VALLIDATION OF ANALYTICAL METHODS

Presented By,Asmita Rajendra MagareM.Pharm 1st year (QAT)

Guided By,Dr. Mrs Sonali Mahaparale

Professor of AAT.

Dr. Dy. Patil college of pharmacy akurdi.

2

“ Action providing that any procedure, process equipment, material, activity or system actually

lead to the expected results.”

VALIDATION

3

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

4

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

5

Characteristics that should be considered during validation of analytical methods include:

Accuracy Precision Specificity Limit of Detection Limit of Quantitation Linearity Range Robustness Ruggedness

6

Accuracy

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

7

• reference material compared with standard.

Assay

• Assed on samples spiked with known amount of impurity.

Impurities'

Determination of accuracy

8

PrecisionThe 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.

9

10

Determination of Precision

11

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

Specificity

12

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

13

LOD and LOQ Estimated by

S = slope of calibration curves = standard deviation of blank readings or standard deviation of regression line

DL = 3.3s QL = 10sS 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

14

Ybl

LOD LOQ

Statistical estimate of LOD & LOQ

LOD = 3.3 Sbl / b LOQ = 10 Sbl / b

Y = b X + a

15noise

Peak ALOD

Peak BLOQ

Baseline

Signal-to Noise-Ratio

16

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

17

By Visual Inspection of plot of signals vs. analyte

concentrationAcceptance criteria: Linear regression r2 > 0.95

Determination of Linearity

18

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

19

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.

20

RuggednessDegree of reproducibility of test results

under a variety of conditions Different Analysts Different Laboratories Different Instruments Different Reagents Different Days

Etc.

21

Equipment validationIt demonstrate that equipment used in validation studies is suitable for use and is comparable to equipment used for routine analysis.

QualificationAction 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.

22

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)

23

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.

24

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.

25

Validation of HPLC

Flow rate accuracy

Injector accuracy

System precision

Wavelength accuracy

Detector linearity

Injector linearity

Gradient performance check

Column oven temperature accuracy

26

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

27

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%

28

Wavelength accuracy:-

Inject blank followed by standard

preparation and note down the height or

absorbance.

Acceptance:-

The maximum absorbance should be ±2

nm.

29

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

30

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

31

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.

32

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.

33

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.

34

Thank you