การประชุมวิชาการเรื่อง Analytical Method Validation for FDA Compliance

สมาคมเภสชั กรอุตสาหการ (ประเทศไทย) 97

5

Example 1. Method verification for Assay

- Specificity
- Linearity and range
- Accuracy
- Precision
- System suitability test
- Stability of analyte solutions

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Appendix 1: Draft of test method
Analysis of Paracetamol in Paracetamol syrup

Chromatographic condition

Column: µBondapak C18, 10 m, 3.9 x 300 mm or equivalent

Mobile phase: 0.01M Sodium butanesulfonate in Formic acid:

Methanol: Water

(4 : 150 : 850)

Flow: 2.0 mL/min

Injection volume: 20 µL

Detector: UV @ 243 nm (Diode array detector)

Based on: Area

Retention time: 4-aminophenol ~ 2 min

Paracetamol ~ 3.7 min

Methylparaben ~ 24 min

Run time: 35 min

7

Preparation of standard and sample solutions

Standard solution (120 µg/mL of Paracetamol)

1. Accurately weigh about 24 mg of Paracetamol WS and transfer into
a 200-mL volumetric flask.

2. Add 100 mL of mobile phase, sonicate for 15 minutes.
3. Adjust to volume with mobile phase.

Sample solution (Nominally 120 µg/ mL of Paracetamol)

1. Accurately weigh about 5 mL of Paracetamol syrup (equivalent to 120
mg of Paracetamol) and transfer into a 100-mL volumetric flask.

2. Dissolve and adjust to volume with mobile phase.
3. Pipette 5.0 mL of the solution in (2) to a 50-mL volumetric flask.
4. Adjust to volume with mobile phase.
5. Filter through 0.45-µm nylon membrane filter and discard the first

portion of the filtrate.

8

สมาคมเภสชั กรอุตสาหการ (ประเทศไทย) 99

1. Specificity

Matrix Standard Sample

9

1.1. Preparation of solutions
Placebo
Prepare placebo without paracetamol.
Standard and Sample solution
Prepare standard and sample as described in
appendix 1: draft of test method.

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1.1. Preparation of solutions
Matrix solution

1. Transfer about 5 mL of Placebo into a 100-mL
volumetric flask.

2. Add 50 mL of mobile phase, sonicate for 15 minutes.
3. Adjust to volume with mobile phase.
4. Pipette 5.0 mL of the solution in (3) to a 50-mL

volumetric flask.
5. Adjust to volume with mobile phase.

6. Filter through a 0.45-µm nylon membrane filter and

discard the first portion of the filtrate.

11

1.2. Samples to be injected into the
chromatographic system

• Mobile phase
• Standard solution (for system suitability test)
• Matrix solution
• Sample solution

12

สมาคมเภสชั กรอุตสาหการ (ประเทศไทย) 101

1.3. Evaluation
The specificity is determined based on

• Comparison of chromatograms obtained from the
matrix solution with those obtained from standard and
sample solution.

• Calculation of Paracetamol peak purity factor (PPF) in
chromatograms obtained from sample using software of
chromatographic data system.

Acceptable limit

• The matrix solution gives no peak eluting at the same time as
Paracetamol.

• Based on sample, peak purity factor of Paracetamol is not
less than 990.

13

2. Linearity and range

50 %LA 75 %LA 100 %LA 125 %LA 150 %LA

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2.1. Preparation of solutions

Standard solution
Described in Appendix 1: Draft of test method.

Standard solution at concentration 50%LA (Std 50), 75%LA
(Std 75), 100%LA (Std 100), 125%LA (Std 125) and 150%LA
(Std 150)
1. Accurately weigh Paracetamol WS as described in Table

1 and transfer into a 100-mL volumetric flask.
2. Add 50 mL of mobile phase, sonicate for 15 minutes.
3. Adjust to volume with mobile phase.

15

2.1. Preparation of solutions

Table 1

Standard solutions Std Std Std Std Std
50 75 100 125 150

Weight (mg) of 6 9 12 15 18
Paracetamol WS
about

16

สมาคมเภสชั กรอุตสาหการ (ประเทศไทย) 103

The other procedure for preparation of
solutions of linearity test

Stock solution of Paracetamol
1. Accurately weigh about 150 mg of Paracetamol WS

and transfer into a 50-mL volumetric flask.
2. Add 25 mL of mobile phase, sonicate for 15

minutes.
3. Adjust to volume with mobile phase.

17

Standard solution at concentration 50%LA (Std 50),
75%LA (Std 75), 100%LA (Std 100), 125%LA (Std 125) and
150%LA (Std 150)
1. Pipette stock solution of Paracetamol as described

in Table 2 into a 100-mL volumetric flask.
2. Adjust to volume with mobile phase.

Table 2

Standard solutions Std Std Std Std Std

50 75 100 125 150

Pipette (mL) stock 2.0 3.0 4.0 5.0 6.0

solution of paracetamol

18

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2.2. Samples to be injected into the
chromatographic system

• Mobile phase
• Standard solution (for system suitability test)
• Std 50
• Std 75
• Std 100
• Std 125
• Std 150

19

2.3. Evaluation

• Plot a linear regression between concentration and
the response, then determine the coefficient of
determination (r2). Residual plot is observed.

Acceptable limit
• For regression equation, r2 > 0.999
• For residual plot, residual values are randomly

spaced around the horizontal axis and no specific
trend of data are observed.

20

สมาคมเภสชั กรอุตสาหการ (ประเทศไทย) 105

3. Accuracy

Prepare duplicate of standard solution and
triplicate x 3 levels of simulated samples

Triplicate of Triplicate of Triplicate of
sim 50 sim 100 sim 150

21

3.1. Preparation of solutions

Simulated sample solution at concentration 50%LA (Sim 50),
100%LA (Sim 100), and 150%LA (Sim 150) (triplicate x 3 levels)
1. Accurately weigh Paracetamol WS as described in Table 3

and transfer into a 50-mL volumetric flask.
2. Add 2.5 mL of Placebo.
3. Add 25 mL of mobile phase, sonicate for 15 minutes.
4. Adjust to volume with mobile phase.

5. Pipette 5.0 mL of the solution in (4) to a 50-mL volumetric

flask.
6. Adjust to volume with mobile phase.
7. Filter through a 0.45-µm nylon membrane filter and discard

the first portion of the filtrate.

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3.1. Preparation of solutions

Table 3 Sim Sim Sim
50 100 150
Simulated sample
solutions 30 60 90
Weight (mg) of
Paracetamol WS about

23

3.2. Samples to be injected into the
chromatographic system

• Mobile phase
• Standard, replicate solution no.1 (for system

suitability test)
• Triplicate of Sim 50
• Triplicate of Sim 100
• Triplicate of Sim 150
• Standard, replicate solution no.2

24

สมาคมเภสชั กรอุตสาหการ (ประเทศไทย) 107

3.3. Evaluation

Determine the found amount (or found concentration)
of Paracetamol in simulated samples based on standard
solutions, then calculate the percentages of recovery
from the below formula:

% recovery = Found concentration or amount x 100
Added concentration or amount

Acceptable limit
Mean recovery = 98.0-102.0%

25

4. Precision

Duplicate of Six replicate of
standard solution sample solutions

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4.1. Preparation of solutions

Prepare standard solution and six replicate of samples
as described in Appendix 1: Draft of test method.

27

4.2. Samples to be injected into the
chromatographic system

• Mobile phase
• Standard, replicate solution no.1 (for system

suitability test)
• Six replicate solutions of samples
• Standard, replicate solution no. 2

28

สมาคมเภสชั กรอุตสาหการ (ประเทศไทย) 109

4.3. Evaluation
Determine the content assay of individual samples and
then calculated %RSD of all results.
Acceptable limit
Repeatability
%RSD < 2.0 % (Six replicate solutions of samples)

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5. System suitability test

Standard solution
Paracetamol peak
- Repeated injection: five injections, %RSD < 2.0%
- Symmetry factor: 0.8-1.5
- Theoretical plate: N > 2000

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6. Stability of analyte solutions

Standard and Sample solution

Insert the standard and sample solution in the sequence every 6 hours for 24
hours

Evaluation
The solution is considered stable if either one or two below
conditions are fulfilled:
- Absolute value of %relative difference

%relative difference = (peak area at elapsed time - peak area at zero time) x 100%
peak area at zero time

- %RSD of repeated injections (during the elapsed time) does not exceed 2%

31

How to inject all samples into HPLC

in the same sequence ?

32

สมาคมเภสชั กรอุตสาหการ (ประเทศไทย) 111

Sequence of injections

Mobile phase

Standard solution no.1 ……………….Standard for t0 *
Six replicate of samples ………………Sample for t0 *
Matrix

Triplicate of sim 50

Standard for t6 hr.* Run time of 1 injection is
Sample for t6 hr.*
Triplicate of sim 100

Triplicate of sim 150 35 minutes

Std 50

Std 75

Std 100

Std 125

Standard for t12 hr.*
Sample for t12 hr.*
Std 150

Standard solution no.2

Run mobile phase 290 minutes

Standard for t24 hr.*
Sample for t24 hr.*
33

Example 2. Method verification for 4-aminophenol

- Specificity
- Limit of detection
- System suitability test
- Stability of analyte solutions

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Appendix 1: Draft of test method
Analysis of 4-aminophenol in Paracetamol syrup

Chromatographic condition

Column: µBondapak C18, 10 m, 3.9 x 300 mm or equivalent

Mobile phase: 0.01M Sodium butanesulfonate in Formic acid:

Methanol: Water

(4 : 150 : 850)

Flow: 2.0 mL/min

Injection volume: 20 µL

Detector: UV @ 272 nm

Based on: Area

Retention time: 4-aminophenol ~ 2 min

Paracetamol ~ 3.7 min

Methylparaben ~ 24 min

Run time: 35 min

35

Preparation of standard and sample solutions

Standard solution (0.024 mg/ mL of 4-aminophenol)

1. Accurately weigh about 24 mg of 4-aminophenol RS and transfer into a
100-mL volumetric flask.

2. Add 50 mL of mobile phase, sonicate for 15 minutes.
3. Adjust to volume with mobile phase.
4. Pipette 5.0 mL of the solution in (3) to a 50-mL volumetric flask.
5. Adjust to volume with mobile phase.

Sample solution (Nominally 4.8 mg/ mL of Paracetamol)

1. Accurately weigh about 5 mL of Paracetamol syrup (equivalent to
120 mg of Paracetamol) and transfer into a 25-mL volumetric flask.

2. Dissolve and adjust to volume with mobile phase.
3. Filter through 0.45-µm nylon membrane filter and discard the first

portion of the filtrate.

36

สมาคมเภสชั กรอุตสาหการ (ประเทศไทย) 113

1. Specificity

Matrix Standard Sample

37

1.1. preparation of solutions

Placebo
Prepare placebo without paracetamol.
Standard and Sample solution
Prepare standard and sample as described in
appendix 1: draft of test method.

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1.1. preparation of solutions
Matrix solution
1. Transfer 5 mL of Placebo into a 25-mL

volumetric flask.
2. Dissolve and adjust to volume with mobile phase.
3. Filter through a 0.45-µm nylon membrane

filter and discard the first portion of the filtrate.

39

1.2. Samples to be injected into the
chromatographic system

• Mobile phase
• Standard solution (for system suitability test)
• Matrix solution
• Sample solution

40

สมาคมเภสชั กรอุตสาหการ (ประเทศไทย) 115

1.3. Evaluation

The specificity/ selectivity is determined based on

• Comparison of chromatograms obtained from the matrix
solution with those obtained from standard and sample
solution.

Acceptable limit

• The matrix solution gives no peak eluting at the same time as
4-aminophenol. Peak of 4-aminophenol is well separated
from the adjacent peaks.

41

2. Limit of detection

Standard solution

0.024 mg/mL of 4-aminophenol

Sample solution

4.8 mg/mL of Paracetamol
Limit of 4-aminophenol in Paracetamol oral solution
is not more than 0.5%.

Calculation of %4-aminophenol in standard solution in
relative to paracetamol in sample solution
= 0.024/4.8 x 100 = 0.5%

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2.1. preparation of solutions

Standard solution of 4-aminophenol is used for test
of limit of detection.
Concentration of 4-aminophenol (0.5%) of standard
solution is at the limit of 4-aminophenol in
paracetamol oral solution.

43

2.2. Samples to be injected into the
chromatographic system

• Mobile phase
• Standard solution (for system suitability test)
• Solution for test of limit of detection

44

สมาคมเภสชั กรอุตสาหการ (ประเทศไทย) 117

2.3. Evaluation

Signal to noise ratio of
4-aminophenol peak is not less than 3.

45

3. System suitability test

Standard solution
4-aminophenol peak
- Repeated injections: six injections, %RSD < 5.0%
- Tailing factor: T < 2.0
- Theoretical plate: N > 2000

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4. Stability of analyte solutions

Standard and Sample solution

Insert the standard and sample solution in the sequence every 6 hours for 24
hours

Evaluation
The solution is considered stable if either one or two below
conditions are fulfilled:
- Absolute value of %relative difference

%relative difference = (peak area at elapsed time - peak area at zero time) x 100%
peak area at zero time

- %RSD of repeated injections (during the elapsed time) does not exceed 5%

47

Deviation of chromatographic condition from
USP monograph

Example: HPLC column (according USP monograph
is C18, 5 µm, 4.6 x 250 mm

If you have C18, 10 µm, 3.9 x
300 mm

Can it be used ?

48

สมาคมเภสชั กรอุตสาหการ (ประเทศไทย) 119

<621> chromatography

49

L/dp = Length/particle size
Limit is within 75-150 %
USP column : 250/5 = 50

Column used : 300/10 = 30 X

(compared with USP is 60%)

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Q&A

THANK YOU
FOR YOUR ATTENTION

ขอบคุณค่ะ

51

สมาคมเภสชั กรอุตสาหการ (ประเทศไทย) 121

ภญ. แคทลียา นิรงั สรรค์
ตาํ แหน่งปัจจบุ นั
หวั หน้าแผนกพฒั นาวธิ วี เิ คราะห์ กองมาตรฐานการผลติ
ฝ่ายประกนั คณุ ภาพ องคก์ ารเภสชั กรรม
ประวตั ิการศึกษา
- เภสชั ศาสตรบ์ ณั ฑติ จฬุ าลงกรณ์มหาวทิ ยาลยั
- เภสชั ศาสตรม์ หาบณั ฑติ จุฬาลงกรณ์มหาวทิ ยาลยั
ประวตั ิการทาํ งาน
- เภสชั กรประจาํ แผนกมาตรฐานผลติ ภณั ฑ์ กองมาตรฐานผลติ ภณั ฑ์

ฝ่ายประกนั คุณภาพ องคก์ ารเภสชั กรรม (เมษายน 2541-กนั ยายน
2547)
- เภสชั กรประจาํ แผนกพฒั นาวธิ วี เิ คราะห์ กองมาตรฐานการผลติ ฝ่าย
ประกนั คุณภาพ องคก์ ารเภสชั กรรม (ตุลาคม 2548-กนั ยายน 2552)
- หวั หน้าแผนกพฒั นาวธิ วี เิ คราะห์ กองมาตรฐานการผลติ ฝ่ายประกนั
คุณภาพ องคก์ ารเภสชั กรรม (ตุลาคม 2552-ปัจจบุ นั )
- เป็นผตู้ รวจประเมนิ ทางวชิ าการ (Technical Assessor) ตามมาตรฐาน
มอก. 17043 (ISO/IEC 17043)ใหก้ บั สาํ นกั งานมาตรฐาน
ผลติ ภณั ฑอ์ ุตสาหกรรม (ตุลาคม 2558 - ปัจจบุ นั )
- สอนเรอ�ื ง analytical method validation คณะเภสชั ศาสตร์
มหาวทิ ยาลยั บรู พา (2561)

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Analytical Method Validation
PROTOCOL & REPORT

Katthaleeya Nirungsan
Nov. 12, 2019

1

Content of 4 applications

• Assay of finished product by HPLC
(example: protocol and report)

• Impurity test of finished product by HPLC
(example: protocol and report part LOQ)

• Dissolution test by UV-spectrophotometer
(example: protocol)

• Assay of Raw material by Titration
(example: protocol)

2

สมาคมเภสชั กรอุตสาหการ (ประเทศไทย) 123

How to start method validation /
verification

Step 1: Prepare Protocol
Step 2: Execute Experiments
Step 3: Prepare Report

3

Example 1

Method verification
Analysis of Haloperidol tablets 2 mg

according to USP 41
Assay by HPLC

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STEP 1: Prepare protocol

Method verification protocol

Name of organization
Protocol no.
Method verification
Analysis of Haloperidol tablets 2 mg

Prepared by Cover page
Reviewed by
Approved by

5

Table of contents

1. OBJECTIVE
2. SCOPE
3. RESPONSIBILITY
4. GENERAL DOCUMENTATION
5. WORKING FORMULA
6. EQUIPMENT
7. CHEMICALS AND REAGENTS
8. VALIDATION EXPERIMENTS
8.1. Preparation of mobile phase and diluting solution
8.2. Chromatographic condition
8.3. Test of specificity
8.4. Test of linearity and range
8.5. Test of accuracy
8.6. Test of precision
8.7. Test of system suitability
8.8. Test of stability of analyte solutions
9. REFERENCES
10. APPENDIX & ATTACHMENT
6

สมาคมเภสชั กรอุตสาหการ (ประเทศไทย) 125

APPENDIX 1 & ATTACHMENT

• APPENDIX 1 (Draft of test method)
• ATTACHMENT 1: Personnel Identification List
• ATTACHMENT 2: Work Instruction Verification
• ATTACHMENT 3: Test Instrument and Equipment
• ATTACHMENT 4: Deviation Report
• ATTACHMENT 5: General Data Worksheet
• ATTACHMENT 6: Data Sheet of Weighing

7

Appendix 1: Draft of test method
Analysis of Haloperidol tablets 2 mg

Chromatographic condition

Column: C18, 5µm, 3.9 x 250 mm or equivalent

Mobile phase: methanol : 0.05 M monobasic potassium phosphate buffer

(60 : 40) a pH of the mixture is 4.0

Flow: 1.0 mL/min

Injection volume: 15 µL

Wavelength of detection: UV @ 254 nm

Retention time:

Haloperidol ~ 5.5 min

Run time: ~ 10 min

8

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Appendix 1: Draft of test method
Analysis of Haloperidol tablets 2 mg (cont.)

Preparation of standard and sample solutions

Standard solution

1. Accurately weigh about 0.010 g of Haloperidol WS and transfer into a
100-mL volumetric flask.

2. Add 50 mL of mobile phase and sonicate for 15 minutes.
3. Adjust to volume with mobile phase.

Sample solution

1. Weigh and powder 20 tablets
2. Accurately weigh about 0.5082 g of sample powder (equivalent to 10 mg

of Haloperidol) and transfer into a 100-mL volumetric flask.
3. Add 50 mL of mobile phase and sonicate for 15 minutes.
4. Adjust to volume with mobile phase.
5. Filter through a 0.45-µm nylon membrane filter and discard the first portion

of the filtrate.

9

Appendix 1: Draft of test method
Analysis of Haloperidol tablets 2 mg (cont.)

Acceptable limit: 90.0-110.0 %LA of Haloperidol

System suitability test

Determine the following parameters from chromatograms of the standard
solution.
1. Column efficiency (N)

Acceptable limit: N  2000
2. Tailing factor (T)

Acceptable limit: T  2.0
3. System precision (Repeatability of injections)

Acceptable limit: %RSD (n = 5)  2.0

10

สมาคมเภสชั กรอุตสาหการ (ประเทศไทย) 127

Protocol part 8.3. Test of specificity

8.3.1. Preparation of solutions

Placebo powder:
Standard solution:
Sample solution:

Matrix solution

1. Accurately weigh and transfer about 0.4982 g of placebo powder into
a 100-mL volumetric flask.

2. Add 50 mL of mobile phase and sonicate for 15 minutes.
3. Adjust to volume with mobile phase.
4. Filter through a 0.45-µm nylon membrane filter and discard the first

portion of the filtrate.

Stability sample

Prepare sample, aging about 2-4 years in analogy to sample solution as
described in Appendix 1: Draft of test method.

11

8.3.2. Samples to be injected into the chromatographic system

- Mobile phase
- Standard solution (for system suitability test)
- Matrix solution
- Sample solution
- Stability sample

8.3.3. Evaluation

- Comparison of chromatograms obtained from diluting solution and matrix
solution with those obtained from standard and sample.
- Calculation of Haloperidol peak purity factor (PPF) in chromatograms
obtained from stability sample using software of chromatographic data
system.
Acceptable limit
- The diluting solution and matrix solution give no peak eluting at the same
time as Haloperidol.
- Based on stability sample, the peak purity factor of Haloperidol is not less
than 990.

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Protocol part 8.4. Test of linearity and range

8.4.1. Preparation of solutions

Standard solution at 50% (Std 50), 75% (Std 75), 100% (Std 100), 125% (Std
125) and 150% (Std150) of the formula amount

1. Accurately weigh Haloperidol WS as described in Table 1 and transfer
into a 100-mL volumetric flask.

2. Add 50 mL of mobile phase and sonicate for 15 minutes.

3. Adjust to volume with mobile phase.

Table 1 Weight of Haloperidol WS for linearity test

Standard Std 50 Std 75 Std 100 Std 125 Std 150
5
solutions 7.5 10 12.5 15
Weight of
Haloperidol
WS (mg)

13

8.4.2. Samples to be injected into the chromatographic system
- Mobile phase
- Std 100 (for system suitability test)
- Std 50
- Std 75
- Std 100
- Std 125
- Std 150

8.4.3. Evaluation
- Plot a linear regression between concentration and the response,
then determine the coefficient of determination (r2). Residual plot is
observed.
Acceptable limit
- For linear regression, r2 > 0.999
- For residual plot, residual values are randomly spaced around the
horizontal axis and no specific trend of data are observed.

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สมาคมเภสชั กรอุตสาหการ (ประเทศไทย) 129

Protocol part 8.5. Test of accuracy

8.5.1. Preparation of solutions

Placebo powder:
Simulated sample at 50% (Sim50), 100% (Sim100) and 150% (Sim150) of the
labeled amount (triplicate x 3 levels)
1. Accurately weigh Haloperidol WS and placebo powder as described in

Table 2 and transfer into a 100-mL volumetric flask.
2. Add 50 mL of mobile phase and sonicate for 15 minutes.
3. Adjust to volume with mobile phase.
4. Filter through a 0.45-µm nylon membrane filter and discarding the first

portion of filtrate.
Table 2 Weight of Haloperidol WS and placebo powder for accuracy test

Simulated sample Sim 50 Sim 100 Sim 150
Weight (mg) of Haloperidol WS about 5 10 15
Weight (g) of placebo powder about
15 0.4982 0.4982 0.4982

8.5.2. Samples to be injected into the chromatographic system

- Mobile phase
- Standard, replicate solution no. 1 (for system suitability test)
- Triplicate solutions of Sim50
- Triplicate solutions of Sim100
- Triplicate solutions of Sim150
- Standard, replicate solution no. 2

8.5.3. Evaluation

Determine the found amount (or found concentration) of Haloperidol
in simulated samples based on standard solutions, then calculate the
percentages of recovery from the below formula:

% recovery = Found concentration or amount x 100
Added concentration or amount

Acceptable limit
Mean recovery = 98.0-102.0%

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130 TIPA | Thai Industrial Pharmacist Association

Protocol part 8.6. Test of precision

8.6.1. Preparation of solutions

Prepare duplicate of standard solutions and six replicate of sample solutions
as described in Appendix 1: Draft of test method.

8.6.2. Samples to be injected into the chromatographic system

- Mobile phase
- Standard, replicate solution no.1 (for system suitability test)
- Six replicate solutions of sample
- Standard, replicate solution no.2

8.6.3. Evaluation

Determine Haloperidol in samples and then calculated %RSD of all results.
Acceptable limit

Repeatability (six samples)
%RSD < 2.0 %

17

Protocol part 8.7. Test of system suitability

Compile the system suitability test results from all the
experimental runs of the validation
as described in Appendix 1: Draft of test method.

18

สมาคมเภสชั กรอุตสาหการ (ประเทศไทย) 131

Protocol part 8.8. Test of stability of analyte solutions

8.8.1. Preparation of solutions

Standard and Sample solution
Insert the standard and sample solution in the sequence of injection every 6
hours for 24 hours

8.8.2. Sample to be injected into the chromatographic system

- Standard
- Sample

8.8.3. Acceptable limit

The solution is considered stable if either one or two below conditions are
fulfilled:
- Absolute value of %relative difference does not exceed 2%.
%relative difference = (Area at elapsed time – Area at zero time) x 100%

Area at zero time
- %RSD of repeated injections (during the elapsed time) does not exceed 2%.

19

STEP 3: Prepare Report

Final report

Name of organization
Protocol no.
Method verification
Analysis of Haloperidol tablets 2 mg

Prepared by Cover page
Reviewed by
Approved by

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132 TIPA | Thai Industrial Pharmacist Association

Table of contents

1. REFERENCES
2. RESULT SUMMARY
3. INTRODUCTION
4. WORKING FORMULA
5. EQUIPMENT
6. CHEMICALS AND REAGENTS
7. VALIDATION EXPERIMENTS
7.1. Preparation of mobile phase and diluting solution
7.2. Chromatographic condition
7.3. Test of specificity
7.4. Test of linearity and range
7.5. Test of accuracy
7.6. Test of precision
7.7. Test of system suitability
7.8. Test for stability of analyte solutions
8. GENERAL CONCLUSION
APPENDIX 1 (Draft of test method)
APPENDIX 2 (Illustration of chromatograms from validation experiments)

21

Result summary

Parameters Results & Conclusion
1. Specificity
The placebo gives no peak eluting at the same
time as Haloperidol. The peak purity is verified to
be a pure single peak.

Conclusion Passed. The test method is specific
regarding to the formulation excipients and the
potential degradation products.

2. System suitability test Column efficiency, N ≥ 13145 (limit: N ≥ 2000)
Tailing factor, T ≤ 1.48 (limit: T ≤ 2.0)
System precision, %RSD = 0.05 (limit: %RSD ≤ 2.0)

Conclusion Passed.

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สมาคมเภสชั กรอุตสาหการ (ประเทศไทย) 133

3. Linearity and range Result summary (cont.)

- regression equation: y = 22658.4439x + 5.3045
- r2 = 1.0000 (limit: r2 > 0.999)

Conclusion Passed. The analyte response is linearly
proportional to the concentration of Haloperidol
within a range of 0.052-0.149 mg/mL (50-150%LA).

4. Accuracy - mean recovery = 99.22 % (%RSD = 0.74, n = 9)
over the concentration range of 0.052-0.155
mg/mL (50-150%LA)

- The 95% confidence interval around the mean
recovery is 99.22 ± 0.57 or 98.65 to 99.79 %.

(limit: Mean recovery is within 98.0-102.0%)

Conclusion Passed.

23

Result summary (cont.)

5. Precision Repeatability:

- mean = 96.89 %LA, %RSD = 0.56 %
(n = 6)

(limit : %RSD < 2.0 %)

Conclusion Passed.

6. Stability of analyte The sample solutions should be
solutions analysed within 24 hours.

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Report part 7.3. Test of specificity
7.3.4. Results
The diluting solution and sample matrix give no peak
eluting at the same time as Haloperidol. Based on the
stability samples, the peak of Haloperidol was also
well resolved from possible degradation products and
the peak purity of Haloperidol is 999.8.
7.3.5. Conclusion
Test of specificity is passed.

25

Ex. Peak purity factor

26

สมาคมเภสชั กรอุตสาหการ (ประเทศไทย) 135

Report part 7.4. Test of linearity and range

7.4.4. Results

Table Results of linearity of Haloperidol

Standard Weight (mg) of Conc. Peak area Mean %RSD
Solutions Haloperidol WS (mg/mL) 1176.47 0.03
1176.26 1685.90 0.11
Std 50 5.198 0.0517 1176.68 2262.00 0.00
2801.79 0.06
Std 75 7.444 0.0740 1687.26 3378.32 0.04
1684.55
Std 100 10.022 0.0997
2261.98
Std 125 12.428 0.1236 2262.01

Std 150 14.952 0.1487 2800.52
27 2803.06

3379.31
3377.33

Fig. 1 Linearity of Haloperidol

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Report part 7.4. Test of linearity and range (cont.)

Table Residual plot

Std Conc. (mg/mL) Area observed Area Predicted a Residuals b

50 0.0517 1176.47 1176.75 -0.2745

75 0.0740 1685.90 1682.03 3.8743

100 0.0997 2262.00 2264.35 -2.3550

125 0.1236 2801.79 2805.89 -4.0984

150 0.1487 3378.32 3374.62 3.7059

a Area predict = (22658.4439)(conc.) + 5.3045
b Residuals = Area observed - Area Predicted

29

Fig. 2 Residual plot

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สมาคมเภสชั กรอุตสาหการ (ประเทศไทย) 137

7.4.5. Conclusion

- regression equation: y = 22658.4439x + 5.3045
- r2 = 1.0000 (limit: r2 > 0.999)
- Test of linearity is passed. The analyte response is
linearly proportional to the concentration of Haloperidol
within a range of 0.052-0.149 mg/mL (50-150%LA).
- For residual plot, residual values are randomly spaced
around the horizontal axis and no specific trend of data are
observed.

31

Report part 7.5. Test of accuracy

7.5.4. Results

Table Results of standard solution

Std Weight Conc. Area Conc./area Mean Grand
(mg) (mg/mL) mean

2261.98 0.0000441 0.0000441 0.0000441
2262.01 0.0000441

Std1 10.022 0.0997 2264.33 0.0000440 %RSD = %RSD =
2261.53 0.0000441 0.05 0.22

2262.27 0.0000441

2263.11 0.0000440

Std2 10.050 0.1000 2261.18 0.0000442 0.0000442
2259.24 0.0000442 %RSD =

2257.42 0.0000443 0.08

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Table Results of accuracy test

Sample Weight Area Average %RSD Conc. Conc.
(mg) 1172.58 0.32 addeda foundb %Recoveryc
sim 50-1 5.266 1175.24 1157.63 0.11 (mg/mL) (mg/mL)
sim 50-1 1169.92 1101.96 0.10 0.0524 0.0517 98.77
sim 50-2 5.168 1158.52 2300.45 0.17
sim 50-2 1156.74 2266.32 0.20 0.0514 0.0511 99.36
sim 50-3 4.972 1101.22 2286.32 0.01
sim 50-3 1102.70 0.0495 0.0486 98.31
sim 100-1 10.348 2297.74
sim 100-1 2303.17 0.1029 0.1015 98.61
sim 100-2 10.046 2269.55
sim 100-2 2263.08 0.0999 0.1000 100.07
sim 100-3 10.128 2286.15
sim 100-3 2286.50 0.1007 0.1009 100.13

33

Table Results of accuracy test (cont.)

Sample Weight Area Average %RSD Conc. Conc.
(mg) 3481.27 0.03 addeda foundb %Recoveryc
3480.45 3443.54 0.03 (mg/mL) (mg/mL)
sim 150-1 15.420 3482.09 3523.38 0.10
sim 150-1 15.416 3442.86 0.1534 0.1536 100.13
sim 150-2 15.868 3444.21
sim 150-2 3526.23 0.1533 0.1519 99.08
sim 150-3 3521.12
sim 150-3 0.1578 0.1555 98.50

Mean 99.22
SD 0.74
0.74
%RSD

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สมาคมเภสชั กรอุตสาหการ (ประเทศไทย) 139

Report part 7.5. Test of accuracy (cont.)

From Table of result of accuracy test

a) conc added = weight of Haloperidol WS (mg) x %purity/100 x dilution
factor,
%purity = 99.46, dilution factor = 1/100

b) conc found = Area of Simulated sample x Grand mean of (conc/area)
obtained from Std1&Std2

c) %recovery = conc found/ conc added x 100

The 95% confidence interval
= mean + (t(0.05,8) x SD)/n = 99.22 ± (2.306 x 0.74)/3
= 99.22 ± 0.57

35

Report part 7.5. Test of accuracy (cont.)

7.5.5. Conclusion
- Mean recovery = 99.22 % (%RSD = 0.74, n = 9)

over the concentration range of 0.052-0.155 mg/mL
(50-150%LA).
- The 95% confidence interval around the mean
recovery is 99.22 ± 0.57 or 98.65 to 99.79 %.
(limit: Mean recovery is within 98.0-102.0%).
- Test of accuracy is passed.

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Report part 7.6. Test of precision

7.6.4. Results

Table Results of standard solution of precision test

Standard Weight Conc. Area Conc./area Mean Grand
(mg) (mg/mL) 0.0000441 mean
2261.98 0.0000441
Std 1 10.022 0.0997 2262.01 0.0000441 %RSD= 0.0000441
2264.33 0.0000440 0.05 %RSD=
2261.53 0.0000441 0.02
2262.27 0.0000441 0.0000442
Std 2 10.050 0.1000 2263.11 0.0000440 %RSD=
37 2261.18 0.0000442 0.08
2259.24 0.0000442
2257.42 0.0000443

Table Results of precision test

Sample Weight (g) Area %LA
sample-1 0.5096 2195.11 96.90
sample-2 0.5088 2184.47 96.59
sample-3 0.5087 2205.71 97.57
sample-4 0.5088 2169.63 96.00
sample-5 0.5088 2197.11 97.24
sample-6 0.5090 2191.70 97.03

Mean 96.89

SD 0.55

%RSD 0.56

7.6.5. Conclusion

- Mean = 96.89 %LA, %RSD = 0.56% (n = 6)
(limit : %RSD < 2.0 %)

- Test of precision is passed.

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สมาคมเภสชั กรอุตสาหการ (ประเทศไทย) 141

Report part 7.8. Test of stability of
analyte solutions

Table Results of stability test of analyte solutions

time standard sample
(hrs.)

Area %rel. dif. Area %rel. dif.

0 2261.98 0.00 2182.27 0.00

6 2269.69 0.34 2183.21 0.04

12 2269.57 0.34 2184.16 0.09

18 2270.34 0.37 2192.23 0.46

24 2272.47 0.46 2194.97 0.58

%RSD 0.18 - 0.27 -

39

Example 2

Method verification
Analysis of Impurities in Ibuprofen suspension

100 mg/5 mL
according to USP 41
Impurity test (quantitative)

By HPLC

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Protocol: Table of content

8. VALIDATION EXPERIMENTS
8.1. Preparation of mobile phase and diluting solution
8.2. Chromatographic condition
8.3. Test of specificity
8.4. Test of limit of quantitation
8.5. Test of linearity and range
8.6. Test of accuracy
8.7. Test of precision
8.8. Test of system suitability
8.9. Test of stability of analyte solutions

41

Appendix 1: Draft of test method
Analysis of Impurities in Ibuprofen suspension

Chromatographic condition

Column: C8, 5m, 4.6 x 150 mm or equivalent

Mobile phase: Phosphate buffer pH 2.05 : Tetrahydrofuran

(56 : 44)

Flow: 1.0 mL/min

Injection volume: 25 µL

Wavelength of detection: UV @ 220 nm for the quantitation of unspecified

degradation products

UV @ 254 nm for Ibuprofen related compound J

and Ibuprofen related compound C

Based on: Area

Retention time: Sodium benzoate ~ 4.3 min

Ibuprofen related compound J ~ 6.6 min

Ibuprofen related compound C ~ 11.4 min

Ibuprofen ~ 13.6 min

Run time: 30 min

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สมาคมเภสชั กรอุตสาหการ (ประเทศไทย) 143

Appendix 1: Draft of test method
Analysis of Impurities in Ibuprofen suspension (cont.)

Stock solution of Ibuprofen related compound C (0.2 mg/mL of Ibuprofen
related compound C)

1. Accurately weigh about 20 mg of USP Ibuprofen related compound C RS and
transfer into a 100-mL volumetric flask.

2. Add 50 mL of diluting solution and sonicate for 15 minutes.
3. Adjust to volume with diluting solution.

Stock solution of Ibuprofen related compound J (0.2 mg/mL of Ibuprofen
related compound J)

1. Accurately weigh about 10 mg of USP Ibuprofen related compound J RS and
transfer into a 50-mL volumetric flask.

2. Add 25 mL of diluting solution and sonicate for 15 minutes.
3. Adjust to volume with diluting solution.

Stock solution of Benzoic acid (0.169 mg/mL of Benzoic acid)

1. Accurately weigh about 16.9 mg of Benzoic acid RS and transfer into a 100-mL
volumetric flask.

2. Add 50 mL of diluting solution and sonicate for 15 minutes.
3. Adjust to volume with diluting solution.

43

System suitability solution (0.004 mg/mL of Ibuprofen related compound C,
0.004 mg/mL of Ibuprofen related compound J, 0.0169 mg/mL of Benzoic
acid, and 0.4 mg/mL of Ibuprofen)

1. Accurately weigh about 20 mg of Ibuprofen RS and transfer into a 50-mL volumetric
flask.

2. Add 25 mL of diluting solution and sonicate for 15 minutes
3. Add 1.0 mL of Stock solution of Ibuprofen related compound C.
4. Add 1.0 mL of Stock solution of Ibuprofen related compound J.
5. Add 5.0 mL of Stock solution of Benzoic acid.
6. Adjust to volume with diluting solution.

Standard solution A (0.0002 mg/mL of Ibuprofen)

1. Accurately weigh about 20 mg of Ibuprofen RS and transfer into a 100-mL
volumetric flask.

2. Add 50 mL of diluting solution and sonicate for 15 minutes.
3. Adjust to volume with diluting solution.
4. Pipette 5.0 mL of the solution in (3) to a 100-mL volumetric flask.
5. Adjust to volume with diluting solution.
6. Pipette 1.0 mL of the solution in (5) to a 50-mL volumetric flask.
7. Adjust to volume with diluting solution.

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Standard solution B (0.004 mg/mL of Ibuprofen related compound C, 0.004
mg/mL of Ibuprofen related compound J)

1. Pipette 1.0 mL of Stock solution of Ibuprofen related compound C into a 50-mL
volumetric flask.

2. Add 1.0 mL of Stock solution of Ibuprofen related compound J.
3. Adjust to volume with diluting solution.

Sensitivity Solution (0.0002 mg/mL of Ibuprofen related compound C, 0.0002
mg/mL of Ibuprofen related compound J)

1. Pipette 5.0 mL of Standard solution B to a 100-mL volumetric flask.
2. Adjust to volume with diluting solution.

Sample Solution (0.4 mg/mL of Ibuprofen)

1. Tare a 50-mL volumetric flask.
2. Transfer 5 mL of Ibuprofen suspension (equivalent to 100 mg of Ibuprofen) into

a 50-mL volumetric flask and record the weight.
3. Add 25 mL of diluting solution and sonicate for 45 minutes.
4. Adjust to volume with diluting solution.
5. Pipette 10.0 mL of the solution in (4) to a 50-mL volumetric flask.
6. Adjust to volume with diluting solution.
7. Filter through a 0.45-µm nylon membrane filter and discard the first portion of
45 the filtrate.

Appendix 1: Draft of test method
Analysis of Impurities in Ibuprofen suspension (cont.)

Acceptable limit

- Disregard any peaks less than 0.05%

%w/w of Ibuprofen related compound C (UV @ 254 nm)

- Acceptable Limit: Not more than 0.25 %w/w.

%w/w of Ibuprofen related compound J (UV @ 254 nm)

- Acceptable Limit: Not more than 0.2 %w/w.

%w/w of Any unspecified degradation product (UV @ 220 nm)

- Acceptable Limit: Not more than 0.2 %w/w.

Total degradation products are sum of individual impurity.

- Acceptable Limit: Not more than 0.9 %w/w.

46

สมาคมเภสชั กรอุตสาหการ (ประเทศไทย) 145

Appendix 1: Draft of test method
Analysis of Impurities in Ibuprofen suspension (cont.)

For the quantitation of unspecified degradation product (UV @ 220 nm)

Determine the following parameters from chromatograms of standard solution A
(Ibuprofen peak).
- Column efficiency (N)

Acceptable limit: N  2000
- Tailing factor (T)

Acceptable limit: T < 2.0
- System precision (Repeatability of injections)

Acceptable limit: %RSD (n = 6)  10.0 %
- Resolution (R) between Benzoic acid and Ibuprofen related compound J,
determine from chromatograms of system suitability solution
- Resolution (R) between Ibuprofen related compound J and Ibuprofen related
compound C, determine from chromatograms of system suitability solution
- Resolution (R) between Ibuprofen related compound C and Ibuprofen,
determine from chromatograms of system suitability solution

Acceptable limit: R > 2.0

47

Appendix 1: Draft of test method
Analysis of Impurities in Ibuprofen suspension (cont.)

For the quantitation of unspecified degradation product (UV @ 254 nm)
Determine the following parameters from chromatograms of standard solution B
(Impurity J and Impurity C peak).
- Column efficiency (N)

Acceptable limit: N  2000
- Tailing factor (T)

Acceptable limit: T < 2.0
- System precision (Repeatability of injections)

Acceptable limit: %RSD (n = 6)  2.0 %
- Resolution (R) between Benzoic acid and Ibuprofen related compound J, determine
from chromatograms of system suitability solution
- Resolution (R) between Ibuprofen related compound J and Ibuprofen related
compound C, determine from chromatograms of system suitability solution
- Resolution (R) between Ibuprofen related compound C and Ibuprofen, determine
from chromatograms of system suitability solution

Acceptable limit: R > 2.0

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PREPARE PROTOCOL

49

Analysis of Impurities in Ibuprofen suspension

Protocol part 8.3. Test of specificity

UV@254nm for quantitation of Impurity J and Impurity C using standard B
(USP Ibuprofen related compound J and C RS)

UV@220nm for quantitation of unspecified impurity using standard A
(Ibuprofen RS)

8.3.2. Samples to be injected into the chromatographic system
UV@254nm and 220 nm
- Diluting solution
- System suitability solution (for calculation of resolution)
- Sensitivity solution (for calculation of signal to noise ratio, UV@254nm)
- Standard A, replicate solution (for system suitability test, UV@220nm)
- Standard B, replicate solution (for system suitability test, UV@254nm)
- Matrix solution
- Sample solution
50