validation of analytical methods rutendo kuwana training workshop: assessment of interchangeable...

Validation of analytical methods

Rutendo Kuwana

Training workshop: Assessment of Interchangeable Multisource Medicines, Kenya, August 2009

Assessment of Interchangeable Multisource Medicines, Kenya, August 20092 | 2

BACKGROUND-LAB METHOD FLOWBACKGROUND-LAB METHOD FLOW

MethodValidation

MethodTransfer

MethodDevelopment

Approved


VALIDATIONVALIDATION

“ THE PROCESS OF PROVIDING DOCUMENTED EVIDENCE THAT SOMETHING DOES WHAT IT IS

INTENDED TO DO”


WHY VALIDATE?WHY VALIDATE?

To demonstrate that the method is suitable for its intended use

Provides assurance of reliability


WHO GMP - 4.11 Analytical methods, computers and cleaning proceduresWHO GMP - 4.11 Analytical methods, computers and cleaning procedures

“It is of critical importance that particular attention is paid to the validation of analytical test methods, automated systems and cleaning procedures.”

Validation of analytical procedures used in the examination of pharmaceutical materials (WHO Expert Committee on Specifications for Pharmaceutical Preparations. 32nd Report. Geneva, WHO, 1992 (WHO Technical Report Series, No. 823)

Text on Validation of Analytical Procedures Q2 (R1) Validation of Analytical Procedures: Text and Methodology. ICH Harmonized Tripartite Guidelines


ANALYTICAL METHOD VALIDATION PERFORMANCE CHARACTERISTICSANALYTICAL METHOD VALIDATION PERFORMANCE CHARACTERISTICS

Range

System Suitability

MethodValidation

Precision

Accuracy

Limit of Detection

Limit of Quantitation

Specificity

Linearity

Robustness

Range

System Suitability

MethodValidation

Precision

Accuracy

Limit of Detection

Limit of Quantitation

Specificity

Linearity

Robustness


TYPES OF ANALYTICAL METHODS TO BE VALIDATED

TYPES OF ANALYTICAL METHODS TO BE VALIDATED

Identification tests

Quantitative tests for impurities’ content

Limit tests for the control of impurities

Quantitative tests of the active in samples of the drug substance (raw material), finished product or other selected components in the drug


ACCURACY ACCURACY

Expresses the CLOSENESS of agreement BETWEEN the value, which is accepted either as a conventional TRUE VALUE or an accepted REFERENCE VALUE and the VALUE FOUND i.e. individual observation or mean of measurements

The closeness of test results to the

true value obtained by the method

(trueness).

– Established across the range


DETERMINATION OF ACCURACYDETERMINATION OF ACCURACY

Drug Substance

– Analysis of reference material

– Compare results to a second, well-characterized method

Drug Product

– Analysis of synthetic mixtures spiked with known quantities of

components

– Compare results to a second, well-characterized method

Determined concurrently with precision, linearity and specificity


DETERMINATION OF ACCURACY cont’DETERMINATION OF ACCURACY cont’

Impurities (Quantitation)

– Analysis of samples (Drug substances/Drug product) spiked with known

amounts of impurities

– If impurities are not available, see specificity

Recommended Data

– Minimum of 9 determinations over a minimum of 3 concentration levels

covering the specified range (e.g. 3 concentrations/3 replicates each)

– Reported as % recovery of known added amount or difference between

the mean and true value, with confidence intervals


PRECISIONPRECISION

Precision

– The measure of the degree of

agreement (degree of scatter)

among test results when the

method is applied repeatedly

to multiple samplings of a

homogeneous sample

– Expressed as %RSD for a

statistically significant number

of samples


Precision (of any process)Precision (of any process)

The precision (VARIABILITY) of an analytical procedure is usually expressed as the standard deviation (S), variance (S2), or coefficient of variation (= relative standard deviation, R.S.D.) of a series of measurements.

The confidence interval should be reported for each type of precision investigated.

Measured mean Real meanMeasured mean Real mean


PRECISION cont’PRECISION cont’

May be considered at 3 levels: – Repeatability, a measure of variability under the same

operating conditions over a short interval (intra-assay precision). Minimum of 9 determinations covering specified range

– Intermediate precision, a measure of within-laboratory variations (different days, different analysts, different equipment)

– Reproducibility, expresses precision between laboratories (e.g. in collaborative studies), also applies to method transfer


Repeatability (of any process)Repeatability (of any process)

Repeatability expresses the precision (spread of the data, variability) under the same operating conditions over a short interval of time. Repeatability is also termed intra-assay precision.

Measured meanMeasured mean


Intermediate Precision and Reproducibility

Intermediate Precision and Reproducibility

Intermediate precision expresses within-laboratories variations. #1, #2 and #3: different days, different analysts, different (manufacturing) equipment, etc.

Reproducibility expresses the precision between laboratories #1, #2 and #3 (collaborative studies, usually applied to standardization of methodology). (Transfer of technology)

Measured meansMeasured means


ACCURACY & PRECISIONACCURACY & PRECISION

Inaccurate &imprecise

Inaccurate butprecise

Accurate butimprecise Accurate and precise


SPECIFICITY/SELECTIVITYSPECIFICITY/SELECTIVITY

– The ability to measure accurately and

specifically the analyte in the presence of

components that may be expected to be

present in the matrix

– The degree of interference

• Active Ingredients

• Excipients

• Impurities (synthetic precursors, enantiomers)

• Degradation Products

• Placebo Ingredients


SPECIFICITY/SELECTIVITYSPECIFICITY/SELECTIVITY

• Combination of 2 or more analytical procedures may be

required to achieve necessary level of discrimination

• Stability indicating analytical methods should always be

specific

• Analysts should ascertain whether the peaks within a sample

chromatogram are pure or consist of more than one compound.

Therefore should know how many compounds are in the

sample or use procedures to detect peak purity


LOD, LOQ and SNRLOD, LOQ and SNR

Limit of Quantitation (LOQ)

Limit of Detection (LOD)

Signal to Noise Ratio (SNR)

noise

Peak ALOD

Peak BLOQ

Baseline


LIMIT OF DETECTIONLIMIT OF DETECTION

The lowest concentration of an analyte in a sample that can be detected, not quantified

Expressed as a concentration at a specified signal:noise ratio


LIMIT OF QUANTIFICATIONLIMIT OF QUANTIFICATION

The lowest concentration of analyte in a sample that can be determined with acceptable precision and accuracy under stated operational conditions

Expressed as concentration of analyte


LINEARITYLINEARITY

– The Ability of the method to obtain test results that are directly

proportional to concentration within a given range

– Method: dilution of stock solution/separate weighings

– Expressed as the variance of the slope of the regression line

– Correlation coefficient, y-intercept, slope of regression line and

residual sum of squares should be presented together with plot of

the data


LinearityLinearity

Linearity expresses differences in precision at different points of a given range.

The linearity of an analytical procedure is its ability (within a given range) to obtain test results, which are directly proportional to the

concentration (amount) of analyte in the sample


RANGERANGE

– Interval between upper and lower levels of analyte demonstrated

by the method

– Confirms that the analytical procedure provides acceptable

degree of linearity, accuracy and precision when applied to

samples containing amounts of analyte within or at the extremes

of the specified range

– Minimum 5 concentrations


Range (minimum requirements)Range (minimum requirements)

Assay of an API or a FPP: ± 20% of the test concentration.

Content uniformity: ± 30% of the test concentration (unless a wider more appropriate range, based on the nature of the dosage form (e.g., metered dose inhalers), is justified).

Dissolution testing: ± 20 % over the specified range.

Impurity: from the reporting level of an impurity to 120% of the specification. (Unusually potent or toxic impurities, LOD and LOQ should be commensurate with ICH requirement.)

If assay and purity are performed together as one test and only a 100% standard is used, linearity should cover the range from the reporting level of the impurities to 120% of the assay specification

Assessment of Interchangeable Multisource Medicines, Kenya, August 200926 |

Typical expected results for Analyte concentration vs. Precision(The AOAC manual for the Peer-Verified Methods program)

Typical expected results for Analyte concentration vs. Precision(The AOAC manual for the Peer-Verified Methods program)

Analyte Conc (%)Analyte RatioUnitRSD%

1001100%1.3

1010-110%2.8

110-21 %2.7

0.110-30.1%3.7

0.0110-4100 ppm5.3

0.00110-510 ppm7.3

0.000110-61 ppm11

0.0000110-7100 ppb15

0.00000110-810 ppb21

0.000000110-91 ppb30


RUGGEDNESSRUGGEDNESS

Ruggedness

– Degree of reproducibility of test

results under a variety of

conditions

• Different Analysts

• Different Laboratories

• Different Instruments

• Different Reagents

• Different Days

• Etc.

– Expressed as %RSD


ROBUSTNESSROBUSTNESS

Measure of the capacity to

remain unaffected by small

(deliberate) variations in

method parameters

– Indication of reliability

during normal use


Sensitivity and robustness

Input-output relationship


Stability of analytical solutionStability of analytical solution

Solutes may readily decompose prior to chromatographic investigations e.g. during sample preparation , extraction, cleanup, phase transfer or storage of prepared vials (refrigerators or automatic sampler). Method development should investigate the stability of the analytes AND standards.

System stability

stability of the samples being analyzed in a sample solution.

Measure of the bias in assay results generated during a preselected time interval e.g. 1 – 48 hours using a single solution

should be determined by replicate analysis of the sample solution.

considered appropriate when the RSD, calculated on the assay results obtained at different time intervals, Less than 20 percent of the corresponding value of the system precision


SYSTEM SUITABILITYSYSTEM SUITABILITY

The checking of a system, before or during analysis of unknowns, to ensure system performance.

• “No sample analysis is acceptable unless the requirements for system suitability have been met.” (USP Chapter 621)

– Plate Count, Tailing, Resolution

– Determination of reproducibility (%RSD)• For %RSD < 2.0%, Five replicates• For %RSD > 2.0%, Six replicates

System Suitability "Sample“ - A mixture of main components and expected by-products utilized to determine system suitability

“Whenever There is a Significant change in Equipment or Reagents System Suitability Testing Should be Performed” (USP Chapter 621)


Classes of analytical testsClasses of analytical tests

“The objective of validation of an analytical procedure is to

demonstrate that it is suitable for its intented purpose.”

Class A: To establish identity

Class B: To detect (Bd) and quantitate (Bq) impurities

Class C: To determine quantitatively the concentration, or assay

Class D: To assess characteristics

Other classes not covered in the guides


Criteria for analytical classesCriteria for analytical classes

CriteriaABqBdCD

AccuracyXXX

PrecisionXXX

RobustnessXXXXX

Linearity and rangeXXX

SpecificityX ?XXXX

Limit of detectionX

Limit of quantitationX


General requirementsGeneral requirements

Qualified and calibrated instruments

Documented methods

Reliable reference standards

Qualified analysts

Sample integrity

Change control (e.g., synthesis, FPP composition)


General requirements (2)General requirements (2)

Analytical methods should be used within GMP and GLP environments, and must be developed using the protocols and acceptance criteria set out in the ICH guidelines Q2

(R1)


General requirements (3)General requirements (3)

Validation Protocol important

Revalidation should accompany

formulation changes (new samples with new compounds or new matrices)

manufacturing batch changes

new analysts with different skills,

new instruments with different characteristics,

new location with different environmental conditions,

new chemicals and/or reference standards and

modification of analytical parameters.


Validation ReportValidation Report

Objective and scope of the method (applicability, type).

Summary of methodology.

Type of compounds and matrix.

All chemicals, reagents, reference standards, QC samples with purity, grade, their source or detailed instructions on their preparation.

Procedures for quality checks of standards and chemicals used.

Method parameters.

Critical parameters taken from robustness testing.

Listing of equipment and its functional and performance requirements, e.g., cell dimensions, baseline noise and column temperature range.


Validation Report (2)Validation Report (2)

Detailed conditions on conduct of experiments, including sample preparation

Statistical procedures and representative calculations.

Procedures for QC in routine analyses, e.g., system suitability tests.

Representative plots, e.g., chromatograms, spectra and calibration curves.

Method acceptance limit performance data and expected uncertainty of measurement results.

Criteria for revalidation.

The person(s) who developed and validated the method.

References (if any).

Summary and conclusions.


Use and Validation of Pharmacopoeial methodsUse and Validation of Pharmacopoeial methods

The degree of validation of a pharmacopoeial method should be adequate for required purpose, and the laboratory be able to match any stated performance data. Following product – specific attributes should be considered

the type of compounds to be analyzed,

matrices,

the type of information required (qualitative or quantitative),

detection and quantitation limits,

Concentration range for analysis based on own product

precision and accuracy (sample preparation critical) as specified by the client of the analytical data and

the type of equipment—its location and environmental conditions.

Therefore PQP three parameters required for PQP – Specificity, Accuracy and Precision


Main Points AgainMain Points Again

Validation of analytical procedures is a critical requirement in risk assessment and management:

– establishment of product-specific acceptance criteria, and– stability of APIs and FPPs.

Validation should demonstrate that the analytical procedure is suitable for its intented purpose.

HPLC systems and method validation deserves special attention during the inspection of QC laboratories.

validation of analytical methods rutendo kuwana training workshop: assessment of interchangeable...

Documents

drug slide

specificity slide

range slide

method trueness

types of analytical

assurance of reliability

cleaning procedures

accepted reference value