67
Analytical Method and Validation Siham Abdoun Mohamed Msc ., PhD

Analytical method validation workshop

  • View
    1.095

  • Download
    12

Embed Size (px)

DESCRIPTION

 

Citation preview

Page 1: Analytical method validation workshop

Analytical Method and Validation

Siham Abdoun MohamedMsc., PhD

Page 2: Analytical method validation workshop

ANALYTICAL PROCEDURE

The analytical procedure refers to the way of performing the

analysis. It should describe in detail the steps necessary to perform

each analytical test. This may include but is not limited to: the

preparation of the sample, the reference standard and the reagents,

use of the apparatus, generation of the calibration curve, use of the

formulae for the calculation, etc.

Page 3: Analytical method validation workshop

Specifications:

A specification is defined as a list of tests, references to analytical procedures,

and appropriate acceptance criteria which are numerical limits, ranges, or other

criteria for the tests described. It establishes the set of criteria to which a new

drug substance or new drug product should conform to be considered acceptable

for its intended use. "Conformance to specifications" means that the drug

substance and / or drug product, when tested according to the listed analytical

procedures, will meet the listed acceptance criteria. Specifications are critical

quality standards that are proposed and justified by the manufacturer and

approved by regulatory authorities as conditions of approval.

Page 4: Analytical method validation workshop

Type of Test

1. General test

1. Description

2. Identification

3. Assay

4. Impurities

.2 Specific test; according to dosage form e.g.

Solid dosage form (tablets and hard gelatin capsules)

In addition to general the following specific tests are required

1. Dissolution

Page 5: Analytical method validation workshop

2. Disintegration

3.Hardness and friability (tablet)

4Uniformity of dosage unit

5. Water content

6. Microbial limit test

Oral liquids: One or more of the following specific tests will normally be applicable to oral liquids and to powders intended for reconstitution as oral liquids.

a) Uniformity of dosage units:

b) pH

c) Microbial limit test

Page 6: Analytical method validation workshop

d) Antimicrobial preservative content

Testing for antimicrobial preservative content should normally be

performed at release. Antimicrobial preservative effectiveness should be

demonstrated during development, and throughout the shelf-life

e) Antioxidant preservative content

f) Extractables

g) Dissolution: for oral suspensions and dry powder products for re-

suspension mainly for insoluble drug substance.

Page 7: Analytical method validation workshop

h) Particle size distribution: Quantitative acceptance criteria and a procedure for determination of particle size distribution may be appropriate for oral suspensions

i) Redispersibility: For oral suspensions which settle on storage (produce sediment),

h) Reconstitution time: Acceptance criteria for reconstitution time should be provided for dry powder products which require reconstitution.

j) Water content: For oral products requiring reconstitution,

Parenteral Drug Products: The following tests may be applicable to parenteral drug products.

a) Uniformity of dosage units: This term includes both the mass of the dosage form and the content of the active substance in the dosage form

Page 8: Analytical method validation workshop

b) pH: Acceptance criteria for pH should be provided where applicable and the proposed range justified.

c) Sterility: All parenteral products should have a test procedure and acceptance criterion for evaluation of sterility.

d) Endotoxins/Pyrogens: A test procedure and acceptance criterion for endotoxins, using a procedure such as the limulus amoebocyte lysate test, should be included in the specification. Pyrogenicity testing may be proposed as an alternative to endotoxin testing where justified.

e) Particulate matter: Parenteral products should have appropriate acceptance criteria for particulate matter. This will normally include acceptance criteria for visible particulates and / or clarity of solution, as well as for sub-visible particulates as appropriate.

Page 9: Analytical method validation workshop

f) Water content: For non-aqueous parenterals, and for parenteral products for

reconstitution, a test procedure and acceptance criterion for water content

should be proposed when appropriate.

g) Antimicrobial preservative content: For parenteral products needing an

antimicrobial preservative, acceptance criteria for preservative content should be

established.

i) Redispersibility: For injectable suspensions which settle on storage (produce

sediment),

h) Reconstitution time: Acceptance criteria for reconstitution time should be

provided for dry powder products which require reconstitution.

Page 10: Analytical method validation workshop

l) Particle size distribution: Quantitative acceptance criteria and a

procedure for determination of particle size distribution may be

appropriate e.g. for injectable suspensions.

i) Extractables: Control of extractables from container/closure systems is

considered significantly more important for parenteral products than for

oral liquids.

h) Antioxidant preservative content: Release testing for antioxidant

content should normally be performed.

Page 11: Analytical method validation workshop

Analytical procedure may be a pharmacopeia or non

pharmacopeia

Methods validation is the process of demonstrating that

analytical procedures are suitable for their intended use.

Validation is performed in order to show that the result(s)

generated by a particular analytical procedure are reliable

and accurate.

Page 12: Analytical method validation workshop

Types of Analytical Procedures to be Validated

• identification tests

• Assay of drug substances and pharmaceutical products

• Content of impurities and limit tests for impurities

• Dissolution testing and determination of particle size

• Uniformity of content of pharmaceutical products

Page 13: Analytical method validation workshop

• Definition of the types of tests considered in the validation

1. Identification tests: are intended to ensure the identity of

an analyte in a sample. This is normally achieved by

comparison of a property of the sample (e.g., spectrum,

chromatographic behavior, chemical reactivity, etc.) to

that of a reference standard;

Page 14: Analytical method validation workshop

2. Testing for impurities can be either a quantitative test or a

limit test for the impurity in a sample. Either test is intended

to accurately reflect the purity characteristics of the sample.

3. Assay procedures are intended to measure the analyte

present in a given sample it represents a quantitative

measurement of the drug substance in the drug product. The

same validation characteristics may also apply to assays

associated with other analytical procedures (e.g., dissolution).

Page 15: Analytical method validation workshop

•Method validation presentation:

1.Protocol: includes procedures and acceptance criteria

2.Report: documented results

3.Justification needed when non-pharmacopoeial methods are

used (if pharmacopoeial methods are available). Justification

to include data, e.g. comparisons with the pharmacopoeial or

other methods

4.Detailed standard test methods include:

chromatographic conditions, reagents and others

Page 16: Analytical method validation workshop

• Validation parameters

• The objective of the analytical procedure should be clearly

understood since this will govern the validation characteristics

which need to be evaluated.

Page 17: Analytical method validation workshop

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

specificity linearity range accuracy

precisiondétection

limitquantitation

limitrobustness

Page 18: Analytical method validation workshop

1/ SPECIFICITY

• Specificity is the ability to assess clearly the analyte in the presence

of components which may be expected to be present. Typically

these might include impurities, degradants, matrix, etc. as follow,

1. Identification: to ensure the identity of an analyte, Suitable

identification tests should be able to discriminate between

compounds of closely related structures which are likely to be

present.

Page 19: Analytical method validation workshop

2.Assay and Impurity Test(s)

• For chromatographic procedures, representative chromatograms should

be used to demonstrate specificity and individual components should

be appropriately labelled. Similar considerations should be given to

other separation techniques.

• In cases where a non-specific assay is used, other supporting analytical

procedures should be used to demonstrate overall specificity.

Page 20: Analytical method validation workshop

• 2.1 Impurities are available

• For the assay , this should involve demonstration of the discrimination

of the analyte in the presence of impurities and/or excipients;

practically, this can be done by spiking pure substances (drug

substance or drug product) with appropriate levels of impurities

and/or excipients

• For the impurity test, the discrimination may be established by spiking

drug substance or drug product with appropriate levels of impurities

Page 21: Analytical method validation workshop

2.2 Impurities are not available

If impurity or degradation product standards are unavailable, specificity may be demonstrated by comparing the test results of samples containing impurities or degradation products to a second well-characterized procedure e.g.: pharmacopoeial method or other validated analytical procedure (independent procedure).

This should include samples stored under relevant stress conditions: light, heat, humidity, acid/base hydrolysis and oxidation.

- for the assay, the two results should be compared;

- for the impurity tests, the impurity profiles should be compared.

Page 22: Analytical method validation workshop

2/ ACCURACY

Accuracy: is the degree of agreement of test results with the true

value, or the closeness of the results obtained by the procedure to

the true value.

Accuracy should be established across the specified range of the

analytical procedure

Page 23: Analytical method validation workshop

2.1. Assay

2.1.1 Drug Substance

Several methods of determining accuracy are available:

a) application of an analytical procedure to an analyte of known purity (e.g.

reference material);

b) comparison of the results of the proposed analytical procedure with those

of a second well-characterized procedure, the accuracy of which is stated

and/or defined

c) accuracy may be determined once precision, linearity and specificity have

been established

Page 24: Analytical method validation workshop

2.1.2 Drug Product

Several methods for determining accuracy are available:

a) application of the analytical procedure to synthetic mixtures of the

drug product components to which known quantities of the drug

substance to be analysed have been added;

b) accuracy may be inferred once precision, linearity and specificity

have been established

Page 25: Analytical method validation workshop

c) in cases where it is impossible to obtain samples of all drug

product components , it may be acceptable either to add known

quantities of the analyte to the drug product or to compare the

results obtained from a second, well characterized procedure, the

accuracy of which is stated and/or defined

Page 26: Analytical method validation workshop

2.2. Impurities (Quantitation)

Accuracy should be assessed on samples (drug substance/drug product)

spiked with known amounts of impurities.

In cases where it is impossible to obtain samples of certain impurities

and/or degradation products, it is considered acceptable to compare

results obtained by an independent procedure.

It should be clear how the individual or total impurities are to be

determined e.g., weight/weight or area percent, in all cases with

respect to the major analyte.

Page 27: Analytical method validation workshop

3/ PRECISION

The precision of an analytical procedure expresses the closeness of

agreement (degree of scatter) between a series of measurements

obtained from multiple sampling of the same homogeneous sample

under the prescribed conditions. Precision may be considered at three

levels: repeatability, intermediate precision and reproducibility.

The precision of an analytical procedure is usually expressed as the

variance, standard deviation or coefficient of variation of a series of

measurements.

Page 28: Analytical method validation workshop

3.1. Repeatability

Repeatability expresses the precision under the same operating conditions

over a short interval of time. Repeatability is also termed intra-assay

precision

Repeatability should be assessed using:

a) a minimum of 9 determinations covering the specified range for the

procedure (e.g., 3 concentrations/3 replicates) b) a minimum of 6

determinations at 100% of the test concentration

Page 29: Analytical method validation workshop

3.2. Intermediate precision

Intermediate precision expresses within-laboratories variations: it study

the effects of random events on the precision of the analytical procedure.

Typical variations to be studied include days, analysts, equipment, etc. It is

not considered necessary to study these effects individually.

Page 30: Analytical method validation workshop

3.3. Reproducibility

Reproducibility expresses the precision between laboratories

(collaborative studies, usually applied to standardization of

methodology) for inclusion of procedures in pharmacopoeias..

Validation of tests for assay and for quantitative determination of

impurities includes an investigation of precision.

d.

Page 31: Analytical method validation workshop

3.4. Recommended Data

The standard deviation, relative standard deviation (coefficient of

variation) and confidence interval should be reported for each type

of precision investigate

Page 32: Analytical method validation workshop

Inaccurate butprecise

Accurate butimprecise

Accurate and Precise

Page 33: Analytical method validation workshop

4/DETECTION LIMIT

The detection limit of an individual analytical procedure is the lowest

amount of analyte in a sample which can be detected but not

necessarily quantitated as an exact value.

Several approaches for determining the detection limit are possible,

depending on whether the procedure is a non-instrumental or

instrumental.

Page 34: Analytical method validation workshop

4.1. Based on Visual Evaluation

Visual evaluation may be used for non-instrumental methods but may

also be used with instrumental methods.

The detection limit is determined by the analysis of samples with

known concentrations of analyte and by establishing the minimum

level at which the analyte can be reliably detected.

Page 35: Analytical method validation workshop

4.2. Based on Signal-to-Noise

This approach can only be applied to analytical procedures which exhibit baseline noise.

Determination of the signal-to-noise ratio is performed by comparing measured signals from samples with known low concentrations of analytewith those of blank samples and establishing the minimum concentration at which the analyte can be reliably detected. A signal-to-noise ratio between 3 or 2:1 is generally considered acceptable for estimating the detection limit.

Page 36: Analytical method validation workshop

5/ QUANTITATION LIMIT

The quantitation limit of an individual analytical procedure is the

lowest amount of analyte in a sample which can be quantitatively

determined with suitable precision and accuracy. The quantitation

limit is a parameter of quantitative assays for low levels of

compounds in sample matrices, and is used particularly for the

determination of impurities and/or degradation products.

Page 37: Analytical method validation workshop

5.1. Based on Visual Evaluation

Visual evaluation may be used for non-instrumental methods but may

also be used with instrumental methods.

The quantitation limit is generally determined by the analysis of

samples with known concentrations of analyte and by establishing the

minimum level at which the analyte can be quantified with acceptable

accuracy and precision.

Page 38: Analytical method validation workshop

5.2. Based on Signal-to-Noise Approach

This approach can only be applied to analytical procedures that exhibit

baseline noise.

Determination of the signal-to-noise ratio is performed by comparing

measured signals from samples with known low concentrations of

analyte with those of blank samples and by establishing the minimum

concentration at which the analyte can be reliably quantified. A typical

signal-to-noise ratio is 10:1.

Page 39: Analytical method validation workshop

5.3. Based on the Standard Deviation of the Response and the Slope

The quantitation limit (QL) may be expressed as:

5.4 Recommended Data

The quantitation limit and the method used for determining the quantitation

limit should be presented.

The limit should be subsequently validated by the analysis of a suitable number

of samples known to be near or prepared at the quantitation limit.

Page 40: Analytical method validation workshop

8/ LINEARITY

• 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.

• It may be demonstrated directly on the drug substance (by dilution of a

standard stock solution) and/or separate weighing's of synthetic

mixtures of the drug product components, using the proposed

procedure.

• For the establishment of linearity, a minimum of 5 concentrations is

recommend

Page 41: Analytical method validation workshop

• Linearity should be evaluated by visual inspection of a plot of signals as

a function of analyte concentration or content. If there is a linear

relationship, test results should be evaluated by appropriate statistical

methods, for example, by calculation of a regression line by the method

of least squares.

• In some cases, to obtain linearity between assays and sample

concentrations, the test data may need to be subjected to a

mathematical transformation prior to the regression analysis.

• Data from the regression line itself may be helpful to provide

mathematical estimates of the degree of linearity.

Page 42: Analytical method validation workshop

Table of values (x,y)

xy

# Reference

material mg/mlCalculated

mg/ml

1 0.0100 0.0101

2 0.0150 0.0145

3 0.0200 0.0210

4 0.0250 0.0260

5 0.0300 0.0294

6 0.0400 0.04100.010

0.015

0.020

0.025

0.030

0.035

0.040

0.01 0.015 0.02 0.025 0.03 0.035 0.04

Calc

ula

ted

an

aly

te i

n m

g/m

L

Reference material mg/ml

Linearity of an analyte in a material

Page 43: Analytical method validation workshop

8./ RANGE

• 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.

• The specified range is normally derived from linearity studies and depends

on the intended application of the procedure. It is established by

confirming that the analytical procedure provides an acceptable degree of

linearity, accuracy and precision when applied to samples

Page 44: Analytical method validation workshop

• The following minimum specified ranges should be considered:

1. For the assay of a drug substance or a finished (drug) product: normally

from 80 to 120 percent of the test concentration;

2. For content uniformity, covering a minimum of 70 to 130 percent 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;

3. For dissolution testing: +/-20 % over the specified range;

Page 45: Analytical method validation workshop

4. For the determination of an impurity: from the reporting level of

an impurity1 to 120% of the specification;

for validation of impurity test procedures carried out during

development, it may be necessary to consider the range around a

suggested (probable) limit.

5. 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 impurities1 to 120% of the assay specification.

Page 46: Analytical method validation workshop

9/ ROBUSTNESS

The robustness of an analytical procedure is a measure of its capacity

to remain unaffected by small, but considered variations in method

parameters and provides an indication of its reliability during normal

usage.

Depends on the type of procedure under study. It should show the

reliability of an analysis with respect to deliberate variations in

method parameters.

Page 47: Analytical method validation workshop

• Examples of typical variations are:

1. stability of analytical solutions;

2. extraction time.

• In the case of liquid chromatography, examples of typical variations are:

1. influence of variations of pH in a mobile phase;

2. influence of variations in mobile phase composition;

3. temperature;

4. flow rate.

Page 48: Analytical method validation workshop

• In the case of gas-chromatography, examples of typical variations are:

1. different columns (different lots and/or suppliers);

2. temperature;

3. flow rate.

Page 50: Analytical method validation workshop

9. SYSTEM SUITABILITY TESTING

System suitability testing is an integral part of many analytical

procedures. The tests are based on the concept that the equipment,

electronics, analytical operations and samples to be analyzed

constitute an integral system that can be evaluated as such.

Page 51: Analytical method validation workshop

CATEGORIES OF ANALYTICAL PROCEDURE AND VALIDATION REQUIRMENT

The following categories of analysis are considered

1. Transfer of a method

2. Screening

3. Development of a new analytical procedure

Page 52: Analytical method validation workshop

1. Transfer of a Method

• In this category validation data for this particular analysis is already available,

so verification of suitability under conditions of use (=method transfer

check)is required

1.1. Pharmacopoeial (compendial) method.

1.1.1 Active substance

The analytical procedures described in a monograph of a pharmacopoiea are

considered to be validated. In this case only system suitability tests are

required.

Page 53: Analytical method validation workshop

• Identification:

no formal validation required;

• Testing for Impurities:

no formal validation required;

• Assay:

no formal validation required.

Page 54: Analytical method validation workshop

1.1.2 Medicinal product

The pharmacopoeial monograph for a specific dosage form is a good

basis for the analysis; however as in many cases there is no indication

about the exact composition of the product (qualitative and quantitative

composition of the excipients), it must at least be made sure that these

do not interfere in the analysis of the active substance,

Page 55: Analytical method validation workshop

The Validation characteristic required in each type of analysis:

• Identification:

no formal validation required;

• Testing for Impurities:

1.specificity: no interference from excipients;

2.the limit of quantification

.

Page 56: Analytical method validation workshop

• Assay:

1.specificity,

2.accuracy: mainly recovery, minimum 1 determination.,

3.precision (repeatability): around the target test concentration

(minimum 2 independent determinations)

4.linearity at three measuring points in the range around the target

value.

Page 57: Analytical method validation workshop

1.2 Method of a manufacturer.

1.2.1: the analytical procedures have been fully validated by the company

• The requirement under pharmacopeia active substance applies for both the

active substance and the medicinal product:

no validation is required for Identification, Testing for Impurities

and Assay:

1.2.2: old application file with no or insufficient validation data This case

should be notified to the authorities. For the validation characteristics to be

considered

Page 58: Analytical method validation workshop

1.3 Non compendial published method.

The validation characteristics to be considered will always depend on the

amount of validation data provided. If the method has been fully validated

and data published in the literature, the same of pharmacopeial (active

substance and medicinal product) is applied. If not, the following has to

be considered:

Identification:

no formal validation required

Page 59: Analytical method validation workshop

1. specificity;

• Testing for Impurities:

1. limit of quantitation;

2. precision/accuracy over the range.

• Assay:

1. specificity: no interference from excipients

2. accuracy: around the target concentration

3. repeatability: around the target concentration (minimum 2 independent determinations)

4. linearity at three measuring points in the range

Page 60: Analytical method validation workshop

1.4 Method of a first manufacturer to be used for a product of a 2nd

manufacturer.

1.4.1 Active substance:

• Identification:

no formal validation required

• Testing for Impurities:

specificity (impurity profile) (if the impurity profile is different, further

validation data might be necessary)

Page 61: Analytical method validation workshop

• Assay:

• no formal validation required in case of a titration;

• Stability indicating: see testing for impurities.

1.4.2 Medicinal product:

A prerequisite is, that we have here comparable formulations (matrix):

• Identification:

no formal validation required

• Testing for Impurities:

1.Specificity (interference of excipients);

2. limit of quantitation;

3.precision/accuracy over the range .

Page 62: Analytical method validation workshop

• Assay:

1. specificity: no interference from excipients

2. accuracy: around the target concentration

3. repeatability: around the target concentration (minimum 2 independent

determinations)

4. linearity at three measuring points in the range around the target value.

Page 63: Analytical method validation workshop

1.5 Method for an active substance to be used for a medicinal product.

The main factor to be considered here is the influence of the matrix on

the analysis including interference from the excipients.

• Identification:

no formal validation required

• Testing for Impurities:

1. specificity;

2. limit of quantitation;

3. precision/accuracy over the range .

Page 64: Analytical method validation workshop

• Assay:

1. specificity: no interference from impurities and excipients

2. accuracy: around the target concentration

3. repeatability: around the target concentration (minimum 2

independent determinations)

4. linearity at three measuring points in the range around the target

value.

Page 65: Analytical method validation workshop

2. Development of a new analytical procedure

This is mainly the case where a product is tested in routine testing

conditions and/or where an in-house analytical procedure is used.

The analytical procedures should be fully validated

Page 66: Analytical method validation workshop

• REVALIDATION

• Revalidation is required when

1. changes in the process for synthesis of the drug substance

2. changes in the composition of the finished product

3. changes in the analytical procedure

4. transfer of methods from one laboratory to another

5. changes in major pieces of equipment, instruments

Page 67: Analytical method validation workshop

• Revalidation should be performed to ensure that the analytical

procedure maintains its characteristics (e.g., specificity) and to

demonstrate that the analytical procedure continues to ensure

the identity, strength, quality, purity, and potency of the drug

substance and drug product, and the bioavailability of the drug

product. The degree of revalidation depends on the nature of

the change.