2. 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.
3. 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.
4. 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
5. 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
6. 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 resuspension mainly for insoluble drug substance.
7. 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
8. 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.
9. 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.
10. 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.
11. 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.
12. 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
13. • 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;
14. 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).
15. • 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
16. • Validation parameters
• The objective of the analytical procedure should be clearly
understood since this will govern the validation characteristics
which need to be evaluated.
17. Characteristics that should be considered during
validation of analytical methods include:
specificity
linearity
range
accuracy
precision
détection
limit
quantitation
limit
robustness
18. 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.
19. 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.
20. • 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
21. 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.
22. 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
23. 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
24. 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
25. 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
26. 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.
27. 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.
28. 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
29. 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.
30. d.
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.
31. 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
33. 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.
34. 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.
35. 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 analyte
with 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.
36. 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.
37. 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.
38. 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.
39. 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.
40. 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
41. • 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.
42. Calculated analyte in mg/mL
Linearity
of an analyte in a material
Table of values (x,y)
x
0.040
#
0.035
Reference
material mg/ml
y
Calculated
mg/ml
1
0.0100
0.0101
2
0.0150
0.0145
3
0.0200
0.0210
4
0.0250
0.0260
5
0.030
0.0300
0.0294
6
0.0400
0.0410
0.025
0.020
0.015
0.010
0.01
0.015
0.02
0.025
0.03
Reference material mg/ml
0.035
0.04
43. 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
44. • 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;
45. 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.
46. 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.
47. • 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.
48. • In the case of gas-chromatography, examples of typical variations are:
1. different columns (different lots and/or suppliers);
2. temperature;
3. flow rate.
49. Table 1. Recommended Validation
Characteristics of the Various Types of Tests.
- signifies that this characteristic is not normally evaluated
+ signifies that this characteristic is normally evaluated
50. 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.
51. 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
52. 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.
53. • Identification:
no formal validation required;
• Testing for Impurities:
no formal validation required;
• Assay:
no formal validation required.
54. 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,
55. 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
.
56. • 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.
57. 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
58. 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
59. 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
60. 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)
61. • 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 .
62. • 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.
63. 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 .
64. • 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.
65. 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
66. • 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
67. • 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.
