the given
range of the procedure (3 concentrations 3
replications) or a minimum of 6 replications at 100
bull Intermediate Precision indicates intra-laboratory
variations different days different analysts different
equipment
bull Reproducibility indicates inter-laboratory variations
Precision
Robustness
Definition ndash A measure of a methodrsquos ability to accept
small but deliberate variations in the parameters of
the method
bull Steps to Consider ndash Design and execution of the experiment
ndash Calculating the effects
ndash Statisticalgraphical analysis of the effects
ndash Relevant conclusions and eventual modifications of the
method
bull Changes in pH column mobile phase etc
Experimental design
bull One factor at a time
bull Factorial Designs bull Full Factorial
bull Fractional Factorial
bull Plackett-Burman
Robustness
Full Factorial Design (2k runs)
pH
Flow
TEA
pH
Flow
TEA
pH
Flow
TEA
pH
Flow
TEA
Particle size
Organic
Fractional Factorial Design ndash 5 factors
pH
Flow
TEA
pH
Flow
TEA
pH
Flow
TEA
pH
Flow
TEA
Particle size
Organic
7 factors (A-G) 2 levels
Exp Factors Result
A B C D E F G
1 + + + + + + + y1
2 + + - + - - - y2
3 + - + - + - - y3
4 + - - - - + + y4
5 - + + - - + - y5
6 - + - - + - + y6
7 - - + + - - + y7
8 - - - + + + - y8
Placket - Burman
Eff = ( y+ - y-)4
25
26
Extract according to the procedure and analyze
Repeat extraction
Combine aliquots and analyze correcting by dilution factor
Use the results for the combined corrected extraction as the
denominator for recovery calculation
Perform statistical analysis
Anderson ML amp Burney DP (1998) J AOAC Int 81
1005ndash1010
Accuracy of extraction procedure
27
28
29
30
Specificity
31
Specificity Phyllanthus amarus by peak purity
32
33
34
35
36
37
Ruggedness and robustness
38
Response factor variation with different columns
39
40
41
42
No instrumental separation
Less specific than chromatographic procedures
Specificity (selectivity) achieved by sample preparation
Specific color reactions
No problems to validate linearity accuracy and precision as per
HPLC
However the higher the complexity in sample preparation the
lowest the precision and ruggedness
Spectrometric analytical procedures
43
Identity well controlled Orthogonal methods with multiple
fingerprints
Ubiquitous markers in high concentration provided ID is well
controlled
Sample preparation with multiple extractions washings acidbasic
Color reactions with high specificity
When is OK to use a less specific quantitative procedure
44
Examples Black Cohosh St John wort Milk Thistle
Often times is not possible to obtain the same results
Results may be correlated but not absolute
Use all known peaks in the chromatogram for the class to
measure and calculate the total (use response factors)
Verify correlation between the two methods
Set new limits according to results
Moving from Spectrophotometric to HPLC
45
46
47
48
Silymarins in Milk Thistle
49
Typical chromatogram
50
Needs redefinition of limits
A more specific test will give consistently lower results
Correlation between tests is recommended
In some cases conversion factors have been proposed
Gaedcke F Pharmeuropa Scientific Notes [2009 2009(1)5-10]
Acceptance criteria after transfer
51
lt1225gt Validation of Pharmacopeial Procedures
Performance Characteristics
Category I Category II Category III Category IV
Qty Limit
Accuracy
Yes
Yes
No
Precision Yes Yes No Yes No
Specificity Yes Yes Yes Yes
LOD No No Yes No
LOQ No Yes No No
Linearity Yes Yes No No
Range Yes Yes No
May be required depending on the type of test
Specificity
Definition ndash The methodrsquos ability to specifically measure the
analyte in the presence of the components in the analytical
matrix
bull Impurities degradation products andor excipients are
available
bull When impurities degradation products or excipients are
not available bull Compare the results with another well-characterised analytical
method
bull Perform an artifical degradation under drastic conditions (light
heat acids y bases etc)
bull Peak purity
bull Peak shape analysis
bull Diode array detection
bull HPLC-MS
Specificity
Linearity
Definition ndash The ability of a method to produce results that are directly proportional to the concentration of the sample at a given interval
bull 5 levels of concentration Different dilutions of an stock
solution or separate weights of the component
bull 3 samples per level of concentration
Linearity
bull Analysis and collection of data
bull Graphical evaluation
bull Esimtating the parameters of the model by least
squares
bull Calculation of the confidence intervals for the estimated
parameters
Coefficient of correlation
i
i
i
i
i
ii
yyxx
yyxx
r
)()(
))((
2
Confidence intervals
2
)ˆ( 2
n
yy
S i
ii
xy
i
i
xy
b
xx
SS
2)(
b plusmn t(n-2) Sb
a plusmn t(n-2) Sa
i
i
i
i
xyaxxn
x
SS2
2
)(
-20
-15
-10
-5
0
5
10
15
20
0 5 10 15 20 25 30 35
Resid
uals
Cc (mgml)
Anaacutelisis de residuos
bull Check that the graph has a random pattern
bull A trend indicates a failure in the chosen model
bull An increase or decrease in residue dispersion indicates that the data
is not homoscedastic
Residue Analysis
Residue Analysis
Accuracy
Definition - The difference between results obtained by
the method and the actual value
bull It is calculated by determining the recovery of the method
bull Three replicates at three levels bull 80 -120 for the principal component bull 50 - 150 for impurities
ad
obs
C
CR
100
Recovery test
Amount added (mg) Amound detected (mg) Recovered
2435 2470 1014
2515 2541 1010
2515 2517 1001
3004 3079 1025
2974 3018 1015
3014 3038 1008
3533 3570 1010
3463 3456 998
3673 3701 1008
Mean 1010
SD 07945
Ecuacion de la recta
Encontrado = a + ( b x Antildeadido)
b= 09989
Ecuacion de la recta
Encontrado = a + ( b x Antildeadido)
b= 09989
a= 03272 Valores de t experimentales
y = 09989x + 03272Rsup2 = 09974
20
25
30
35
40
20 25 30 35 40
ob
serv
ed
added
Accuracy
Fraction Unit Range of Recovery ()
100 100 98-102
01 01 95-105
001 100 ppm 90-107
000000001 1 ppb 40-120
Acceptable Recovery depending on the analyte level
Gonzaacutelez et al Trends in Anal Chem 26 (3) 2007
Precision
Definition - The degree of agreement between results
when a procedure is repeatedly applied to a
homogenous sample
bull Repeatability Intermediate Precision Reproducibility
bull Calculation of the SD or RSD
bull Repeatability is precision under the same operating
conditions for a short period of time It is determined
by a minimum of 9 measurements within the given
range of the procedure (3 concentrations 3
replications) or a minimum of 6 replications at 100
bull Intermediate Precision indicates intra-laboratory
variations different days different analysts different
equipment
bull Reproducibility indicates inter-laboratory variations
Precision
Robustness
Definition ndash A measure of a methodrsquos ability to accept
small but deliberate variations in the parameters of
the method
bull Steps to Consider ndash Design and execution of the experiment
ndash Calculating the effects
ndash Statisticalgraphical analysis of the effects
ndash Relevant conclusions and eventual modifications of the
method
bull Changes in pH column mobile phase etc
Experimental design
bull One factor at a time
bull Factorial Designs bull Full Factorial
bull Fractional Factorial
bull Plackett-Burman
Robustness
Full Factorial Design (2k runs)
pH
Flow
TEA
pH
Flow
TEA
pH
Flow
TEA
pH
Flow
TEA
Particle size
Organic
Fractional Factorial Design ndash 5 factors
pH
Flow
TEA
pH
Flow
TEA
pH
Flow
TEA
pH
Flow
TEA
Particle size
Organic
7 factors (A-G) 2 levels
Exp Factors Result
A B C D E F G
1 + + + + + + + y1
2 + + - + - - - y2
3 + - + - + - - y3
4 + - - - - + + y4
5 - + + - - + - y5
6 - + - - + - + y6
7 - - + + - - + y7
8 - - - + + + - y8
Placket - Burman
Eff = ( y+ - y-)4
25
26
Extract according to the procedure and analyze
Repeat extraction
Combine aliquots and analyze correcting by dilution factor
Use the results for the combined corrected extraction as the
denominator for recovery calculation
Perform statistical analysis
Anderson ML amp Burney DP (1998) J AOAC Int 81
1005ndash1010
Accuracy of extraction procedure
27
28
29
30
Specificity
31
Specificity Phyllanthus amarus by peak purity
32
33
34
35
36
37
Ruggedness and robustness
38
Response factor variation with different columns
39
40
41
42
No instrumental separation
Less specific than chromatographic procedures
Specificity (selectivity) achieved by sample preparation
Specific color reactions
No problems to validate linearity accuracy and precision as per
HPLC
However the higher the complexity in sample preparation the
lowest the precision and ruggedness
Spectrometric analytical procedures
43
Identity well controlled Orthogonal methods with multiple
fingerprints
Ubiquitous markers in high concentration provided ID is well
controlled
Sample preparation with multiple extractions washings acidbasic
Color reactions with high specificity
When is OK to use a less specific quantitative procedure
44
Examples Black Cohosh St John wort Milk Thistle
Often times is not possible to obtain the same results
Results may be correlated but not absolute
Use all known peaks in the chromatogram for the class to
measure and calculate the total (use response factors)
Verify correlation between the two methods
Set new limits according to results
Moving from Spectrophotometric to HPLC
45
46
47
48
Silymarins in Milk Thistle
49
Typical chromatogram
50
Needs redefinition of limits
A more specific test will give consistently lower results
Correlation between tests is recommended
In some cases conversion factors have been proposed
Gaedcke F Pharmeuropa Scientific Notes [2009 2009(1)5-10]
Acceptance criteria after transfer
51
Specificity
Definition ndash The methodrsquos ability to specifically measure the
analyte in the presence of the components in the analytical
matrix
bull Impurities degradation products andor excipients are
available
bull When impurities degradation products or excipients are
not available bull Compare the results with another well-characterised analytical
method
bull Perform an artifical degradation under drastic conditions (light
heat acids y bases etc)
bull Peak purity
bull Peak shape analysis
bull Diode array detection
bull HPLC-MS
Specificity
Linearity
Definition ndash The ability of a method to produce results that are directly proportional to the concentration of the sample at a given interval
bull 5 levels of concentration Different dilutions of an stock
solution or separate weights of the component
bull 3 samples per level of concentration
Linearity
bull Analysis and collection of data
bull Graphical evaluation
bull Esimtating the parameters of the model by least
squares
bull Calculation of the confidence intervals for the estimated
parameters
Coefficient of correlation
i
i
i
i
i
ii
yyxx
yyxx
r
)()(
))((
2
Confidence intervals
2
)ˆ( 2
n
yy
S i
ii
xy
i
i
xy
b
xx
SS
2)(
b plusmn t(n-2) Sb
a plusmn t(n-2) Sa
i
i
i
i
xyaxxn
x
SS2
2
)(
-20
-15
-10
-5
0
5
10
15
20
0 5 10 15 20 25 30 35
Resid
uals
Cc (mgml)
Anaacutelisis de residuos
bull Check that the graph has a random pattern
bull A trend indicates a failure in the chosen model
bull An increase or decrease in residue dispersion indicates that the data
is not homoscedastic
Residue Analysis
Residue Analysis
Accuracy
Definition - The difference between results obtained by
the method and the actual value
bull It is calculated by determining the recovery of the method
bull Three replicates at three levels bull 80 -120 for the principal component bull 50 - 150 for impurities
ad
obs
C
CR
100
Recovery test
Amount added (mg) Amound detected (mg) Recovered
2435 2470 1014
2515 2541 1010
2515 2517 1001
3004 3079 1025
2974 3018 1015
3014 3038 1008
3533 3570 1010
3463 3456 998
3673 3701 1008
Mean 1010
SD 07945
Ecuacion de la recta
Encontrado = a + ( b x Antildeadido)
b= 09989
Ecuacion de la recta
Encontrado = a + ( b x Antildeadido)
b= 09989
a= 03272 Valores de t experimentales
y = 09989x + 03272Rsup2 = 09974
20
25
30
35
40
20 25 30 35 40
ob
serv
ed
added
Accuracy
Fraction Unit Range of Recovery ()
100 100 98-102
01 01 95-105
001 100 ppm 90-107
000000001 1 ppb 40-120
Acceptable Recovery depending on the analyte level
Gonzaacutelez et al Trends in Anal Chem 26 (3) 2007
Precision
Definition - The degree of agreement between results
when a procedure is repeatedly applied to a
homogenous sample
bull Repeatability Intermediate Precision Reproducibility
bull Calculation of the SD or RSD
bull Repeatability is precision under the same operating
conditions for a short period of time It is determined
by a minimum of 9 measurements within the given
range of the procedure (3 concentrations 3
replications) or a minimum of 6 replications at 100
bull Intermediate Precision indicates intra-laboratory
variations different days different analysts different
equipment
bull Reproducibility indicates inter-laboratory variations
Precision
Robustness
Definition ndash A measure of a methodrsquos ability to accept
small but deliberate variations in the parameters of
the method
bull Steps to Consider ndash Design and execution of the experiment
ndash Calculating the effects
ndash Statisticalgraphical analysis of the effects
ndash Relevant conclusions and eventual modifications of the
method
bull Changes in pH column mobile phase etc
Experimental design
bull One factor at a time
bull Factorial Designs bull Full Factorial
bull Fractional Factorial
bull Plackett-Burman
Robustness
Full Factorial Design (2k runs)
pH
Flow
TEA
pH
Flow
TEA
pH
Flow
TEA
pH
Flow
TEA
Particle size
Organic
Fractional Factorial Design ndash 5 factors
pH
Flow
TEA
pH
Flow
TEA
pH
Flow
TEA
pH
Flow
TEA
Particle size
Organic
7 factors (A-G) 2 levels
Exp Factors Result
A B C D E F G
1 + + + + + + + y1
2 + + - + - - - y2
3 + - + - + - - y3
4 + - - - - + + y4
5 - + + - - + - y5
6 - + - - + - + y6
7 - - + + - - + y7
8 - - - + + + - y8
Placket - Burman
Eff = ( y+ - y-)4
25
26
Extract according to the procedure and analyze
Repeat extraction
Combine aliquots and analyze correcting by dilution factor
Use the results for the combined corrected extraction as the
denominator for recovery calculation
Perform statistical analysis
Anderson ML amp Burney DP (1998) J AOAC Int 81
1005ndash1010
Accuracy of extraction procedure
27
28
29
30
Specificity
31
Specificity Phyllanthus amarus by peak purity
32
33
34
35
36
37
Ruggedness and robustness
38
Response factor variation with different columns
39
40
41
42
No instrumental separation
Less specific than chromatographic procedures
Specificity (selectivity) achieved by sample preparation
Specific color reactions
No problems to validate linearity accuracy and precision as per
HPLC
However the higher the complexity in sample preparation the
lowest the precision and ruggedness
Spectrometric analytical procedures
43
Identity well controlled Orthogonal methods with multiple
fingerprints
Ubiquitous markers in high concentration provided ID is well
controlled
Sample preparation with multiple extractions washings acidbasic
Color reactions with high specificity
When is OK to use a less specific quantitative procedure
44
Examples Black Cohosh St John wort Milk Thistle
Often times is not possible to obtain the same results
Results may be correlated but not absolute
Use all known peaks in the chromatogram for the class to
measure and calculate the total (use response factors)
Verify correlation between the two methods
Set new limits according to results
Moving from Spectrophotometric to HPLC
45
46
47
48
Silymarins in Milk Thistle
49
Typical chromatogram
50
Needs redefinition of limits
A more specific test will give consistently lower results
Correlation between tests is recommended
In some cases conversion factors have been proposed
Gaedcke F Pharmeuropa Scientific Notes [2009 2009(1)5-10]
Acceptance criteria after transfer
51
bull Peak purity
bull Peak shape analysis
bull Diode array detection
bull HPLC-MS
Specificity
Linearity
Definition ndash The ability of a method to produce results that are directly proportional to the concentration of the sample at a given interval
bull 5 levels of concentration Different dilutions of an stock
solution or separate weights of the component
bull 3 samples per level of concentration
Linearity
bull Analysis and collection of data
bull Graphical evaluation
bull Esimtating the parameters of the model by least
squares
bull Calculation of the confidence intervals for the estimated
parameters
Coefficient of correlation
i
i
i
i
i
ii
yyxx
yyxx
r
)()(
))((
2
Confidence intervals
2
)ˆ( 2
n
yy
S i
ii
xy
i
i
xy
b
xx
SS
2)(
b plusmn t(n-2) Sb
a plusmn t(n-2) Sa
i
i
i
i
xyaxxn
x
SS2
2
)(
-20
-15
-10
-5
0
5
10
15
20
0 5 10 15 20 25 30 35
Resid
uals
Cc (mgml)
Anaacutelisis de residuos
bull Check that the graph has a random pattern
bull A trend indicates a failure in the chosen model
bull An increase or decrease in residue dispersion indicates that the data
is not homoscedastic
Residue Analysis
Residue Analysis
Accuracy
Definition - The difference between results obtained by
the method and the actual value
bull It is calculated by determining the recovery of the method
bull Three replicates at three levels bull 80 -120 for the principal component bull 50 - 150 for impurities
ad
obs
C
CR
100
Recovery test
Amount added (mg) Amound detected (mg) Recovered
2435 2470 1014
2515 2541 1010
2515 2517 1001
3004 3079 1025
2974 3018 1015
3014 3038 1008
3533 3570 1010
3463 3456 998
3673 3701 1008
Mean 1010
SD 07945
Ecuacion de la recta
Encontrado = a + ( b x Antildeadido)
b= 09989
Ecuacion de la recta
Encontrado = a + ( b x Antildeadido)
b= 09989
a= 03272 Valores de t experimentales
y = 09989x + 03272Rsup2 = 09974
20
25
30
35
40
20 25 30 35 40
ob
serv
ed
added
Accuracy
Fraction Unit Range of Recovery ()
100 100 98-102
01 01 95-105
001 100 ppm 90-107
000000001 1 ppb 40-120
Acceptable Recovery depending on the analyte level
Gonzaacutelez et al Trends in Anal Chem 26 (3) 2007
Precision
Definition - The degree of agreement between results
when a procedure is repeatedly applied to a
homogenous sample
bull Repeatability Intermediate Precision Reproducibility
bull Calculation of the SD or RSD
bull Repeatability is precision under the same operating
conditions for a short period of time It is determined
by a minimum of 9 measurements within the given
range of the procedure (3 concentrations 3
replications) or a minimum of 6 replications at 100
bull Intermediate Precision indicates intra-laboratory
variations different days different analysts different
equipment
bull Reproducibility indicates inter-laboratory variations
Precision
Robustness
Definition ndash A measure of a methodrsquos ability to accept
small but deliberate variations in the parameters of
the method
bull Steps to Consider ndash Design and execution of the experiment
ndash Calculating the effects
ndash Statisticalgraphical analysis of the effects
ndash Relevant conclusions and eventual modifications of the
method
bull Changes in pH column mobile phase etc
Experimental design
bull One factor at a time
bull Factorial Designs bull Full Factorial
bull Fractional Factorial
bull Plackett-Burman
Robustness
Full Factorial Design (2k runs)
pH
Flow
TEA
pH
Flow
TEA
pH
Flow
TEA
pH
Flow
TEA
Particle size
Organic
Fractional Factorial Design ndash 5 factors
pH
Flow
TEA
pH
Flow
TEA
pH
Flow
TEA
pH
Flow
TEA
Particle size
Organic
7 factors (A-G) 2 levels
Exp Factors Result
A B C D E F G
1 + + + + + + + y1
2 + + - + - - - y2
3 + - + - + - - y3
4 + - - - - + + y4
5 - + + - - + - y5
6 - + - - + - + y6
7 - - + + - - + y7
8 - - - + + + - y8
Placket - Burman
Eff = ( y+ - y-)4
25
26
Extract according to the procedure and analyze
Repeat extraction
Combine aliquots and analyze correcting by dilution factor
Use the results for the combined corrected extraction as the
denominator for recovery calculation
Perform statistical analysis
Anderson ML amp Burney DP (1998) J AOAC Int 81
1005ndash1010
Accuracy of extraction procedure
27
28
29
30
Specificity
31
Specificity Phyllanthus amarus by peak purity
32
33
34
35
36
37
Ruggedness and robustness
38
Response factor variation with different columns
39
40
41
42
No instrumental separation
Less specific than chromatographic procedures
Specificity (selectivity) achieved by sample preparation
Specific color reactions
No problems to validate linearity accuracy and precision as per
HPLC
However the higher the complexity in sample preparation the
lowest the precision and ruggedness
Spectrometric analytical procedures
43
Identity well controlled Orthogonal methods with multiple
fingerprints
Ubiquitous markers in high concentration provided ID is well
controlled
Sample preparation with multiple extractions washings acidbasic
Color reactions with high specificity
When is OK to use a less specific quantitative procedure
44
Examples Black Cohosh St John wort Milk Thistle
Often times is not possible to obtain the same results
Results may be correlated but not absolute
Use all known peaks in the chromatogram for the class to
measure and calculate the total (use response factors)
Verify correlation between the two methods
Set new limits according to results
Moving from Spectrophotometric to HPLC
45
46
47
48
Silymarins in Milk Thistle
49
Typical chromatogram
50
Needs redefinition of limits
A more specific test will give consistently lower results
Correlation between tests is recommended
In some cases conversion factors have been proposed
Gaedcke F Pharmeuropa Scientific Notes [2009 2009(1)5-10]
Acceptance criteria after transfer
51
Linearity
Definition ndash The ability of a method to produce results that are directly proportional to the concentration of the sample at a given interval
bull 5 levels of concentration Different dilutions of an stock
solution or separate weights of the component
bull 3 samples per level of concentration
Linearity
bull Analysis and collection of data
bull Graphical evaluation
bull Esimtating the parameters of the model by least
squares
bull Calculation of the confidence intervals for the estimated
parameters
Coefficient of correlation
i
i
i
i
i
ii
yyxx
yyxx
r
)()(
))((
2
Confidence intervals
2
)ˆ( 2
n
yy
S i
ii
xy
i
i
xy
b
xx
SS
2)(
b plusmn t(n-2) Sb
a plusmn t(n-2) Sa
i
i
i
i
xyaxxn
x
SS2
2
)(
-20
-15
-10
-5
0
5
10
15
20
0 5 10 15 20 25 30 35
Resid
uals
Cc (mgml)
Anaacutelisis de residuos
bull Check that the graph has a random pattern
bull A trend indicates a failure in the chosen model
bull An increase or decrease in residue dispersion indicates that the data
is not homoscedastic
Residue Analysis
Residue Analysis
Accuracy
Definition - The difference between results obtained by
the method and the actual value
bull It is calculated by determining the recovery of the method
bull Three replicates at three levels bull 80 -120 for the principal component bull 50 - 150 for impurities
ad
obs
C
CR
100
Recovery test
Amount added (mg) Amound detected (mg) Recovered
2435 2470 1014
2515 2541 1010
2515 2517 1001
3004 3079 1025
2974 3018 1015
3014 3038 1008
3533 3570 1010
3463 3456 998
3673 3701 1008
Mean 1010
SD 07945
Ecuacion de la recta
Encontrado = a + ( b x Antildeadido)
b= 09989
Ecuacion de la recta
Encontrado = a + ( b x Antildeadido)
b= 09989
a= 03272 Valores de t experimentales
y = 09989x + 03272Rsup2 = 09974
20
25
30
35
40
20 25 30 35 40
ob
serv
ed
added
Accuracy
Fraction Unit Range of Recovery ()
100 100 98-102
01 01 95-105
001 100 ppm 90-107
000000001 1 ppb 40-120
Acceptable Recovery depending on the analyte level
Gonzaacutelez et al Trends in Anal Chem 26 (3) 2007
Precision
Definition - The degree of agreement between results
when a procedure is repeatedly applied to a
homogenous sample
bull Repeatability Intermediate Precision Reproducibility
bull Calculation of the SD or RSD
bull Repeatability is precision under the same operating
conditions for a short period of time It is determined
by a minimum of 9 measurements within the given
range of the procedure (3 concentrations 3
replications) or a minimum of 6 replications at 100
bull Intermediate Precision indicates intra-laboratory
variations different days different analysts different
equipment
bull Reproducibility indicates inter-laboratory variations
Precision
Robustness
Definition ndash A measure of a methodrsquos ability to accept
small but deliberate variations in the parameters of
the method
bull Steps to Consider ndash Design and execution of the experiment
ndash Calculating the effects
ndash Statisticalgraphical analysis of the effects
ndash Relevant conclusions and eventual modifications of the
method
bull Changes in pH column mobile phase etc
Experimental design
bull One factor at a time
bull Factorial Designs bull Full Factorial
bull Fractional Factorial
bull Plackett-Burman
Robustness
Full Factorial Design (2k runs)
pH
Flow
TEA
pH
Flow
TEA
pH
Flow
TEA
pH
Flow
TEA
Particle size
Organic
Fractional Factorial Design ndash 5 factors
pH
Flow
TEA
pH
Flow
TEA
pH
Flow
TEA
pH
Flow
TEA
Particle size
Organic
7 factors (A-G) 2 levels
Exp Factors Result
A B C D E F G
1 + + + + + + + y1
2 + + - + - - - y2
3 + - + - + - - y3
4 + - - - - + + y4
5 - + + - - + - y5
6 - + - - + - + y6
7 - - + + - - + y7
8 - - - + + + - y8
Placket - Burman
Eff = ( y+ - y-)4
25
26
Extract according to the procedure and analyze
Repeat extraction
Combine aliquots and analyze correcting by dilution factor
Use the results for the combined corrected extraction as the
denominator for recovery calculation
Perform statistical analysis
Anderson ML amp Burney DP (1998) J AOAC Int 81
1005ndash1010
Accuracy of extraction procedure
27
28
29
30
Specificity
31
Specificity Phyllanthus amarus by peak purity
32
33
34
35
36
37
Ruggedness and robustness
38
Response factor variation with different columns
39
40
41
42
No instrumental separation
Less specific than chromatographic procedures
Specificity (selectivity) achieved by sample preparation
Specific color reactions
No problems to validate linearity accuracy and precision as per
HPLC
However the higher the complexity in sample preparation the
lowest the precision and ruggedness
Spectrometric analytical procedures
43
Identity well controlled Orthogonal methods with multiple
fingerprints
Ubiquitous markers in high concentration provided ID is well
controlled
Sample preparation with multiple extractions washings acidbasic
Color reactions with high specificity
When is OK to use a less specific quantitative procedure
44
Examples Black Cohosh St John wort Milk Thistle
Often times is not possible to obtain the same results
Results may be correlated but not absolute
Use all known peaks in the chromatogram for the class to
measure and calculate the total (use response factors)
Verify correlation between the two methods
Set new limits according to results
Moving from Spectrophotometric to HPLC
45
46
47
48
Silymarins in Milk Thistle
49
Typical chromatogram
50
Needs redefinition of limits
A more specific test will give consistently lower results
Correlation between tests is recommended
In some cases conversion factors have been proposed
Gaedcke F Pharmeuropa Scientific Notes [2009 2009(1)5-10]
Acceptance criteria after transfer
51
Linearity
bull Analysis and collection of data
bull Graphical evaluation
bull Esimtating the parameters of the model by least
squares
bull Calculation of the confidence intervals for the estimated
parameters
Coefficient of correlation
i
i
i
i
i
ii
yyxx
yyxx
r
)()(
))((
2
Confidence intervals
2
)ˆ( 2
n
yy
S i
ii
xy
i
i
xy
b
xx
SS
2)(
b plusmn t(n-2) Sb
a plusmn t(n-2) Sa
i
i
i
i
xyaxxn
x
SS2
2
)(
-20
-15
-10
-5
0
5
10
15
20
0 5 10 15 20 25 30 35
Resid
uals
Cc (mgml)
Anaacutelisis de residuos
bull Check that the graph has a random pattern
bull A trend indicates a failure in the chosen model
bull An increase or decrease in residue dispersion indicates that the data
is not homoscedastic
Residue Analysis
Residue Analysis
Accuracy
Definition - The difference between results obtained by
the method and the actual value
bull It is calculated by determining the recovery of the method
bull Three replicates at three levels bull 80 -120 for the principal component bull 50 - 150 for impurities
ad
obs
C
CR
100
Recovery test
Amount added (mg) Amound detected (mg) Recovered
2435 2470 1014
2515 2541 1010
2515 2517 1001
3004 3079 1025
2974 3018 1015
3014 3038 1008
3533 3570 1010
3463 3456 998
3673 3701 1008
Mean 1010
SD 07945
Ecuacion de la recta
Encontrado = a + ( b x Antildeadido)
b= 09989
Ecuacion de la recta
Encontrado = a + ( b x Antildeadido)
b= 09989
a= 03272 Valores de t experimentales
y = 09989x + 03272Rsup2 = 09974
20
25
30
35
40
20 25 30 35 40
ob
serv
ed
added
Accuracy
Fraction Unit Range of Recovery ()
100 100 98-102
01 01 95-105
001 100 ppm 90-107
000000001 1 ppb 40-120
Acceptable Recovery depending on the analyte level
Gonzaacutelez et al Trends in Anal Chem 26 (3) 2007
Precision
Definition - The degree of agreement between results
when a procedure is repeatedly applied to a
homogenous sample
bull Repeatability Intermediate Precision Reproducibility
bull Calculation of the SD or RSD
bull Repeatability is precision under the same operating
conditions for a short period of time It is determined
by a minimum of 9 measurements within the given
range of the procedure (3 concentrations 3
replications) or a minimum of 6 replications at 100
bull Intermediate Precision indicates intra-laboratory
variations different days different analysts different
equipment
bull Reproducibility indicates inter-laboratory variations
Precision
Robustness
Definition ndash A measure of a methodrsquos ability to accept
small but deliberate variations in the parameters of
the method
bull Steps to Consider ndash Design and execution of the experiment
ndash Calculating the effects
ndash Statisticalgraphical analysis of the effects
ndash Relevant conclusions and eventual modifications of the
method
bull Changes in pH column mobile phase etc
Experimental design
bull One factor at a time
bull Factorial Designs bull Full Factorial
bull Fractional Factorial
bull Plackett-Burman
Robustness
Full Factorial Design (2k runs)
pH
Flow
TEA
pH
Flow
TEA
pH
Flow
TEA
pH
Flow
TEA
Particle size
Organic
Fractional Factorial Design ndash 5 factors
pH
Flow
TEA
pH
Flow
TEA
pH
Flow
TEA
pH
Flow
TEA
Particle size
Organic
7 factors (A-G) 2 levels
Exp Factors Result
A B C D E F G
1 + + + + + + + y1
2 + + - + - - - y2
3 + - + - + - - y3
4 + - - - - + + y4
5 - + + - - + - y5
6 - + - - + - + y6
7 - - + + - - + y7
8 - - - + + + - y8
Placket - Burman
Eff = ( y+ - y-)4
25
26
Extract according to the procedure and analyze
Repeat extraction
Combine aliquots and analyze correcting by dilution factor
Use the results for the combined corrected extraction as the
denominator for recovery calculation
Perform statistical analysis
Anderson ML amp Burney DP (1998) J AOAC Int 81
1005ndash1010
Accuracy of extraction procedure
27
28
29
30
Specificity
31
Specificity Phyllanthus amarus by peak purity
32
33
34
35
36
37
Ruggedness and robustness
38
Response factor variation with different columns
39
40
41
42
No instrumental separation
Less specific than chromatographic procedures
Specificity (selectivity) achieved by sample preparation
Specific color reactions
No problems to validate linearity accuracy and precision as per
HPLC
However the higher the complexity in sample preparation the
lowest the precision and ruggedness
Spectrometric analytical procedures
43
Identity well controlled Orthogonal methods with multiple
fingerprints
Ubiquitous markers in high concentration provided ID is well
controlled
Sample preparation with multiple extractions washings acidbasic
Color reactions with high specificity
When is OK to use a less specific quantitative procedure
44
Examples Black Cohosh St John wort Milk Thistle
Often times is not possible to obtain the same results
Results may be correlated but not absolute
Use all known peaks in the chromatogram for the class to
measure and calculate the total (use response factors)
Verify correlation between the two methods
Set new limits according to results
Moving from Spectrophotometric to HPLC
45
46
47
48
Silymarins in Milk Thistle
49
Typical chromatogram
50
Needs redefinition of limits
A more specific test will give consistently lower results
Correlation between tests is recommended
In some cases conversion factors have been proposed
Gaedcke F Pharmeuropa Scientific Notes [2009 2009(1)5-10]
Acceptance criteria after transfer
51
Coefficient of correlation
i
i
i
i
i
ii
yyxx
yyxx
r
)()(
))((
2
Confidence intervals
2
)ˆ( 2
n
yy
S i
ii
xy
i
i
xy
b
xx
SS
2)(
b plusmn t(n-2) Sb
a plusmn t(n-2) Sa
i
i
i
i
xyaxxn
x
SS2
2
)(
-20
-15
-10
-5
0
5
10
15
20
0 5 10 15 20 25 30 35
Resid
uals
Cc (mgml)
Anaacutelisis de residuos
bull Check that the graph has a random pattern
bull A trend indicates a failure in the chosen model
bull An increase or decrease in residue dispersion indicates that the data
is not homoscedastic
Residue Analysis
Residue Analysis
Accuracy
Definition - The difference between results obtained by
the method and the actual value
bull It is calculated by determining the recovery of the method
bull Three replicates at three levels bull 80 -120 for the principal component bull 50 - 150 for impurities
ad
obs
C
CR
100
Recovery test
Amount added (mg) Amound detected (mg) Recovered
2435 2470 1014
2515 2541 1010
2515 2517 1001
3004 3079 1025
2974 3018 1015
3014 3038 1008
3533 3570 1010
3463 3456 998
3673 3701 1008
Mean 1010
SD 07945
Ecuacion de la recta
Encontrado = a + ( b x Antildeadido)
b= 09989
Ecuacion de la recta
Encontrado = a + ( b x Antildeadido)
b= 09989
a= 03272 Valores de t experimentales
y = 09989x + 03272Rsup2 = 09974
20
25
30
35
40
20 25 30 35 40
ob
serv
ed
added
Accuracy
Fraction Unit Range of Recovery ()
100 100 98-102
01 01 95-105
001 100 ppm 90-107
000000001 1 ppb 40-120
Acceptable Recovery depending on the analyte level
Gonzaacutelez et al Trends in Anal Chem 26 (3) 2007
Precision
Definition - The degree of agreement between results
when a procedure is repeatedly applied to a
homogenous sample
bull Repeatability Intermediate Precision Reproducibility
bull Calculation of the SD or RSD
bull Repeatability is precision under the same operating
conditions for a short period of time It is determined
by a minimum of 9 measurements within the given
range of the procedure (3 concentrations 3
replications) or a minimum of 6 replications at 100
bull Intermediate Precision indicates intra-laboratory
variations different days different analysts different
equipment
bull Reproducibility indicates inter-laboratory variations
Precision
Robustness
Definition ndash A measure of a methodrsquos ability to accept
small but deliberate variations in the parameters of
the method
bull Steps to Consider ndash Design and execution of the experiment
ndash Calculating the effects
ndash Statisticalgraphical analysis of the effects
ndash Relevant conclusions and eventual modifications of the
method
bull Changes in pH column mobile phase etc
Experimental design
bull One factor at a time
bull Factorial Designs bull Full Factorial
bull Fractional Factorial
bull Plackett-Burman
Robustness
Full Factorial Design (2k runs)
pH
Flow
TEA
pH
Flow
TEA
pH
Flow
TEA
pH
Flow
TEA
Particle size
Organic
Fractional Factorial Design ndash 5 factors
pH
Flow
TEA
pH
Flow
TEA
pH
Flow
TEA
pH
Flow
TEA
Particle size
Organic
7 factors (A-G) 2 levels
Exp Factors Result
A B C D E F G
1 + + + + + + + y1
2 + + - + - - - y2
3 + - + - + - - y3
4 + - - - - + + y4
5 - + + - - + - y5
6 - + - - + - + y6
7 - - + + - - + y7
8 - - - + + + - y8
Placket - Burman
Eff = ( y+ - y-)4
25
26
Extract according to the procedure and analyze
Repeat extraction
Combine aliquots and analyze correcting by dilution factor
Use the results for the combined corrected extraction as the
denominator for recovery calculation
Perform statistical analysis
Anderson ML amp Burney DP (1998) J AOAC Int 81
1005ndash1010
Accuracy of extraction procedure
27
28
29
30
Specificity
31
Specificity Phyllanthus amarus by peak purity
32
33
34
35
36
37
Ruggedness and robustness
38
Response factor variation with different columns
39
40
41
42
No instrumental separation
Less specific than chromatographic procedures
Specificity (selectivity) achieved by sample preparation
Specific color reactions
No problems to validate linearity accuracy and precision as per
HPLC
However the higher the complexity in sample preparation the
lowest the precision and ruggedness
Spectrometric analytical procedures
43
Identity well controlled Orthogonal methods with multiple
fingerprints
Ubiquitous markers in high concentration provided ID is well
controlled
Sample preparation with multiple extractions washings acidbasic
Color reactions with high specificity
When is OK to use a less specific quantitative procedure
44
Examples Black Cohosh St John wort Milk Thistle
Often times is not possible to obtain the same results
Results may be correlated but not absolute
Use all known peaks in the chromatogram for the class to
measure and calculate the total (use response factors)
Verify correlation between the two methods
Set new limits according to results
Moving from Spectrophotometric to HPLC
45
46
47
48
Silymarins in Milk Thistle
49
Typical chromatogram
50
Needs redefinition of limits
A more specific test will give consistently lower results
Correlation between tests is recommended
In some cases conversion factors have been proposed
Gaedcke F Pharmeuropa Scientific Notes [2009 2009(1)5-10]
Acceptance criteria after transfer
51
Confidence intervals
2
)ˆ( 2
n
yy
S i
ii
xy
i
i
xy
b
xx
SS
2)(
b plusmn t(n-2) Sb
a plusmn t(n-2) Sa
i
i
i
i
xyaxxn
x
SS2
2
)(
-20
-15
-10
-5
0
5
10
15
20
0 5 10 15 20 25 30 35
Resid
uals
Cc (mgml)
Anaacutelisis de residuos
bull Check that the graph has a random pattern
bull A trend indicates a failure in the chosen model
bull An increase or decrease in residue dispersion indicates that the data
is not homoscedastic
Residue Analysis
Residue Analysis
Accuracy
Definition - The difference between results obtained by
the method and the actual value
bull It is calculated by determining the recovery of the method
bull Three replicates at three levels bull 80 -120 for the principal component bull 50 - 150 for impurities
ad
obs
C
CR
100
Recovery test
Amount added (mg) Amound detected (mg) Recovered
2435 2470 1014
2515 2541 1010
2515 2517 1001
3004 3079 1025
2974 3018 1015
3014 3038 1008
3533 3570 1010
3463 3456 998
3673 3701 1008
Mean 1010
SD 07945
Ecuacion de la recta
Encontrado = a + ( b x Antildeadido)
b= 09989
Ecuacion de la recta
Encontrado = a + ( b x Antildeadido)
b= 09989
a= 03272 Valores de t experimentales
y = 09989x + 03272Rsup2 = 09974
20
25
30
35
40
20 25 30 35 40
ob
serv
ed
added
Accuracy
Fraction Unit Range of Recovery ()
100 100 98-102
01 01 95-105
001 100 ppm 90-107
000000001 1 ppb 40-120
Acceptable Recovery depending on the analyte level
Gonzaacutelez et al Trends in Anal Chem 26 (3) 2007
Precision
Definition - The degree of agreement between results
when a procedure is repeatedly applied to a
homogenous sample
bull Repeatability Intermediate Precision Reproducibility
bull Calculation of the SD or RSD
bull Repeatability is precision under the same operating
conditions for a short period of time It is determined
by a minimum of 9 measurements within the given
range of the procedure (3 concentrations 3
replications) or a minimum of 6 replications at 100
bull Intermediate Precision indicates intra-laboratory
variations different days different analysts different
equipment
bull Reproducibility indicates inter-laboratory variations
Precision
Robustness
Definition ndash A measure of a methodrsquos ability to accept
small but deliberate variations in the parameters of
the method
bull Steps to Consider ndash Design and execution of the experiment
ndash Calculating the effects
ndash Statisticalgraphical analysis of the effects
ndash Relevant conclusions and eventual modifications of the
method
bull Changes in pH column mobile phase etc
Experimental design
bull One factor at a time
bull Factorial Designs bull Full Factorial
bull Fractional Factorial
bull Plackett-Burman
Robustness
Full Factorial Design (2k runs)
pH
Flow
TEA
pH
Flow
TEA
pH
Flow
TEA
pH
Flow
TEA
Particle size
Organic
Fractional Factorial Design ndash 5 factors
pH
Flow
TEA
pH
Flow
TEA
pH
Flow
TEA
pH
Flow
TEA
Particle size
Organic
7 factors (A-G) 2 levels
Exp Factors Result
A B C D E F G
1 + + + + + + + y1
2 + + - + - - - y2
3 + - + - + - - y3
4 + - - - - + + y4
5 - + + - - + - y5
6 - + - - + - + y6
7 - - + + - - + y7
8 - - - + + + - y8
Placket - Burman
Eff = ( y+ - y-)4
25
26
Extract according to the procedure and analyze
Repeat extraction
Combine aliquots and analyze correcting by dilution factor
Use the results for the combined corrected extraction as the
denominator for recovery calculation
Perform statistical analysis
Anderson ML amp Burney DP (1998) J AOAC Int 81
1005ndash1010
Accuracy of extraction procedure
27
28
29
30
Specificity
31
Specificity Phyllanthus amarus by peak purity
32
33
34
35
36
37
Ruggedness and robustness
38
Response factor variation with different columns
39
40
41
42
No instrumental separation
Less specific than chromatographic procedures
Specificity (selectivity) achieved by sample preparation
Specific color reactions
No problems to validate linearity accuracy and precision as per
HPLC
However the higher the complexity in sample preparation the
lowest the precision and ruggedness
Spectrometric analytical procedures
43
Identity well controlled Orthogonal methods with multiple
fingerprints
Ubiquitous markers in high concentration provided ID is well
controlled
Sample preparation with multiple extractions washings acidbasic
Color reactions with high specificity
When is OK to use a less specific quantitative procedure
44
Examples Black Cohosh St John wort Milk Thistle
Often times is not possible to obtain the same results
Results may be correlated but not absolute
Use all known peaks in the chromatogram for the class to
measure and calculate the total (use response factors)
Verify correlation between the two methods
Set new limits according to results
Moving from Spectrophotometric to HPLC
45
46
47
48
Silymarins in Milk Thistle
49
Typical chromatogram
50
Needs redefinition of limits
A more specific test will give consistently lower results
Correlation between tests is recommended
In some cases conversion factors have been proposed
Gaedcke F Pharmeuropa Scientific Notes [2009 2009(1)5-10]
Acceptance criteria after transfer
51
-20
-15
-10
-5
0
5
10
15
20
0 5 10 15 20 25 30 35
Resid
uals
Cc (mgml)
Anaacutelisis de residuos
bull Check that the graph has a random pattern
bull A trend indicates a failure in the chosen model
bull An increase or decrease in residue dispersion indicates that the data
is not homoscedastic
Residue Analysis
Residue Analysis
Accuracy
Definition - The difference between results obtained by
the method and the actual value
bull It is calculated by determining the recovery of the method
bull Three replicates at three levels bull 80 -120 for the principal component bull 50 - 150 for impurities
ad
obs
C
CR
100
Recovery test
Amount added (mg) Amound detected (mg) Recovered
2435 2470 1014
2515 2541 1010
2515 2517 1001
3004 3079 1025
2974 3018 1015
3014 3038 1008
3533 3570 1010
3463 3456 998
3673 3701 1008
Mean 1010
SD 07945
Ecuacion de la recta
Encontrado = a + ( b x Antildeadido)
b= 09989
Ecuacion de la recta
Encontrado = a + ( b x Antildeadido)
b= 09989
a= 03272 Valores de t experimentales
y = 09989x + 03272Rsup2 = 09974
20
25
30
35
40
20 25 30 35 40
ob
serv
ed
added
Accuracy
Fraction Unit Range of Recovery ()
100 100 98-102
01 01 95-105
001 100 ppm 90-107
000000001 1 ppb 40-120
Acceptable Recovery depending on the analyte level
Gonzaacutelez et al Trends in Anal Chem 26 (3) 2007
Precision
Definition - The degree of agreement between results
when a procedure is repeatedly applied to a
homogenous sample
bull Repeatability Intermediate Precision Reproducibility
bull Calculation of the SD or RSD
bull Repeatability is precision under the same operating
conditions for a short period of time It is determined
by a minimum of 9 measurements within the given
range of the procedure (3 concentrations 3
replications) or a minimum of 6 replications at 100
bull Intermediate Precision indicates intra-laboratory
variations different days different analysts different
equipment
bull Reproducibility indicates inter-laboratory variations
Precision
Robustness
Definition ndash A measure of a methodrsquos ability to accept
small but deliberate variations in the parameters of
the method
bull Steps to Consider ndash Design and execution of the experiment
ndash Calculating the effects
ndash Statisticalgraphical analysis of the effects
ndash Relevant conclusions and eventual modifications of the
method
bull Changes in pH column mobile phase etc
Experimental design
bull One factor at a time
bull Factorial Designs bull Full Factorial
bull Fractional Factorial
bull Plackett-Burman
Robustness
Full Factorial Design (2k runs)
pH
Flow
TEA
pH
Flow
TEA
pH
Flow
TEA
pH
Flow
TEA
Particle size
Organic
Fractional Factorial Design ndash 5 factors
pH
Flow
TEA
pH
Flow
TEA
pH
Flow
TEA
pH
Flow
TEA
Particle size
Organic
7 factors (A-G) 2 levels
Exp Factors Result
A B C D E F G
1 + + + + + + + y1
2 + + - + - - - y2
3 + - + - + - - y3
4 + - - - - + + y4
5 - + + - - + - y5
6 - + - - + - + y6
7 - - + + - - + y7
8 - - - + + + - y8
Placket - Burman
Eff = ( y+ - y-)4
25
26
Extract according to the procedure and analyze
Repeat extraction
Combine aliquots and analyze correcting by dilution factor
Use the results for the combined corrected extraction as the
denominator for recovery calculation
Perform statistical analysis
Anderson ML amp Burney DP (1998) J AOAC Int 81
1005ndash1010
Accuracy of extraction procedure
27
28
29
30
Specificity
31
Specificity Phyllanthus amarus by peak purity
32
33
34
35
36
37
Ruggedness and robustness
38
Response factor variation with different columns
39
40
41
42
No instrumental separation
Less specific than chromatographic procedures
Specificity (selectivity) achieved by sample preparation
Specific color reactions
No problems to validate linearity accuracy and precision as per
HPLC
However the higher the complexity in sample preparation the
lowest the precision and ruggedness
Spectrometric analytical procedures
43
Identity well controlled Orthogonal methods with multiple
fingerprints
Ubiquitous markers in high concentration provided ID is well
controlled
Sample preparation with multiple extractions washings acidbasic
Color reactions with high specificity
When is OK to use a less specific quantitative procedure
44
Examples Black Cohosh St John wort Milk Thistle
Often times is not possible to obtain the same results
Results may be correlated but not absolute
Use all known peaks in the chromatogram for the class to
measure and calculate the total (use response factors)
Verify correlation between the two methods
Set new limits according to results
Moving from Spectrophotometric to HPLC
45
46
47
48
Silymarins in Milk Thistle
49
Typical chromatogram
50
Needs redefinition of limits
A more specific test will give consistently lower results
Correlation between tests is recommended
In some cases conversion factors have been proposed
Gaedcke F Pharmeuropa Scientific Notes [2009 2009(1)5-10]
Acceptance criteria after transfer
51
Residue Analysis
Accuracy
Definition - The difference between results obtained by
the method and the actual value
bull It is calculated by determining the recovery of the method
bull Three replicates at three levels bull 80 -120 for the principal component bull 50 - 150 for impurities
ad
obs
C
CR
100
Recovery test
Amount added (mg) Amound detected (mg) Recovered
2435 2470 1014
2515 2541 1010
2515 2517 1001
3004 3079 1025
2974 3018 1015
3014 3038 1008
3533 3570 1010
3463 3456 998
3673 3701 1008
Mean 1010
SD 07945
Ecuacion de la recta
Encontrado = a + ( b x Antildeadido)
b= 09989
Ecuacion de la recta
Encontrado = a + ( b x Antildeadido)
b= 09989
a= 03272 Valores de t experimentales
y = 09989x + 03272Rsup2 = 09974
20
25
30
35
40
20 25 30 35 40
ob
serv
ed
added
Accuracy
Fraction Unit Range of Recovery ()
100 100 98-102
01 01 95-105
001 100 ppm 90-107
000000001 1 ppb 40-120
Acceptable Recovery depending on the analyte level
Gonzaacutelez et al Trends in Anal Chem 26 (3) 2007
Precision
Definition - The degree of agreement between results
when a procedure is repeatedly applied to a
homogenous sample
bull Repeatability Intermediate Precision Reproducibility
bull Calculation of the SD or RSD
bull Repeatability is precision under the same operating
conditions for a short period of time It is determined
by a minimum of 9 measurements within the given
range of the procedure (3 concentrations 3
replications) or a minimum of 6 replications at 100
bull Intermediate Precision indicates intra-laboratory
variations different days different analysts different
equipment
bull Reproducibility indicates inter-laboratory variations
Precision
Robustness
Definition ndash A measure of a methodrsquos ability to accept
small but deliberate variations in the parameters of
the method
bull Steps to Consider ndash Design and execution of the experiment
ndash Calculating the effects
ndash Statisticalgraphical analysis of the effects
ndash Relevant conclusions and eventual modifications of the
method
bull Changes in pH column mobile phase etc
Experimental design
bull One factor at a time
bull Factorial Designs bull Full Factorial
bull Fractional Factorial
bull Plackett-Burman
Robustness
Full Factorial Design (2k runs)
pH
Flow
TEA
pH
Flow
TEA
pH
Flow
TEA
pH
Flow
TEA
Particle size
Organic
Fractional Factorial Design ndash 5 factors
pH
Flow
TEA
pH
Flow
TEA
pH
Flow
TEA
pH
Flow
TEA
Particle size
Organic
7 factors (A-G) 2 levels
Exp Factors Result
A B C D E F G
1 + + + + + + + y1
2 + + - + - - - y2
3 + - + - + - - y3
4 + - - - - + + y4
5 - + + - - + - y5
6 - + - - + - + y6
7 - - + + - - + y7
8 - - - + + + - y8
Placket - Burman
Eff = ( y+ - y-)4
25
26
Extract according to the procedure and analyze
Repeat extraction
Combine aliquots and analyze correcting by dilution factor
Use the results for the combined corrected extraction as the
denominator for recovery calculation
Perform statistical analysis
Anderson ML amp Burney DP (1998) J AOAC Int 81
1005ndash1010
Accuracy of extraction procedure
27
28
29
30
Specificity
31
Specificity Phyllanthus amarus by peak purity
32
33
34
35
36
37
Ruggedness and robustness
38
Response factor variation with different columns
39
40
41
42
No instrumental separation
Less specific than chromatographic procedures
Specificity (selectivity) achieved by sample preparation
Specific color reactions
No problems to validate linearity accuracy and precision as per
HPLC
However the higher the complexity in sample preparation the
lowest the precision and ruggedness
Spectrometric analytical procedures
43
Identity well controlled Orthogonal methods with multiple
fingerprints
Ubiquitous markers in high concentration provided ID is well
controlled
Sample preparation with multiple extractions washings acidbasic
Color reactions with high specificity
When is OK to use a less specific quantitative procedure
44
Examples Black Cohosh St John wort Milk Thistle
Often times is not possible to obtain the same results
Results may be correlated but not absolute
Use all known peaks in the chromatogram for the class to
measure and calculate the total (use response factors)
Verify correlation between the two methods
Set new limits according to results
Moving from Spectrophotometric to HPLC
45
46
47
48
Silymarins in Milk Thistle
49
Typical chromatogram
50
Needs redefinition of limits
A more specific test will give consistently lower results
Correlation between tests is recommended
In some cases conversion factors have been proposed
Gaedcke F Pharmeuropa Scientific Notes [2009 2009(1)5-10]
Acceptance criteria after transfer
51
Accuracy
Definition - The difference between results obtained by
the method and the actual value
bull It is calculated by determining the recovery of the method
bull Three replicates at three levels bull 80 -120 for the principal component bull 50 - 150 for impurities
ad
obs
C
CR
100
Recovery test
Amount added (mg) Amound detected (mg) Recovered
2435 2470 1014
2515 2541 1010
2515 2517 1001
3004 3079 1025
2974 3018 1015
3014 3038 1008
3533 3570 1010
3463 3456 998
3673 3701 1008
Mean 1010
SD 07945
Ecuacion de la recta
Encontrado = a + ( b x Antildeadido)
b= 09989
Ecuacion de la recta
Encontrado = a + ( b x Antildeadido)
b= 09989
a= 03272 Valores de t experimentales
y = 09989x + 03272Rsup2 = 09974
20
25
30
35
40
20 25 30 35 40
ob
serv
ed
added
Accuracy
Fraction Unit Range of Recovery ()
100 100 98-102
01 01 95-105
001 100 ppm 90-107
000000001 1 ppb 40-120
Acceptable Recovery depending on the analyte level
Gonzaacutelez et al Trends in Anal Chem 26 (3) 2007
Precision
Definition - The degree of agreement between results
when a procedure is repeatedly applied to a
homogenous sample
bull Repeatability Intermediate Precision Reproducibility
bull Calculation of the SD or RSD
bull Repeatability is precision under the same operating
conditions for a short period of time It is determined
by a minimum of 9 measurements within the given
range of the procedure (3 concentrations 3
replications) or a minimum of 6 replications at 100
bull Intermediate Precision indicates intra-laboratory
variations different days different analysts different
equipment
bull Reproducibility indicates inter-laboratory variations
Precision
Robustness
Definition ndash A measure of a methodrsquos ability to accept
small but deliberate variations in the parameters of
the method
bull Steps to Consider ndash Design and execution of the experiment
ndash Calculating the effects
ndash Statisticalgraphical analysis of the effects
ndash Relevant conclusions and eventual modifications of the
method
bull Changes in pH column mobile phase etc
Experimental design
bull One factor at a time
bull Factorial Designs bull Full Factorial
bull Fractional Factorial
bull Plackett-Burman
Robustness
Full Factorial Design (2k runs)
pH
Flow
TEA
pH
Flow
TEA
pH
Flow
TEA
pH
Flow
TEA
Particle size
Organic
Fractional Factorial Design ndash 5 factors
pH
Flow
TEA
pH
Flow
TEA
pH
Flow
TEA
pH
Flow
TEA
Particle size
Organic
7 factors (A-G) 2 levels
Exp Factors Result
A B C D E F G
1 + + + + + + + y1
2 + + - + - - - y2
3 + - + - + - - y3
4 + - - - - + + y4
5 - + + - - + - y5
6 - + - - + - + y6
7 - - + + - - + y7
8 - - - + + + - y8
Placket - Burman
Eff = ( y+ - y-)4
25
26
Extract according to the procedure and analyze
Repeat extraction
Combine aliquots and analyze correcting by dilution factor
Use the results for the combined corrected extraction as the
denominator for recovery calculation
Perform statistical analysis
Anderson ML amp Burney DP (1998) J AOAC Int 81
1005ndash1010
Accuracy of extraction procedure
27
28
29
30
Specificity
31
Specificity Phyllanthus amarus by peak purity
32
33
34
35
36
37
Ruggedness and robustness
38
Response factor variation with different columns
39
40
41
42
No instrumental separation
Less specific than chromatographic procedures
Specificity (selectivity) achieved by sample preparation
Specific color reactions
No problems to validate linearity accuracy and precision as per
HPLC
However the higher the complexity in sample preparation the
lowest the precision and ruggedness
Spectrometric analytical procedures
43
Identity well controlled Orthogonal methods with multiple
fingerprints
Ubiquitous markers in high concentration provided ID is well
controlled
Sample preparation with multiple extractions washings acidbasic
Color reactions with high specificity
When is OK to use a less specific quantitative procedure
44
Examples Black Cohosh St John wort Milk Thistle
Often times is not possible to obtain the same results
Results may be correlated but not absolute
Use all known peaks in the chromatogram for the class to
measure and calculate the total (use response factors)
Verify correlation between the two methods
Set new limits according to results
Moving from Spectrophotometric to HPLC
45
46
47
48
Silymarins in Milk Thistle
49
Typical chromatogram
50
Needs redefinition of limits
A more specific test will give consistently lower results
Correlation between tests is recommended
In some cases conversion factors have been proposed
Gaedcke F Pharmeuropa Scientific Notes [2009 2009(1)5-10]
Acceptance criteria after transfer
51
Recovery test
Amount added (mg) Amound detected (mg) Recovered
2435 2470 1014
2515 2541 1010
2515 2517 1001
3004 3079 1025
2974 3018 1015
3014 3038 1008
3533 3570 1010
3463 3456 998
3673 3701 1008
Mean 1010
SD 07945
Ecuacion de la recta
Encontrado = a + ( b x Antildeadido)
b= 09989
Ecuacion de la recta
Encontrado = a + ( b x Antildeadido)
b= 09989
a= 03272 Valores de t experimentales
y = 09989x + 03272Rsup2 = 09974
20
25
30
35
40
20 25 30 35 40
ob
serv
ed
added
Accuracy
Fraction Unit Range of Recovery ()
100 100 98-102
01 01 95-105
001 100 ppm 90-107
000000001 1 ppb 40-120
Acceptable Recovery depending on the analyte level
Gonzaacutelez et al Trends in Anal Chem 26 (3) 2007
Precision
Definition - The degree of agreement between results
when a procedure is repeatedly applied to a
homogenous sample
bull Repeatability Intermediate Precision Reproducibility
bull Calculation of the SD or RSD
bull Repeatability is precision under the same operating
conditions for a short period of time It is determined
by a minimum of 9 measurements within the given
range of the procedure (3 concentrations 3
replications) or a minimum of 6 replications at 100
bull Intermediate Precision indicates intra-laboratory
variations different days different analysts different
equipment
bull Reproducibility indicates inter-laboratory variations
Precision
Robustness
Definition ndash A measure of a methodrsquos ability to accept
small but deliberate variations in the parameters of
the method
bull Steps to Consider ndash Design and execution of the experiment
ndash Calculating the effects
ndash Statisticalgraphical analysis of the effects
ndash Relevant conclusions and eventual modifications of the
method
bull Changes in pH column mobile phase etc
Experimental design
bull One factor at a time
bull Factorial Designs bull Full Factorial
bull Fractional Factorial
bull Plackett-Burman
Robustness
Full Factorial Design (2k runs)
pH
Flow
TEA
pH
Flow
TEA
pH
Flow
TEA
pH
Flow
TEA
Particle size
Organic
Fractional Factorial Design ndash 5 factors
pH
Flow
TEA
pH
Flow
TEA
pH
Flow
TEA
pH
Flow
TEA
Particle size
Organic
7 factors (A-G) 2 levels
Exp Factors Result
A B C D E F G
1 + + + + + + + y1
2 + + - + - - - y2
3 + - + - + - - y3
4 + - - - - + + y4
5 - + + - - + - y5
6 - + - - + - + y6
7 - - + + - - + y7
8 - - - + + + - y8
Placket - Burman
Eff = ( y+ - y-)4
25
26
Extract according to the procedure and analyze
Repeat extraction
Combine aliquots and analyze correcting by dilution factor
Use the results for the combined corrected extraction as the
denominator for recovery calculation
Perform statistical analysis
Anderson ML amp Burney DP (1998) J AOAC Int 81
1005ndash1010
Accuracy of extraction procedure
27
28
29
30
Specificity
31
Specificity Phyllanthus amarus by peak purity
32
33
34
35
36
37
Ruggedness and robustness
38
Response factor variation with different columns
39
40
41
42
No instrumental separation
Less specific than chromatographic procedures
Specificity (selectivity) achieved by sample preparation
Specific color reactions
No problems to validate linearity accuracy and precision as per
HPLC
However the higher the complexity in sample preparation the
lowest the precision and ruggedness
Spectrometric analytical procedures
43
Identity well controlled Orthogonal methods with multiple
fingerprints
Ubiquitous markers in high concentration provided ID is well
controlled
Sample preparation with multiple extractions washings acidbasic
Color reactions with high specificity
When is OK to use a less specific quantitative procedure
44
Examples Black Cohosh St John wort Milk Thistle
Often times is not possible to obtain the same results
Results may be correlated but not absolute
Use all known peaks in the chromatogram for the class to
measure and calculate the total (use response factors)
Verify correlation between the two methods
Set new limits according to results
Moving from Spectrophotometric to HPLC
45
46
47
48
Silymarins in Milk Thistle
49
Typical chromatogram
50
Needs redefinition of limits
A more specific test will give consistently lower results
Correlation between tests is recommended
In some cases conversion factors have been proposed
Gaedcke F Pharmeuropa Scientific Notes [2009 2009(1)5-10]
Acceptance criteria after transfer
51
Accuracy
Fraction Unit Range of Recovery ()
100 100 98-102
01 01 95-105
001 100 ppm 90-107
000000001 1 ppb 40-120
Acceptable Recovery depending on the analyte level
Gonzaacutelez et al Trends in Anal Chem 26 (3) 2007
Precision
Definition - The degree of agreement between results
when a procedure is repeatedly applied to a
homogenous sample
bull Repeatability Intermediate Precision Reproducibility
bull Calculation of the SD or RSD
bull Repeatability is precision under the same operating
conditions for a short period of time It is determined
by a minimum of 9 measurements within the given
range of the procedure (3 concentrations 3
replications) or a minimum of 6 replications at 100
bull Intermediate Precision indicates intra-laboratory
variations different days different analysts different
equipment
bull Reproducibility indicates inter-laboratory variations
Precision
Robustness
Definition ndash A measure of a methodrsquos ability to accept
small but deliberate variations in the parameters of
the method
bull Steps to Consider ndash Design and execution of the experiment
ndash Calculating the effects
ndash Statisticalgraphical analysis of the effects
ndash Relevant conclusions and eventual modifications of the
method
bull Changes in pH column mobile phase etc
Experimental design
bull One factor at a time
bull Factorial Designs bull Full Factorial
bull Fractional Factorial
bull Plackett-Burman
Robustness
Full Factorial Design (2k runs)
pH
Flow
TEA
pH
Flow
TEA
pH
Flow
TEA
pH
Flow
TEA
Particle size
Organic
Fractional Factorial Design ndash 5 factors
pH
Flow
TEA
pH
Flow
TEA
pH
Flow
TEA
pH
Flow
TEA
Particle size
Organic
7 factors (A-G) 2 levels
Exp Factors Result
A B C D E F G
1 + + + + + + + y1
2 + + - + - - - y2
3 + - + - + - - y3
4 + - - - - + + y4
5 - + + - - + - y5
6 - + - - + - + y6
7 - - + + - - + y7
8 - - - + + + - y8
Placket - Burman
Eff = ( y+ - y-)4
25
26
Extract according to the procedure and analyze
Repeat extraction
Combine aliquots and analyze correcting by dilution factor
Use the results for the combined corrected extraction as the
denominator for recovery calculation
Perform statistical analysis
Anderson ML amp Burney DP (1998) J AOAC Int 81
1005ndash1010
Accuracy of extraction procedure
27
28
29
30
Specificity
31
Specificity Phyllanthus amarus by peak purity
32
33
34
35
36
37
Ruggedness and robustness
38
Response factor variation with different columns
39
40
41
42
No instrumental separation
Less specific than chromatographic procedures
Specificity (selectivity) achieved by sample preparation
Specific color reactions
No problems to validate linearity accuracy and precision as per
HPLC
However the higher the complexity in sample preparation the
lowest the precision and ruggedness
Spectrometric analytical procedures
43
Identity well controlled Orthogonal methods with multiple
fingerprints
Ubiquitous markers in high concentration provided ID is well
controlled
Sample preparation with multiple extractions washings acidbasic
Color reactions with high specificity
When is OK to use a less specific quantitative procedure
44
Examples Black Cohosh St John wort Milk Thistle
Often times is not possible to obtain the same results
Results may be correlated but not absolute
Use all known peaks in the chromatogram for the class to
measure and calculate the total (use response factors)
Verify correlation between the two methods
Set new limits according to results
Moving from Spectrophotometric to HPLC
45
46
47
48
Silymarins in Milk Thistle
49
Typical chromatogram
50
Needs redefinition of limits
A more specific test will give consistently lower results
Correlation between tests is recommended
In some cases conversion factors have been proposed
Gaedcke F Pharmeuropa Scientific Notes [2009 2009(1)5-10]
Acceptance criteria after transfer
51
Precision
Definition - The degree of agreement between results
when a procedure is repeatedly applied to a
homogenous sample
bull Repeatability Intermediate Precision Reproducibility
bull Calculation of the SD or RSD
bull Repeatability is precision under the same operating
conditions for a short period of time It is determined
by a minimum of 9 measurements within the given
range of the procedure (3 concentrations 3
replications) or a minimum of 6 replications at 100
bull Intermediate Precision indicates intra-laboratory
variations different days different analysts different
equipment
bull Reproducibility indicates inter-laboratory variations
Precision
Robustness
Definition ndash A measure of a methodrsquos ability to accept
small but deliberate variations in the parameters of
the method
bull Steps to Consider ndash Design and execution of the experiment
ndash Calculating the effects
ndash Statisticalgraphical analysis of the effects
ndash Relevant conclusions and eventual modifications of the
method
bull Changes in pH column mobile phase etc
Experimental design
bull One factor at a time
bull Factorial Designs bull Full Factorial
bull Fractional Factorial
bull Plackett-Burman
Robustness
Full Factorial Design (2k runs)
pH
Flow
TEA
pH
Flow
TEA
pH
Flow
TEA
pH
Flow
TEA
Particle size
Organic
Fractional Factorial Design ndash 5 factors
pH
Flow
TEA
pH
Flow
TEA
pH
Flow
TEA
pH
Flow
TEA
Particle size
Organic
7 factors (A-G) 2 levels
Exp Factors Result
A B C D E F G
1 + + + + + + + y1
2 + + - + - - - y2
3 + - + - + - - y3
4 + - - - - + + y4
5 - + + - - + - y5
6 - + - - + - + y6
7 - - + + - - + y7
8 - - - + + + - y8
Placket - Burman
Eff = ( y+ - y-)4
25
26
Extract according to the procedure and analyze
Repeat extraction
Combine aliquots and analyze correcting by dilution factor
Use the results for the combined corrected extraction as the
denominator for recovery calculation
Perform statistical analysis
Anderson ML amp Burney DP (1998) J AOAC Int 81
1005ndash1010
Accuracy of extraction procedure
27
28
29
30
Specificity
31
Specificity Phyllanthus amarus by peak purity
32
33
34
35
36
37
Ruggedness and robustness
38
Response factor variation with different columns
39
40
41
42
No instrumental separation
Less specific than chromatographic procedures
Specificity (selectivity) achieved by sample preparation
Specific color reactions
No problems to validate linearity accuracy and precision as per
HPLC
However the higher the complexity in sample preparation the
lowest the precision and ruggedness
Spectrometric analytical procedures
43
Identity well controlled Orthogonal methods with multiple
fingerprints
Ubiquitous markers in high concentration provided ID is well
controlled
Sample preparation with multiple extractions washings acidbasic
Color reactions with high specificity
When is OK to use a less specific quantitative procedure
44
Examples Black Cohosh St John wort Milk Thistle
Often times is not possible to obtain the same results
Results may be correlated but not absolute
Use all known peaks in the chromatogram for the class to
measure and calculate the total (use response factors)
Verify correlation between the two methods
Set new limits according to results
Moving from Spectrophotometric to HPLC
45
46
47
48
Silymarins in Milk Thistle
49
Typical chromatogram
50
Needs redefinition of limits
A more specific test will give consistently lower results
Correlation between tests is recommended
In some cases conversion factors have been proposed
Gaedcke F Pharmeuropa Scientific Notes [2009 2009(1)5-10]
Acceptance criteria after transfer
51
bull Repeatability is precision under the same operating
conditions for a short period of time It is determined
by a minimum of 9 measurements within the given
range of the procedure (3 concentrations 3
replications) or a minimum of 6 replications at 100
bull Intermediate Precision indicates intra-laboratory
variations different days different analysts different
equipment
bull Reproducibility indicates inter-laboratory variations
Precision
Robustness
Definition ndash A measure of a methodrsquos ability to accept
small but deliberate variations in the parameters of
the method
bull Steps to Consider ndash Design and execution of the experiment
ndash Calculating the effects
ndash Statisticalgraphical analysis of the effects
ndash Relevant conclusions and eventual modifications of the
method
bull Changes in pH column mobile phase etc
Experimental design
bull One factor at a time
bull Factorial Designs bull Full Factorial
bull Fractional Factorial
bull Plackett-Burman
Robustness
Full Factorial Design (2k runs)
pH
Flow
TEA
pH
Flow
TEA
pH
Flow
TEA
pH
Flow
TEA
Particle size
Organic
Fractional Factorial Design ndash 5 factors
pH
Flow
TEA
pH
Flow
TEA
pH
Flow
TEA
pH
Flow
TEA
Particle size
Organic
7 factors (A-G) 2 levels
Exp Factors Result
A B C D E F G
1 + + + + + + + y1
2 + + - + - - - y2
3 + - + - + - - y3
4 + - - - - + + y4
5 - + + - - + - y5
6 - + - - + - + y6
7 - - + + - - + y7
8 - - - + + + - y8
Placket - Burman
Eff = ( y+ - y-)4
25
26
Extract according to the procedure and analyze
Repeat extraction
Combine aliquots and analyze correcting by dilution factor
Use the results for the combined corrected extraction as the
denominator for recovery calculation
Perform statistical analysis
Anderson ML amp Burney DP (1998) J AOAC Int 81
1005ndash1010
Accuracy of extraction procedure
27
28
29
30
Specificity
31
Specificity Phyllanthus amarus by peak purity
32
33
34
35
36
37
Ruggedness and robustness
38
Response factor variation with different columns
39
40
41
42
No instrumental separation
Less specific than chromatographic procedures
Specificity (selectivity) achieved by sample preparation
Specific color reactions
No problems to validate linearity accuracy and precision as per
HPLC
However the higher the complexity in sample preparation the
lowest the precision and ruggedness
Spectrometric analytical procedures
43
Identity well controlled Orthogonal methods with multiple
fingerprints
Ubiquitous markers in high concentration provided ID is well
controlled
Sample preparation with multiple extractions washings acidbasic
Color reactions with high specificity
When is OK to use a less specific quantitative procedure
44
Examples Black Cohosh St John wort Milk Thistle
Often times is not possible to obtain the same results
Results may be correlated but not absolute
Use all known peaks in the chromatogram for the class to
measure and calculate the total (use response factors)
Verify correlation between the two methods
Set new limits according to results
Moving from Spectrophotometric to HPLC
45
46
47
48
Silymarins in Milk Thistle
49
Typical chromatogram
50
Needs redefinition of limits
A more specific test will give consistently lower results
Correlation between tests is recommended
In some cases conversion factors have been proposed
Gaedcke F Pharmeuropa Scientific Notes [2009 2009(1)5-10]
Acceptance criteria after transfer
51
Robustness
Definition ndash A measure of a methodrsquos ability to accept
small but deliberate variations in the parameters of
the method
bull Steps to Consider ndash Design and execution of the experiment
ndash Calculating the effects
ndash Statisticalgraphical analysis of the effects
ndash Relevant conclusions and eventual modifications of the
method
bull Changes in pH column mobile phase etc
Experimental design
bull One factor at a time
bull Factorial Designs bull Full Factorial
bull Fractional Factorial
bull Plackett-Burman
Robustness
Full Factorial Design (2k runs)
pH
Flow
TEA
pH
Flow
TEA
pH
Flow
TEA
pH
Flow
TEA
Particle size
Organic
Fractional Factorial Design ndash 5 factors
pH
Flow
TEA
pH
Flow
TEA
pH
Flow
TEA
pH
Flow
TEA
Particle size
Organic
7 factors (A-G) 2 levels
Exp Factors Result
A B C D E F G
1 + + + + + + + y1
2 + + - + - - - y2
3 + - + - + - - y3
4 + - - - - + + y4
5 - + + - - + - y5
6 - + - - + - + y6
7 - - + + - - + y7
8 - - - + + + - y8
Placket - Burman
Eff = ( y+ - y-)4
25
26
Extract according to the procedure and analyze
Repeat extraction
Combine aliquots and analyze correcting by dilution factor
Use the results for the combined corrected extraction as the
denominator for recovery calculation
Perform statistical analysis
Anderson ML amp Burney DP (1998) J AOAC Int 81
1005ndash1010
Accuracy of extraction procedure
27
28
29
30
Specificity
31
Specificity Phyllanthus amarus by peak purity
32
33
34
35
36
37
Ruggedness and robustness
38
Response factor variation with different columns
39
40
41
42
No instrumental separation
Less specific than chromatographic procedures
Specificity (selectivity) achieved by sample preparation
Specific color reactions
No problems to validate linearity accuracy and precision as per
HPLC
However the higher the complexity in sample preparation the
lowest the precision and ruggedness
Spectrometric analytical procedures
43
Identity well controlled Orthogonal methods with multiple
fingerprints
Ubiquitous markers in high concentration provided ID is well
controlled
Sample preparation with multiple extractions washings acidbasic
Color reactions with high specificity
When is OK to use a less specific quantitative procedure
44
Examples Black Cohosh St John wort Milk Thistle
Often times is not possible to obtain the same results
Results may be correlated but not absolute
Use all known peaks in the chromatogram for the class to
measure and calculate the total (use response factors)
Verify correlation between the two methods
Set new limits according to results
Moving from Spectrophotometric to HPLC
45
46
47
48
Silymarins in Milk Thistle
49
Typical chromatogram
50
Needs redefinition of limits
A more specific test will give consistently lower results
Correlation between tests is recommended
In some cases conversion factors have been proposed
Gaedcke F Pharmeuropa Scientific Notes [2009 2009(1)5-10]
Acceptance criteria after transfer
51
Experimental design
bull One factor at a time
bull Factorial Designs bull Full Factorial
bull Fractional Factorial
bull Plackett-Burman
Robustness
Full Factorial Design (2k runs)
pH
Flow
TEA
pH
Flow
TEA
pH
Flow
TEA
pH
Flow
TEA
Particle size
Organic
Fractional Factorial Design ndash 5 factors
pH
Flow
TEA
pH
Flow
TEA
pH
Flow
TEA
pH
Flow
TEA
Particle size
Organic
7 factors (A-G) 2 levels
Exp Factors Result
A B C D E F G
1 + + + + + + + y1
2 + + - + - - - y2
3 + - + - + - - y3
4 + - - - - + + y4
5 - + + - - + - y5
6 - + - - + - + y6
7 - - + + - - + y7
8 - - - + + + - y8
Placket - Burman
Eff = ( y+ - y-)4
25
26
Extract according to the procedure and analyze
Repeat extraction
Combine aliquots and analyze correcting by dilution factor
Use the results for the combined corrected extraction as the
denominator for recovery calculation
Perform statistical analysis
Anderson ML amp Burney DP (1998) J AOAC Int 81
1005ndash1010
Accuracy of extraction procedure
27
28
29
30
Specificity
31
Specificity Phyllanthus amarus by peak purity
32
33
34
35
36
37
Ruggedness and robustness
38
Response factor variation with different columns
39
40
41
42
No instrumental separation
Less specific than chromatographic procedures
Specificity (selectivity) achieved by sample preparation
Specific color reactions
No problems to validate linearity accuracy and precision as per
HPLC
However the higher the complexity in sample preparation the
lowest the precision and ruggedness
Spectrometric analytical procedures
43
Identity well controlled Orthogonal methods with multiple
fingerprints
Ubiquitous markers in high concentration provided ID is well
controlled
Sample preparation with multiple extractions washings acidbasic
Color reactions with high specificity
When is OK to use a less specific quantitative procedure
44
Examples Black Cohosh St John wort Milk Thistle
Often times is not possible to obtain the same results
Results may be correlated but not absolute
Use all known peaks in the chromatogram for the class to
measure and calculate the total (use response factors)
Verify correlation between the two methods
Set new limits according to results
Moving from Spectrophotometric to HPLC
45
46
47
48
Silymarins in Milk Thistle
49
Typical chromatogram
50
Needs redefinition of limits
A more specific test will give consistently lower results
Correlation between tests is recommended
In some cases conversion factors have been proposed
Gaedcke F Pharmeuropa Scientific Notes [2009 2009(1)5-10]
Acceptance criteria after transfer
51
Full Factorial Design (2k runs)
pH
Flow
TEA
pH
Flow
TEA
pH
Flow
TEA
pH
Flow
TEA
Particle size
Organic
Fractional Factorial Design ndash 5 factors
pH
Flow
TEA
pH
Flow
TEA
pH
Flow
TEA
pH
Flow
TEA
Particle size
Organic
7 factors (A-G) 2 levels
Exp Factors Result
A B C D E F G
1 + + + + + + + y1
2 + + - + - - - y2
3 + - + - + - - y3
4 + - - - - + + y4
5 - + + - - + - y5
6 - + - - + - + y6
7 - - + + - - + y7
8 - - - + + + - y8
Placket - Burman
Eff = ( y+ - y-)4
25
26
Extract according to the procedure and analyze
Repeat extraction
Combine aliquots and analyze correcting by dilution factor
Use the results for the combined corrected extraction as the
denominator for recovery calculation
Perform statistical analysis
Anderson ML amp Burney DP (1998) J AOAC Int 81
1005ndash1010
Accuracy of extraction procedure
27
28
29
30
Specificity
31
Specificity Phyllanthus amarus by peak purity
32
33
34
35
36
37
Ruggedness and robustness
38
Response factor variation with different columns
39
40
41
42
No instrumental separation
Less specific than chromatographic procedures
Specificity (selectivity) achieved by sample preparation
Specific color reactions
No problems to validate linearity accuracy and precision as per
HPLC
However the higher the complexity in sample preparation the
lowest the precision and ruggedness
Spectrometric analytical procedures
43
Identity well controlled Orthogonal methods with multiple
fingerprints
Ubiquitous markers in high concentration provided ID is well
controlled
Sample preparation with multiple extractions washings acidbasic
Color reactions with high specificity
When is OK to use a less specific quantitative procedure
44
Examples Black Cohosh St John wort Milk Thistle
Often times is not possible to obtain the same results
Results may be correlated but not absolute
Use all known peaks in the chromatogram for the class to
measure and calculate the total (use response factors)
Verify correlation between the two methods
Set new limits according to results
Moving from Spectrophotometric to HPLC
45
46
47
48
Silymarins in Milk Thistle
49
Typical chromatogram
50
Needs redefinition of limits
A more specific test will give consistently lower results
Correlation between tests is recommended
In some cases conversion factors have been proposed
Gaedcke F Pharmeuropa Scientific Notes [2009 2009(1)5-10]
Acceptance criteria after transfer
51
Fractional Factorial Design ndash 5 factors
pH
Flow
TEA
pH
Flow
TEA
pH
Flow
TEA
pH
Flow
TEA
Particle size
Organic
7 factors (A-G) 2 levels
Exp Factors Result
A B C D E F G
1 + + + + + + + y1
2 + + - + - - - y2
3 + - + - + - - y3
4 + - - - - + + y4
5 - + + - - + - y5
6 - + - - + - + y6
7 - - + + - - + y7
8 - - - + + + - y8
Placket - Burman
Eff = ( y+ - y-)4
25
26
Extract according to the procedure and analyze
Repeat extraction
Combine aliquots and analyze correcting by dilution factor
Use the results for the combined corrected extraction as the
denominator for recovery calculation
Perform statistical analysis
Anderson ML amp Burney DP (1998) J AOAC Int 81
1005ndash1010
Accuracy of extraction procedure
27
28
29
30
Specificity
31
Specificity Phyllanthus amarus by peak purity
32
33
34
35
36
37
Ruggedness and robustness
38
Response factor variation with different columns
39
40
41
42
No instrumental separation
Less specific than chromatographic procedures
Specificity (selectivity) achieved by sample preparation
Specific color reactions
No problems to validate linearity accuracy and precision as per
HPLC
However the higher the complexity in sample preparation the
lowest the precision and ruggedness
Spectrometric analytical procedures
43
Identity well controlled Orthogonal methods with multiple
fingerprints
Ubiquitous markers in high concentration provided ID is well
controlled
Sample preparation with multiple extractions washings acidbasic
Color reactions with high specificity
When is OK to use a less specific quantitative procedure
44
Examples Black Cohosh St John wort Milk Thistle
Often times is not possible to obtain the same results
Results may be correlated but not absolute
Use all known peaks in the chromatogram for the class to
measure and calculate the total (use response factors)
Verify correlation between the two methods
Set new limits according to results
Moving from Spectrophotometric to HPLC
45
46
47
48
Silymarins in Milk Thistle
49
Typical chromatogram
50
Needs redefinition of limits
A more specific test will give consistently lower results
Correlation between tests is recommended
In some cases conversion factors have been proposed
Gaedcke F Pharmeuropa Scientific Notes [2009 2009(1)5-10]
Acceptance criteria after transfer
51
7 factors (A-G) 2 levels
Exp Factors Result
A B C D E F G
1 + + + + + + + y1
2 + + - + - - - y2
3 + - + - + - - y3
4 + - - - - + + y4
5 - + + - - + - y5
6 - + - - + - + y6
7 - - + + - - + y7
8 - - - + + + - y8
Placket - Burman
Eff = ( y+ - y-)4
25
26
Extract according to the procedure and analyze
Repeat extraction
Combine aliquots and analyze correcting by dilution factor
Use the results for the combined corrected extraction as the
denominator for recovery calculation
Perform statistical analysis
Anderson ML amp Burney DP (1998) J AOAC Int 81
1005ndash1010
Accuracy of extraction procedure
27
28
29
30
Specificity
31
Specificity Phyllanthus amarus by peak purity
32
33
34
35
36
37
Ruggedness and robustness
38
Response factor variation with different columns
39
40
41
42
No instrumental separation
Less specific than chromatographic procedures
Specificity (selectivity) achieved by sample preparation
Specific color reactions
No problems to validate linearity accuracy and precision as per
HPLC
However the higher the complexity in sample preparation the
lowest the precision and ruggedness
Spectrometric analytical procedures
43
Identity well controlled Orthogonal methods with multiple
fingerprints
Ubiquitous markers in high concentration provided ID is well
controlled
Sample preparation with multiple extractions washings acidbasic
Color reactions with high specificity
When is OK to use a less specific quantitative procedure
44
Examples Black Cohosh St John wort Milk Thistle
Often times is not possible to obtain the same results
Results may be correlated but not absolute
Use all known peaks in the chromatogram for the class to
measure and calculate the total (use response factors)
Verify correlation between the two methods
Set new limits according to results
Moving from Spectrophotometric to HPLC
45
46
47
48
Silymarins in Milk Thistle
49
Typical chromatogram
50
Needs redefinition of limits
A more specific test will give consistently lower results
Correlation between tests is recommended
In some cases conversion factors have been proposed
Gaedcke F Pharmeuropa Scientific Notes [2009 2009(1)5-10]
Acceptance criteria after transfer
51
25
26
Extract according to the procedure and analyze
Repeat extraction
Combine aliquots and analyze correcting by dilution factor
Use the results for the combined corrected extraction as the
denominator for recovery calculation
Perform statistical analysis
Anderson ML amp Burney DP (1998) J AOAC Int 81
1005ndash1010
Accuracy of extraction procedure
27
28
29
30
Specificity
31
Specificity Phyllanthus amarus by peak purity
32
33
34
35
36
37
Ruggedness and robustness
38
Response factor variation with different columns
39
40
41
42
No instrumental separation
Less specific than chromatographic procedures
Specificity (selectivity) achieved by sample preparation
Specific color reactions
No problems to validate linearity accuracy and precision as per
HPLC
However the higher the complexity in sample preparation the
lowest the precision and ruggedness
Spectrometric analytical procedures
43
Identity well controlled Orthogonal methods with multiple
fingerprints
Ubiquitous markers in high concentration provided ID is well
controlled
Sample preparation with multiple extractions washings acidbasic
Color reactions with high specificity
When is OK to use a less specific quantitative procedure
44
Examples Black Cohosh St John wort Milk Thistle
Often times is not possible to obtain the same results
Results may be correlated but not absolute
Use all known peaks in the chromatogram for the class to
measure and calculate the total (use response factors)
Verify correlation between the two methods
Set new limits according to results
Moving from Spectrophotometric to HPLC
45
46
47
48
Silymarins in Milk Thistle
49
Typical chromatogram
50
Needs redefinition of limits
A more specific test will give consistently lower results
Correlation between tests is recommended
In some cases conversion factors have been proposed
Gaedcke F Pharmeuropa Scientific Notes [2009 2009(1)5-10]
Acceptance criteria after transfer
51
26
Extract according to the procedure and analyze
Repeat extraction
Combine aliquots and analyze correcting by dilution factor
Use the results for the combined corrected extraction as the
denominator for recovery calculation
Perform statistical analysis
Anderson ML amp Burney DP (1998) J AOAC Int 81
1005ndash1010
Accuracy of extraction procedure
27
28
29
30
Specificity
31
Specificity Phyllanthus amarus by peak purity
32
33
34
35
36
37
Ruggedness and robustness
38
Response factor variation with different columns
39
40
41
42
No instrumental separation
Less specific than chromatographic procedures
Specificity (selectivity) achieved by sample preparation
Specific color reactions
No problems to validate linearity accuracy and precision as per
HPLC
However the higher the complexity in sample preparation the
lowest the precision and ruggedness
Spectrometric analytical procedures
43
Identity well controlled Orthogonal methods with multiple
fingerprints
Ubiquitous markers in high concentration provided ID is well
controlled
Sample preparation with multiple extractions washings acidbasic
Color reactions with high specificity
When is OK to use a less specific quantitative procedure
44
Examples Black Cohosh St John wort Milk Thistle
Often times is not possible to obtain the same results
Results may be correlated but not absolute
Use all known peaks in the chromatogram for the class to
measure and calculate the total (use response factors)
Verify correlation between the two methods
Set new limits according to results
Moving from Spectrophotometric to HPLC
45
46
47
48
Silymarins in Milk Thistle
49
Typical chromatogram
50
Needs redefinition of limits
A more specific test will give consistently lower results
Correlation between tests is recommended
In some cases conversion factors have been proposed
Gaedcke F Pharmeuropa Scientific Notes [2009 2009(1)5-10]
Acceptance criteria after transfer
51
Extract according to the procedure and analyze
Repeat extraction
Combine aliquots and analyze correcting by dilution factor
Use the results for the combined corrected extraction as the
denominator for recovery calculation
Perform statistical analysis
Anderson ML amp Burney DP (1998) J AOAC Int 81
1005ndash1010
Accuracy of extraction procedure
27
28
29
30
Specificity
31
Specificity Phyllanthus amarus by peak purity
32
33
34
35
36
37
Ruggedness and robustness
38
Response factor variation with different columns
39
40
41
42
No instrumental separation
Less specific than chromatographic procedures
Specificity (selectivity) achieved by sample preparation
Specific color reactions
No problems to validate linearity accuracy and precision as per
HPLC
However the higher the complexity in sample preparation the
lowest the precision and ruggedness
Spectrometric analytical procedures
43
Identity well controlled Orthogonal methods with multiple
fingerprints
Ubiquitous markers in high concentration provided ID is well
controlled
Sample preparation with multiple extractions washings acidbasic
Color reactions with high specificity
When is OK to use a less specific quantitative procedure
44
Examples Black Cohosh St John wort Milk Thistle
Often times is not possible to obtain the same results
Results may be correlated but not absolute
Use all known peaks in the chromatogram for the class to
measure and calculate the total (use response factors)
Verify correlation between the two methods
Set new limits according to results
Moving from Spectrophotometric to HPLC
45
46
47
48
Silymarins in Milk Thistle
49
Typical chromatogram
50
Needs redefinition of limits
A more specific test will give consistently lower results
Correlation between tests is recommended
In some cases conversion factors have been proposed
Gaedcke F Pharmeuropa Scientific Notes [2009 2009(1)5-10]
Acceptance criteria after transfer
51
28
29
30
Specificity
31
Specificity Phyllanthus amarus by peak purity
32
33
34
35
36
37
Ruggedness and robustness
38
Response factor variation with different columns
39
40
41
42
No instrumental separation
Less specific than chromatographic procedures
Specificity (selectivity) achieved by sample preparation
Specific color reactions
No problems to validate linearity accuracy and precision as per
HPLC
However the higher the complexity in sample preparation the
lowest the precision and ruggedness
Spectrometric analytical procedures
43
Identity well controlled Orthogonal methods with multiple
fingerprints
Ubiquitous markers in high concentration provided ID is well
controlled
Sample preparation with multiple extractions washings acidbasic
Color reactions with high specificity
When is OK to use a less specific quantitative procedure
44
Examples Black Cohosh St John wort Milk Thistle
Often times is not possible to obtain the same results
Results may be correlated but not absolute
Use all known peaks in the chromatogram for the class to
measure and calculate the total (use response factors)
Verify correlation between the two methods
Set new limits according to results
Moving from Spectrophotometric to HPLC
45
46
47
48
Silymarins in Milk Thistle
49
Typical chromatogram
50
Needs redefinition of limits
A more specific test will give consistently lower results
Correlation between tests is recommended
In some cases conversion factors have been proposed
Gaedcke F Pharmeuropa Scientific Notes [2009 2009(1)5-10]
Acceptance criteria after transfer
51
29
30
Specificity
31
Specificity Phyllanthus amarus by peak purity
32
33
34
35
36
37
Ruggedness and robustness
38
Response factor variation with different columns
39
40
41
42
No instrumental separation
Less specific than chromatographic procedures
Specificity (selectivity) achieved by sample preparation
Specific color reactions
No problems to validate linearity accuracy and precision as per
HPLC
However the higher the complexity in sample preparation the
lowest the precision and ruggedness
Spectrometric analytical procedures
43
Identity well controlled Orthogonal methods with multiple
fingerprints
Ubiquitous markers in high concentration provided ID is well
controlled
Sample preparation with multiple extractions washings acidbasic
Color reactions with high specificity
When is OK to use a less specific quantitative procedure
44
Examples Black Cohosh St John wort Milk Thistle
Often times is not possible to obtain the same results
Results may be correlated but not absolute
Use all known peaks in the chromatogram for the class to
measure and calculate the total (use response factors)
Verify correlation between the two methods
Set new limits according to results
Moving from Spectrophotometric to HPLC
45
46
47
48
Silymarins in Milk Thistle
49
Typical chromatogram
50
Needs redefinition of limits
A more specific test will give consistently lower results
Correlation between tests is recommended
In some cases conversion factors have been proposed
Gaedcke F Pharmeuropa Scientific Notes [2009 2009(1)5-10]
Acceptance criteria after transfer
51
30
Specificity
31
Specificity Phyllanthus amarus by peak purity
32
33
34
35
36
37
Ruggedness and robustness
38
Response factor variation with different columns
39
40
41
42
No instrumental separation
Less specific than chromatographic procedures
Specificity (selectivity) achieved by sample preparation
Specific color reactions
No problems to validate linearity accuracy and precision as per
HPLC
However the higher the complexity in sample preparation the
lowest the precision and ruggedness
Spectrometric analytical procedures
43
Identity well controlled Orthogonal methods with multiple
fingerprints
Ubiquitous markers in high concentration provided ID is well
controlled
Sample preparation with multiple extractions washings acidbasic
Color reactions with high specificity
When is OK to use a less specific quantitative procedure
44
Examples Black Cohosh St John wort Milk Thistle
Often times is not possible to obtain the same results
Results may be correlated but not absolute
Use all known peaks in the chromatogram for the class to
measure and calculate the total (use response factors)
Verify correlation between the two methods
Set new limits according to results
Moving from Spectrophotometric to HPLC
45
46
47
48
Silymarins in Milk Thistle
49
Typical chromatogram
50
Needs redefinition of limits
A more specific test will give consistently lower results
Correlation between tests is recommended
In some cases conversion factors have been proposed
Gaedcke F Pharmeuropa Scientific Notes [2009 2009(1)5-10]
Acceptance criteria after transfer
51
Specificity
31
Specificity Phyllanthus amarus by peak purity
32
33
34
35
36
37
Ruggedness and robustness
38
Response factor variation with different columns
39
40
41
42
No instrumental separation
Less specific than chromatographic procedures
Specificity (selectivity) achieved by sample preparation
Specific color reactions
No problems to validate linearity accuracy and precision as per
HPLC
However the higher the complexity in sample preparation the
lowest the precision and ruggedness
Spectrometric analytical procedures
43
Identity well controlled Orthogonal methods with multiple
fingerprints
Ubiquitous markers in high concentration provided ID is well
controlled
Sample preparation with multiple extractions washings acidbasic
Color reactions with high specificity
When is OK to use a less specific quantitative procedure
44
Examples Black Cohosh St John wort Milk Thistle
Often times is not possible to obtain the same results
Results may be correlated but not absolute
Use all known peaks in the chromatogram for the class to
measure and calculate the total (use response factors)
Verify correlation between the two methods
Set new limits according to results
Moving from Spectrophotometric to HPLC
45
46
47
48
Silymarins in Milk Thistle
49
Typical chromatogram
50
Needs redefinition of limits
A more specific test will give consistently lower results
Correlation between tests is recommended
In some cases conversion factors have been proposed
Gaedcke F Pharmeuropa Scientific Notes [2009 2009(1)5-10]
Acceptance criteria after transfer
51
Specificity Phyllanthus amarus by peak purity
32
33
34
35
36
37
Ruggedness and robustness
38
Response factor variation with different columns
39
40
41
42
No instrumental separation
Less specific than chromatographic procedures
Specificity (selectivity) achieved by sample preparation
Specific color reactions
No problems to validate linearity accuracy and precision as per
HPLC
However the higher the complexity in sample preparation the
lowest the precision and ruggedness
Spectrometric analytical procedures
43
Identity well controlled Orthogonal methods with multiple
fingerprints
Ubiquitous markers in high concentration provided ID is well
controlled
Sample preparation with multiple extractions washings acidbasic
Color reactions with high specificity
When is OK to use a less specific quantitative procedure
44
Examples Black Cohosh St John wort Milk Thistle
Often times is not possible to obtain the same results
Results may be correlated but not absolute
Use all known peaks in the chromatogram for the class to
measure and calculate the total (use response factors)
Verify correlation between the two methods
Set new limits according to results
Moving from Spectrophotometric to HPLC
45
46
47
48
Silymarins in Milk Thistle
49
Typical chromatogram
50
Needs redefinition of limits
A more specific test will give consistently lower results
Correlation between tests is recommended
In some cases conversion factors have been proposed
Gaedcke F Pharmeuropa Scientific Notes [2009 2009(1)5-10]
Acceptance criteria after transfer
51
33
34
35
36
37
Ruggedness and robustness
38
Response factor variation with different columns
39
40
41
42
No instrumental separation
Less specific than chromatographic procedures
Specificity (selectivity) achieved by sample preparation
Specific color reactions
No problems to validate linearity accuracy and precision as per
HPLC
However the higher the complexity in sample preparation the
lowest the precision and ruggedness
Spectrometric analytical procedures
43
Identity well controlled Orthogonal methods with multiple
fingerprints
Ubiquitous markers in high concentration provided ID is well
controlled
Sample preparation with multiple extractions washings acidbasic
Color reactions with high specificity
When is OK to use a less specific quantitative procedure
44
Examples Black Cohosh St John wort Milk Thistle
Often times is not possible to obtain the same results
Results may be correlated but not absolute
Use all known peaks in the chromatogram for the class to
measure and calculate the total (use response factors)
Verify correlation between the two methods
Set new limits according to results
Moving from Spectrophotometric to HPLC
45
46
47
48
Silymarins in Milk Thistle
49
Typical chromatogram
50
Needs redefinition of limits
A more specific test will give consistently lower results
Correlation between tests is recommended
In some cases conversion factors have been proposed
Gaedcke F Pharmeuropa Scientific Notes [2009 2009(1)5-10]
Acceptance criteria after transfer
51
34
35
36
37
Ruggedness and robustness
38
Response factor variation with different columns
39
40
41
42
No instrumental separation
Less specific than chromatographic procedures
Specificity (selectivity) achieved by sample preparation
Specific color reactions
No problems to validate linearity accuracy and precision as per
HPLC
However the higher the complexity in sample preparation the
lowest the precision and ruggedness
Spectrometric analytical procedures
43
Identity well controlled Orthogonal methods with multiple
fingerprints
Ubiquitous markers in high concentration provided ID is well
controlled
Sample preparation with multiple extractions washings acidbasic
Color reactions with high specificity
When is OK to use a less specific quantitative procedure
44
Examples Black Cohosh St John wort Milk Thistle
Often times is not possible to obtain the same results
Results may be correlated but not absolute
Use all known peaks in the chromatogram for the class to
measure and calculate the total (use response factors)
Verify correlation between the two methods
Set new limits according to results
Moving from Spectrophotometric to HPLC
45
46
47
48
Silymarins in Milk Thistle
49
Typical chromatogram
50
Needs redefinition of limits
A more specific test will give consistently lower results
Correlation between tests is recommended
In some cases conversion factors have been proposed
Gaedcke F Pharmeuropa Scientific Notes [2009 2009(1)5-10]
Acceptance criteria after transfer
51
35
36
37
Ruggedness and robustness
38
Response factor variation with different columns
39
40
41
42
No instrumental separation
Less specific than chromatographic procedures
Specificity (selectivity) achieved by sample preparation
Specific color reactions
No problems to validate linearity accuracy and precision as per
HPLC
However the higher the complexity in sample preparation the
lowest the precision and ruggedness
Spectrometric analytical procedures
43
Identity well controlled Orthogonal methods with multiple
fingerprints
Ubiquitous markers in high concentration provided ID is well
controlled
Sample preparation with multiple extractions washings acidbasic
Color reactions with high specificity
When is OK to use a less specific quantitative procedure
44
Examples Black Cohosh St John wort Milk Thistle
Often times is not possible to obtain the same results
Results may be correlated but not absolute
Use all known peaks in the chromatogram for the class to
measure and calculate the total (use response factors)
Verify correlation between the two methods
Set new limits according to results
Moving from Spectrophotometric to HPLC
45
46
47
48
Silymarins in Milk Thistle
49
Typical chromatogram
50
Needs redefinition of limits
A more specific test will give consistently lower results
Correlation between tests is recommended
In some cases conversion factors have been proposed
Gaedcke F Pharmeuropa Scientific Notes [2009 2009(1)5-10]
Acceptance criteria after transfer
51
36
37
Ruggedness and robustness
38
Response factor variation with different columns
39
40
41
42
No instrumental separation
Less specific than chromatographic procedures
Specificity (selectivity) achieved by sample preparation
Specific color reactions
No problems to validate linearity accuracy and precision as per
HPLC
However the higher the complexity in sample preparation the
lowest the precision and ruggedness
Spectrometric analytical procedures
43
Identity well controlled Orthogonal methods with multiple
fingerprints
Ubiquitous markers in high concentration provided ID is well
controlled
Sample preparation with multiple extractions washings acidbasic
Color reactions with high specificity
When is OK to use a less specific quantitative procedure
44
Examples Black Cohosh St John wort Milk Thistle
Often times is not possible to obtain the same results
Results may be correlated but not absolute
Use all known peaks in the chromatogram for the class to
measure and calculate the total (use response factors)
Verify correlation between the two methods
Set new limits according to results
Moving from Spectrophotometric to HPLC
45
46
47
48
Silymarins in Milk Thistle
49
Typical chromatogram
50
Needs redefinition of limits
A more specific test will give consistently lower results
Correlation between tests is recommended
In some cases conversion factors have been proposed
Gaedcke F Pharmeuropa Scientific Notes [2009 2009(1)5-10]
Acceptance criteria after transfer
51
37
Ruggedness and robustness
38
Response factor variation with different columns
39
40
41
42
No instrumental separation
Less specific than chromatographic procedures
Specificity (selectivity) achieved by sample preparation
Specific color reactions
No problems to validate linearity accuracy and precision as per
HPLC
However the higher the complexity in sample preparation the
lowest the precision and ruggedness
Spectrometric analytical procedures
43
Identity well controlled Orthogonal methods with multiple
fingerprints
Ubiquitous markers in high concentration provided ID is well
controlled
Sample preparation with multiple extractions washings acidbasic
Color reactions with high specificity
When is OK to use a less specific quantitative procedure
44
Examples Black Cohosh St John wort Milk Thistle
Often times is not possible to obtain the same results
Results may be correlated but not absolute
Use all known peaks in the chromatogram for the class to
measure and calculate the total (use response factors)
Verify correlation between the two methods
Set new limits according to results
Moving from Spectrophotometric to HPLC
45
46
47
48
Silymarins in Milk Thistle
49
Typical chromatogram
50
Needs redefinition of limits
A more specific test will give consistently lower results
Correlation between tests is recommended
In some cases conversion factors have been proposed
Gaedcke F Pharmeuropa Scientific Notes [2009 2009(1)5-10]
Acceptance criteria after transfer
51
Ruggedness and robustness
38
Response factor variation with different columns
39
40
41
42
No instrumental separation
Less specific than chromatographic procedures
Specificity (selectivity) achieved by sample preparation
Specific color reactions
No problems to validate linearity accuracy and precision as per
HPLC
However the higher the complexity in sample preparation the
lowest the precision and ruggedness
Spectrometric analytical procedures
43
Identity well controlled Orthogonal methods with multiple
fingerprints
Ubiquitous markers in high concentration provided ID is well
controlled
Sample preparation with multiple extractions washings acidbasic
Color reactions with high specificity
When is OK to use a less specific quantitative procedure
44
Examples Black Cohosh St John wort Milk Thistle
Often times is not possible to obtain the same results
Results may be correlated but not absolute
Use all known peaks in the chromatogram for the class to
measure and calculate the total (use response factors)
Verify correlation between the two methods
Set new limits according to results
Moving from Spectrophotometric to HPLC
45
46
47
48
Silymarins in Milk Thistle
49
Typical chromatogram
50
Needs redefinition of limits
A more specific test will give consistently lower results
Correlation between tests is recommended
In some cases conversion factors have been proposed
Gaedcke F Pharmeuropa Scientific Notes [2009 2009(1)5-10]
Acceptance criteria after transfer
51
Response factor variation with different columns
39
40
41
42
No instrumental separation
Less specific than chromatographic procedures
Specificity (selectivity) achieved by sample preparation
Specific color reactions
No problems to validate linearity accuracy and precision as per
HPLC
However the higher the complexity in sample preparation the
lowest the precision and ruggedness
Spectrometric analytical procedures
43
Identity well controlled Orthogonal methods with multiple
fingerprints
Ubiquitous markers in high concentration provided ID is well
controlled
Sample preparation with multiple extractions washings acidbasic
Color reactions with high specificity
When is OK to use a less specific quantitative procedure
44
Examples Black Cohosh St John wort Milk Thistle
Often times is not possible to obtain the same results
Results may be correlated but not absolute
Use all known peaks in the chromatogram for the class to
measure and calculate the total (use response factors)
Verify correlation between the two methods
Set new limits according to results
Moving from Spectrophotometric to HPLC
45
46
47
48
Silymarins in Milk Thistle
49
Typical chromatogram
50
Needs redefinition of limits
A more specific test will give consistently lower results
Correlation between tests is recommended
In some cases conversion factors have been proposed
Gaedcke F Pharmeuropa Scientific Notes [2009 2009(1)5-10]
Acceptance criteria after transfer
51
40
41
42
No instrumental separation
Less specific than chromatographic procedures
Specificity (selectivity) achieved by sample preparation
Specific color reactions
No problems to validate linearity accuracy and precision as per
HPLC
However the higher the complexity in sample preparation the
lowest the precision and ruggedness
Spectrometric analytical procedures
43
Identity well controlled Orthogonal methods with multiple
fingerprints
Ubiquitous markers in high concentration provided ID is well
controlled
Sample preparation with multiple extractions washings acidbasic
Color reactions with high specificity
When is OK to use a less specific quantitative procedure
44
Examples Black Cohosh St John wort Milk Thistle
Often times is not possible to obtain the same results
Results may be correlated but not absolute
Use all known peaks in the chromatogram for the class to
measure and calculate the total (use response factors)
Verify correlation between the two methods
Set new limits according to results
Moving from Spectrophotometric to HPLC
45
46
47
48
Silymarins in Milk Thistle
49
Typical chromatogram
50
Needs redefinition of limits
A more specific test will give consistently lower results
Correlation between tests is recommended
In some cases conversion factors have been proposed
Gaedcke F Pharmeuropa Scientific Notes [2009 2009(1)5-10]
Acceptance criteria after transfer
51
41
42
No instrumental separation
Less specific than chromatographic procedures
Specificity (selectivity) achieved by sample preparation
Specific color reactions
No problems to validate linearity accuracy and precision as per
HPLC
However the higher the complexity in sample preparation the
lowest the precision and ruggedness
Spectrometric analytical procedures
43
Identity well controlled Orthogonal methods with multiple
fingerprints
Ubiquitous markers in high concentration provided ID is well
controlled
Sample preparation with multiple extractions washings acidbasic
Color reactions with high specificity
When is OK to use a less specific quantitative procedure
44
Examples Black Cohosh St John wort Milk Thistle
Often times is not possible to obtain the same results
Results may be correlated but not absolute
Use all known peaks in the chromatogram for the class to
measure and calculate the total (use response factors)
Verify correlation between the two methods
Set new limits according to results
Moving from Spectrophotometric to HPLC
45
46
47
48
Silymarins in Milk Thistle
49
Typical chromatogram
50
Needs redefinition of limits
A more specific test will give consistently lower results
Correlation between tests is recommended
In some cases conversion factors have been proposed
Gaedcke F Pharmeuropa Scientific Notes [2009 2009(1)5-10]
Acceptance criteria after transfer
51
42
No instrumental separation
Less specific than chromatographic procedures
Specificity (selectivity) achieved by sample preparation
Specific color reactions
No problems to validate linearity accuracy and precision as per
HPLC
However the higher the complexity in sample preparation the
lowest the precision and ruggedness
Spectrometric analytical procedures
43
Identity well controlled Orthogonal methods with multiple
fingerprints
Ubiquitous markers in high concentration provided ID is well
controlled
Sample preparation with multiple extractions washings acidbasic
Color reactions with high specificity
When is OK to use a less specific quantitative procedure
44
Examples Black Cohosh St John wort Milk Thistle
Often times is not possible to obtain the same results
Results may be correlated but not absolute
Use all known peaks in the chromatogram for the class to
measure and calculate the total (use response factors)
Verify correlation between the two methods
Set new limits according to results
Moving from Spectrophotometric to HPLC
45
46
47
48
Silymarins in Milk Thistle
49
Typical chromatogram
50
Needs redefinition of limits
A more specific test will give consistently lower results
Correlation between tests is recommended
In some cases conversion factors have been proposed
Gaedcke F Pharmeuropa Scientific Notes [2009 2009(1)5-10]
Acceptance criteria after transfer
51
No instrumental separation
Less specific than chromatographic procedures
Specificity (selectivity) achieved by sample preparation
Specific color reactions
No problems to validate linearity accuracy and precision as per
HPLC
However the higher the complexity in sample preparation the
lowest the precision and ruggedness
Spectrometric analytical procedures
43
Identity well controlled Orthogonal methods with multiple
fingerprints
Ubiquitous markers in high concentration provided ID is well
controlled
Sample preparation with multiple extractions washings acidbasic
Color reactions with high specificity
When is OK to use a less specific quantitative procedure
44
Examples Black Cohosh St John wort Milk Thistle
Often times is not possible to obtain the same results
Results may be correlated but not absolute
Use all known peaks in the chromatogram for the class to
measure and calculate the total (use response factors)
Verify correlation between the two methods
Set new limits according to results
Moving from Spectrophotometric to HPLC
45
46
47
48
Silymarins in Milk Thistle
49
Typical chromatogram
50
Needs redefinition of limits
A more specific test will give consistently lower results
Correlation between tests is recommended
In some cases conversion factors have been proposed
Gaedcke F Pharmeuropa Scientific Notes [2009 2009(1)5-10]
Acceptance criteria after transfer
51
Identity well controlled Orthogonal methods with multiple
fingerprints
Ubiquitous markers in high concentration provided ID is well
controlled
Sample preparation with multiple extractions washings acidbasic
Color reactions with high specificity
When is OK to use a less specific quantitative procedure
44
Examples Black Cohosh St John wort Milk Thistle
Often times is not possible to obtain the same results
Results may be correlated but not absolute
Use all known peaks in the chromatogram for the class to
measure and calculate the total (use response factors)
Verify correlation between the two methods
Set new limits according to results
Moving from Spectrophotometric to HPLC
45
46
47
48
Silymarins in Milk Thistle
49
Typical chromatogram
50
Needs redefinition of limits
A more specific test will give consistently lower results
Correlation between tests is recommended
In some cases conversion factors have been proposed
Gaedcke F Pharmeuropa Scientific Notes [2009 2009(1)5-10]
Acceptance criteria after transfer
51
Examples Black Cohosh St John wort Milk Thistle
Often times is not possible to obtain the same results
Results may be correlated but not absolute
Use all known peaks in the chromatogram for the class to
measure and calculate the total (use response factors)
Verify correlation between the two methods
Set new limits according to results
Moving from Spectrophotometric to HPLC
45
46
47
48
Silymarins in Milk Thistle
49
Typical chromatogram
50
Needs redefinition of limits
A more specific test will give consistently lower results
Correlation between tests is recommended
In some cases conversion factors have been proposed
Gaedcke F Pharmeuropa Scientific Notes [2009 2009(1)5-10]
Acceptance criteria after transfer
51
46
47
48
Silymarins in Milk Thistle
49
Typical chromatogram
50
Needs redefinition of limits
A more specific test will give consistently lower results
Correlation between tests is recommended
In some cases conversion factors have been proposed
Gaedcke F Pharmeuropa Scientific Notes [2009 2009(1)5-10]
Acceptance criteria after transfer
51
47
48
Silymarins in Milk Thistle
49
Typical chromatogram
50
Needs redefinition of limits
A more specific test will give consistently lower results
Correlation between tests is recommended
In some cases conversion factors have been proposed
Gaedcke F Pharmeuropa Scientific Notes [2009 2009(1)5-10]
Acceptance criteria after transfer
51
48
Silymarins in Milk Thistle
49
Typical chromatogram
50
Needs redefinition of limits
A more specific test will give consistently lower results
Correlation between tests is recommended
In some cases conversion factors have been proposed
Gaedcke F Pharmeuropa Scientific Notes [2009 2009(1)5-10]
Acceptance criteria after transfer
51
Silymarins in Milk Thistle
49
Typical chromatogram
50
Needs redefinition of limits
A more specific test will give consistently lower results
Correlation between tests is recommended
In some cases conversion factors have been proposed
Gaedcke F Pharmeuropa Scientific Notes [2009 2009(1)5-10]
Acceptance criteria after transfer
51
Typical chromatogram
50
Needs redefinition of limits
A more specific test will give consistently lower results
Correlation between tests is recommended
In some cases conversion factors have been proposed
Gaedcke F Pharmeuropa Scientific Notes [2009 2009(1)5-10]
Acceptance criteria after transfer
51
Needs redefinition of limits
A more specific test will give consistently lower results
Correlation between tests is recommended
In some cases conversion factors have been proposed
Gaedcke F Pharmeuropa Scientific Notes [2009 2009(1)5-10]
Acceptance criteria after transfer
51