Analytical

Method Validation

Paul R. Johnson

Campbell University

Pharmaceutical Sciences-PERC

QC/R&D Analytical Lab Manager

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Guidance for Industry

Q2B Validation of Analytical

Procedures: MethodologyCenter for Drug Evaluation and Research (CDER),

5600 Fishers Lane, Rockville, MD 20857 (Tel) 301-827-4573

http://www.fda.gov/cder/guidance/index.htm

or

Center for Biologics Evaluation and Research (CBER)

1401 Rockville Pike, Rockville, MD 20852-1448,

http://www.fda.gov/cber/guidelines.htm

U.S. Department of Health and Human Services

Food and Drug Administration

November 1996

ICH

http://www.ich.org/products/guidelines/quality/quality-single/article/validation-of-analytical-procedures-text-and-methodology.html

USP 37- NF 32<1225> VALIDATION OF COMPENDIAL PROCEDURES

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Components of Data Quality

Quality of

analysis

before

sample

analysis

Quality of

analysis

results

immediately

before and

during

sample

analysis

From: AAPS “A Scientific Approach to Analytical Instrument Validation”

3

4

Qualified vs. Validated

Instruments are “Qualified”.

Analytical Instrument Qualification (AIQ)

Processes are “Validated”.

Validation reserved for processes that include

analytical procedures (methods) and software

development (applications).

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Analytical Instrument Qualification

Documented evidence that an

instrument performs suitably for

its intended purpose and is

properly maintained and

calibrated.

Quality Standards and Regulations

All GMP,GLP and GCP regulations require analytical

equipment to be qualified and calibrated.

Example: FDA cGMP 21 CFR 211.160 i(4) requires

The calibration of instruments, apparatus, gauges, and recording

devices at suitable intervals in accordance with an established

written program containing specific directions, schedules, limits

for accuracy and precision.

Instruments not meeting established specifications shall not be

used.

CFR: Code of Federal Regulations

GMP: Good Manufacturing Practices

GLP: Good Laboratory Practices

GCP: Good Clinical Practices

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7

Analytical Instruments

Provides Valid Data for Research, Development,

Manufacturing and Quality Control (FDA 21 CFR 210 and 211)

cGMP regulations require equipment supporting the

product testing is “fit for use”.

Demonstrate the precision and accuracy of analytical

Instruments used.

Demonstrate/record training of operators.

AAPS “A Scientific Approach to Analytical Instrument

Validation” March 2003

USP Chapter <1058> 2008

ICH Q7A

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Instrument Categories

Group A Instruments Simple instruments whose conformance to user requirements is

determined by visual observation.

No independent qualification process is required.

Group B Instruments Instruments whose conformance to user requirements is

performed according to the instruments’ SOP. Conformity assessments are usually unambiguous.

Installation is relatively simple and instrument failure is readily detected by simple observations.

Group C Instruments Complex instruments whose conformance to user requirements

is highly method specific.

Installation can be complicated and may require specialists.

A full-qualification process must be applied to these instruments.

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Instrument Categories

Group A Instruments Light Microscope

Magnetic Stirrer

Mortar & Pestle

Spatula

Vortex Mixers

Group B Instruments Balances

Incubators

Melting Point Apparatus

Muffle Furnace

Ovens

Group B Instruments (continued) pH Meters

Pipettes

Refractometers

Refrigerators-Freezers

Thermocouples

Thermometers

Titrators

Vacuum Ovens

Viscometers

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Instrument Categories Group C Instruments

Atomic Absorption Spectrometers

Densitometers

Differential Scanning Calorimeters

Diode-Array/UV-Vis Detectors

Dissolution Apparatus

Electron Microscope

Elemental Analyzers

Flame Absorption Spectrometers

Gas Chromatographs

HPLCs

Group C Instruments (continued) NIRs

FTIRs

Mass Spectrometers

Raman Spectrometers

Micro-plate Readers

UV/Vis Spectrometers

Thermal Gravimetric Analyzers

Inductively Coupled Plasma Emission Spectrometers

X-ray Fluorescence Spectrometers

ValidationFDA-guidelines:

Validation is establishing documented evidence which provides a

high degree of assurance that a specific process will consistently

produce a product meeting its pre-determined specifications and

quality attributes

EU-guidelines

Action of proving, in accordance with GMP-principles that any

procedure, process, equipment, material, activity or system actually

leads to the expected results

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What Methods to be Validated?

Defined for:

- identification

- quantitative tests for content of impurities

- limit tests for control of impurities

- quantitative tests for active moiety in drug

substances and drug products

Referred to:

- dissolution testing

- particle size determination (drug substance)

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Components of Data Quality

Analytical Methods Validation

Documented evidence that an analytical method does

what it purports to do and delivers the required

attributes.

Defined procedures from User Groups & Regulatory

Agencies. (See References)

Common Parameters:

Accuracy Sensitivity Specificity

Precision/Repeatability Linearity

Analyte Stability Limits of Detection and Quantitation

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Recommended Validation characteristics of various Types of Tests

Type of tests/Characteristics IdentificationTesting for impurities

Assay/

Dissolution Specific

Tests

Quantitative Limits

Accuracy - + - + +

Precision-repeatability - + - + +

Precision-Intermediate precision

(Ruggedness)- + - + +

Specificity + - + + +

Detection limit - + + - -

Quantitation Limit - + - - -

Linearity - + - + -

Range - + - + -

Robustness - + - + +

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Components of Data Quality

System Suitability Tests

Documented evidence that the system works

according to the performance expectations

and criteria set forth in the method at the time

of the analysis

Quality Control Checks

Analyses of reference or calibration

standards.

Documented evidence that results on quality

control check samples yield expected results.

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What is a Method

A method is a set of instrument

parameters that define the conditions of

the instrument during the time the data is

collected.

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HPLC Method Mobile Phases: Water, Buffer, Acetonitrile, Methanol

Flow Rate of Mobile Phase (1mL/min)

Composition of Mobile Phase (gradient, isocratic)

Run Time

Column:

Type (C18, C8, CN, NH2, SAX)

Dimensions and particle size (150mm x 4.6mm, 3m)

Temperature (40C)

Detector Type (UV-Vis, ELSD, RI, Fl, MS)

Wavelength for UV or Fl

Other parameters for ELSD or MS)

Injection Volume

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Typical HPLC MethodMethod Name MA000157X

Column Phenomenex Aqua C18 150x4.6mm 3m

Temp 40°C

Mobile Phase A Water w/0.05%TFA

Mobile Phase B Acetonitrile w/0.05%TFA

Flow Rate 1mL/min

Gradient

0 min 20% B

20 min 80% B

21min 20% B

Run Time 25min

Detector Diode Array UV-Vis

Wavelength 235nm

Injection Volume 5mL

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Method Validation Linearity (R2 ≥ 0.998)

Range (80% to 120% of nominal concentration)

Accuracy (98% to 102%)

Precision/Repeatability (%RSD ≤1%)

Detection Limit (TBD)

Quantitation Limit (TBD)

Robustness

Ruggedness

Specificity (no co-elution)

System Suitability Testing http://www.fda.gov/cder/guidance/cmc3.pdf

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Method Validation

LinearityThe linear range of detectability that obeys Beer's Law is dependent on the compound analyzed and detector used. The working sample concentration and samples tested should be in the linear range.

A linear relationship should be evaluated across the range of the analytical method. It may be demonstrated directly on the drug substance (by dilution of a standard stock solution) and/or separate weighings of the drug product components.

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Method Validation

Linearity

21

0.00, 0.00000

0.0606, 596

0.1401, 1388

0.2007, 2055

0.2272, 2345

0.2651, 2731

y = 10243xR² = 0.9994

0

500

1000

1500

2000

2500

3000

0.00 0.05 0.10 0.15 0.20 0.25 0.30

Are

a R

esp

on

se (

mA

U*s

)

Concentration (mg/mL)

Calibration curve

Acceptable Linearity (R2 ≥ 0.998)

Method ValidationRange

Range is the interval between the high and low levels of analyte

studied.

The specified range is normally derived from linearity studies and

depends on the intended application of the method. It is established

by confirming that the analytical method provides an acceptable

degree of linearity, accuracy, and precision when applied to samples

containing amounts of analyte within or at the extremes of the

specified range of the analytical method.

The following minimum specified ranges should be considered.

For the content assay of a drug substance or a finished (drug)

product: From 80 to 120 percent of the product label strength.

For content uniformity: Covering a minimum of 70 to 130 percent of

the product label strength.

For some methods may be much lower depending on needs of

assay

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Method Validation

Accuracy

Accuracy is the measure of how close the

experimental value is to the true value.

For the drug product, this is performed

frequently by the addition of known amounts of

drug by weight or volume (dissolved in diluent)

to the placebo formulation working in the linear

range of detection of the analyte.

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Method ValidationAccuracy

Theoretical 0.862 mg/mL

Inj 1 1492.23 (AUC)

Inj 2 1514.52

Inj 3 1484.93

Ave 1497.23

SD 15.41

%RSD 1.03

weight mg/mL 0.855

Accuracy % 99.18%

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Acceptable Accuracy (98% to 102%)

Method Validation

PrecisionPrecision is the measure of how close the data values are to each other for a number of measurements under the same analytical conditions. ICH has defined precision to contain three components: repeatability, intermediate precision and reproducibility.

Injection Repeatability (Precision)

The precision as measured by multiple injections of a homogeneous sample (prepared solution) indicates the performance of the HPLC instrument under the chromatographic conditions and day tested. The specification, as the coefficient of variation in % or relative standard deviation (RSD), set here will determine the variation limit of the analysis.

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Injection AUC

Inj 1 843.00

Inj 2 852.84

Inj 3 837.84

Inj 4 836.44

Inj 5 833.81

Inj 6 833.59

Ave 839.59

SD 7.34

%RSD 0.87

Precision Data

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(Acceptable %RSD ≤1%)

Method Validation

Limit of Detection and Limit of QuantitationThese limits are normally applied to related substances in the drug substance or drug product. Specifications on these limits are submitted with the regulatory impurities method relating to release and stability of both drug substance and drug product.

Detection limit (LOD) is the lowest concentration of analyte in a sample that can be detected, but not necessarily quantified, under the stated experimental conditions.

Quantitation limit (LOQ) is the lowest concentration of analyte in a sample that can be determined with acceptable precision and accuracy under the stated experimental conditions.

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LOD & LOQ

Method 1: Determine the peak to peak noise on Y scale and

determine from the calibration linearity curve of conc. vs.

height, the concentrations where:

LOD = 3.3 times the noise

LOQ = 10 times the noise

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AU

LOD & LOQ

Plot Peak Height vs Concentration

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Method Validation

Robustness

Robustness is a measure of the method's capability to remain

unaffected by small, but deliberate variations in method parameters.

Robustness can be partly assured by good system suitability

specifications. Thus, it is important to set tight, but realistic, system

suitability specifications.

Testing varying some or all conditions, e.g., age of columns, column

type, column temperature, pH of buffer in mobile phase, reagents, is

normally performed.

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Method Validation

Intermediate Precision (Ruggedness)

Ruggedness evaluates the reliability of the method in a

different environment other than that used during

development of the method. The objective is to ensure

that the method will provide the same results when

similar samples are analyzed once the method

development phase is over.

The method can be tested on multiple days, analysts,

instruments, etc.

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Method ValidationSpecificity/Selectivity (Peak Purity)

The representative analyte should have no interference from other extraneous

components and be well resolved from them. A chromatogram or profile should be

generated and submitted to show that the extraneous peaks either by addition of

known compounds or samples from stress testing are baseline resolved from the

parent analyte.

Examples of the extraneous peaks are as follows:

For the drug substance or raw material, the related substances to consider are

process impurities from the synthesis process. For the drug product, the related

substances may be impurities present in the active drug, degradation products,

interaction of the active drug with excipients, extraneous components.

Submission of data from stress testing of the drug substance using acid and base

hydrolysis, temperature, photolysis and oxidation.

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Method ValidationSpecificity

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The system must be shown to be suitable for performing the

analytical testing during the assay by employing discriminating

limits to assays of the standard or other reference solutions. The

parameters employed function on the entire system as a discrete

unit and are normally determined by empirical testing. No sample

analysis is acceptable unless the requirements of system

suitability have been met.

1. Resolution

2. Precision/Injection repeatability

3. Retention time reproducibility

4. Tailing Factor

5. Standard Accuracy (Check)

System Suitability

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Typical SequenceSequence

name

vial sample inj/vial type method

1 MP blank 2 blank MA00001

2 Std A 3 Calib MA00001

3 Std B 3 Calib MA00001

4 Stdrepeat 6 Calib MA00001

5 Sample 1 3 sample MA00001

6 Sample 2 3 sample MA00001

7 Sample 3 3 sample MA00001

8 Sample 4 3 sample MA00001

9 Sample 5 3 sample MA00001

10 Sample 6 3 sample MA00001

11 Std A 3 Calib MA00001

12 Sample 7 3 sample MA00001

13 Sample 8 3 sample MA00001

14 Sample 9 3 sample MA00001

15 Sample 10 3 sample MA00001

16 Sample 11 3 sample MA00001

17 Sample 12 3 sample MA00001

18 Std A 3 Calib MA00001

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ResolutionThe resolution, R,, is a measure of how well two peaks

are separated and is a function of column efficiency, and

is specified to ensure that closely eluting compounds are

resolved from each other.

R of > 2 between the peak of interest and the closest potential

interfering peak (impurity, excipient, degradation product, internal

standard, etc.) is desirable.

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Precision/Injection Repeatability

The area under the curve (AUC) for replicate

injections of a standard preparation used in the

assay are compared to ascertain whether

requirements for precision are met. Unless

otherwise specified in the individual monograph,

data from five replicate injections of the analyte

are used to calculate the relative standard

deviation, %RSD. Typically the requirement is

1.0%

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Injection and Retention Time

Reproducibility

AUC Ret. Time

Inj 1 1361.25 3.182

Inj 2 1375.16 3.184

Inj 3 1371.77 3.183

Inj 4 1357.26 3.182

Inj 5 1358.23 3.180

Inj 6 1381.02 3.177

Ave 1367.45 3.181

Std Dev 11.42 0.003

%RSD 0.83% 0.079%

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≤ 1% ≤ 1%

Tailing Factor

The tailing factor, T, a measure of peak symmetry, is unity for

perfectly symmetrical peaks and its value increases as tailing

becomes more pronounced. As peak asymmetry increases,

integration, and hence precision, becomes less reliable.

T = W0.05 / 2f T 2 is recommended

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System Suitability Report

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Standard Check

Conc Std A = Conc Std B

AUC Std A AUC Std B

AUC Std A x Conc Std B = 98.0% to 102.0%

Conc Std A AUC Std B

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Questions on Method Validation

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