Analytical methods

By: Kemal H. (Bpharm, MSC)

March 2021
2. Specifications
 Outline of the Presentation

1. Objectives and expected outcomes

2. Specifications

3. The Rational for Analytical methods development

4. Analytical Methods

5. Analytical method validation/verification

 1. Objectives and expected
outcomes
 At the end of the presentation participant shall be able to
understand
 analytical methods,

 analytical method validation/verification

 At the end of the presentation participant should also be
able to
 Properly validate Analytical Methods
 review and identify deficiency/ies with regard to
analytical method.
 Specifications
What is Specification?
• A specification is defined as a list of tests, references to analytical
procedures, and with appropriate acceptance criteria, which are
numerical limits, ranges, or other criteria for the tests described.

• It establishes the set of criteria to which a drug substance or drug

product should conform to be considered acceptable for its
intended use
Ser. No Test Type Specifications Limit Analytical Procedures
1 Description SOP---
2 Identification USP----
3 Assay USP----
 Specification...contd

• Specifications are critical quality standards that are proposed

and justified by the manufacturer and approved by regulatory
authorities as conditions of approval, for MA purpose

• Specifications are chosen to confirm the quality of the drug

substance and drug product.
 Specification...contd
Setting Specification
• When a specification is first proposed, It should be justified:
• The justification should refer:
 to relevant Pharmaceutical development data,
 Pharmacopoeial standards,
 test data for drug substances and drug products used in
toxicology and clinical studies,
 results from accelerated and long term stability studies,
 A reasonable range of expected analytical and manufacturing
variability should be considered.
 It is important to consider all of this information.
Therefore, properly Developed and Validated Analytical Method
is required to test a drug substance or drug Product against the
Specification Set
3. The Rational For Analytical method
development

8
 Analytical Method Development……ctd

• Analytical method development plays important role in the

discovery, development, and manufacturing of
pharmaceuticals.
• The number of drugs introduced into the market is increasing
every year. These drugs may be either new entities or partial
structural modification of the existing one.
• Therefore, it becomes necessary to develop newer analytical
methods for such drugs.
• The official analytical methods are results of these processes
 are used by the manufactures and quality control
laboratories to ensure the identity, purity, potency, and
performance of drug products.
 Basic reason for new method development :-

• The drug or drug combinations may not be official in any

pharmacopoeias,

• A proper analytical procedure for the drugs may not be

available in the literature due to patent rights,

• Analytical methods may not be available for the drug in the

form of a formulation.
 Basic reason……ctd

• Analytical methods for the quantitation of the drug in

biological fluids may not be available,

• Analytical methods for a drug in combination with other

drugs may not be available,

• The existing analytical procedures may require expensive

reagents and solvents.

• It may also involve extraction and separation procedures and

these may not be reliable.
 Basic reason…….ctd
• Existing methods may have error, artifact , or contamination
prone, or they may be unreliable (have poor accuracy or
precision).

• Existing methods may not provide adequate sensitivity or

analyte selectivity in samples of interest. Example, method
used for cleaning procedures.
 Basic reason…….ctd

• Newer instrumentation and techniques may have evolved that

provide opportunities for improved methods, including:

 improved analyte identification or detection limits,

 greater accuracy or precision, or better return on

investment
5.Analytical Methods
 Analytical Methods
• Instrumental or non instrumental methods that are used
for:

– Determination of potency/assay, which can directly

relate to a known dose or formulation
strength/concentration

– Determination of impurities, which can relate to the

safety profile of the drug

– Evaluation of degradation products/stability profile of

pharmaceutical products
Analytical Methods cont…

– Evaluation of key drug characteristics such as crystal

form, drug release, drug uniformity, properties which
can compromise bioavailability

– Evaluation of key manufacturing parameters/Process,

to ensure that production of drug substances/ drug
products is consistent.
 Types of Analytical Methods

A. Regulatory Analytical Methods

• Are analytical Procedures found in pharmacopeias of
different countries (Examples: USP, BP, EP, JP and so on)

• A regulatory analytical methods is the analytical

procedure used to evaluate a defined characteristic of
the drug substance or drug product which are accepted
by the authority
 B. Alternative Analytical Procedure
• An alternative analytical procedure is an analytical
procedure proposed by the applicant for use instead of
the regulatory analytical procedure.

• A validated alternative analytical procedure should be

used only if it is shown to perform equal to or better
than the regulatory analytical procedure.
 Alternative Analytical Procedure cont..

• If an alternative analytical procedure is used, the

manufacture should have/provide:

 A rationale for its inclusion and identify its use (e.g.,

release, stability testing),

 Validation data,

 Comparative data to the regulatory analytical

procedure.
 C. (None) Stability Indicative Method
• A stability-indicating assay is a validated quantitative analytical
procedure that can detect the changes with time in the
properties of the drug substance and drug product.

• A stability-indicating assay accurately measures the active

ingredient(s), without interference from:

 degradation products,

 process impurities,

 excipients,

 or other potential impurities.

 Stability Indicative Method cont…

• If a manufacturer uses a non-stability-indicating analytical

procedure for release testing,

 then an analytical procedure capable of qualitatively and

quantitatively monitoring the impurities, including
degradation products, should complement it.

• Assay analytical procedures for stability studies should be

stability-indicating
 6. Analytical Method
Validation/Verification
 Method Validation
Method validation can be defined as:

 Establishing documented evidence, which provides a high

degree of assurance that a specific activity will consistently
produce a desired result or product meeting its
predetermined specifications and quality characteristics.

 It is the process of demonstrating that analytical procedures

are suitable for their intended use.

23
 Content and format of analytical procedures

• Any analytical procedure:

should be described in sufficient detail to allow a

competent analyst to reproduce the necessary conditions
and obtain results comparable to the applicant.

• .

24
 Content and format of analytical procedures cont…
• The following is a list of information that should typically
be included in a description of an analytical procedure.
 Principle
 Sampling and Preparation of Samples
 Equipment and Equipment Parameters
 Reagents
 Preparation of Standards
 Procedure
 System Suitability Testing
 Calculations
 Reporting of Results
 Qualitative and quantitative limits
25
 Compendial methods-Verification
All methods are appropriately validated as specified under
Validation of Compendial Procedures (1225).
A validated method may be used to test a new formulation only
after confirming that the new formulation does not interfere with
the accuracy, linearity, or precision of the method (i.e.
Verification).
It may not be assumed that a validated method could correctly
measure the active ingredient in a formulation that is different
from the one used in establishing the original validity of the
method
Therefore, assay method for APIs may not be required?!
 Compendial methods….ctd
Compendial methods should be verified for its proper
performance especially for dosage forms.

The parameters to be verified for formulations depends on:

 Nature of Matrices,

 Intended applications,

 USP general chapter <1226> VERIFICATION OF

COMPENDIAL PROCEDURES
 Compendial methods…..ctd
When a manufacturer claims/uses a compendial method, there

should be no change in:

The type of column i.e the stationary phases

Detector wavelength

Components in Mobile phase

System suitability testing and criteria

 Compendial methods………..ctd
ratio of components in mobile phase

flow rate

column temp, dimension of column, particle size

Adjustments to: Eg USP general chapter <621>.

Full validation is required for specified impurities that are not

included in the monograph

 specificity, linearity, accuracy, repeatability, intermediate

precision, LOD/LOQ

What if there is change from these Conditions?

 Non-compendial methods-Validation
Full validation is required for these type of methods

Performance characteristics are discrete attributes, obtained

by actual execution of an analytical method using controlled
samples are used for method validation.

Demonstrate the suitability and proper use of the method for

a specific application

Specificity Precision

Accuracy LOD/LOQ
Robustness
Linearity
 Pre-validation Works

• Equipment: Suitable for expected task?

• Reference Materials and reagents: Suitability
• Personnel’s: Is trained and qualified
• Analytical Method: Is procedure finalized?
• Validation Protocol: Management / Approval
 Validation Protocols
• The validation Protocol document should at. least
contains:
– Description of the analytical method and its intended
use (purpose)
– Lists of instruments/apparatus
– Personnel competency
– List of applicable performance characteristics
– Appropriate acceptance criteria
– Review and approval date/signatures

32
 Table: Test parameters with acceptance criteria

Ser. Parameter Acceptance Criteria

No.
1 Linearity
 Correlation coefficient > 0.99
 Y-intercept (relative to the 100% calculated value) ±2.0%
 Visual Linear
2 Accuracy Percent Recovery
 Recovery of each over the whole range 96.0–104.0%
 Mean recovery per concentration 98.0–102.0%
 RSD (n=9) ≤2.0%
3 Precision RSD
 System Repeatability ≤1.0%
 Method Repeatability ≤2.0%
 Intermediate Precision Pooled RSD ≤3.0% + Absolute mean
assay difference ≤ 3.0%
4 Specificity
 using DAD Peak Purity Index ≥ 0.999
5 Robustness Not to affect the system suitability
criteria
6 LOD and LOQ To report the calculated values
7 Sample Solution Stability RPD ≤ 2.0%
8 Filtration Study RPD ≤ 2.0%
 Performance Characteristics cont…
Validation Identification Testing for Impurities Assay, Dissolution
Characteristics (measurement
Quantitative Limit
only),
Content/Potency
Accuracy - + - +

Precision -

Repeatability - + - +

Interm. precision - + - +

Specificity + + + +

Detection limit - - + -

Quantitation limit - + - -

Linearity - + - +

Range - + - +

Robustness - + - +
34
 1.Specificity
• The ability to assess unequivocally the analyte in the
presence of components that may be expected to be
present, such as impurities, degradation products, and
matrix components

• Lack of specificity of an individual analytical procedure

may be compensated by other supporting analytical
procedure(s)

• Specificity is concentration-dependent and should be

determined at the low end of the calibration range.
35
 Specificity Cont…

Specificity Applied
• Identification
– to ensure the identity of the analyte
• Purity Test
– accurate statement of the content of impurities of an
analyte (related substances, residual solvents, etc.)
• Assay
– an exact result which allows an accurate statement on the
content of potency of the analyte in a sample

36
 Specificity Cont..

• Some analytical procedures are not sufficiently specific

for the intended purpose

– Assay by titration

– Assay of enantiomer by HPLC method

– Identification by UV absorbance

• A combination of two or more analytical procedures is

recommended to achieve sufficient specificity under
such condition
37
 Specificity Cont..

• When the criteria are not met, this indicates that the
method is not sufficiently developed

• Poor specificity can impact accuracy, precision and

linearity

38
 Samples for Specificity Testing

• Blank solution to show no interference with any HPLC system

artifact peak.

• Placebo to demonstrate the lack of interference from excipients.

• Drug substance to show that all other substances are resolved

from the drug substance.

• Authentic samples of critical related substances to show that all

known related substances are resolved from each other
 How Specificity?
• The specificity of a test method is determined by comparing test
results from an analysis of samples containing impurities,
degradation products, or placebo ingredients with those
obtained from an analysis of samples without impurities,
degradation products, or placebo ingredients.

• For the purpose of a stability indicating assay method,

degradation peaks need to be resolved from the drug substance.
However, they do not need to be resolved from each other.

• Specificity can best be demonstrated by the resolution of two

chromographic peaks that elute close to each other. In the
potency assay, one of the peaks would be the analyte peak.
Example,
 Approaches
1. When authentic samples of related substance are available.

• Analyze stressed drug product, placebo, drug substance, stressed

placebo, and solutions spiked with authentic samples of related
substances.

• The HPLC chromatograms are used to show the resolution

among related substances, drug substance, and other potential
interferences.
 Approaches……
2. When authentic samples of impurities are not available.

A stressed drug product can be analyzed to show separation

of the most significant related substances

The peak homogeneity of the stressed sample should be

investigated by PDA or mass spectrometry

Typically, a stressed sample of about 10 % to 20%

degradation is used. A 10 to 20% degraded sample is used
because it has a sufficiently high concentration level of
critical related substance.
 2. Linearity
• Linearity is the ability of analytical procedure to produce test
results which are proportional to the concentration (amount)
of analyte in samples within a given concentration range.

• Justifies the use of a single point standard to quantitative

across the range

• Performed directly on the drug substance (dilution of

standard stock solution)

• Minimum of five concentration is recommended from 80% to

120 % of the analytical concentration
44
 Linearity Cont…

Validation Principles Experimental Acceptance criteria

Characteristics condition
 Prepare standard • A plot of the data
stock solution should be reported
(different weighing
Direct relation of of different
Linearity response with substance)
concentration  The slope of the
 Prepare serial line , intercept
dilution (5-6) from and correlation
stock solution coefficient
(80%-120%) of the (>/=0.99)
expected conc.

45
 Linearity, practical example
Calibration Levels Concentration (micgr/mL) Response (Peak Area) Response factor
1 5 301568 60313.6
2 10 567413 56741.3
3 20 1126639 56332.0
4 40 2248305 56207.6
5 60 3421719 57028.7
6 80 4544013 56800.2
7 100 5702036 57020.4
8 120 6743868 56198.9
9 140 7885679 56326.3
10 160 8846651 55291.6
11 180 10002721 55570.7
12 200 10915064 54575.3
13 240 13286836 55361.8
14 280 15360306 54858.2
46
15 320 19269779 56024.1
16 360 21170824 58807.8
1.8
 Linearity, practical example (Excluding
the reds)

Chart Title
18000000

16000000 y = 54836x + 92588

R² = 0.9997
14000000

12000000
Axis Title

10000000

Series1
8000000
Linear (Series1)
6000000

4000000

2000000

0
0 50 100 150 200 250 300
Axis Title 47
 Linearity, practical example (Including
the reds)

Chart Title
25000000

y = 57901x - 165414
R² = 0.9965

20000000

15000000
Axis Title

Series1
10000000 Linear (Series1)

5000000

0
0 50 100 150 200 250 300 350 400
Axis Title 48
 3. Accuracy

• The accuracy of an analytical method is defined as the degree

to which the determined value of analyte in a sample
corresponds to the true value.

• ICH recommends a minimum of 9 determinations over a

minimum of 3 concentrations covering the stated
quantitation range (3 test at 3 conc.) (Ex. For Assay: 80, 100
and 120%)

49
 Accuracy…….
• For a drug substance, the common method of determining
accuracy is to apply the analytical procedure to the drug
substance and to quantitate it against a reference standard of
known purity.
• For the drug product, accuracy is usually determined by
application of the analytical procedure to synthetic mixtures of
the drug product components or placebo dosage form to
which known quantities of drug substance of known purity
have been added
• Experimental Considerations.
 Typically, known amounts of related substances and the drug
substance in placebo are spiked to prepare an accuracy sample
of known concentration of related substance and drug product
 Accuracy Cont…

Acceptance criteria

% Active/impurity content Acceptable mean recovery

≥ 10 98 –102%
1 -10 90 –110%
0.1 – 1 80 – 120%
< 0.1 75 – 125%

51
 Table Recovery of D1 and D2 three concentration levels 50 %, 100 % and 150 % of
the analytical concentration in triplicate preparations at each level

Levels Average peak area spiked Average peak area of Amount add (mg) Amount recovered (mg) Percent
sample standard recovered recovered
D1 D2 D1 D2 D1 D2 D1 D2 D1 D2
50 % - 1 3585970.0 7113609.0 1333420.3 2353278.9 2.0015 2.0027 2.0067 2.0349 100.3 101.6
50 % - 2 3615666.0 7162589.0 1351445.1 2377594.0 2.0015 2.0027 2.0344 2.0552 101.6 102.6
50 % - 3 3642196.5 7170121.5 1354633.1 2335796.7 2.0015 2.0027 2.0393 2.0203 101.9 100.9
100 % - 1 6252466.0 12056729.5 4011587.6 7321064.3 6.0044 6.0080 6.1114 6.1811 101.8 102.9
100 % - 2 6207407.5 12050038.5 3919844.1 7215713.7 6.0044 6.0080 5.9708 6.0932 99.4 101.4
100 % - 3 6207264.0 11942704.0 3954714.3 7182373.9 6.0044 6.0080 6.0243 6.0653 100.3 101.0
150 % - 1 9349575.0 17734261.0 7132039.0 13047925.7 11.0080 11.0147 10.8910 10.9608 99.0 99.5
150 % - 2 9509192.5 17917261.0 7256642.8 13156930.9 11.0080 11.0147 11.0850 11.0518 100.7 100.3
150 % - 3 9404765.5 17781983.0 7163887.1 13046317.8 11.0080 11.0147 10.9428 10.9595 99.4 99.5
Average 100.5 101.1
SD 1.1 1.2
RSD 1.1 1.2
Minimum 99.0 99.5
Maximum 101.9 102.9
 4. Precision
• The precision of an analytical procedure expresses the
closeness of agreement (degree of scatter) between a series
of measurements obtained from multiple sampling of the
same homogeneous sample under the prescribed conditions.
• Determination
– Assay individual samples of a homogeneous preparation
– Calculate Standard Deviation or Relative Standard
Deviation
• Precision is expressed as RSD
53
 Precision Cont…
Level of precision
A. Repeatability
– Agreement within a short period of the same analyst and
instrumentation
– System and method precision
– n=6, 100% level
B. Intermediate precision
– Agreement in results intra-laboratory but from different
days, analysts and equipment (as appropriate)
C. Reproducibility
– Agreement in results between laboratories (as in a
collaboration study)…..(If C is done, B is not required) 54
 Precision Cont…

Acceptance criteria (Repeatability)

Component measured in sample Precision (in RSD)

>10.0% ≤ 2%
1.0 up to 10.0% ≤ 5%
0.1 up to 1.0% ≤ 10%
< 0.1% ≤ 20%

55
:
Table: System precision data of performed at the analytical
concentration (n=6) of the standard solutions

Ser. No. Peak area of Peak area of

D1 D2
1 6497180 11697872
2 6468308 11682527
3 6530359 11756621
4 6484146 11739370
5 6475588 11756981
6 6494368 11741426
Average 6491658.2 11729132.8
SD 21888.4 31421.0
RSD 0.3 0.3
Table : Method Precision data of method validation performed at the
analytical concentration on multiple preparation (n=6) of the same
homogenous sample

Replication Average peak area

D1 D2
Preparation 1 7559824.6 16089877
Preparation 2 7542248.4 15949232
Preparation 3 7693793.2 16438709
Preparation 4 7454508.2 15896539
Preparation 5 7371967.2 15913313
Preparation 6 7525189.9 16243777
Average 7524588.6 16088574.5
SD 120818.2 226268.0
RSD 1.6 1.4
 Precision Cont…

Intermediate Precision
• FDA recommends a minimum of 2 different days with
different analysts
• Acceptance criteria
– Perform F-test
– results between analysts should not be statistically
different
– Typically about 2x repeatability (2 x RSD), or pooled RSD
and Absolute mean difference

58
Table Intermediate Precision data of method validation performed by multiple analyses
(n = 6) of the same homogenous sample by two analysts on two different days, using
independently prepared reagents and sample preparations, and on two different
equipment setups

Ser No. Percent Recovered (n=6) Percent Recovered (n=6)

Day I & Analyst I Day II and Analyst II
D1 D2 D1 D2
1 115.5 96.4 115.9 98.2
2 115.2 95.6 115.7 98.6
3 117.6 98.5 116.0 98.4
4 113.9 95.3 116.7 98.9
5 112.6 95.4 115.2 98.5
6 115.0 97.3 115.7 97.7
Average 115.0 96.4 115.8 98.4
RSD 1.4 1.3 0.4 0.4
Pooled RSD of n=12 D1 1.1
D2 1.4
Absolute mean assay D1 0.8
difference D2 2.0
 Precision Cont…

Reproducibility

• Reproducibility with the same samples using different

laboratories, analysts, days, reagent, and environmental
conditions

• Analysis of an homogeneous sample in different laboratories,

by different analysts, using the specified parameters

• Mainly applied for method Transfer

60
 5. Range

• The interval between the upper and lower quantitation levels of

analyte (including these levels) demonstrated by suitable
precision, accuracy, and linearity

Example 50 µg/mL – 150 µg/mL

61
 Range Cont…

• The following minimum specified ranges should be

considered

• Assay of drug substance or finished drug product

– from 80% to 120% of the test concentration

• Content Uniformity and dissolution

– minimum of 70% to 130% of the test concentration,

unless otherwise justified

62
 6. Limit of Detection / Quantitation

• Characteristic for low level impurity assays

 LOD

• The lowest amount of any analyte which can be

detected but not necessarily quantitated

 LOQ

• The lowest amount of analyte that can be determined

with acceptable precision and accuracy
63
 Limit of Detection

• Instrumental/Chromatographic

– Can be made at a value at which the signal to noise ratio

(S/N) is 3:1

• Non-instrumental Methods

 Analysis of samples with known concentrations of analyte

to establish the minimum concentration at which the
analyte can be detected

 Examples: TLC, color comparison

64
 Limit of Quantitation

• The lowest amount of analyte in a sample that can be

determined with acceptable precision and accuracy.

– Can be made at a value at which the signal to noise ratio

(S/N) is 10:1

65
Detection Limit/ Quantitation Limit

66
 7. System Suitability

System suitability tests are an integral part of

chromatographic methods.
These tests are used to verify that the chromatographic
system is adequate for the intended analysis.
Prior to injecting a standard solution in creating the standard
plot, it is essential to ensure that the system is performing
adequately for its intended purpose.
This function is fulfilled by the use of a solution of the system
suitability.
The tests are based on the concept that the equipment
electronics, analytical operations, and samples analyzed
constitute an integral system that can be evaluated as such.
 System Suitability

The following notes should be given due consideration when

evaluating a system suitability:

 System suitability is a measure of the performance of a given

system on a given day within a particular sample analysis set.

 The main objective of system suitability is to recognize whether

or not system operation is adequate given such variability as
chromatographic columns, column aging, mobile-phase
variations, and variations in instrumentation.
 System Suitability
 System suitability is part of method validation. Experience gained
during method development will give insights to help determine
the system suitability requirements of the final method.
 Example, the hydrolysis of acetylsalicylic acid to salicylic acid in
acidic media. Separation of this degradation peak from the
analyte could be one criterion for the system suitability of an
acetylsalicylic acid assay.
 A system suitability test should be performed in full each time a
system is used for an assay.
 If the system is in continuous use for the same analysis over an
extended period, system suitability should be reevaluated at
appropriate intervals (bracketing).
 System Suitability
Factors that may affect chromatographic behavior include the
following
Composition, ionic strength, temperature, and apparent pH
of the mobile phase (external Factors)
Flow rate, column dimensions, column temperature, and
pressure (internal Factor)
Stationary phase characteristics, including type of
chromatographic support (particle-based or monolithic),
particle or macropore size, porosity, and specific surface area
Reverse-phase and other surface modification of the
stationary phases, the extent of chemical modification (as
expressed by end-capping, carbon loading, etc.)
 System Suitability
• System suitability should be based on criteria and parameters
collected as a group that will be able to define the performance
of the system.
• Some of the common parameters used includes:
precision of repetitive injections (usually five or six)
Resolution (R),
Tailing factor (T )
Number of theoretical plates (N), and
Capacity factor (k).
 System suitability testing (SST)
1. Precision:
– Assay: RSD ≤1% (API) or ≤ 2% (FPP), n ≥ 5
– Impurities: in general, RSD ≤ 5% at the limit level, up to
10% or higher at LOQ, n ≥ 6
2. Resolution (R): >2
 System suitability testing (SST)
3. Tailing factor/peak asymmetry: (≤ 2)
 System suitability testing (SST)

4. Number of theoretical plates (N): column efficiency ≥ 2000

 System suitability testing (SST)

A SST should contain:

• For Assay:
Precision + one or more other parameter
• For impurity test:
Resolution + precision + one or more other parameter
 Example System suitability of an analytical
method
The resolution (R) between the two analytical peak pairs is not less
than 8.0; theoretical plate number is not less than 6000 for the
two analytes and tailing factor should be between 0.9 and 1.5 for
the two analytes.

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 7. Robustness

• The ability of the method to remain unaffected by small

but deliberate variations in method parameters
• Provides an indication of the reliability of the method
during normal usage.
• Evaluated by varying method parameters and
determining the effect (if any) on the results of the
method.
• If the results are susceptible to parameter variations,
these parameters should be adequately controlled and a
precautionary statement included in the method

77

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 Robustness Cont…

• Two approaches

A. Uni-variable (One factor at a time)

– Systematically varying each parameter sequentially

– Typically, each parameter may be varied by 5 – 10% above

and below the value set in the method

B. Multi-variable

– Use of the statistical design

78

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 Table: Internal variables (column temperature, mobile phase flow rate,
detector wavelength and injection volume) of robustness testing of the
proposed method.

Peak Column Temperature in Mobile Phase flow rate Detector Wavelength in Injection
Characteri 0C in mL per minute (nm) Volume in µL
stics (25) (1.0) (220) (20)

20 30 0.9 1.1 217 223 10 30

R 16.5 13.6 15.5 14.8 15.1 15.1 16.5 13.0

T* 1.3/1.1 1.2/1.1 1.3/1.1 1.2/1.1 1.2/1.1 1.3/1.1 1.2/1.1 1.2/1.1

N** 6421/655 6618/666 6646/704 6421/689 6282/6644 6326/6658 7458/8008 6050/6104

7 3 4 0

And ** for D1/D2 respectively

 Table: External variables (pH, Ion pair concentration, organic con and
triethylamine concentration of the mobile phase) of robustness testing
of the proposed method.

Peak pH of the mobile Ion pair con. of the Organic con. of the Triethylamine con.
Characte phase Mobile Phase Mobile Phase of the Mobile Phase
ristics (7.0) (30) (51) (5)
6.8 7.2 27 mM 33 mM 50 % 52 % 4 mL 6 mL

R 16.9 11.8 13.7 13.4 16.8 13.8 13.0 16.1

T*/** 1.3/1.1 1.2/1.1 1.2/1.1 1.2/1.1 1.2/1.1 1.2/1.1 1.2/1.1 1.2/1.1

N*/** 6315/7263 6568/6261 6511/6539 6471/6508 6663/7249 6390/6555 6447/6392 6479/7069

And ** for D1/D2 respectively

 Revalidation

 Verification or revalidation should be performed when relevant, for

example:
– Changes in the synthesis of the drug substance

– Changes in the composition of the drug product

– Changes in the analytical procedure

– when major pieces of equipment instruments change to another

– when analytical methods are transferred.

 The verification or degree of revalidation depend on the nature of
the change(s).
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THANK YOU!

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