การประชุมวิชาการเรื่อง Analytical Method Validation for FDA Compliance

สมาคมเภสชั กรอุตสาหการ (ประเทศไทย) 147

Analysis of Impurities in Ibuprofen suspension

Protocol part 8.3. Test of specificity (cont.)

8.3.3. Evaluation
UV@254 nm and UV@220 nm
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 sample matrix gives no peak eluting at the same time as
Ibuprofen, Sodium benzoate, Ibuprofen related compound J and
Ibuprofen related compound C.

51

Analysis of Impurities in Ibuprofen suspension

Protocol part 8.4. Test of limit of quantitation

8.4.2. Samples to be injected into the chromatographic system
- Diluting solution
- System suitability solution (for calculation of resolution)
- Standard solution A, no.1 (for system suitability test)
- Six replicate of Simulated sample_LOQ that give the signal to noise ratio

about 10:1
- Standard solution A, no.2

8.4.3. Evaluation (Disregard limit 0.05% should be tested)
- Calculate signal to noise ratio of Ibuprofen, Impurity J and Impurity C peak
in chromatograms obtained from sample solution at LOQ level using
software of chromatographic data system.
- Calculate %recovery.
- Calculate %RSD.
Acceptable limit
- Each of %recovery is within 80.0-120.0 %
- Signal to noise ratio > 10 and %RSD < 10.0 %

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Analysis of Impurities in Ibuprofen suspension

Protocol part 8.5. Test of linearity and range

8.5.2. Samples to be injected into the chromatographic system
- Diluting solution
- System suitability solution (for calculation of resolution)
- Sensitivity solution (for calculation of signal to noise ratio)
Ibuprofen + Impurity J + Impurity C solution at 0.05-0.3 %w/w in relative to Ibuprofen
in sample solution
- Std 0.25 % (for system suitability test)
- Std 0.05
- Std 0.1
- Std 0.15
- Std 0.2
- Std 0.25
- Std 0.3
8.5.3. Evaluation (UV@254 and 220 nm)
- Plot a linear regression between concentration and the response, then determine
the coefficient of determination (r2). Residual plot is observed.
Acceptable limit
- For regression line, r2 > 0.999
- For residual plot, residual values are randomly spaced around the horizontal axis
and no specific trend of data are observed.
53

Analysis of Impurities in Ibuprofen suspension

Protocol part 8.6. Test of accuracy test

8.6.2. Samples to be injected into the chromatographic system

- Diluting solution
- System suitability solution (for calculation of resolution)
- Sensitivity solution (for calculation of signal to noise ratio)
- Standard solution A, replicate solution no.1 (for system suitability

test)
- Standard solution B, replicate solution no.1 (for system suitability

test)
- Triplicate of simulated samples at 0.1 %w/w
- Triplicate of simulated samples at 0.2 %w/w
- Triplicate of simulated samples at 0.3 %w/w
- Standard solution A, replicate solution no.2
- Standard solution B, replicate solution no.2

54

สมาคมเภสชั กรอุตสาหการ (ประเทศไทย) 149

Analysis of Impurities in Ibuprofen suspension
Protocol part 8.6. Test of accuracy test (cont.)

8.6.3. Evaluation UV@254nm and 220nm

Determine the found amount (or found concentration) of
Ibuprofen, Ibuprofen related compound J and Ibuprofen related
compound C 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

Each of % recovery is within 90.0 - 110.0 %.

55

Analysis of Impurities in Ibuprofen suspension

Protocol part 8.7. Test of precision
8.7.2. Samples to be injected into the chromatographic system

- Diluting solution
- System suitability solution (for calculation of resolution)
- Sensitivity solution (for calculation of signal to noise ratio)
- Standard solution A, replicate solution no.1 (for system suitability test)
- Standard solution B, replicate solution no.1 (for system suitability test)
- Six replicate of samples
- Standard solution A, replicate solution no.1
- Standard solution B, replicate solution no.2

8.7.3. Evaluation (UV@254nm and 220nm)

Determine Ibuprofen related impurities in samples and then calculated SD of
all results.
Acceptable limit

Repeatability (six samples)
SD < 0.02 %w/w

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PREPARE REPORT

57

Example: report part LOQ

7.5.4. Results
Table Results of limit of quantitation for Ibuprofen (UV @ 220 nm)

Solution Weight (mg) Conc. (mg/mL) a Area Signal to

noise ratio

1 0.1855 22.6

2 0.1849 13.6

3 10 0.0002 0.1922 12.0

4 0.1887 12.0

5 0.2009 9.6

6 0.1921 13.7

Mean 0.1907

a Conc. = weight (mg) x %purity/100 x %diRluStDion factor 3.08
%purity of Ibuprofen RS = 99.80 %, dilution factor = 1/50 x 5/100 x 1/50

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สมาคมเภสชั กรอุตสาหการ (ประเทศไทย) 151

Table Results of limit of quantitation for Ibuprofen related compound J
(UV @ 254 nm)

Solution Weight (mg) Conc. (mg/mL) a Area Signal to noise ratio

1 7.1602 4134

2 7.2655 4433

3 0.0002 7.2740 4654
10 7.3000 6457

4

5 7.3293 4302

6 7.3483 3787

Mean 7.2796

%RSD 0.91
a Conc. = weight (mg) x %purity/100 x dilution factor

%purity of USP Ibuprofen related compound J RS = 100 %,

dilution factor = 1/50 x 1/50 x 5/100

59

Table Results of limit of quantitation for Ibuprofen related compound C
(UV @ 254 nm)

Solution Weight (mg) Conc. (mg/mL) a Area Signal to noise ratio

1 8.8488 3154

2 8.8454 3335

3 0.0002 8.8468 3496
20 8.8403 4834

4

5 8.8494 3210

6 9.8536 2829

Mean 8.8474

%RSD 0.05

a Conc. = weight (mg) x %purity/100 x dilution factor

%purity of USP Ibuprofen related compound C RS = 100 %,

dilution factor = 1/100 x 1/50 x 5/100

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0.0002 mg/mL / 0.4 mg/mL of Ibuprofen in
sample solution x 100 = 0.05 %

7.5.5. Conclusion

- For unspecified impurities,
limit of quantitation is 0.0002 mg/mL (0.05 %w/w).
- For Ibuprofen related compound J,
limit of quantitation is 0.0002 mg/mL (0.05 %w/w).
- For Ibuprofen related compound C,
limit of quantitation is 0.0002 mg/mL (0.05 %w/w).

61

Example 3

Method verification
Dissolution test of Chloroquine phosphate

tablets 250 mg
according to USP 41

Dissolution Test by UV-spectrophotometer

62

สมาคมเภสชั กรอุตสาหการ (ประเทศไทย) 153

Protocol: Table of content

8. VALIDATION EXPERIMENTS

8.1. Test of specificity
8.2. Test of linearity and range
8.3. Test of accuracy
8.4. Test of precision
8.5. Test of stability of analyte solutions

63

Appendix 1: Draft of test method
Dissolution test of Chloroquine phosphate tablets 250 mg

Dissolution condition

Medium : Water; 900 mL

Apparatus 2 : 100 rpm

Time : 45 minutes

Limit : Not less than 75 % (Q) of the labeled amount of
Chloroquine phosphate is dissolved.

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Appendix 1: Draft of test method
Dissolution test of Chloroquine phosphate tablets 250 mg

Standard solution

1. Accurately weigh about 28 mg of Chloroquine Phosphate WS and transfer
into a 100-mL volumetric flask.

2. Add 50 mL of medium, sonicate for 15 minutes.
3. Adjust to volume with medium.
4. Pipette 2.0 mL of the solution in (3) to a 25-mL volumetric flask.
5. Adjust to volume with medium.

Sample solution

1. Pipette 2.0 mL of the solution under test to a 25-mL volumetric flask.
2. Adjust to volume with medium.

Measurement

Measure the absorbance of the resulting solutions by a spectrophotometer
at the maximum at about 343 nm, using medium as a blank.

65

PREPARE PROTOCOL

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สมาคมเภสชั กรอุตสาหการ (ประเทศไทย) 155

Protocol part 8.3. Test of specificity

8.3.2. Samples to be measured absorbance and spectra

- Standard solution
- Matrix solution
- Sample solution

8.3.3. Evaluation

The specificity/ selectivity is determined based on
- Comparison of Spectra obtained from the matrix solution with those

obtained from standard and sample solution.
- For identification, the spectra of standard solution and sample solution

are compared.
Acceptable limit
- The matrix solution give the absorbance, not more than 2.0%, compared

with standard solution at the wavelength of measurement.
- For identification, the spectra of standard solution and sample solution

are identical.

67

Protocol part 8.4. Test of linearity and range

8.4.2. Samples to be measured the absorbance

- Std 20
- 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 regression line, 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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Protocol part 8.5. Test of accuracy

8.5.2. Samples to be measured the absorbance

- Standard, replicate solution no. 1
- Triplicate solutions of Sim 20
- Triplicate solutions of Sim 50
- Triplicate solutions of Sim 100
- Triplicate solutions of Sim 150
- Standard, replicate solution no.2

8.5.3. Evaluation

Determine the found amount (or found concentration) of Chloroquine
Phosphate 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 = 95.0-105.0%

69

Protocol part 8.6. Test of precision
8.6.1. Preparation of solutions

Standard solution

Prepare duplicate of standard solutions as described in Appendix 1: Draft of
test method.

Sample solution

- Put only one tablet into 900 mL of medium and perform the dissolution
test as described in Appendix 1: Draft of test method. After 45 minutes,
withdraw 6 samples of dissolution medium, each of 10 mL. The
withdrawal of 6 samples should be completed within 2 minutes
to minimize the possible variation due to different dissolution time.

- Pipette 2.0 mL of the solution under test to a 25-mL volumetric flask.
- Adjust to volume with medium.

70

สมาคมเภสชั กรอุตสาหการ (ประเทศไทย) 157

Protocol part 8.6. Test of precision (cont.)

8.6.2. Samples to be measured the absorbance
- Standard, replicate solution no.1
- Six replicate solutions of sample
- Standard, replicate solution no.2
8.6.3. 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)

71

Example 4

Method verification

Analysis of Methyl salicylate
according to BP 2016

Assay of raw material
by titration

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Protocol: Table of content

1. OBJECTIVE
2. SCOPE
3. RESPONSIBILITY
4. GENERAL DOCUMENTATION
5. CHEMICALS AND REAGENTS
6. VALIDATION EXPERIMENTS
6.1. Test of linearity and range
6.2. Test of accuracy
6.3. Test of precision
7. REFERENCES
8. APPENDIX & ATTACHMENT

73

Appendix 1: Draft of test method
Analysis of Methyl salicylate

Procedure

1. Weigh accurately about 0.500 g of sample and transfer into a
250-mL Stopper Erlenmeyer flask.

2. Add 25 ml of Ethanol (96%) and swirl until dissolve.
3. Add 0.05 mL of phenol red solution and neutralize with 0.1 N

sodium hydroxide.
4. Add 50.0 mL of 0.1 N sodium hydroxide VS.
5. Heat under a reflux condenser on a water-bath for 30 minutes

and then cool it to room temperature.
6. Titrate with 0.1 N hydrochloric acid VS until the solution is

changed from red color to colorless.
7. Calculate the volume of 0.1 N Sodium Hydroxide used in

saponification. Carry out a blank titration.

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สมาคมเภสชั กรอุตสาหการ (ประเทศไทย) 159

PREPARE PROTOCOL

75

Protocol part 6.1. Test of linearity and range

6.1.1. Procedure

Proceed the analyses of five sample solutions, spanning over the
concentration range of 80-120% level (Sam80, Sam90, Sam100, Sam110 and
Sam120) as per draft test method (see Appendix 1) with “Methyl Salicylate”.
See Table 1 for the amount of Methyl Salicylate.

Table 1 Weight (mg) of Methyl Salicylate for the linearity test

Standard solutions Sam80 Sam90 Sam100 Sam110 Sam120
Weight (mg) of Methyl 400 450 500 550 600

Salicylate about

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Protocol part 6.1. Test of linearity and range (cont.)

6.1.2. Evaluation

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

Acceptable limit
- For regression line: r2 > 0.999
- For residual plot: residual values are randomly spaced around the

horizontal axis and no specific trend of data are observed.

77

Protocol part 6.2. Test of accuracy

6.2.1. Procedure

Proceed the analyses of 3 x 3 samples (3 concentration levels, 3 replicates
per level), spanning over the concentration range of 80-120 %level (Sam80,
Sam100 and Sam120), as per draft test method (see Appendix 1) with
“Methyl Salicylate”. See Table 2 for the amount of Methyl Salicylate.

Table 2 Weight (mg) of Methyl Salicylate for accuracy test

Samples Sam80 Sam100 Sam120
Weight (mg) of Methyl 400 500 600

Salicylate about

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สมาคมเภสชั กรอุตสาหการ (ประเทศไทย) 161

Protocol part 6.2. Test of accuracy (cont.)

6.2.2. Evaluation

Determine the found amount (or found concentration) of
Methyl Salicylate in 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%

79

Protocol part 6.3. Test of precision

6.3.1. Procedure
Repeatability:
Proceed the analyses of 6 replicate samples as per draft test
method (see Appendix 1).

6.3.2. 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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Q&A

THANK YOU
FOR YOUR ATTENTION

ขอบคุณค่ะ

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ภญ. นพวรรณ องั กรู ศนั สนีย
การศึกษา
• เภสชั ศาสตรบ์ ณั ฑติ จฬุ าลงกรณ์มหาวทิ ยาลยั
• วศิ วกรรมศาสตรม์ หาบณั ฑติ สาขาวศิ วอตุ สาหการและ

การผลติ มหาวทิ ยาลยั เทคโนโยลพี ระจอมเกลา้ ธนบรุ ี
ตาํ แหน่งปัจจบุ นั : รกั ษาการในตาํ แหน่งผอู้ าํ นวยการกองบรหิ ารระบบคณุ ภาพ การ

ประกนั คณุ ภาพโรงงานผลติ ยารงั สติ 1 องคก์ ารเภสชั กรรม
ประวตั ิการทาํ งาน
• เภสชั กรประจาํ แผนกมาตรฐานวตั ถุดบิ 1 กองมาตรฐานวตั ถดุ บิ ฝ่าย
ประกนั คณุ ภาพ องคก์ ารเภสชั กรรม (เมษายน 2546 – กนั ยายน 2550)
• เภสชั กรประจาํ โครงการ Fast Track ฝ่ายประกนั คณุ ภาพ องคก์ ารเภสชั
กรรม (ตุลาคม 2550 – กรกฎาคม 2553) รบั ผดิ ชอบการควบคมุ คณุ ภาพวตั ถดุ บิ
และยาสาํ เรจ็ รปู
• รกั ษาการในตาํ แหน่งผอู้ าํ นวยการกองการควบคมุ คณุ ภาพ การประกนั
คณุ ภาพโรงงานผลติ ยารงั สติ 1 องคก์ ารเภสชั กรรม (สงิ หาคม 2553 – 15
พฤษภาคม 2562) รบั ผดิ ชอบงานควบคมุ คณุ ภาพทกุ กระบวนการ
• รกั ษาการในตาํ แหน่งผอู้ าํ นวยการกองบรหิ ารระบบคณุ ภาพ การประกนั
คณุ ภาพโรงงานผลติ ยารงั สติ 1 องคก์ ารเภสชั กรรม (16 พฤษภาคม 2562–
ปัจจบุ นั ) รบั ผดิ ชอบงานบรหิ ารระบบคณุ ภาพทกุ กระบวนการ
• เป็นผปู้ ระเมนิ ทางวชิ าการ (Technical Assessor) ตามมาตรฐาน มอก.
17043 (ISO/IEC 17043) ใหก้ บั สาํ นกั งานมาตรฐานผลติ ภณั ฑอ์ ตุ สาหกรรม
(ตลุ าคม 2560 – ปัจจบุ นั )
• เป็นผตู้ รวจประเมนิ GMP Self - inspection ของโรงงานผลติ ยารงั สติ 1
(กมุ ภาพนั ธ์ 2561 - ปัจจุบนั )
• เป็นผตู้ รวจประเมนิ Supplier audit วตั ถุดบิ ยาและบรรจุภณั ฑ์

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Differences between
Method Verification

and
Method Validation

Noppawan Angkulsansanee
12 November 2019

1

Significance of
Validation/Verification

• ICH guidelines (Q2A & Q2B) >> primarily addressed
to pharmaceutical industry indicating which
validation data need to be provided in an application
file.

• These data should demonstrate that the proposed
testing and acceptance criteria are sufficiently under
control to guarantee reproducible quality of the
products at release and adequate control during
shelf-life (stability).

2

สมาคมเภสชั กรอุตสาหการ (ประเทศไทย) 165

Consideration on
Validation/Verification

Correctness Time

Workload Budget

3

To avoid failure

• Clearly define what has to be done
• Validation VS Verification
• Good planning
• Complete protocol

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Differences between
Validation & Verification

• New method is developed • Compendial / Official /
• Compendial / Official / Existing method unchanged

Existing method • Compendial method
modification adjustment within
• Extension of scope (i.e., allowance
additional matrices not
evaluated, changes in the • Method transfer
intended use) • Demonstration that a
• Significant changes in
instrument parameters, previously validated method
reagents, time, temperature, can meet the analytical
etc. requirements and
• A change in technology/ suitability in YOUR lab
instrumentation • Fit for use
• Validation has already been
5 performed and the method
is well established

VALIDATION

verification



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สมาคมเภสชั กรอุตสาหการ (ประเทศไทย) 167

VALIDATION � �
� �
verification � �
� �
� �

7

Available Guidelines (USP)

• USP General Chapters <1225>
Validation of Compendial Procedures

• USP General Chapters <1226>
Verification of Compendial Procedures

• USP General Chapters <621>
Chromatography

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USP General Chapters <1226>

Verification of Compendial Procedures
=1=

The intent of this general information chapter is to provide
general information on the verification of compendial

procedures that are being performed for the first time to
yield acceptable results utilizing the personnel, equipment,

and reagents available.

9

USP General Chapters <1226>

Verification of Compendial Procedures
=2=

This chapter is not intended for retroactive application to
already successfully established laboratory procedures. The
chapter Validation of Compendial Procedures <1225> provides
general information on characteristics that should be considered

for various test categories and on the documentation that
should accompany analytical procedures submitted for inclusion

in USP-NF.

10

สมาคมเภสชั กรอุตสาหการ (ประเทศไทย) 169

USP General Chapters <1226>

Verification of Compendial Procedures
=3=

Verification consists of assessing selected analytical
performance characteristics, such as those that are described in
chapter <1225>, to generate appropriate, relevant data rather

than repeating the validation process.

Users of compendial analytical procedures are not required to
validate these procedures when first used in their laboratories,

but documented evidence of suitability should be established
under actual conditions of use.

11

USP General Chapters <1226>

Verification of Compendial Procedures
=4=

In the United States, this requirement is established in 21
CFR 211.194(a)(2) of

the current Good Manufacturing Practice regulations,
which states that the “suitability of all testing methods
used shall be verified under actual conditions of use.”

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USP General Chapters <1226>

Verification of Compendial Procedures
=exception=

Verification of microbiological procedures is not covered in
this chapter because it is covered in USP General Test
Chapters
• Antimicrobial Effectiveness Testing <51>
• Microbiological Examination of Nonsterile Products:

Microbial Enumeration Tests <61>
• Microbiological Examination of Nonsterile Products: Test

for Specified Microorganisms <62>
• Sterility Tests <71>
• Validation of Microbial Recovery from Pharmacopeial

Articles <1227>

13

USP General Chapters <1226>

Verification Requirements (1)

Although complete revalidation

of a compendial method is not required

to verify the suitability of a procedure under actual
actual conditions of use, some of the analytical

performance characteristics listed in chapter <1225>,
may be used for the verification process.

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USP General Chapters <1226>

Verification Requirements (2)

• Assessment the suitability of use for drug substance/drug
product matrix (drug substance’s synthetic route, method of
manufacture for the drug product) >> Specificity

• Assessment of the effect of the matrix on the recovery of
impurities and drug substances from the drug product matrix
>> Accuracy (Recovery)

• Assessment of suitability of chromatographic conditions and
column, the appropriateness of detector signal response >>
System suitability

15

USP General Chapters <1226>

Verification Requirements (3)

• Impurity test
– Limit of detection
– Limit of quantitation
– Precision

• Demonstrate the stability of standard and sample
preparations throughout the duration of the procedure

16

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172 TIPA | Thai Industrial Pharmacist Association

https://www.gmp-compliance.org/gmp-news/revised-usp-chapter-1226-verification-of-compendial-procedures

17

USP General Chapters <1226>

Verification exclusion

• Verification is not required for basic compendial test
procedures

– Loss on drying
– Residue on ignition
– Various wet chemical procedures (such as Acid value)
– pH measurement

• Intermediate precision is not required

18

สมาคมเภสชั กรอุตสาหการ (ประเทศไทย) 173

USP General Chapters <621> Chromatography
Adjustment Allowances (1)

If adjustments of operating conditions are necessary in
order to meet system suitability requirements, each of
the items in the following lists is the maximum variation
that can be considered, unless otherwise directed in the

monograph; these changes may require additional
verification data.

To verify the suitability of the method under the new
conditions, assess the relevant analytical performance

characteristics potentially affected by the change.

19

USP General Chapters <621> Chromatography
Adjustment Allowances (2)

20

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USP General Chapters <621> Chromatography
Adjustment Allowances (3)

21

USP General Chapters <621> Chromatography
Adjustment Allowances (4)

22

สมาคมเภสชั กรอุตสาหการ (ประเทศไทย) 175

USP General Chapters <621> Chromatography
Adjustment Allowances (5)

23

USP General Chapters <621> Chromatography
Adjustment Allowances (6)

24

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176 TIPA | Thai Industrial Pharmacist Association

USP General Chapters <621> Chromatography
Adjustment Allowances (7)

25

Available Guidelines

• Validation of Analytical Procedures
PA/PH/OMCL (13) 82 2R, OMCL
Network/EDQM of the Council of
Europe

26

สมาคมเภสชั กรอุตสาหการ (ประเทศไทย) 177

OMCL/EDQM

Method Types to be Transferred (1)

1.1 Pharmacopoeial (compendial) method
1.2 Method of a manufacturer
1.3 Non compendial published method
1.4 Method of a first manufacturer to be used for a

product of a 2nd manufacturer
1.5 Method for an active substance to be used for a

medicinal product
1.6 Methods validated to reduce, refine or replace

animal use

27

OMCL/EDQM

Method Types to be Transferred (2)

1.1 Pharmacopoeial (compendial) method
1.1.1 Active substance

• The analytical procedures described in a monograph of a
pharmacopoiea are considered to be validated. In this case it
should be made sure that all reference materials needed are
available and the required system suitability tests are
performed.

• Nevertheless, it should also be considered that a
pharmacopoeial monograph is only considered validated
(related substances test) when it is applicable to the control of
the listed impurities (specific source material, see PhEur).

28

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178 TIPA | Thai Industrial Pharmacist Association

OMCL/EDQM

Method Types to be Transferred (3)

1.1 Pharmacopoeial (compendial) method
1.1.1 Active substance
• Identification:

– no formal validation required;
• Testing for Impurities:

– no formal validation required;
• Assay:

– no formal validation required.

“No formal validation required” indicates that the respective validation
characteristics have already been considered by others. However a
verification of suitability under conditions of use (=method transfer
check) has to be done.

29

OMCL/EDQM

Method Types to be Transferred (4)

1.1 Pharmacopoeial (compendial) method
1.1.2 Medicinal product
The pharmacopoeial monograph for a specific dosage form is a
good basis for the analysis; however as in many cases there is no
indication about the exact composition of the product (qualitative
and quantitative composition of the excipients), it must at least
be made sure that these do not interfere in the analysis of the
active substance, unless addressed in the monograph.

30

สมาคมเภสชั กรอุตสาหการ (ประเทศไทย) 179

OMCL/EDQM

Method Types to be Transferred (5)

1.1 Pharmacopoeial (compendial) method
1.1.2 Medicinal product
• Identification:

– no formal validation required;

• Testing for Impurities:

– specificity: no interference from excipients;
– reporting threshold (at least the quantitation limit)

• Assay:

– specificity,
– accuracy: mainly recovery, minimum 1 determination.,
– precision (repeatability): around the target test concentration (minimum 2 independent

determinations)
– linearity at three measuring points in the range around the target value.

31

THANK YOU

32

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180 TIPA | Thai Industrial Pharmacist Association

ภญ. พรรณรชั ต์ พรหมเพญ็

การศึกษา
• เภสชั ศาสตรบ์ ณั ฑติ มหาวทิ ยาลยั สงขลานครนิ ทร์
• วศิ วกรรมศาสตรม์ หาบณั ฑติ สาขาวศิ วอุตสาหการ

และการผลติ มหาวทิ ยาลยั เทคโนโยลพี ระจอมเกลา้ ธนบุรี
ตาํ แหน่งปัจจบุ นั หวั หน้าแผนกงานชวี วเิ คราะห์ กองการควบคุม
คุณภาพ การประกนั คณุ ภาพโรงงานผลติ ยารงั สติ 1 องคก์ ารเภสชั กรรม
ประวตั ิการทาํ งาน
• เภสชั กรประจาํ แผนกตรวจสอบคุณภาพทางจลุ ชวี วทิ ยา 2 กอง
ตรวจสอบคุณภาพทางจลุ ชวี วทิ ยา ฝ่ายประกนั คณุ ภาพ องคก์ ารเภสชั กรรม
(ตุลาคม 2552 – ธนั วาคม 2553)
• หวั หน้าแผนกงานชวี วเิ คราะห์ กองการควบคมุ คุณภาพ การประกนั
คณุ ภาพโรงงานผลติ ยารงั สติ 1 องคก์ ารเภสชั กรรม (ธนั วาคม 2553 –
ปัจจบุ นั ) รบั ผดิ ชอบงานตรวจสอบคณุ ภาพทางจลุ ชวี วทิ ยาของวตั ถุดบิ ยา
สาํ เรจ็ รปู ระบบน�ํา ระบบอากาศและตวั อยา่ งอ�นื ๆทก�ี าํ หนดใหม้ กี ารตรวจสอบ
คุณภาพทางจลุ ชวี วทิ ยา
• เป็นผตู้ รวจประเมนิ GMP Self - inspection ของโรงงานผลติ ยารงั สติ
1 (กุมภาพนั ธ์ 2561 - ปัจจบุ นั )

สมาคมเภสชั กรอุตสาหการ (ประเทศไทย) 181

SUITABILITY TEST FOR
MICROBIOLOGICAL TEST

PHANNARAT PHROMPHEN

THE GOVERNMENT PHARMACEUTICAL ORGANIZATION

1

SCOPE

Microbiological examination of non-sterile products
• Microbial enumeration tests

– Membrane Filtration method
– Plate-Count methods

• Pour plate
• Spread plate
• Test for specified microorganisms

2

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182 TIPA | Thai Industrial Pharmacist Association

REFERENCE

• USP 42-NF37 <61> MICROBIOLOGICAL EXAMINATION OF NON-
STERILE PRODUCTS: MICROBIAL ENUMERATION TESTS

• USP 42-NF37 <62> MICROBIOLOGICAL EXAMINATION OF NON-
STERILE PRODUCTS: TESTS FOR SPECIFIED MICROORGANISMS

• USP 42-NF37 <1111> MICROBIOLOGICAL EXAMINATION OF
NONSTERILE PRODUCTS: ACCEPTANCE CRITERIA FOR
PHARMACEUTICAL PREPARATIONS AND SUBSTANCES FOR
PHARMACEUTICAL USE

3

SUITABILITY TEST

Negative control

Positive control

Product Positive control

Testing of Product

4

สมาคมเภสชั กรอุตสาหการ (ประเทศไทย) 183

ENUMERATION METHODS

1. MEMBRANE FILTRATION METHOD
2. PLATE COUNT METHODS

• POUR PLATE
• SURFACE-SPREAD
3. MOST PROBABLE NUMBER (MPN) METHOD

THE CHOICE OF METHOD BASED ON

1. The Nature of product

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THE CHOICE OF METHOD BASED ON (CONT.)

2. Required limit of micro-organisms

Method Detection Required Limit (CFU/g or ml)

(Dilution 1 in 10) limit ≤ 2x103 ≤ 2x102 ≤ 2x10

MEMBRANE 1 √ √ √

FILTRATION

(10 ml)

POUR PLATE 10 √ √ X

(1 ml)

SURFACE-SPREAD 1oo √ X X

(0.1 ml)

7

THE CHOICE OF METHOD BASED ON (CONT.)

The suitability of the method chosen must be established

The ability of the test to detect microorganisms in the
presence of product to be tested

8

สมาคมเภสชั กรอุตสาหการ (ประเทศไทย) 185

TEST STRAINS FOR GROWTH PROMOTION AND
SUITABILITY OF THE COUNTING METHOD

TEST STRAINS

Staphylococcus aureus ATCC 6538, NCIMB 9518, CIP 4.83, or
NBRC 13276

Pseudomonas aeruginosa ATCC 9027, NCIMB 8626, CIP 82.118,
or NBRC 13275

Bacillus subtilis ATCC 6633, NCIMB 8054, CIP 52.62,
or NBRC 3134

Candida albicans ATCC 10231, NCPF 3179, IP 48.72, or
NBRC 1594

Aspergillus brasiliensis ATCC 16404, IMI 149007, IP 1431.83,
or NBRC 9455

REF: USP 42-NF37 <61> MICROBIOLOGICAL EXAMINATION OF NON-STERILE PRODUCTS: MICROBIAL ENUMERATION TESTS

PREPARATION OF TEST STRAINS

• Not more than 5 passages(From ATCC) : Seed-lot systems.

• Use standardized stable suspensions.

• Use BSP or PB to make test suspensions.

• Use within 2 hr or 24 hr if stored at 2-8 ˚C.

• An alternative for A. brasiliensis, B. subtilis stable spore
suspension is used and maintained at 2-8 ˚C for a validated
period of time.

Original
inoculum

ATCC BSP,PB/+
Polysorbate 80

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186 TIPA | Thai Industrial Pharmacist Association

0.1 0.1 ml 0.1 ml 0.1 ml 0.1 ml

ml

>300 CFU 80 CFU 6 CFU

11

Table 1 PREPARATION AND USED OF TEST MICRO-ORGANISMS

Preparation Growth Promotion/ Positive Suitability of Counting
of Test control Method in the Presence of
Strain
Microorganism Product
TSA or TSB
Staphylococcus 30 –35 ˚C Total Aerobic Total Yeasts and Total Aerobic Total Yeasts
aureus 18–24 hours Microbial Molds Count Microbial and Molds
Count Count
Pseudomonas Count
aeruginosa
TSA or TSB TSA or TSB
Bacillus ≤ 100 cfu ≤100 cfu
subtilis 30 –35 ˚C 30 –35 ˚C
3 days
3 days

สมาคมเภสชั กรอุตสาหการ (ประเทศไทย) 187

Table 1 PREPARATION AND USED OF TEST MICRO-ORGANISMS (cont.)

Preparation Growth Promotion/ Positive Suitability of Counting Method
Microorganism of Test control in the Presence of Product

Strain Total Aerobic Total Yeasts Total Aerobic Total Yeasts
Microbial and Molds Microbial and Molds
Candida SDA or SDB Count Count
albicans 20 –25 ˚C Count Count
2–3 days TSA TSA SDA
≤ 100 cfu SDA ≤ 100 cfu ≤ 100 cfu
30 –35 ˚C ≤ 100 cfu 30 –35 ˚C 20 –25 ˚C
20 –25 ˚C 5 days
5 days 5 days
5 days MPN: not SDA
applicable ≤ 100 cfu
Aspergillus SDA or PDA TSA SDA 20 –25 ˚C
brasiliensis 20 –25˚C ≤ 100 cfu ≤ 100 cfu TSA
30 –35 ˚C 20 –25 ˚C ≤ 100 cfu 5 days
5–7 days, or 30 –35 ˚C
until good 5 days 5 days
sporulation is 5 days
achieved MPN: not
applicable

INOCULATION AND DILUTION

Add test organism
– To the lowest possible dilution factor of prepared sample
and to control
– Volume test organism ≤ 1% of the volume of diluted
product to obtain an inoculum ≤ 100 cfu (but NMT
required limit)

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GROWTH PROMOTION OF THE MEDIA

• Inoculate portions of test strains described in Table 1.
• Using a separate portion
• For solid media:

– Standardized inoculum: Not differ by a factor greater than 2
– Freshly prepared inoculum: comparable to previously tested
• For liquid media:
– Comparable to previously tested
• Test each batch of medium

PREPARATION OF THE SAMPLE

Sample Prepared 1 : 10 dilution

Sample 10 g 10 ml (Filtration)
= x CFU/g or ml

90 ml 1 ml (Pour plate)
= x CFU*10 /g or ml
BSP/PB/TSB
0.1 ml (Spread plate)
= x CFU*100 /g or ml

สมาคมเภสชั กรอุตสาหการ (ประเทศไทย) 189

PREPARATION OF THE SAMPLE

• WATER-SOLUBLE PRODUCTS:
– 10 g or 10 ml dissolve in BSP/PB/TSB (1 in 10)

• NON-FATTY PRODUCTS INSOLUBLE IN WATER:
– 10 g or 10 ml suspend in (BSP/PB/TSB+ surface active agent)
(1 in 10)

• FATTY PRODUCTS:
– 10 g or 10 ml dissolve in isoproply myristate
– 10 g or 10 ml Mix with minimum polysorbate 80 or non-
inhibitory surface active agent then add sufficient of pre –
warmed BSP/PB/TSB (1 in 10)

PREPARATION OF THE SAMPLE (CONT.)

• FLUIDS OR SOLIDS IN AEROSOL FORM:
– 10 Containers (use total contents/a defined number of
meter doses) aseptically transfer into membrane filter
apparatus or sterile container

• TRANSDERMAL PATCHS:
– 10 patches : cover adhesive surface sheet with a sterile
porous material and transfer to diluent containing
inactivatators (polysorbate 80 and/or lecithin), Shake
vigoriously at least 30 mins.

SURFACE-ACTIVE SUBSTANCES : ABSENCE TOXICITY FOR MICRO –
ORGANISMS AND COMPATIBILITY WITH INACTIVATORS

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RECOVERY OF MICRO-ORGANISM IN THE
PRESENCE OF PRODUCT

Sample

Inoculum Diluent

Product
positive
control

RECOVERY OF MICRO-ORGANISM IN THE
PRESENCE OF PRODUCT

1. Membrane filtration
– Pore size ≤ 0.45 µm
– Transfer the sample prepared (representing 1 g or 1 patch)
– Rinse the membrane filter with an appropriate volume of diluent
– For each micro-organisms use one membrane filter

2. Plate-count methods: at least in duplicate
2.1 Pour plate method
• Add 1 ml of the sample prepared and 15-20 ml of TSA or SDA
2.2 Surface-spread method
• Add 15-20 ml of TSA or SDA: Solidify, dry
• Spread NLT 0.1 ml of the sample prepared

สมาคมเภสชั กรอุตสาหการ (ประเทศไทย) 191

RECOVERY OF MICRO-ORGANISM IN THE
PRESENCE OF PRODUCT(CONT.)

TAMC Media Incubation Temp. Incubation Periods
TYMC TSA 30-35˚C NMT 3 days

SDA 20-25˚C NMT 5 days

RESULTS AND INTERPRETATION

Not differing by a factor greater than 2 from the value of the
control defined in Inoculation and Dilution in the absence of
product must be obtained.

TESTING OF PRODUCTS

Sample Diluent

Testing of
products

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TESTING OF PRODUCTS

• AMOUNT USED FOR THE TEST
– Unless otherwise prescribed, use 10 g or 10 ml
– Fluids or solids in aerosol form, use 10 containers
– Transdermal patches, use 10 patches
– Active substances may be reduced
• The amount per dosage unit ≤ 1 mg – NLT the amount
present in 10 dosage unit
• Batch size is extremely small (<1000 g or 1000 ml) Shall
be 1 % of batch

EXAMINATION OF THE PRODUCT

1. Membrane filtration
2. Plate count method

2.1 Pour plate method, ≤ 250
2.2 Surface spread method, ≤ 50

TAMC Media Incubation Temp. Incubation Periods
TSA 30-35˚C NMT 3 days

TYMC SDA 20-25˚C NMT 5 days

สมาคมเภสชั กรอุตสาหการ (ประเทศไทย) 193

INTERPRETATION AND THE RESULTS

• TAMC : The number of CFU found on TSA
• TYMC : The number of CFU found on SDA
arithmetic mean, calculate CFU per g or ml of the product

Maximum acceptance count
101 cfu: maximum acceptable count = 20
102 cfu: maximum acceptable count = 200
103 cfu: maximum acceptable count = 2000

NEGATIVE CONTROL

Diluent • Use the chosen diluent in place
of the test preparation.
Negative
control • Verify testing and also testing
the products.

• There must be no growth of the
micro- organisms.

• A failed negative control

requires an investigation.

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POSITIVE CONTROL

Inoculum Diluent

Positive
control

POSITIVE CONTROL

• Use the chosen diluent in place of the test preparation, add test
organisms

• Verify testing and also testing the products.
• There must be growth of the micro- organisms.

สมาคมเภสชั กรอุตสาหการ (ประเทศไทย) 195

INTERPRETATION

Positive Control Acceptance Limit
Positive Product NMT 100 cfu

Control Not differing by a factor greater than 2 from
the value of the control
Negative Control
Must be no growth of the micro- organisms.

Testing of Products Follow products specification

29

Positive Product Negative Testing
Control Positive Control Product

Control

Test Strains Acceptance Conclusion
Limit
Result

Acceptance
Limit
Result

Acceptance
Limit
Result

Acceptance
Limit
Result

Staphylococcus aureus 10 -100 CFU  Suitable
Not detected  Unsuitable
Total Pseudomonas aeruginosa <2,000 CFU/g
Aerobic  Suitable
Microbial  Unsuitable

Bacillus subtilis  Suitable
Count  Unsuitable
(TAMC)
 Suitable
Candida albicans  Unsuitable

Aspergillus brasiliensis  Suitable
 Unsuitable

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Positive Product Negative Testing
Control Positive Control Product

Control

Test Strains Acceptance Conclusion
Limit
Result

Acceptance
Limit
Result

Acceptance
Limit
Result

Acceptance
Limit
Result

Total 10 -100 CFU  Suitable
Candida albicans Not detected  Unsuitable
<200 CFU/g
Combine  Suitable
 Unsuitable
Yeast and

Mould
Count Aspergillus brasiliensis

(TYMC)

INTERPRETATION

Suitable The test of product may be carried out
without further modification

Unsuitable Modify the conditions in order to

eliminate the antimicrobial activity,
and repeat the method suitability test