Vallidation 25

VALLIDATION OF ANALYTICAL METHODS
Presented By,
Asmita Rajendra Magare
M.Pharm 1st year (QAT)
Guided By,
Dr. Mrs Sonali Mahaparale
Professor of AAT.
Dr. Dy. Patil college of pharmacy akurdi.

“ Action providing that any procedure, process
equipment, material, activity or system actually
lead to the expected results.”
VALIDATION
2

Provides assurance of reliability The field of
validation is divided into a number of subsections as
follows-
 Cleaning validation
 Process validation
 Analytical method validation
 Computer system validation
Equipment validation
3

Validation of Analytical procedure is the
process by which it is established, by
laboratory studies ,that the performance
characteristics of the analytical procedure
meet the requirement for its use
Validation of Analytical Method
4

Characteristics that should be considered during
validation of analytical methods include:
 Accuracy
 Precision
 Specificity
 Limit of Detection
 Limit of Quantitation
 Linearity
 Range
 Robustness
 Ruggedness
5

Accuracy
Closeness of Test result obtained between the
true value .This accuracy should be established
across its range.
6

• reference material
compared with
standard.
Assay
• Assed on samples
spiked with known
amount of
impurity.
Impurities'
Determination of accuracy
7

Precision
The measure of the degree of agreement
(degree of scatter) between a series of
measurement obtain from multiple sampling
of the same sample under the prescribe
conditions.
8

Specificity is the ability to detect analyte of
interest in the presence of interfering
subestance . Typically these might include
impurities, degradation product etc.
Specificity
11

LOD
Lowest amount of
analyte in a sample that
can be detected but not
necessarily quantitated.
Estimated by Signal to
Noise Ratio of 3:1.
LOQ
Lowest amount of
analyte in a sample that
can be quantified with
suitable accuracy and
precision.
Estimated by Signal to
Noise Ratio of 10:1.
LOD and LOQ
12

LOD and LOQ Estimated by
S = slope of calibration curve
s = standard deviation of blank readings or
standard deviation of regression line
DL =
3.3s
QL =
10s
S S
1. Based in Visual Evaluations
- Used for non-instrumental methods
2. Based on Signal-to Noise-Ratio
- 3:1 for Detection Limit
- 10:1 for Quantitation Limit
3. Based on Standard Deviation of the
Response and the Slope
13

Ybl
LOD LOQ
Statistical estimate of LOD & LOQ
LOD = 3.3 Sbl / b LOQ = 10 Sbl / b
Y = b X + a
14

noise
Peak A
LOD
Peak B
LOQ
Baseline
Signal-to Noise-Ratio
15

Linearity
Linearity- Its ability to obtain results that are directly or by
a well defined mathematical transformations proportional to
concentration of analyte in sample within a given range.
 A minimum of five concentrations should be used
16

By Visual Inspection of plot of signals vs. analyte
concentration
Acceptance criteria: Linear regression r2 > 0.95
Determination of Linearity
17

The range of an analytical procedure is the interval
between the upper and lower concentration
(amounts) of analyte in the sample (including these
concentrations) for which it has been demonstrated
that the analytical procedure has a suitable level of
precision, accuracy and linearity.
Range
18

Robustness
Definition: Capacity to remain unaffected by small but
deliberate variations in method parameters.
Determination: Comparison results under differing
conditions with precision under normal conditions.
19

Ruggedness
Degree of reproducibility of test results
under a variety of conditions
Different Analysts
Different Laboratories
Different Instruments
Different Reagents
Different Days
Etc. 20

Equipment validation
It demonstrate that equipment used in validation studies is
suitable for use and is comparable to equipment used for routine
analysis.
Qualification
Action of proving and documenting that equipment or
ancillary systems are properly installed, work correctly, and
actually lead to the expected results.
Qualification is part of validation , but the individual
qualification steps alone do not constitute process validation.
21

Parts of qualification
The activity of qualifying system of equipment is
divided into four subtypes-
Design qualification (DQ)
 Installation qualification (IQ)
 Operational qualification (OQ)
 Performance qualification (PQ)
22

Design qualification (DQ)- Demonstrates that the proposed
design will satisfy all the requirements.
Installation qualification (IQ)- Demonstrates that the
process or equipment meets all specifications, is
installed correctly.
23

Operational qualification (OQ)- Demonstrates that all
facets of the process or equipment are operating
correctly.
Performance qualification (PQ)- Demonstrates that the
process or equipment performs as intended in a
consistent manner over time.
24

Validation of HPLC
Flow rate accuracy
Injector accuracy
System precision
Wavelength accuracy
Detector linearity
Injector linearity
Gradient performance check
Column oven temperature accuracy
25

Flow rate accuracy:-
The performance of pump module is the ability to maintain
accurate and consistent flow of the mobile phase. Poor flow rate will
affect the retention time and resolution of the separation.
Test:-
The flow rate accuracy at 2 ml/min can be verified by using a
calibrated stopwatch to measure the time it takes to collect 25 ml of
effluent from the pump into a 25 ml volumetric flask
Acceptance:-
±2.0% of the set flow rate
26

System precision:-
 Weigh accurately 60 mg of caffeine into 100 ml volumetric
flask.
 Dissolve and dilute to the volume with mobile phase.
Transfer 10 ml of this solution into 100 ml flask and make up with
mobile phase.
 Inject blank followed by standard preparation in 6
replicates. Note down the areas and retention times.
Acceptance:-
The % RSD of retention time and peak area should be
<1.0%
27

Wavelength accuracy:-
Inject blank followed by standard
preparation and note down the height or
absorbance.
Acceptance:-
The maximum absorbance should be ±2
nm. 28

Detector linearity:-
 The linearity of the detector response can be checked by
pumping or by filling the flow cell with a series of standard
solution of various concentration.
 Aqueous caffeine solutions are convenient for the linearity
measurement.
The correlation coefficient between sample concentration and
response can be calculated to determine the linearity.
Acceptance:-
R≥ 0.999
29

Injector linearity:-
 Linearity is important for methods that require the use of variable
injection volumes.
 The linearity of the injector can be demonstrated by making
injections, typically 5,10,20,50 and 100 μl to cover the range 0 to 100 μl.
 The response of the injection is plotted against the injection volume.
 The correlation coefficient of the plot is used in evaluation of the
injector linearity.
Acceptance:-
R≥0.999 30

Column oven temperature accuracy:-
 The efficiency of HPLC column varies with column temperature.
 The temperature accuracy of the column heater is evaluated by
placing a calibrated thermometer in the column compartment
to measure the actual compartment temperature.
 The thermometer readings are compared to the preset
temperature at 40 and 60°C.
Acceptance:-
The resulting oven temperature from the thermometer
display should be within ±2°C of the set temperature.
31

REFERENCES
Dr. Sohan S.Chitlange,Dr.Pravin Dchaudhari Pharmaceutical
Validation Pharmatree Education First Edition April 2014,
Page no: 1.1-4.4
Manohar A.Potdar Pharmaceutical Quality Assurance Nirali
Prakashan Third Edition Octomber 2013.Page no ;8.1-8.44.
www.Pharmaweb.net case study on HPLC document.
32

Journal of Biomedical sciences and research-volume
2(2), 2010, page no:- 89-99.
 Practical pharmaceutical chemistry by A.H.Beckett,
J.B.Stenlake, Part Two, page no:- 326.
 Analytical Method Validation And Instrument
Performance Verification by Herman lam., page no:-
153-186. 33

Vallidation 25

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Vallidation 25