25/06/12 Module 1, Part 4 focuses on Quality Control-related validation . The suggested time for Part 4 is: 60-90 minutes. (Note for the trainer: the times noted are very approximate.)
25/06/12 Introduction : Analytical monitoring of a pharmaceutical product, or of specific ingredients within the product, is necessary to ensure its safety and efficacy throughout all phases of its shelf-life, including storage, distribution, and use. This monitoring should be conducted in accordance with specifications validated during product development. The principal purpose of analytical validation is to ensure that a selected analytical procedure will give reproducible and reliable results that are adequate for the intended purpose. It is necessary to define properly both the conditions in which the procedure is to be used and the purpose for which it is intended. These principles apply to all procedures described in a pharmacopoeia and to non-pharmacopoeia procedures used by a manufacturing company. These guidelines apply to procedures used to examine chemical and physicochemical attributes, but many are equally applicable to microbiological and biological procedures.
25/06/12 Extent of validation required: New (from manufacturer/literature) methods require complete validation. Methods in pharmacopoeias require partial validation, if the method has not been previously validated for that specific drug product. Manufacturers should validate pharmacopoeial methods to ensure they work with their own products - as a minimum accuracy and specificity. The USP monograph states: “Already established general assays and tests - should also be validated to verify their accuracy (and absence of possible interference) when used for a new product or starting materials.” At least partial revalidation is required whenever significant changes are made which could reasonably be expected to affect the results obtained, e.g. in case of instrument change, product formula change, changed suppliers of critical reagents, method.
25/06/12 The relationship between accuracy and precision can be represented by arrows being shot at a target. The first small target at the top shows the arrows have landed indiscriminately. This is neither accurate nor precise. The second target on the left shows the arrows have grouped together nicely but are not on the bullseye. This is precise but inaccurate. This is sometimes called analytical bias and sometimes a correction factor can be applied. The third, small target shows the arrows AVERAGE is on the bullseye, but the precision is unacceptable. The fourth, large target shows the arrows are all clustered on or in the bullseye; this shows accuracy and precision.
25/06/12 Characteristics of analaytical procedures: (Contd) Ruggedness and Robustness Robustness, and ruggedness, of an analytical procedures is a measure of its capacity to remain unaffected by small, but deliberate variations in method parameters, and thus provides an indication of the reliability of the method during normal usage, under various conditions. Ruggedness is due to factors external to the method; robustness is due to factors internal to the method. Things that may cause variability include: Day-to-day variations in e.g. temperature, relative humidity, etc. Analyst-to-analyst Laboratory-to-laboratory Instrument-to-instrument Chromatographic column-to-column Reagent kit-to-kit or lot-to-lot variation Time from sample preparation to assay Instability of analytical reagents
25/06/12 Following a system suitability test, the actual analytical method is then validated by checking: Specificity: by checking that the method is free of interference from excipients, impurities, etc. Accuracy: by checking that the method gives closeness to true results. Precision: by checking that the method is precise. Linearity: by checking that the method will produce results that are directly proportional to the concentration of analyte in the samples. Robustness: by checking that the method will withstand deliverate changes.
25/06/12 The system suitability tests are carried out during the method development phase, prior to method validation. These tests are designed to evaluate the performance of the entire system. It is done by analysing a “system suitability” sample, which consists of the main components, including impurities. This may also contain excipients, which may interfere with peaks of interest. The system suitability is evaluated in terms of the following parameters: - system precision - column efficiency (usually >2000) - symmetry factor (acceptance criteria 0.9 to 2.5) - capacity factor (acceptance criteria NLT 1.5)
analytical method validation and validation of hplc
SEMINAR ONAnalytical Method Validation& Validation of HPLC• GUIDE: • Presented by: MR. Ishaq Ahmed T.VENKATESH (Asst. Proff) M. Pharmacy (pharmaceutics) Sri Kakatiya Institute of Pharmaceutical Science.
CONTENTS• INTRODUCTION• PARAMETERS FOR METHOD VALIDATION -AS PER USP/BP -AS PER ICH• VALIDATION OF HPLC -TYPICAL HPLC DESIGN -VALIDATION PARAMETERS• CONCLUSION• REFERENCES
INTRODUCTION Validation Establish a documented evidence which provides a high degree of assurance that a specific process will consistently produce a product meeting its predetermined specifications and quality attributes Analytical Validation The principle purpose of analytical validation is to ensure that the selected analytical procedure will give reproducible and reliable results that are adequate for the intended purpose.
Why validation is necessary? It is an important element of quality control. Validation helps provide assurance that a measurement will be reliable. In some fields, validation of methods is a regulatory requirement.
When is validation needed? Before introduction of a new method in to routine use. Whenever condition change for which method has been validation e.g. instrument with different characteristics. Whenever the method is changed and the change is outside the scope of the original method
When Revalidation To Be Done? • Equipment changes • Formula changed • Changed suppliers of critical reagents
Parameters For Method ValidationComparison BP/USP/ICH As per USP and BP As per ICH
AccuracyAccuracy The accuracy is the closeness of the test results obtained by themethod to he true value. Accuracy should be established across its range. Accuracy assessed using a minimum of 9 determinations over a minimum of 3 concentration levels
Precision Precision : The precision of an analytical method is the degree of agreement between a series of measurements obtained from multiple sampling of the same homogeneous sample. Repeatability : Repeatability expresses the precision under the same operating conditions over a short interval of time. Repeatability is also termed intra-assay precision . a minimum of 9 determinations covering the specified range for the procedure ( e.g., 3 concentrations/3 replicates each); or a minimum of 6 determinations at 100% of the test concentration.
PrecisionIntermediate Precision: Intermediate precision expresses within-laboratories variations, different days, different analysts, different equipment, etc.Reproducibility: Reproducibility expresses the precision between laboratories (collaborative studies, usually applied to standardization of methodology).
Relationship between Accuracy and Precision Inaccurate & impreciseInaccurate but Accurate but Accurate AND Precise precise imprecise
Linearity The linearity of an analytical procedure is its ability to obtain test results that are directly proportional to the concentration of the analyte in the sample. Linearity is usually demonstrated by the analysis of various concentrations of the analyte (s) across the indented range and represented graphically. A statistical analysis of the data is usually required, such as the calculation of a regression line using the method of least square . A minimum of 5 concentration is recommended.
Range Range of the analytical procedure is the interval between the upper and the lower concentration of the analyte for which it has been demonstrated that the analytical procedure has a suitable precision, accuracy and linearity. For assay the range is usually not less than 80 to 120% of the test concentration. For determination of content uniformity the range is usually not less than 70 to 130% or the test concentration. For determination of impurities the range is usually not less than the reporting limit of the impurity to 120% or the specification. For dissolution testing the range is usually ±20% over the expected concentration.
Specificity/Selectivity The ability to assess unequivocally the analyte in the presence of components that may be expected to be present. – Impurities – degradants – excipients Specificity must be demonstrated for: – Identification – Impurities Test – Assay Test
Detection limit (limit of detection) Definition : Limit of Detection is the smallest quantity of an analyte that can be detected, but not necessarily quantified. Approaches to calculation :• visual evaluation• signal to noise ratio• standard deviation of the response and the slope of the calibration curve
Calculation 1. visual evaluation- DL is determined by the analysis of a series of samples with known concentrations and establishing the minimum level at which the analyte can be reliably detected. 2. signal to noise ratio- For instrumental procedures that exhibit background noise, it is common to compare measured signals from samples with known low concentrations of analyte with those of the blank samples. The minimum concentration at which the analyte can reliably be detected is established using an acceptable signal - to - noise ratio of 2 : 1 or 3 : 1. 3. standard deviation of the response and the slope of the calibration curve DL=3σ/S where σ is the standard deviation of the response and S is the slope of the calibration curve
Limit of Quantitation• The quantitation limit is the lowest amount of analyte in a sample which can be quantitatively determined with suitable precision and accuracy.• Used particularly for the determination of impurities and/or degradation products.
Limit of Quantitation Various approaches of determining the Quantitation Limits are- Based on visual evaluation- Based on signal-to-noise- Based On Standard Deviation Of Response And Slope DL= 10 σ/S σ = the standard deviation of the response S = the slope of the calibration curve LOQ vary with detector sensitivity. Lamp aging, different manufacturer of detector.
Ruggedness Ruggedness: The ruggedness of an analytical method is the degree of reproducibility of the test results obtained by the analysis of the same samples under a variety of conditions, such as Day-to-day variations Analyst-to-analyst Laboratory-to-laboratory Instrument-to-instrument Chromatographic column-to-column Reagent kit-to-kit Instability of analytical reagents
Robustness The robustness of an analytical procedure is a measure of its capacity to remain unaffected by small but deliberate variations in method parameters and provides an indication of its reliability during use.
VALIDATION OF HPLCThe goal of equipment validation is to produce constant result withminimal variation with out compromising the product and performance ofequipment.
Qualification Qualification is a subset of the validation process that verifies module and systemperformance prior to the instrument being placed on-line.
DesignQualification(DQ) •For setting the functional and performance specifications •DQ can be very simple for similar equipment e.g. just another HPLC system
InstallationQualification(IQ)•for performing anddocumenting theinstallation in the selecteduser environment•safety, servicerequirements
OperationalQualification(OQ)•For testing theequipment in theselected userenvironment to ensure itmeets our definedfunctional andperformancespecifications
PerformanceQualification(PQ)•For testing that the systemconsistently performs as intendedfor the selected application•Periodic calibration /maintenance•Must be signed back by user•May use Method SystemSuitability Checks as part of PQ
System suitability The simplest form of an HPLC system suitability test involves a comparison of chromatogram trace with a standard trace. This allows a comparison of the peak shape, peak width, baseline resolution. Parameters to be calculated to provide a system suitability test report.
System Suitability Number of theoretical plates (efficiency) Capacity factor Separation (relative retention) Resolution Tailing factor Relative standard deviation These are measured on a peak or peaks of known retention time and peak width.
Retention Factor or capacity factor •The capacity factor is a measure of how long each component is retained on the column. • k is used in preference to retention time. •Generally the value ok K’ is > 2. •Inpractice the k value for the first peak of interest should be >1 to assure that it is separated from solvent. •Hear Tr is retention time of peak of interest & T0 is unretained peaks retention time.
Relative Retention/ Separation•This describes the relative position of two adjacent peaks. Ideally it iscalculated using the capacity factor•Because the peak separation depends on the components interction withthe stationary phase.
Number of theoretical plate •Thisis a measure of sharpness of the peaks and therefore the efficiency of the column that is , how many peaks can be located per unit run time of the chromatogram. •This cam be calculated in various ways. •ex: USP uses the peak width at base and BP at half the height. •The theoretical plate number depends on elution time but in general should be > 2000
Tailing factor T • This is a measure for asymmetry of peak. • Peak asymmetry is measured at 5% of full peak height.
Peak Resolution R• This is not only a measure of separation between twopeaks, but also the efficiency of the column.• It is expressed as ratio of the distance between the twopeak maxima to mean value of peak width.
Precision• Injection repeatability (i.e. 6)• If RSD (relative standard deviation) of ≤ 2% is required then 5 replicate injections should be used.• Data from six injections are used if the RSD is more than 2.0%
CONCLUSION This summarizes the validation parameters that are required according to the requirement of ICH/USP&BP. Summarized the extent, need & necessary of validation. Validation may cost initially but it avoids the risk of breaking down, for this reason even small industries are concentrating more on validation their by fulfilling goal of GMP. Validation when done according to standard protocol and used it always produce a product which meets its predetermined specifications and quality.
References Article on Validation of Analytical Procedures: comparison of ICH vs Pharmacopoeia by katrai Sahil. British Pharmacopoeia, 2007 (4) A523 and A159-163 United states pharmacopoeia 30, 1920-1924 and 2149-2152. www.ich.org CDER guideline Nov 1994 validation of chromatographic methods