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