INTRODUCTION

The objective of any analytical measurement is to obtain consistent, reliable and accurate data. Validated analytical methods play a major role in achieving this goal. The results from method validation can be used to judge the quality, reliability and consistency of analytical results, which is an integral part of anygood analytical practice. Validation of analytical methods is also required by most regulations and quality standards that impact laboratories. Analytical methods need to be validated, verified, or revalidated in the following instances: o Before initial use in routine testing o When transferred to another laboratory o Whenever the conditions or method parameters for which the method has been validated change ,for example, an instrument with different characteristics or samples with a different matrix.
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If the change is outside the original scope of the method.

There is a wide variety of information and guidance available on analytical method validation, published as a result of work done in task forces and by private authors, a list of available data is presented as follows:.

The Laboratory of the Government Chemist (LGC) developed a guide for internal method validation. It includes a discussion of related laboratory accreditation requirements.

The United States Food and Drug Administration developed two industry guidelines: one for the validation of analytical methods2 and one for the validation of bioanalytical methods.

ICH published two guidelines for method validation. Q2A4 describes terminology and definitions for eight validation parameters that should be considered for validation. Q2B5 includes methodology but allows flexibility through the statement It is the responsibility of the applicant to choose the validation procedure and the protocol most suitable for their product. IUPAC6 published Harmonized Guidelines for Single-Laboratory Validation of Methods of Analysis. EURACHEM published a detailed guide for method validation. This is the most detailed official guide for theory and practice of method validation. It has been primarily developed for ISO/IEC accredited laboratories but because of its completeness it is also a good source for (bio)pharmaceutical laboratories.

Data Quality in Laboratories: VALIDATION

During method development, analysts establish the most suitable steps of the analytical process that will lead to the information required: sample pretreatment, when necessary, separation technique, and the detection system, among others. The best analytical conditions for obtaining good results are also considered. The information gathered after the analysis may have several goals: to take decisions involving the control of the manufacturing process of a product, to assess whether a product complies with regulatory limits, to take decisions about legal affairs, international trade, health problems or the environment, etc. Therefore, the analytical information must be of sufficient quality, which means that it must be reliable and match the purposes of the analysis. To meet these premises, analysts must define the purposes of the analysis and the requirements that the method should fulfil. Therefore, the validation of the method of analysis will provide, according to the ISO definition the confirmation by examination and provision of evidences that the particular requirements for a specified intended use are fulfilled .Another definition given in the Handbook for the Quality Assurance of Metrological Measurements states that method validation consists of documenting the quality of an analytical procedure, by establishing adequate requirements for performance criteria, such as accuracy, precision, detection limit, etc. and by measuring the values of these criteria . In general terms, then, the requirements and performance parameters must first be defined for every analytical method and purpose of analysis; and second, the
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value for these parameters must be estimated and checked to see if they really meet the criteria. This is an essential condition if the results provided are to be used. The process of assessing the performance criteria is closely related to the concept of fitness-for-purpose , which is defined by IUPAC in the Orange Book as the degree to which data produced by a measurement process enables a user to make technically and administratively correct decisions for a stated purpose . Hence, it is important, first, to consider the necessary conditions related to the problem at hand, second to choose the method of analysis that best fits the necessities, and, finally, to validate it as is shown in Figure below:

The validation process must satisfy three requirements 1) The whole method must be validated. It is quite usual to focus on the detection technique or the instrumental measurement, which often means that just this stage is validated. However, the previous steps of sample pre-treatment, extraction or preconcentration also belong to the method of analysis and are of utmost importance. So they must all be validated. 2) The whole range of concentrations must be validated. It is difficult to comply with this condition because a method may work very well in one particular concentration range but not in others. 3) The whole range of matrices must be validated. It is well known that the matrix can have a decisive effect on the analysis. Therefore, and for the sake of representativeness, several matrices must be submitted to method validation.

THERE ARE TWO TYPES OF PERFORMANCE CRITERIA: Primary and secondary. Precision, bias, accuracy, trueness and the detection limit belong to the first group while the other parameters that can influence these primary criteria belong to the second that is Linearity, The range of linearity, the quantification limit, selectivity, and sensitivity or ruggedness.

Parameters and Tests for Method Validation

Validation

of an analytical procedure is the process by which it is established,

by laboratory studies, that the performance characteristics of the procedure meet the requirements for the intended analytical applications. The required laboratory tests for method validation have been defined in different working groups of national and international committees and are described in the literature. Unfortunately, some of the definitions vary between the different organizations. Therefore, laboratories should have a glossary with definitions on their understanding of the terms. In an attempt to standardize, representatives from the industry and regulatory agencies from the United States, Europe and Japan defined parameters, requirements and methodology for analytical methods validation through the ICH.

Specificity/Selectivity
ICH defines specificity as the ability to assess unequivocally the analyte in the presence of components which may be expected to be present. Typically this might include impurities, degradants, matrix, etc.USP refers to the same definition but also comments that other reputable authorities such as IUPAC and AOAC use the term selectivity for the same meaning. This reserves the use of specific for those procedures that produce a response for a single analyte only. ISO/IEC most likely has the same understanding because it requires a method to be selective rather than specific. Our goal is to distinguish and quantify the response of the target compounds from the responses of all other compounds. Analytical techniques that can measure the analyte response in the presence of all potential sample components should be used for specificity validation. It is not always possible to demonstrate that a single analytical procedure is specific for a particular analyte. In this case a combination of two or more analytical procedures is recommended to achieve the necessary level of discrimination. A frequently used technique in pharmaceutical laboratories is high performance liquid chromatography (HPLC) and to some extent gas chromatography (GC). In practice, a test mixture is prepared that contains the analyte and all potential sample components. The result is compared with the response of the analyte. In pharmaceutical test mixtures, components can come from synthesis intermediates , excipients and degradation products. Generation of degradation products can be accelerated by putting the sample under stress conditions, such as elevated temperature, humidity or light. Specificity in liquid chromatography is obtained by choosing optimal columns and setting chromatographic conditions, such as mobile phase composition, column temperature and detector
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wavelength. Besides chromatographic separation, the sample preparation step can also be optimized for best selectivity. It is a difficult task in chromatography to ascertain whether the peaks within a sample chromatogram are pure or consist of more than one compound. The analyst should know how many compounds are in the sample which is not always possible. Therefore, the target compound peak should be evaluated for purity. Although in the past chromatographic parameters such as mobile phase composition or the column were modified, the current practice is the use of spectroscopic detectors coupled online to the chromatograph. UV-visible diodearray detectors and mass spectrometers acquire spectra on-line throughout the entire chromatogram. The spectra acquired during the elution of a peak are compared. If the spectra are different, the peak consists of at least two compounds. The principles of using spectral detectors for specificity evaluation is shown in Figure below using a high performance liquid chromatography (HPLC) UVvisible diode array detector as an example. The figure shows examples for a specific and non-specific chromatographic method. The two peaks look very similar. From the peak shape it is not obvious whether the peak consists of one or more compounds. For both examples UV spectra are taken at the peak upslope and downslope, normalized and compared. In the example on the left, the spectra are identical indicating that the peak consists of single compound, or the peak is spectrally pure. In the example on the right, the peak is clearly impure which is demonstrated by two different UV spectra. Modern diode array detectors compare the spectra automatically and print a match factor for each peak. This, in combination with the graphical visualization helps to assess selectivity without any additional workload.

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Precision

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ICH defines the precision of an analytical procedure as 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. Repeatability expresses the precision under the same operating conditions over a short interval of time. Repeatability is also termed intra-assay precision. Intermediate precision expresses variations within laboratories, such as different days, different analysts, different equipment, and so forth. Reproducibility expresses the precision between laboratories, collaborative studies usually applied to standardization of methodology. The ICH requires repeatability to be tested from at least six replications measured at 100 per cent of the test target concentration or from at least nine replications covering the complete specified range. For example, the results can be obtained at three concentrations with three injections at each concentration. Intermediate precision is determined by comparing the results of a method run within a single laboratory over a number of days. A methods intermediate precision may reflect discrepancies in results obtained from :

Different operators Inconsistent working practice Different instruments

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 Standards and reagents from different suppliers Columns from different batches a combination The objective of intermediate precision validation is to verify that in the same laboratory the method will provide the same results once the development phase is over. The objective of reproducibility is to verify that the method will provide the same results in different laboratories. The reproducibility of an analytical method is determined by analysing aliquots from homogeneous lots indifferent laboratories with different analysts. In addition, typical variations of operational and environmental conditions that may differ from, but are still within, the specified parameters of the method are used. Validation of reproducibility is important if the method is to be used in different laboratories. Factors that can influence reproducibility include differences in

Room temperature and humidity

Equipment with different characteristics: such as delay volume of an HPLC Columns from different suppliers or different batches

Operators with different experience and thoroughness.

Accuracy and Recovery

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ICH defines the accuracy of an analytical procedure as the closeness of agreement between the conventional true value or an accepted reference value and the value found. Accuracy can also be described as the extent to which test results generated by the method and the true value agree. The true value for accuracy assessment can be obtained in several ways. One alternative is to compare the results of the method with results from an established reference method. This approach assumes that the uncertainty of the reference method is known. Secondly, accuracy can be assessed by analysing a sample with known concentrations (for example, a control sample or certified reference material) and comparing the measured value with the true value as supplied with the material. If certified reference materials or control samples are not available, a blank sample matrix of interest can be spiked with a known concentration by weight or volume. After extraction of the analyte from the matrix and injection into the analytical instrument, its recovery can be determined by comparing the response of the extract with the response of the reference material dissolved in a pure solvent. Because this accuracy assessment measures the effectiveness of sample preparation, care should be taken to mimic the actual sample preparation as closely as possible. If validated correctly ,the recovery factor determined for different concentrations can be used to correct the final results.

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The concentration should cover the range of concern and should include concentrations close to the quantitation limit, one in the middle of the range and one at the high end of the calibration curve. Another approach is to use the critical decision value as the concentration point that must be the point of greatest accuracy. The ICH document on validation methodology recommends accuracy to be assessed using a minimum of nine determinations over a minimum of three concentration levels covering the specified range (for example, three concentrations with three replicates each).

Accuracy should be reported as per cent recovery by the assay of known added amount of analyte in the sample or as the difference between the mean and the accepted true value, together with the confidence intervals.

Linearity

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ICH defines linearity of an analytical procedure as its ability to obtain test results that are directly proportional to the concentration of analyte in the sample. Linearity may be demonstrated directly on the test substance by dilution of a standard stock solution or by separately weighing synthetic mixtures of the test product components. Linearity is determined by a series of five to six injections of five or more standards whose concentrations span 80120 per cent of the expected concentration range. The response should be directly proportional to the concentrations of the analytes or proportional by means of a well-defined mathematical calculation. A linear regression equation applied to the results should have an intercept not significantly different from zero. If a significant nonzero intercept is obtained, it should be demonstrated that this has no effect on the accuracy of the method. Frequently, the linearity is evaluated graphically, in addition to or as an alternative to mathematical evaluation. The evaluation is made by visually inspecting a plot of signal height or peak area as a function of analyte concentration. Because deviations from linearity are sometimes difficult to detect, two additional graphical procedures can be used. The first is to plot the deviations from the regression line versus the concentration or versus the logarithm of the concentration if the concentration range covers several decades. For linear ranges, the deviations should be equally distributed between positive and negative values. Another approach is to divide signal data by their respective concentrations, yielding the relative responses. A graph is plotted with the relative responses on the y-axis and the corresponding concentrations on the x-axis, on a log scale.
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The obtained line should be horizontal over the full linear range. At higher concentrations, there will typically be a negative deviation from linearity. Parallel horizontal lines are drawn on the graph corresponding to, for example, 95 per cent and 105 per cent of the horizontal line. The method is linear up to the point where the plotted relative response line intersects the 95 per cent line. Figure below shows a comparison of the two graphical evaluations using HPLC.

Plotting the sensitivity (response/amount) gives a clear indication of the linear

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range. Plotting the amount on a logarithmic scale has a significant advantage for wide linear ranges. Rc designates the line of constant response. ICH recommends the linearity curves correlation coefficient, y-intercept, slope of the regression line and residual sum of squares for accuracy reporting. A plot of the data should be included in the report. In addition, an analysis of the deviation of the actual data points from the regression line may also be helpful for evaluating linearity. Some analytical procedures, such as immunoassays, do not demonstrate linearity after any transformation. In this case, the analytical response should be described by an appropriate function of the concentration (amount) of an analyte in a sample. In order to establish linearity, a minimum of five concentrations is recommended. Other approaches should be justified.

Range

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ICH defines the range of an analytical procedure as the interval from the upper to the lower concentration 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 range of an analytical method is the interval from the upper to the lower levels (including these levels) that have been demonstrated to be determined with precision, accuracy and linearity using the method as written. The range is normally expressed in the same units as the test results (for example percentage, parts per million) obtained by the analytical method. For assay tests, ICH requires the minimum specified range to be 80 to120 per cent of the test concentration. It also requires the range for the determination of an impurity to extend from the limit of quantitation or from 50 per cent of the specification of each impurity, whichever is greater, to 120 per cent of the specification. Figure below shows graphical definition of linearity and range.

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Limit of Detection

ICH defines the detection limit of an individual analytical procedure as the lowest amount of analyte in a sample which can be detected but not necessarily quantitated as an exact value. The limit of detection (LOD) is the point at which a measured value is larger than the uncertainty associated with it. It is the lowest concentration of analyte in a sample that can be detected but not necessarily quantified. The limit of detection is frequently confused with the sensitivity of the method. The sensitivity of an analytical method is the capability of the method to discriminate small differences in concentration or mass of the test analyte. In practical terms, sensitivity is the slope of the calibration curve that is obtained by plotting the response against the analyte concentration or mass. In chromatography, the detection limit is the injected amount that results in a peak with a height at least two or three times as high as the baseline noise level. Besides this signal-to-noise method, there are three more methods: Visual evaluation: The detection limit is determined by the analysis ofsamples with known concentrations of analyte and by establishing the minimum level at which the analyte can be reliably detected.

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Standard deviation of the response based on the standard deviation of the blank: Measurement of the magnitude of analytical background response is performed by analysing an appropriate number of blank samples and calculating the standard deviation of these responses.

Standard deviation of the response based on the slope of the calibration curve A specific calibration curve is studied using samples containing an analyte in the range of the limit of detection. The residual standard deviation of a regression line, or the standard deviation of y-intercepts of regression lines, may be used as the standard deviation. Figure below illustrates the graphical evaluations of LOD and LOQ via signalto-noise.

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Limit of Quantitation

ICH defines the limit of quantitation (LOQ) of an individual analytical procedure as 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 issued particularly for the determination of impurities or degradation products. 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. If the required precision of the method at the limit of quantitation has been specified, 5 or 6 samples with decreasing amounts of the analyte are injected six times. The amounts range from the known LOD as determined above to 20 times the LOD. The calculated relative standard deviation (RSD) per cent of the precision of six repetitive injections is plotted against the analyte amount. The amount that corresponds to the previously defined required precision is equal to the limit of quantitation. It is important to use not only pure standards for this test but also spiked matrices that closely represent the unknown samples. Figure below shows required experimental steps and a typical graph.

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For chromatographic methods, the LOQ can also be determined through comparing measured signals from samples with known low concentrations of analyte with those of blank samples. This establishes the minimum concentration at which the analyte can be reliably quantified. A typical signalto-noise ratio is 10:1. Any results of limits of detection and quantitation measurements must be verified by experimental tests with samples containing the analytes at levels across the two regions. It is equally important to assess other method validation parameters, such as precision, reproducibility and accuracy, close to the limits of detection and quantitation.

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Ruggedness
Ruggedness is not addressed in the ICH documents. Its definition has been replaced by reproducibility, which has the same meaning. Ruggedness is defined by the USP as the degree of reproducibility of results obtained under a variety of conditions, such as different laboratories, analysts, instruments, environmental conditions, operators and materials. Ruggedness is a measure of the reproducibility of test results under normal, expected operational conditions from laboratory to laboratory and from analyst to analyst. Ruggedness is determined by the analysis of aliquots from homogeneous lots in different laboratories.

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Robustness
ICH defines the robustness of an analytical procedure as a measure of its capacity to remain unaffected by small, but deliberate variations in method parameters. It provides an indication of the procedures reliability during normal usage. Robustness tests examine the effect that operational parameters have on the analysis results. For the determination of a methods robustness, a number of method parameters, such as pH, flow rate, column temperature, injection volume, detection wavelength or mobile phase composition, are varied within a realistic range, and the quantitative influence of the variables is determined. If the influence of the parameter is within a previously specified tolerance, the parameter is said to be within the methods robustness range. Obtaining data on these effects helps to assess whether a method needs to be revalidated when one or more parameters are changed, for example, to compensate for column performance over time. In the ICH document ,it is recommended to consider the evaluation of a methods robustness during the development phase, and any results that are critical for the method should be documented.

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Stability
Chemical compounds can decompose prior to chromatographic investigations, for example, during the preparation of the sample solutions, extraction, clean up, phase transfer or storage of prepared vials (in refrigerators or in an automatic sampler). Under these circumstances, method development should investigate the stability of the analytes and standards. It is a measure of the bias in assay results generated during a preselected time interval, for example, every hour up to46 hours, using a single solution. Stability testing is important for estimating the allowed time span between sample collection and sample analysis. It is also important to evaluate an analytical methods ability to measure drug products in the presence of its degradation products. Experiments should be conducted under real sample storage conditions because the stability of drug substances is a function of the storage conditions, the chemical properties of the drug, the matrix, and the container system stability. The studies should evaluate the stability of the analytes during sample collection and handling after typical storage scenarios such as long term storage (when frozen at intended storage temperatures), short term storage (during a series of sample analyses at room temperature), and after freeze and thaw cycles. Conditions used in stability experiments should reflect situations likely to be encountered during actual sample handling, storage and analysis. All stability determinations should use a set of samples prepared from a freshly made stock solution of the analyte in the appropriate analyte-free, interference-free matrix. Stock solutions of the analyte for stability evaluation are prepared in an appropriate solvent at known concentrations. The stability of the stock solutions of the drug and the internal standard should be evaluated at room temperature for at least six hours. After completion of the desired storage
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time, the stability is tested by comparing the instrument response with that of freshly prepared solutions. System stability is determined by replicate analysis of the sample solution and calculation of the RSD of the responses. System stability is considered appropriate when the RSD does not exceed more than 20 per cent of the corresponding value of the short-term system precision. If the value is higher on plotting the assay results as a function of time, the maximum duration of the sample solution usability can be calculated. To force degradation, ICH also recommends conducting stress studies, in conditions such as elevated temperature, humidity or light to demonstrate the specificity of the assay in presence of degradation products. The goal is to generate typical degradation products that may be expected. As a rule of thumb, stress conditions should be selected so that 5-20 per cent of the drug substances are degraded. In addition, it is recommended to measure the stability under different freeze and thaw cycles, both short and long term. Below are example conditions suggested for bio analytical studies. Exact conditions depend on applicationspecific storage conditions. Freeze and Thaw Stability Analyte stability should be determined after three freeze and thaw cycles. At least three aliquots at each of the low and high concentrations should be stored at the intended storage temperature for 24 hours and thawed unassisted at room temperature.

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Short-Term Temperature Stability Three aliquots of each of the low and high concentrations should be thawed at room temperature and kept at this temperature from 4 to 24hours (based on the expected duration that samples will be maintained at room temperature in the intended study) and analysed. Long-Term Stability The storage time in a long-term stability evaluation should exceed the time between the date of first sample collection and the date of last sample analysis. Long-term stability should be determined by storing at least three aliquots of each of the low and high concentrations under the same conditions as the study samples. The concentrations of all the stability samples should be compared to the mean of back-calculated values for the standards at the appropriate concentrations from the first day of long-term stability testing. Stability of Processed Samples The stabilities of processed samples, including the resident time in the auto sampler, should be determined. The stability of the drug and the internal standard should be assessed over the anticipated run time for the batch size in validation samples by determining concentrations on the basis of original calibration standards.

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Method Validation Process

The validity of a specific method should be demonstrated in laboratory experiments using samples or standards that are similar to unknown samples analysed routinely. The preparation and execution should follow a validation protocol, preferably written in a step-by-step instruction format. The approach and steps to validate nonstandard analytical procedures and methods. The proposed procedure assumes that the type of instrument has been selected and the method has been developed. It meets criteria such as ease of use; ability to be automated and to be controlled by computer systems; costs per analysis; sample throughput; turnaround time; and environmental, health and safety requirements. As with equipment qualifications or computer system validations, method validation is not a single event. It begins when somebody wants to implement a new method in a laboratory and ends when the method is no longer in use. The process is broken down in phases because of the length of time and complexity. The process is illustrated in Figure below.

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A validation plan is developed, including owners, responsibilities and deliverables. The first step is to define the scope of the method. This includes the compounds with concentration range, the sample matrix, the specific equipment that should be used and the location where the method should be performed for sample analysis. Once we know what should be analysed we define performance characteristics, performance tests and acceptance criteria. Test protocols are then developed with all experimental details and the tests are executed according to protocols. Tests results are compared with acceptance criteria. Finally, routine method procedures are developed to verify continual system performance at the time of analysis. Tests may include system suitability testing and the analysis of quality control samples. All experimental conditions and validation results are documented in a validation report. Once tests and acceptance criteria have been defined, experiments fortesting should be thoroughly prepared, executed and documentedaccording to a validation protocol.

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Preparation
Good preparation work is important for efficient experiment execution. Most important are the use of qualified material, qualified equipment, trained people and well documented procedures. Any chemicals used to determine critical validation parameters, such as reagents and reference standards, should be available in sufficient quantities, accurately identified, sufficiently stable and checked for exact composition and purity according to specifications. Any other materials and consumables, for example, chromatographic columns, should be new and qualified to meet the columns performance criteria. This ensures that one set of consumables can be used for most experiments and avoid unpleasant surprises during method validation. Analytical equipment should be clearly defined, well characterized, qualified or calibrated to make sure that it meets the functional and performance specifications as required by the analytical method. The selected equipment should have average performance rather than selecting best performing equipment. Otherwise there may be problems with intermediate precision and reproducibility studies to meet acceptance criteria with different equipment. Operators should be sufficiently familiar with the technique and equipment. This will allow them to identify and diagnose unforeseen problems more easily and to run the entire process more efficiently.

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If there is little or no information on the methods performance characteristics, it is recommended that the suitability of the method be proven for its intended use either during development or in initial validation experiments. These studies should include the approximate precision, working range and detection limits. If the preliminary validation data appear to be in appropriate ,the method itself, the specifications and the equipment or the analysis technique should be changed. Method development and validation are, therefore, an iterative process. For example, in liquid chromatography, selectivity is achieved through the selection of mobile phase composition. For quantitative measurements, the resolution factor between two peaks should be 2 or higher. If this value is not achieved, the mobile phase composition needs further optimization. The influence of operating parameters such as pH, mobile composition, or flow rate on the methods performance should be assessed at this stage if this was not done during development and optimization of the method.

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Test Execution
There are no official guidelines for the sequence of validation testing. The optimal sequence may depend on the method itself. Based on the authors experience, for a liquid chromatographic method, the following sequence has proven to be useful: 1. Specificity/selectivity 2. Repeatability of retention times and peak areas 3. Linearity, limit of quantitation, limit of detection, range 4. Accuracy at different concentrations 5. Intermediate precision 6. Reproducibility The more time-consuming experiments, such as intermediate precision and reproducibility, are included towards the end. Some of the parameters, as listed under points 2-4, can be measured in combined experiments .For example, when the precision of peak areas is measured over the full concentration range, the data can be used to validate the linearity.

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System Suitability Tests

Once an analytical procedure has been validated the method can be transferred for routine use. However, to do so and to verify the validation status each time the analytical procedure is applied, system suitability tests are to be included in the written procedure. These tests are to be developed during the validation of the analytical method and are to measure parameters, which are critical to the conduct of the method, and the limits set should ensure the adequate performance of the analytical procedure. In the pharmacopoeias, general methods, when referenced in an individual monograph, have been demonstrated to be applicable to that substance. Revalidation is not required but only compliance to any prescribed system suitability criteria .These requirements are usually given in the general method unless otherwise prescribed in the individual monograph. System suitability criteria are limits applied to various analytical procedures designed to ensure their adequate performance. These criteria are to be fulfilled before and/or during the analyses of the samples. Failure to comply with system suitability criteria during an analytical run will render the results obtained un-useable and an investigation into the cause of the poor performance must be undertaken. Corrective action is to be taken before continuing the analysis. Compliance with the criteria of the system suitability tests will ensure transferability of the method and increase the reliability of the results obtained by better control of the procedure.

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Selectivity

COLUMN PERFORMANCE
A number of different approaches are valid to ensure the selectivity of the method. The column performance (apparent efficiency) with regard to selectivity may be calculated as the apparent number of theoretical plates (N).

The disadvantage of this method of indicating selectivity is that it varies depending on the stationary phase employed and the retention time of the analyte. It also varies with the usage or the extent of use of the stationary phase so this term is not very reliable as a measure of the separation to be achieved

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Resolution
The resolution is calculated from the following formula :

The resolution is calculated by using two closely eluting peaks (critical pair) usually of similar height, preferably corresponding to the substance itself and an impurity. When the elution times of the peaks are very different and the resolution is large (> 5.0) the use of the resolution factor as a performance test has little value. It is preferable to use another impurity or another substance, perhaps chemically related to the original substance, which will give a more meaningful resolution.

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Peak-To-Valley Ratio
When the chromatography is such that there is incomplete separation of the impurity and the availability of the impurity is restricted, the peak-to-valley ratio (p/v) can be employed to define the selectivity.

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Symmetry

Peak shape is an important contributor to the selectivity of the method. It is therefore necessary to include a system suitability criterion for symmetry, which is generally applicable to the appropriate reference solution either in the assay method or in the procedure for the test for related substances. In the latter case, the symmetry factor does not apply to the principal peak of the test solution since it will either be asymmetric due to overloading or it cannot be calculated because of detector saturation. Unless otherwise stated in the monograph, the symmetry factor should fall between 0.8 and 1.6 (a value of 1.0 signifies ideal symmetry).

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Retention Time/Relative Retention

Although not generally given as suitability requirements in monographs, for guidance the approximate retention time of the substance as well as the relative retention of the impurities, should be indicated. Nonetheless, it has been shown that, even when the selectivity requirements are fulfilled, there can be a wide variation in the retention time of the main component depending on the stationary phase employed . In a collaborative study to assess a liquid chromatographic method for the assay, and a related substances test for dicloxacillin sodium the retention times of the peak due to dicloxacillin were in the range 739 minutes! This is dramatic and will have an effect on the selectivity of the method. Such disparity in retention times between different columns when using an isocratic elution indicates that a resolution criterion is insufficient on its own and should be supplemented by an indicated retention time which should be within pre-defined limits .Although not considered as system suitability criteria, the expected retention time of the main component and the relative retentions of other compounds should be given for information.

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System Precision
System suitability criteria for system precision are included in the description of the assay procedures prescribed in specifications or in individual monographs. Often the test requires six replicate injections of the same solution and the RSD should not exceed 1.0 or 2.0 per cent. It was demonstrated, however, that there was no apparent link between the precision requirement and the limits set for the assay. The repeatability requirements were often incompatible with the assay limits given. As a result, the European Pharmacopoeia introduced system precision criteria which were dependent on the number of replicate injections performed and the reproducibility of the method. It had been proposed that maximum permitted relative standard deviations can be calculated for a system suitability requirement for precision, taking into account repeatability reported in inter-laboratory studies in a similar manner as had been described for setting assay limits . It was shown that a maximum RSD of 0.6 after six replicate injections was required to set a limit of 1.0 per cent for direct methods such as volumetric titration. For comparative assay methods this value is to be divided by 2. To assure the same level of precision if less replicates (n) are performed it is necessary to adjust the calculation

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The relationship between the number of replicate injections, the maximum permitted relative standard deviation and the upper content limit is given as follows: A* (%) 1.0 1.5 2.0 2.5 3.0 Maximum permitted related standard deviation (RSD max ) N=2 N=3 N=4 N=5 N=6 0.08 0.21 0.30 0.37 0.42 0.12 0.31 0.44 0.55 0.64 0.16 0.20 0.23 0.41 0.52 0.62 0.59 0.7 0.89 0.73 0.92 1.10 0.85 1.06 1.27

* A: Upper specification limit (%) 100

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Any decision to accept or reject analytical results should include assessment of the system suitability criteria to ensure that adequate precision is achieved. The maximum number of replicate injections to be performed is six but fewer may be performed provided the system precision RSD is equal to or less than RSD max given in the table for the appropriate number of injections. These levels of precision can be easily achieved with modern chromatographic equipment, provided that the concentration of the analyte is sufficiently above the quantitation limit, to reflect the Relationship between the number of injections, the maximum permitted relative standard deviation and the content limit injection precision.

REFERENCES
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LGC,

In-House

Method

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Guide

for

Chemical

Laboratories,2003.
2.

U.S. FDA Guidance for Industry (draft): Analytical Procedures and Methods Validation: Chemistry, Manufacturing, and Controls and Documentation, 2000. ICH Q2A, Validation of Analytical Procedures: Definitions and Terminology, Geneva, 1995, in 2005 incorporated in Q2 ICH Q2B, Validation of Analytical Procedures: Methodology, adopted in 1996, Geneva Q2B, in 2005 incorporated in Q2(R1).

3.

4. IUPAC Technical Report, Harmonized Guidelines for Single-Laboratory Validation of Methods of Analysis.
5.

Eurachem The Fitness for Purpose of Analytical Methods A Laboratory Guide to Method Validation and Related Topics, 1998.

6. L. Huber, Validation and Qualification in Analytical Laboratories, Informa Healthcare, New York, USA, 2007.
7.

AOAC, How to Meet ISO 17025 Requirements for Methods Verification, 2007

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