Understanding the Analytical method validation in a Practical Perspective

Professor, Department of Pharmaceutical Analysis,
Santhiram College of Pharmacy, Nandyal, Kurnool Dist. AP. India.
drbmdishaq@gmail.com
Sat Dec 12,th 2020
http://srcpnandyal.com/
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Santhiram College of Pharmacy, Nandyal, Kurnool Dist. AP. 518501.
INTRODUCITON
 Analytical method
development, validation
and transfer play
important roles in the
discovery, development
and Manufacture of
pharmaceuticals.
 Method development is
the process of proving
that an analytical method
is acceptable for use to
measure the concentr-
ation of an API in a
dosage form, accurately
and consistently will
deliver a reliable
measurement of an active
ingredient in it.

 Effective method development ensures that laboratory resources are optimized,
while methods meet the objectives required at each stage of drug development.
 Method validation, as required by regulatory agencies at certain stages of the
drug approval process, is deﬁned as the “process of demonstrating that
analytical procedures are suitable for their intended use”.
 Method transfer is the formal process of assessing the suitability of methods in
another laboratory. Each of these processes contributes to continual
improvement of methods and results in more efficient drug development.
 Analytical methods are intended to establish the identity, purity, physical
characteristics and potency of drugs.
 Methods are developed to support drug testing against speciﬁcations during
manufacturing and quality release operations, as well as during long-term
stability studies.
 Methods may also support safety and characterization studies or evaluations of
drug performance.

Choice of Method
It is important to appreciate the difference between an
‘analytical method’ (combination of steps illustrated by the
‘analytical process’) and an ‘analytical technique’ (chemical
or instrumental procedure by which analytical data is
eventually obtained). In selecting a method we shall need
to consider the following parameters:
• sample type (matrix) and size (lot or a little);
• data required (qualitative/quantitative);
• expected level(s) of analyte(s);
• precision & accuracy expected;
• likely interferences;
• number and frequency of samples for analysis.

S. No Parameter Description/Value
1 Stationary Phase Zorbax SB-C18 C18 (250X4.6 mmX3µ)
2 Mobile Phase
8 mM ammonium acetate, pH: 5.4:
acetonitrile (66:34)
3 Flow rate 0.4 ml/min
4 Detection Wavelength 272 nm
5 Detector Photo diode array
6 Injection Autosampler -Waters, model 717 plus
8 Injection volume 3 μl
9 Column Temperature Ambient
10 Run time 5 mins
11 Diluent Mobile Phase
Optimized Chromatographic conditions

Analytical Method Validation
 Method validation (MV) is the process used to confirm that
the analytical procedure employed for a specific test is
suitable for its intended use.
 Results from method validation can be used to judge the
quality, reliability and consistency of analytical results; it is an
integral part of any good analytical practice.
 Definition: MV is the process of “establishing documented
evidence” which provides high degree of assurance that
product (equipment) will meet the requirements for the
intended analytical applications.
 Why Validate Analytical Procedures?
Regulatory requirements, good science, and quality control
requirements.

 Types of Analytical procedures
to be Validated:
• Identification tests
• Quantitative tests for
impurities content
• Limit tests for the control of
impurities
• Quantitative tests of the
active moiety in samples of
drug
Method Validation Parameters

Type of analytical
Procedure Identification
Testing for Impurities ASSAY - dissolution
(measurement only) -
content/potency
Quantitative Limit
Characteristics
Accuracy X  X 
Precision
Repeatability X  X 
Interm. Precision X  X 
Specificity    
Detection Limit X X  X
Quantitation Limit X  X X
Linearity X  X 
Range X  X 
Degree of Validation depends upon the type of analytical test/procedure

Methodology
 No exact methodology is available for each parameter.
 Only ICH and FDA provides the guidelines but not to the extent of 100%.
 Good understanding of each performance characteristics is mot important. This
understanding must be beyond the basic definition of each parameter.
 Understanding must be anchored by sufficient years of practical experience and
knowledge. It will permit sound and local decision, even under the most
intense situations.

SYSTEM SUITABILITY
Definition:
Its an integral part of each method/procedure to ensure that all analytical
operations, electronics and equipments are working properly at the time of
analysis.
Methodology:
 Prepare 6 replicates at Target concentration and inject into chromatographic
system or check for absorbance in UV-Vis spectrophotometer.
Evaluation:
 Peak area, Rt, Tailing factor, Peak height, Resolution (for two or more no. of
Analytes), No of theoretical plates.
 Absorbance and λmax
 Calculate Average (Mean), SD and % RSD of all the parameters.

SYSTEM SUITABILITY
Result:
S. No Peak Area Rt Plate Count Peak Height Tailing
1 7356579 3.835 6891 1142687 1.01
2 7348022 3.832 6839 1165338 1.00
3 7338022 3.832 6839 1165338 1.00
4 7351189 3.833 6755 1152145 1.00
5 7351189 3.833 6755 1152145 1.00
6 7340002 3.836 6616 1137949 0.99
Average 7347500.5 3.8335 6782.50 1152600.333 1.00
STDEV 7155.007 0.002 97.357 11294.660 0.006
%RSD 0.10 0.20 1.44 0.98 0.63
Acceptance Criteria:
 Mean Plate count: more than 3000.
 Tailing factor: less than 2
 % RSD of all the parameters: less than 2%.

SPECIFICITY
Definition:
Specificity of analytical method is its ability to measure accurately an analyte in
the presence of interference, such as synthetic precursors, excipients, enantiomers
and know (or likely) degradation product that may be expected to be present in
the sample matrix.
Discussion:
 Specificity generally refers to a method that produces a response for a single
analyte only.
 Selectivity refers to a method which provides responses for a Multiple
chemical entities that may/may not be distinguished from each other.
 If each response is distinguished from all other responses, then the method is
said to be selective.
 Use of the term selectivity is appropriate for the methods based on
chromatographic techniques like HPLC, UPLC and GC etc.

SPECIFICITY
Methodology:
 Inject/analyze Blank, Placebo (If known) Standard (API) and Sample (Dosage
form) at target concentration for 6 replicates.
Evaluation:
 Identification tests: Response for compound of interest (Analyte) only.
 Assay: Peak Purity of analyte peak
 Impurities: Resolution between within the impurity(s) and/or degradants and
from analyte.

 Identification tests: Positive response for
compound of interest only.
 Assay: No interfering peaks should be found at
the RT of analyte peak.
 Impurities: Should pass peak purity of main
analyte and impurity peaks.
No interfering peaks should be found at the
RT of analyte peak.
 Dissolution: interfering peaks should be found
at the RT of analyte peak.
In case of UV method, %difference between λmax
of API and sample should not be more than 2%
Placebo
API
Tablet
SPECIFICITY

LINEARITY
Definition:
Linearity of an analytical procedure is its ability (within a given range) to obtain
test results that are directly proportional to the concentration of analyte in the
sample.
Range: The Interval between the upper and lower level that have been
demonstrated to be determined with precision, accuracy and linearity using this
method .
Methodology:
 Prepare a series of solutions (5 to 8 non zero standard solutions) at 25 to 200%
of target concentration.
Inject into Chromatographic system or check the absorbance in UV-Vis
Spectrophotometer.

S. No Linearity Level Concentration (μg/mL) Peak Area
1 25 % 35.00 1841901
2 50 % 70.00 3697872
3 75 % 105.00 5547026
4 100 % 140.00 7394914
5 125 % 175.00 9352643
6 150 % 210.00 11084071
7 175 % 245.00 12988710
8 200 % 280.00 14846511
 All the response points must be
on line. (Direct proportional)
 R2 (correlation co-efficient)
must be more than 0.999.

PRECISION
Definition:
Closeness of agreement (degree of scatter) between a series of measurements
obtained from multiple sampling of the same homogenous sample under the
prescribed conditions.
Precision may be considered at three levels.
 System precision (System suitability)
 Method Repeatability
 Intermediate precision
 Reproducibility

 Repeatability: Precision under same operating conditions (with in a laboratory
over a short period of time by the same analyst with same equipment)
Injection repeatability – System precision.
Method Repeatability – Method Precision.
Intermediate Precision: Precision under different laboratory conditions (with in
laboratory variation, as on different days or with different analysts or equipment
within the same laboratory).
Reproducibility: Precision between laboratories/Intermediate Precision can be
considered during the standardization of a procedure before it is submitted to the
Pharmacopoeia.

Methodology:
 Six Replicates of target concentration were
prepared using the dosage form and
injected - HPLC or determined the
absorbance – UV-VIs.
 Six Replicates of target concentration were
prepared through the complete analytical
procedure from sample preparation to
final result for method precision.
 Six replicates of target concentration were
prepared using dosage form by two
different analysts, analysed on two
different columns or instruments and on
different days for Ruggedness.
S. No
Analyte
Peak Area % Assay
1 7432699 101.03
2 7357606 100.01
3 7398974 100.57
4 7349367 99.90
5 7390881 100.46
6 7412006 100.75
Average 7390255.50 100.45
STDEV 31902.34 0.43
% RSD 0.43 0.43
 % RSD of Response and %
Assay should be less than
2%.

ACCURACY
Definition:
Closeness of agreement between the standard / reference value and the value
found experimentally.
Methodology:
Assay/Dissolution: Know amount of drug sample spiked with synthetic mixture of
drug product components (excipients) – Minimum at three levels
80%. 100% and 120% of test concentration
Or
50%, 100% and 150%
Evaluation:
% Recovery from the amount added and found was determined.
% Mean recovery of three levels was determined.

Recovery Studies (Accuracy):
Amount Added:
Amount Found:
% Recovery:

Accuracy Level Wt. of Sample Peak Area Amount Added Amount Found % Recovery Mean % Recovery
50%
10.506 3631749 69.69 69.11 99.17
100.00
10.506 3711606 69.69 70.63 100.35
10.506 3673683 69.69 69.91 100.32
10.506 3730428 69.69 70.99 100.87
10.506 3713458 69.69 70.66 100.40
10.506 3702716 69.69 70.46 100.11
100%
21.011 7253479 139.38 138.03 99.03
99.74
21.011 7351061 139.38 139.89 100.37
21.011 7311150 139.38 139.13 99.82
150%
31.517 11039927 209.06 210.08 100.49
100.00
31.517 11088563 209.06 211.01 100.93
31.517 11105958 209.06 211.34 100.09
31.517 11205024 209.06 213.22 100.99
31.517 11003028 209.06 209.38 100.15
31.517 11104679 209.06 211.31 100.08
 Mean % Recovery at 50%, 100% and 150% should be in between 98-100%

LOD & LOQ
Definition:
LOD: Lowest amount of analyte in a sample which can be detected but not
necessarily be quantitated.
LOQ: Lowest amount of analyte in a sample which can be quantitatively
determined.

Methodology:
 Calculate LOD and LOQ using Slope from Linearity and SD from Precision.
Prepare the solutions as per the concentration obtained
Inject into chromatographic system or check the absorbance.
Evaluation:
Response at LOD and LOQ.
Result:
 Same as Linearity and Precision.
Parameter Result LOD LOQ
Slope 53080
STDEV 31902.34 1.80μg/mL 6.01μg/mL

ROBUSTNESS
Definition:
Ability to remain unaffected by small but deliberate changes in method
parameters and provides indication of its reliability during its normal usage.
Typical variations include:
 Flow rate: ±10%
 Wavelength: ±2nm
 Organic component of Mobile phase: ±2%
 Temperature: ±5°C
 pH of the mobile phase: ±0.2 Units.
Procedure:
 Prepare the three replicate sample at target concentration and determine the
response (peak area or absorbance) under changed method parameters.

Evaluation:
 System suitability parameters and % Assay
Result:
Robustness
Parameter
Condition
Analyte
System suitability parameters
Peak Area % Assay
RT
Theoretical
Plates
Tailing
Flow
0.3 ml/min 0.995 3630 1.01 3988943 100.07
0.5 ml/min 0.816 3623 1.02 3986771 100.01
0.7 ml/min 0.779 3689 1.02 3987160 100.02
Temp
25 °C 0.817 3768 1.01 3978156 99.79
30 °C 0.816 3564 1.02 3986771 100.01
35 °C 0.810 3573 1.02 3987612 100.03
Wave length
256 nm 0.817 3267 1.02 3957612 99.28
258 nm 0.816 3674 1.01 3986771 100.01
260 nm 0.814 3890 1.01 3987612 100.03
 System suitability parameters must pass with % Assay in between 98-100%

FORCED DEGRADATION STUDIES
Definition:
 Forced degradation or stress testing is undertaken to demonstrate specificity
when developing stability indicating methods.
 A stability indicating method is one that accurately quantitates the active
ingredient without interference from degradation products, process impurities,
excipients, or other potential impurities.
Why to perform?
 Address the stability of the compound
 Establish the degradation pathway
 Identify the degradation products.
 Validate the stability indicating nature of the analytical procedure used.
Forced degradation studies should mimic the conditions to which the drug
substance/ drug product is actually exposed during its shelf life.

Procedure
Perform analysis for each stressed sample as per methodology using the initial
stress conditions viz.,
Acid: 1M HCl
Base: 1M NaOH
Peroxide: 10% H2O2
Thermal: 105°C for 72 H
Photolytic: 12000 Lux for 72 H
Humidity: 92% RH at 25°C for 72 H

Evaluation:
% Difference of assay for control (Unstressed, Normal) and each stressed
samples.
Peak Purity of analyte peak for control and stressed sample.
Points to be remembered
 If the degradation condition degrades the drug substance/drug product to too greater
extent or do not degrade at all, then alternative action should be taken (eg: change the
strength of the degradation medium or exposure time or heat applied or period of
exposure etc).
 5-10 % of API must degrade.
 Some compounds may not necessarily degrade under a given stress condition. No
further stressing is advised in these cases.
 Over stressing may lead to the formation of secondary degradants, causes disturbance
to the selectivity of the method, requires further development.

S. No Condition Peak Area % Assay % Degradation
1 Acid 6549579 89.02 10.98
2 Base 6689022 90.92 9.08
3 H2O2 6789022 92.28 7.72
4 UV 6614583 89.91 10.09
5 Heat 6616189 89.93 10.07
 At least 5-10% of drug substance should degrade.
Stability indicating power of the method demonstrates the ability of a method to
separate the analyte of interest from its degradation or impurities formed if any,
caused by quality of chemicals used in the manufacturing process, packaging,
storage, shipment and/or any unexpected exposure during shelf life of the drug
substance or drug product.
Result

References
1. FDA Guidance for Industry – Analytical Procedures and Method Validation,
Chemistry, Manufacturing, and Controls Documentation, Center for Drug
Evaluation and Re- search (CDER) and Center for Biologics Evaluation and Research
(CBER), August 2000.
2. International Conference on Harmonization Quality Guidelines Q2(R1), Validation
of Analytical Procedures, Text and Methodology, Parent guideline dated 27 Oct
1994, Complementary guideline on methodology dated 6 Nov 1996, incorporated
November 2005
3. USP 31 (2009): General Tests, Chapter 621 – Chromatography System Suitability,
United States Pharmacopeial Convention (USP), Rockville, MD.
4. Chan, C. C. et. al. (ed.), (2004), Analytical Method Validation and Instrument
Performance Veriﬁcation, Hoboken, NJ: John Wiley & Sons (Wiley Interscience).

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Understanding the Analytical method validation in a Practical Perspective

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