Validation of analytical methods in accordance with ICH guidelines Q2(R1)

This paper describes in detail the validation requirements of the mentioned ICH guideline suitable for a data package of a registration application. It is focused on chromatographic procedures.
LinkCitationEmailPrint FavoriteCollect this page 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 within the European Community, Japan and the USA. General validation characteristics: ICH Q2(R1) Accuracy Precision/repeatability Precision/intermediate precision Precision/reproducibility Specificity Detection limit Quantitation limit Linearity Range USP <1225> Accuracy Precision/repeatability Precision/intermediate precision Precision/reproducibility Specificity Detection limit Quantitation limit Linearity Range Robustness Robustness:

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 of system 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, is a 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 the drug. 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]

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 calculate the 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.

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 solutions as 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 system suitability 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 analytical performance 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.

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 minor component 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 maximum permitted 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 third component, 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 the method 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 smallest particle) particle size GC mesh support is acceptable, provided the chromatography meets the requirements of the system suitability.

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 is recommended 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 changed from 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 an appropriate 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 critical reagent, 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 analyses obtained 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 are usually 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;

 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 individual monograph 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 a series of injections of the reference solution using the following equation:

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

K B n t90%,n1

= = = =

constant (0.349), 6 injections, B= 1.0 upper limit given in the definition of the individual monograph minus 100 per cent; number of replicate injections of the reference solution (3 n 6); Students t at the 90 per cent probability level (double sided) with n1 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 related substances. Table 2.2.46.-1. Repeatability requirements Number of individual injections 3 4 5 Maximum permitted relative standard deviation 0.41 0.59 0.73 0.52 0.74 0.92 0.62 0.89 1.10

6 0.85 1.06 1.27

B (per cent) 2.0 2.5 3.0

in a related substances test, the limit of quantification (corresponding to a signal-to-noise ratio of 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

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 incorrect assignment 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 the monograph. 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 be necessary to achieve the prescribed system suitability requirements. With reversed-phase liquid chromatographic methods in particular, adjustment of the various parameters will not always result 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 contains information 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 adjusted by 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 value therefore 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.

Application volume: 10-20 per cent of the prescribed volume if using fine particle size plates (210 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 F2 l1

= = =

flow rate indicated in the monograph, in millilitres per minute; adjusted flow rate, in millilitres per minute; length of the column indicated in the monograph, in millimetres;

l2 d1 d2

= = =

length of the column used, in millimetres; internal diameter of the column indicated in the monograph, in millimetres; 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 account of 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 (t min) stated in the gradient table may be replaced by adapted time points (tc min), calculated using the following equation:

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

D D0 F

= = =

dwell volume, in millilitres; dwell volume used for development of the method, in millilitres; 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 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: no adjustment 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 F2 l1 l2 d1 d2

= = = = = =

flow rate indicated in the monograph, in millilitres per minute; adjusted flow rate, in millilitres per minute; length of the column indicated in the monograph, in millimetres; length of the column used, in millimetres; internal diameter of the column indicated in the monograph, in millimetres; internal diameter of the column used, in millimetres.

Temperature: 5 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. 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: length: 70 per cent; internal diameter: 50 per cent. 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. Detector wavelength: no adjustment permitted. Column parameters Stationary phase: particle size: maximum reduction of 50 per cent; no increase permitted (packed columns). Column dimensions:

 length: 70 per cent; 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 confidence interval 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 both methods; 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 <1010> 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

Accuracy / assay / drug product: option 1: matrix (placebo)-method application of the analytical method to synthetic mixtures of the drug product components (excipients,dye stuffs, preservatives, antioxidants, coating material, gelatin etc.) to which known quantities 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 medicinal products;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 known quantities 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 additional labwork 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 out the 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 <1010> 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 a convincing explanation regarding accuracy

Accuracy / impurities (quantitative) option 1: impurities are available add known amounts of impurities to drug substance or drug product (3 concentrations, 3 replicates per 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) and make 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 (exposure to 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 (active pharmaceutical 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;

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 as possible (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 and error 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 are not 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 the chromatogram 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 and specified unidentified related substances in the release - and shelf life specification < identification threshold, analyze the samples at the different time points of the stability study according to the specification and determine unknown related substances on the basis of the response factor of the reference substance of the active ingredient (as long as they are < identification threshold); if an unidentified related substance exceeds the identification threshold, it must be identified and so you have to complete the validation work for this special substance with option 1 (impurities available)

Precision/repeatability: Prerequisites: same lab, same method, same analyst, same analytical equipment, same day option 1: minimum of 9 individual determinations covering the specified range (3 concentrations, 3 replicates each); the easiest way is to use the results of the accuracy, so there is no additional labwork; calculate the (arithmetic) mean, the standard deviation, the variance, the relative standard

deviation, the confidence interval of the mean (two sided t-distribution, degrees of freedom f= n-1, significance level 5%)

option 2: minimum of 6 individual determinations at the 100% level (injection concentration as described in the analytical method);there is additional labwork; calculate the (arithmetic) mean, the standard deviation, the variance, the relative standard deviation, the confidence interval of the mean (two sided t-distribution, degrees of freedom f= n-1, significance level 5%)

Precision/intermediate precision: Prerequisites: same lab, same method, different analyst, different analytical equipment, different day (so, it is an intra lab precision); it is not necessary to investigate these effects individually; option 1: you need at least two data groups created by two different analysts on two different days using different HPLC (GC- etc.) systems; data group 1 is normally the one resulting from "precision/repeatability"; data group 2 is a new one; calculate for both groups separately the (arithmetic) mean, the standard deviation, the variance, the relative standard deviation, the confidence interval of the mean (two sided t-distribution, degrees of freedom f = n-1, significance level 5%); now conduct a F-test in order to demonstrate statistically comparable variances of the groups; if this test is o.k., then conduct a t-test in order to demonstrate that the deviation between the means is random option 2: if you decide to generate more than two data groups created by more than two different analysts on different days using different HPLC (GC- etc.) systems, you have to apply in principle the same procedure; the statistical evaluation differs; calculate for all groups separately the (arithmetic) mean, the standarddeviation, the variance, the relative standard deviation, the confidence interval of the mean (two sided t-distribution, degrees of freedom f = n-1, significance level 5%); now conduct a one factor ANOVA using all data groups; the outcome should be that all data groups belong to the same population and differ only randomly Precision/reproducibility: Prerequisites: different labs, same method, different analysts, different analytical equipment, diferent days (so, it is an inter lab precision); it is not necessary to investigate these effects individually; this test is not part of a marketing authorization dossier

Specificity: i recommend to start the validation work with "specificity" Verify that the analytical procedure is specific for the analyte under investigation; record chromatograms of: solvent, mobile phase, matrix (placebo/excipients), related substances, by products, degradation products (don't forget the stressed samples) establish acceptance criteria for critical separations (maybe together with tailing factors and number of theoretical plates) Detection limit: option 1: signal to noise ratio a signal to noise ratio of about 3:1 is considered as acceptable (more details and a picture is presented for instance in Ph.Eur. chapter 2.2.46) option 2: regression line and standard deviation detection limit (DL) is calculated as follows DL = (3.3 x sigma) / S S = slope of the calibration line (derived from linear regression) sigma = standard deviation of the response of replicate injections of a blank solution or sigma = residual standard deviation of the linear regression line or sigma = standard deviation of the y-intercepts of several regression lines Quantitation limit: option 1: signal to noise ratio a signal to noise ratio of about 10:1 is considered as acceptable (more details and a picture is presented for instance in Ph.Eur. chapter 2.2.46) option 2: regression line and standard deviationquantitation limit (QL) is calculated as follows QL = (10 x sigma) / S S = slope of the calibration line (derived from linear regression) sigma = standard deviation of the response of replicate injections of a blank solution or

sigma = residual standard deviation of the linear regression line or sigma = standard deviation of the y-intercepts of several regression lines Linearity: Evaluate a linear relationship between peak areas (or the ratio (peak area analyte) / (peak area internal standard)) and different concentrations of the analyte in the injection solution. Inject a minimum of 5 different concentrations, covering at least the specified range (not resulting from the same stock solution), measure the responses (peak areas or peak area ratios), calculate the regression line (method of least squares) and document slope and intercept. Calculate the correlation coefficient and the residual sum of squares and the residual standard deviation. Also submit a plot of the regression line (including confidence intervals) and a plot of the residues. Calculate the adequacy of the model (lack of fit, goodness of fit) in order to prove that the linear model represents the best fit and not a model of higher order. For this task conduct ANOVA or at least a Mandel test. Furthermore show that the matrix of the drug product has no impact on the slope or intercept of theregression line (constant - or proportional systematic errors), otherwise it would be necessary to calibrate with synthetic mixtures which contain the matrix components. And show that the confidence interval of the intercept includes the origin of the coordinate system in order to be allowed to apply a one point calibration for routine work instead of a regression line. Range: The specified range of an analytical method is derived from linearity studies. Within the specified rangethe analytical procedure has to provide linearity, accuracy and precision. The recommended minimum specified ranges should be: 80% to 120%, related to the injection concentration, described in the analytical method (for assay of a drug substance or a drug product); 70% to 130%, related to the injection concentration, described in the analytical method (for content uniformity testing); + / - 20% over the specified range, related to the injection concentration, described in the analytical method (for dissolution testing); reporting threshold to 120% of the specification, related to the injection concentration, described in the analytical method (for impurity testing).

Comments
Sign in to write a comment

Krishan Maggon Add References, Mass Spectrometry, Analytical standards and photos, graphics etc It is good start and glad to see ICH validation articles, I am sure you are going to add more stuff. Wait for your additions