Validation of Analytical Methods in Accordance With ICH Guidelines Q2

8/2/2019 Validation of Analytical Methods in Accordance With ICH Guidelines Q2

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Validation of analytical methods in

accordance with ICH guidelines Q2(R1)

This paper describes in detail the validation requirements ofthe mentioned ICH guideline suitable for a data package of a

registration application. It is focused on chromatographic

procedures.

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Validation of an analytical procedure to demonstrate that it is suitable for its intended purpose;

provides an assurance of reliability during normal (routine) use.

The described extent of validation work could be part of a registration application submitted withinthe

European Community, Japan and the USA.

General validation characteristics:

ICH Q2(R1) USP

Accuracy Accuracy

Precision/repeatability Precision/repeatability

Precision/intermediate precision Precision/intermediate precision

Precision/reproducibility Precision/reproducibility

Specificity Specificity

Detection limit Detection limit

Quantitation limit Quantitation limit

Linearity Linearity

Range Range

Robustness

Robustness:
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should be tested and verified during method development

it is defined as the capacity of an analytical procedure to remain unaffected by small variations in

method variables. One consequence of the evaluation of robustness should be that a series ofsystem suitability parameters is established to ensure that the validity of the analytical procedure is

maintained whenever used.

In principle the following variations are tested:

HPLC:

stationary phase (different columns of different lots and / or from different suppliers),

mobile phase (including pH value, if applicable),

temperature,

flow rate (gradient, if applicable)

GC:

stationary phase (different columns of different lots and / or from different suppliers),

temperature (program),

flow rate of carrier gas

SYSTEM SUITABILITY, recommended by USP

The resolution,R, isa function of column efficiency,N (number of theoretical plates), and is

specified to ensure that closely eluting compounds are resolved from each other, to establish the

general resolving power of the system, and to ensure that internal standards are resolved from thedrug. Column efficiency may be specified also as a system suitability requirement, especially if

there is only one peak of interest in the chromatogram; however, it is a less reliable means to

ensure resolution than direct measurement. Column efficiency is a measure of peak sharpness,which is important for the detection of trace components.

R = [2 (t2 - t1)] / [w1 + w2]


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Replicate injections of a standard preparation used in the assay or other standard solution 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 calculatethe relative standard deviation,SR, if the requirement is 2.0% or less; data from six replicate

injections are used if the relative standard deviation requirement is more than 2.0%.The tailing factor, T, a measure of peak symmetry, is unity for perfectly symmetrical peaks and its

value increases as tailing becomes more pronounced. In some cases, values less than unity may be

observed. As peak asymmetry increases, integration, and hence precision, becomes less reliable.
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The picture shows details for calculating an asymmetry factor according to Ph. Eur. The equation

is:

As = W 0.05 / 2 d

where W (0.05) is the peak width at 5% of peak height

These tests are performed by collecting data from replicate injections of standard or other solutionsas specified in the individual monograph. The specification of definitive parameters in a

monograph does not preclude the use of other suitable operating conditions. If adjustments of

operating conditions to meet system suitability requirements are necessary, each of the following is

the maximum variation that can be considered, unless otherwise directed in the monograph.Adjustments are permitted only when suitable standards (including Reference Standards) are

available for all compounds used in the suitability test and only when those standards are used to

show that the adjustments have improved the quality of the chromatography in meeting systemsuitability requirements. Adjustments to chromatographic systems performed in order to comply

with system suitability requirements are not to be made to compensate for column failure or system

malfunction. The changes described below may require additional validation data. The user should

verify the suitability of the method under the new conditions by assessing the relevant analyticalperformance characteristics potentially affected by the change. Multiple adjustments can have a

cumulative effect in the performance of the system and should be considered carefully before

implementation.

pH of mobile Phase (HPLC):The pH of the aqueous buffer used in the preparation of the mobile

phase can be adjusted to within 0.2 units of the value or range specified.
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Concentration of Salts in Buffer (HPLC):The concentration of the salts used in the preparation of

the aqueous buffer used in the mobile phase can be adjusted to within 10%, provided the

permitted pH variation (see above) is met.

Ratio of Components in Mobile Phase (HPLC):The following adjustment limits apply to minor

components of the mobile phase (specified at 50% or less). The amount(s) of these component(s)can be adjusted by 30% relative. However, the change in any component cannot exceed 10%

absolute (i.e., in relation to the total mobile phase). Adjustment can be made to one minorcomponent in a ternary mixture. Examples of adjustments for binary and ternary mixtures are

given below.

Binary Mixtures

specified ratio of 50:50:Thirty percent of 50 is 15% absolute, but this exceeds the maximumpermitted change of 10% absolute in either component. Therefore, the mobile phase ratio may be

adjusted only within the range of 40:60 to 60:40.

specified ratio of 2:98:Thirty percent of 2 is 0.6% absolute. Therefore the maximum allowed

adjustment is within the range of 1.4:98.6 to 2.6:97.4.

Ternary Mixtures

specified ratio of 60:35:5:For the second component, 30% of 35 is 10.5% absolute, which exceeds

the maximum permitted change of 10% absolute in any component. Therefore the second

component may be adjusted only within the range of 25% to 45% absolute. For the thirdcomponent, 30% of 5 is 1.5% absolute. In all cases, a sufficient quantity of the first component

is used to give a total of 100%. Therefore, mixture ranges of 50:45:5 to 70:25:5 or 58.5:35:6.5 to

61.5:35:3.5 would meet the requirement.

Wavelength of UV-Visible Detector (HPLC):Deviations from the wavelengths specified in themethod are not permitted. The procedure specified by the detector manufacturer, or another

validated procedure, is to be used to verify that error in the detector wavelength is, at most, 3 nm.

Column Length (GC, HPLC): can be adjusted by as much as 70%.

Column Inner Diameter (GC, HPLC): can be adjusted by as much as 25% for HPLC and 50%

for GC.

Film Thickness (Capillary GC): can be adjusted by as much as 50% to 100%.

Particle Size (HPLC): can be reduced by as much as 50%.

Particle Size (GC): going from a larger to a smaller or a smaller to a larger (if it is the same

Range Ratio, which is the diameter of the largest particle divided by the diameter of the smallestparticle) particle size GC mesh support is acceptable, provided the chromatography meets the

requirements of the system suitability.


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Flow Rate (GC, HPLC): can be adjusted by as much as 50%.

Injection Volume (GC, HPLC): can be reduced as far as is consistent with accepted precision and

detection limits.

Column Temperature (HPLC): can be adjusted by as much as 10 . Column thermostating isrecommended to improve control and reproducibility of retention time.

Oven Temperature (GC): can be adjusted by as much as 10%.

Oven Temperature Program (GC)Adjustment of temperatures is permitted as stated above.

For the times specified for the temperature to be maintained or for the temperature to be changedfrom one value to another, an adjustment of up to 20% is permitted.

Unless otherwise directed in the monograph, system suitability parameters are determined from the

analyte peak.

Relative retention times may be provided in monographs for informational purposes only, to aid in

peak identification. There are no acceptance criteria applied to relative retention times.

To ascertain the effectiveness of the final operating system, it should be subjected to suitability

testing. Replicate injections of the standard preparation required to demonstrate adequate system

precision may be made before the injection of samples or may be interspersed among sample

injections. System suitability must be demonstrated throughout the run by injection of anappropriate control preparation at appropriate intervals, including at the end of the analysis. The

control preparation can be a standard preparation or a solution containing a known amount of

analyte and any additional materials useful in the control of the analytical system, such as

excipients or impurities. Whenever there is a significant change in equipment or in a criticalreagent, suitability testing should be performed before the injection of samples. No sample analysis

is acceptable unless the requirements of system suitability have been met. Sample analysesobtained while the system fails system suitability requirements are unacceptable.

SYSTEM SUITABILITY, recommended by Ph. Eur.

The system suitability tests represent an integral part of the method and are used to ensure

adequate performance of the chromatographic system. Apparent efficiency, retention factor (mass

distribution ratio), resolution, relative retention and symmetry factor are the parameters that areusually employed in assessing the performance of the column. Factors that may affect the

chromatographic behavior include:

the composition, ionic strength, temperature and apparent pH of the mobile phase;

flow rate, column dimensions, column temperature and pressure;

stationary phase characteristics including type of chromatographic support (particle-based or

monolithic), particle or macropore size, porosity, specific surface area;


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reversed-phase and other surface-modification of the stationary phases, the extent of chemical

modification (as expressed by end-capping, carbon loading etc.).

The following requirements and any supplementary requirements given in the individualmonograph are to be fulfilled unless otherwise prescribed:

in a related substances test or assay, for a peak in the chromatogram obtained with a reference

solution used for quantification, the symmetry factor is 0.8 to 1.5, unless otherwise prescribed;

in an assay of an active substance where the value is 100 per cent for a pure substance, the

maximum permitted relative standard deviation (sr(%)max) for the defined limits is calculated for aseries of injections of the reference solution using the following equation:

sr(%) max = [K * B * (n)1/2] / t 90%, n-1

K = constant (0.349), 6 injections, B= 1.0B = upper limit given in the definition of the individual monograph minus

100 per cent;

n = number of replicate injections of the reference solution (3 n 6);

t90%,n1 = Students tat the 90 per cent probability level (double sided) withn1 degrees of freedom.

Unless otherwise prescribed, the maximum permitted relative standard deviation does not exceed

the appropriate value given in Table 2.2.46.-1. This requirement does not apply to tests for relatedsubstances.

Table 2.2.46.-1. Repeatability requirements

Number of individual injections

3 4 5 6

B (per cent) Maximum permitted relative standard deviation

2.0 0.41 0.59 0.73 0.85

2.5 0.52 0.74 0.92 1.06

3.0 0.62 0.89 1.10 1.27

in a related substances test, the limit of quantification (corresponding to a signal-to-noise ratioof 10) is equal to or less than the disregard limit.

Compliance with the system suitability criteria is required throughout the chromatographic

procedure. Depending on various factors, such as the frequency of use of the procedure and


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experience with the chromatographic system, the analyst chooses an appropriate verification

scheme to monitor this.

ADJUSTMENT OF CHROMATOGRAPHIC CONDITIONS

The extent to which the various parameters of a chromatographic test may be adjusted to satisfy

the system suitability criteria without fundamentally modifying the methods are listed below.Adjustment of conditions with gradient elutions is more critical than with isocratic elutions, since

it may lead to shifts in peaks to a different step of the gradient, thus leading to the incorrectassignment of peaks, and to the masking of peaks or a shift such that elution occurs beyond the

prescribed elution time. Changes other than those indicated require revalidation of the method. The

chromatographic conditions described have been validated during the elaboration of themonograph.

The system suitability tests are included to verify that the separation required for satisfactory

performance of the test or assay is achieved. Nonetheless, since the stationary phases are described

in a general way and there is such a variety available commercially, with differences in

chromatographic behavior, some adjustments of the chromatographic conditions may benecessary to achieve the prescribed system suitability requirements. With reversed-phase

liquid chromatographic methods in particular, adjustment of the various parameters will not alwaysresult in satisfactory chromatography. In that case, it may be necessary to replace the column with

another of the same type (e.g. octadecylsilyl silica gel), which exhibits the desired

chromatographic behavior. The Knowledge database on the EDQM website usually containsinformation on the column(s) used during monograph elaboration.

For critical parameters the adjustments are defined clearly in the monograph to ensure the system

suitability.

Thin-layer chromatography and paper chromatography

Composition of the mobile phase: the amount of the minor solvent component may be adjustedby 30 per cent relative or 2 per cent absolute, whichever is the larger; for a minor component at

10 per cent of the mobile phase, a 30 per cent relative adjustment allows a range of 7-13 per cent

whereas a 2 per cent absolute adjustment allows a range of 8-12 per cent, the relative valuetherefore being the larger; for a minor component at 5 per cent of the mobile phase, a 30 per cent

relative adjustment allows a range of 3.5-6.5 per cent whereas a 2 per cent absolute adjustment

allows a range of 3-7 per cent, the absolute value being the larger in this case; no other component

is altered by more than 10 per cent absolute.

pH of the aqueous component of the mobile phase: 0.2 pH, unless otherwise prescribed, or

1.0 pH when non-ionisable substances are to be examined.

Concentration of salts in the buffer component of a mobile phase: 10 per cent.


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Application volume: 10-20 per cent of the prescribed volume if using fine particle size plates (2-

10 m).

Liquid chromatography: isocratic elution

Composition of the mobile phase: the amount of the minor solvent component may be adjusted

by 30 per cent relative or 2 per cent absolute, whichever is the larger (see example above); no

other component is altered by more than 10 per cent absolute.

pH of the aqueous component of the mobile phase: 0.2 pH, unless otherwise prescribed, or 1.0 pH when non-ionisable substances are to be examined.

Concentration of salts in the buffer component of a mobile phase: 10 per cent.

Flow rate: 50 per cent; a larger adjustment is acceptable when changing the column dimensions(see the formula below).

Column parameters

Stationary phase:

no change of the identity of the substituent of the stationary phase permitted (e.g. no

replacement of C18 by C8);

particle size: maximum reduction of 50 per cent; no increase permitted.

Column dimensions:

length: 70 per cent;

internal diameter: 25 per cent.

When column dimensions are changed, the flow rate may be adjusted as necessary using the

following equation:

F2 = F1 * [(l2 * d2) / (l1 * d1)]

F1 = flow rate indicated in the monograph, in millilitres per minute;F2 = adjusted flow rate, in millilitres per minute;

l1 = length of the column indicated in the monograph, in millimetres;


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l2 = length of the column used, in millimetres;

d1 = internal diameter of the column indicated in the monograph, in millimetres;

d2 = internal diameter of the column used, in millimetres.

Temperature: 10 C, where the operating temperature is specified, unless otherwise prescribed.

Detector wavelength: no adjustment permitted.

Injection volume: may be decreased, provided detection and repeatability of the peak(s) to be

determined are satisfactory; no increase permitted.

Liquid chromatography: gradient elution

Adjustment of chromatographic conditions for gradient systems requires greater caution than for

isocratic systems.

Composition of the mobile phase/gradient elution: minor adjustments of the composition of the

mobile phase and the gradient are acceptable provided that:

the system suitability requirements are fulfilled;

the principal peak(s) elute(s) within 15 per cent of the indicated retention time(s);

the final composition of the mobile phase is not weaker in elution power than the prescribed

composition.

Where compliance with the system suitability requirements cannot be achieved, it is often

preferable to consider the dwell volume or to change the column.

Dwell volume. The configuration of the equipment employed may significantly alter the

resolution, retention time and relative retentions described. Should this occur, it may be due to

excessive dwell volume. Monographs preferably include an isocratic step before the start of the

gradient programme so that an adaptation can be made to the gradient time points to take accountof differences in dwell volume between the system used for method development and that actually

used. It is the users responsibility to adapt the length of the isocratic step to the analytical

equipment used. If the dwell volume used during the elaboration of the monograph is given in the

monograph, the time points (tmin) stated in the gradient table may be replaced by adapted timepoints (tc min), calculated using the following equation:

tc = t - [(D - D0) / F]


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D = dwell volume, in millilitres;

D0 = dwell volume used for development of the method, in millilitres;

F = flow rate, in millilitres per minute.

The isocratic step introduced for this purpose may be omitted if validation data for application of

the method without this step is available.

pH of the aqueous component of the mobile phase: no adjustment permitted.

Concentration of salts in the buffer component of a mobile phase: no adjustment permitted.

Flow rate: adjustment is acceptable when changing the column dimensions (see the formulabelow).

Column parameters

Stationary phase:

no change of the identity of the substituent of the stationary phase permitted (e.g. no

replacement of C18 by C8);

particle size: no adjustment permitted.

Column dimensions:



When column dimensions are changed, the flow rate may be adjusted as necessary using the

following equation:

F2 = F1 * [(l2 * d2) / (l1 * d1)]

F1 = flow rate indicated in the monograph, in millilitres per minute;F2 = adjusted flow rate, in millilitres per minute;

l1 = length of the column indicated in the monograph, in millimetres;l2 = length of the column used, in millimetres;

d1 = internal diameter of the column indicated in the monograph, in millimetres;

d2 = internal diameter of the column used, in millimetres.

Temperature: 5 C, where the operating temperature is specified, unless otherwise prescribed.


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Injection volume: may be decreased, provided detection and repeatability of the peak(s) to be

determined are satisfactory; no increase permitted.

Gas chromatography

Column parameters

Stationary phase:

particle size: maximum reduction of 50 per cent; no increase permitted (packed columns);

film thickness: 50 per cent to + 100 per cent (capillary columns).

Column dimensions:



Flow rate: 50 per cent.

Temperature: 10 per cent.

Injection volume and split volume: may be adjusted, provided detection and repeatability are

satisfactory.

Supercritical fluid chromatography

Composition of the mobile phase: for packed columns, the amount of the minor solvent

component may be adjusted by 30 per cent relative or 2 per cent absolute, whichever is the

larger; no adjustment is permitted for a capillary column system.


Column parameters

Stationary phase:

particle size: maximum reduction of 50 per cent; no increase permitted (packed columns).

Column dimensions:


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internal diameter:

25 per cent (packed columns);

50 per cent (capillary columns).

Flow rate: 50 per cent.

Temperature: 5 C, where the operating temperature is specified.

Injection volume: may be decreased, provided detection and repeatability are satisfactory; no

increase permitted.

Accuracy / assay / drug substance:

option 1: application of the method to an analyte of known purity

use for instance a primary reference standard (or equivalent quality) with a documented purity and

apply your method to it (3 x 3 separate weighings covering the range of your method, maybe 80 to

120% of injection concentration); calculate the mean, the standard deviation and the confidenceinterval of the mean(two sided t-distribution, significance level 1%);

the accuracy is verified, if the confidence interval contains the (true) value of the primary reference

standard

option 2: application of a second, well characterised (validated) method

3 x 3 samples of the drug substance, covering the range of the method; carry out the test with bothmethods; the outcome is two groups of data (9 results each); compare the two groups statistically

on the basis of F- and t-Test or an ANOVA test or follow the instructions given in the general

chapter of the USP

option 3: accuracy is established, if precision, linearity and specificity are verified

verify precision, linearity and specificity over the specified range of the method and give a

convincing explanation regarding accuracy


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Accuracy / assay / drug product:

option 1: matrix (placebo)-methodapplication of the analytical method to synthetic mixtures of the drug product components

(excipients,dye stuffs, preservatives, antioxidants, coating material, gelatin etc.) to which knownquantities of the drug substance (API) have been added (range: 80 to 120% of the labeled amount/

be aware of overages (for stability reasons or manufacturing reasons));a minimum of 9 determinations is required (3 concentrations, 3 replicates per concentration,

covering the specified range);

the results are reported as "percent recovery" (mean, standard deviation, coefficient of variation,confidence interval of the mean, in which the true "100" value should fall).

Note: the 9 recoveries should also be used for the "precision/repeatability" test, so no additional

labwork is necessary.

option 2: spiking method

the method of choice for instance for herbal drug preparations and related herbal medicinalproducts;option 1 is also possible (add the herbal drug preparation to synthetic mixtures of the

herbal medicinal product and conduct recovery experiments);the spiking is done by adding knownquantities of the marker substance or the constituents of known therapeutic activity (not the herbal

drug preparation itself, but individual components of it); prepare the samples according to the

instructions of the analytical procedure and make recovery experiments;

a minimum of 9 determinations is required (3 concentrations, 3 replicates per concentration,covering the specified range);

the results are reported as "percent recovery" (mean, standard deviation, coefficient of variation,

confidence interval of the mean, in which the true "100" value should fall).

Note: the 9 recoveries should also be used for the "precision/repeatability" test, so no additionallabwork is necessary.

option 3: application of a second, well characterised (validated) method

minimum of 6 individual samples of the drug product at 100% of the test concentration; carry outthe test with both method; the outcome is two groups of data (6 results each); compare the two

groups statistically on the basis of F- and t-Test or an ANOVA test or follow the instructions given

in the

general chapter of the USP

option 4: accuracy is established, if precision, linearity and specificity are verified

verify precision, linearity and specificity over the specified range of the method and give aconvincing

explanation regarding accuracy


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Accuracy / impurities (quantitative)

option 1: impurities are available

add known amounts of impurities to drug substance or drug product (3 concentrations, 3 replicatesper concentration, covering the range, usually from reporting threshold to 120% of the

specification of the individual impurity);

the results are reported as "percent recovery" (mean, standard deviation, coefficient of variation,confidence interval of the mean, in which the true "100" value should fall).

Note: the 9 recoveries should also be used for the "precision/repeatability" test, so no additional

labwork is necessary.

option 2: impurities are not available

application of a second, well characterised (validated) method and compare the results, obtained by

the two analytical methods.My personal proposal to proceed on this complex task:

As soon as the method is involved in a stability study, it has to be stability indicating. As a

consequence it has to be able to deal with known and unknown related substances and degradation

products. In my opinion an appropriate approach for the first step could be:make yourself familiar with the meaning of:

detection limit,

limit of decision,quantitation limit,

reporting threshold,

identification threshold,

qualification threshold;use the reference substance of the active pharmaceutical ingredient to establish detection limit,

limit of decision and quantitation limit (prerequisite: quantitation limit < reporting threshold) andmake an extra effort to sort it out and fill the categories:

1) what are reagent (solvent) peaks,

2) how does the chromatogram of the mobile phase look like (including the profile of a dradient, if

applicable),3) what are matrix (placebo) peaks,

4) what peaks are related to the API itself (related substances, resulting from synthesis),

5) what peaks are related to degradation products (generate them using stress conditions (exposureto light, heat, acid, base,oxidation, humidity))

as a second step i would strongly recommend to invest time in your office before using the

sophisticated and expensive resources of your laboratory;contact your supplier of the API (activepharmaceutical ingredient) to get as much information as possible, i.e. synthesis, by products,

intermediates, catalysts,solvents (potential residual solvents), stability of the API, potential

degradation products;


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now you should be able to fill category 4 and remember that there is no need during a stability

study to specify or monitor substances, related to the API, unless they are also degradation

products (but it is necessary to specify them in the chromatogram, if they appear, for instance "A1"for API peak 1 and "A2" for API peak 2,...);

furthermore use the scientific literature and the internet to learn as much about your molecule aspossible (stability, degradation products, behavior when exposed to light, oxygen, humidity,..)

before you start to fill category 5, in order to avoid an expensive and time consuming trial anderror procedure in your lab; become familiar with the molecule, find out, what reference

substances are commercially available, plan the stress tests in order to generate degradation

products (exposure to light, heat, acid, base,oxidation, humidity) and be aware that these tests arenot necessary, if degradation products are described in pharmacopoeias or scientific literature;

now proceed with categories 1, 2 and 3; at the end of this procedure you should be able to focus

your labwork for stability studies on the real degradation products; my recommendation at this

stage is (on the basis of your broad basis of knowledge on the molecule, the chromatographic

method and thechromatogram itself):

establish the specificity of the method (details later),establish relative retention times of the potential degradation products,

determine detection limit and quantitation limit (< reporting threshold) using the reference

substance of the active ingredient (details later),set an acceptance criterion for unspecified andspecified unidentified related substances in the release - and shelf life specification

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Validation of Analytical Methods in Accordance With ICH Guidelines Q2