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ANALYTICAL METHOD VALIDATIONSri NoegrohatiGood practices in productionPrinciple. Production operations must follow clearly defined procedures in accordance with manufacturing and marketing authorizations, with the objective of obtaining products of the requisite quality.All handling of materials and products, such as receipt cleaningquarantine, processing, sampling, packaging and labellingstorage, distribution,dispensing should be done in accordance with written procedures or instructions and, where necessary, recorded.as far as possible no deviation from instructions or procedures should be avoided. Any If deviations occur, they should be done in accordance with an approved procedure.MonitorRoutine Commercial ManufacturingMonitor critical operating and performance parameters Utilize appropriate tools, e.g., Statistical Process ControlMonitor product characteristics (e.g., stability, product specifications)Monitor state of personnel training and material, facility/equipment and SOP changesInvestigate OOS for root cause and implement corrective action. Validated Manufacturing ProcessA validated manufacturing process has a high level of scientific assurance that it will reliably produce acceptable product

Measurement, Data Analysis & EvaluationMeasurementAppropriate systems, variables and attributes Data AnalysisPeriodic analysis to allow for trending on a periodic basis to detect problems as early as possible and implement corrective actionStatistical Process Control - preventative actions Selection of Critical Quality Attributes Meaningful specifications/ limits

The objective of an Analytical Work is:The achievement of reliable and dependable analytical informationQuality Assurance in Analytical ChemistryFor conducting nonclinical laboratory studies that support or intended to support applications for research or marketing permits for products regulated by the National Food and Drug Administration, Good Laboratory Practices should be followed in a strict senseCompliance with this part is intended to assure the quality and integrity of the safety dataTherefore: analytical method validation is required Method validation provides an assurance of reliability during normal use, and is sometime referred to as "the process of providing documented evidence that the method does what it is intended to do."

Analytical Method ValidationValidation the process of demonstrating that a laboratory procedure is robust, reliable, and reproducible in the hands of the personnel performing the test in that LaboratoryMethod validation is the process of establishing the performance characteristics and limitations of a method and the identification of the influences which may change these characteristics and to what extent. It is also the process of verifying that a method is fit for purpose, i.e., for use for solving a particular analytical problem.ISO 17025:Validation is the confirmation by examination and the provision of objective evidence that the particular requirements for a specific intended use are fulfilled

Why is validation important?Validation is part of overall quality assurance program in a laboratory to have:the correct answer when collecting datato have confidence that the sample contains no un-safe compound rather than there might have been something wrong with the detection method

How does validation help with quality assurance within a laboratory?Costs Test materials Standards Quality assurance equipment Analysis of QA/QC samples Quality assurance official Committee Work Interlab Studies Travel to meetingsBenefits More efficient outputs Fewer replicates for same reliability Fewer do-overs Greater confidence of: Staff Laboratory Customers

Costs/Benefits of Quality AssuranceWhy is Method Validation 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.The validation of methods is good science.

When is Validation needed?Before introduction of a new method into routine useWhenever the conditions change for which a method has been validated, e.g., instrument with different characteristicsWhenever the method is changed, and the change is outside the original scope of the methodWho carries out method validation?Can laboratories validate methods on their own, in-house validationThe in-house methods should demonstrate that a. the required tolerances of all measurements undertaken within the method (volume, temperatures, masses etc.);b. the forms of the determinand measured, including speciation;c. the effect of interferences has been widely investigated and quantified;d. significant sources of error have been identified and adequate means of controlling them have been identified.questions Will methods validated in this way be recognized by other laboratories?What sort of recognition can be expected for in-house methods used in a regulatory environment?

Error in Chemical AnalysisGross errorErrors which are so serious that there is no real alternative to abandoning the experiment and making a complete startRandom errorError that cause the individual result to fall on both side of the average value affect the precision of an experiment Systematic errorError that cause all the result to be in error in the same sense affect the accuracy, i.e. proximity to the true value Real Population Accurate Precise Error: Systematic Random

Estimate of

Sample X SReliable method the obtained results are accurate and correctly reflect the sample being testedChoosing, developing and evaluating methods

Analytical MethodSteps to have an Analytical Information:Sampling and sample storage Preparation of SamplesMeasurementEquipment and Equipment parameters ReagentsSystem Suitability testing Analytical Method Preparation of Standards Analytical Procedure ProcedureEvaluation Analytical PrincipleCalculationsReporting of resultsDocumentationQUALITY ASSURANCEAssure that:What is reported there is really thereThe level is exactly as what you reportThere is no addition through contaminationIf reported as negative, it is really not thereMethod SelectionAppropriate for intended usePreferable standard methodPublished in international, regional national standard, technical journalsNon Standard methods required validationJournal article In-house method Validation Parameters

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For a large number of replicates the results approach GAUSSIAN or NORMAL DISTRIBUTION CURVEThe mean value xgives the center of the distributionThe standard deviation s measures the width of the distribution

Experimental results are commonly expressed in the form: mean standard deviation18Sri Noegrohati, UGMNormal Distribution

Curve is symmetrical and centred at m. The greater the value of , the greater the spread of the curve.Whatever values of and , 68.27% of observations are within 95.45% of observations are within 2 99.97% of observations are within 3

19Sri Noegrohati, UGMDefinitionsArithmetic mean: average of all observations

If the sample is random then the arithmetic mean is the best estimate of the population (true) mean, m

Variance: measures the extent to which the data differs in relation to itself. Variance of population is the mean squared deviation from the population mean, denoted 2, while the variance of the sample data is denoted s2.AccuracyThe closeness of agreement between a test result and the accepted reference value.Accuracy of a measuring instrument is the ability of a measuring instrument to give responses close to a true value.

EURACHEM Guide (1998) The Fitness for Purpose of Analytical Methods: A Laboratory Guide to Method Validation and Related Topics, pp. 39, 41; available at http://www.eurachem.ul.pt/guides/valid.pdf

21Sri Noegrohati, Fac of Pharmacy GMUSYSTEMATIC ERROR Reproducible under the same conditions in the same experiment Can be detected and corrected for It is always positive or always negativeTo detect a systematic error: Use Standard Reference Materials Run a blank sample Use different analytical methods Participate in round robin experiments (different labs and people running the same analysis)PrecisionThe closeness of agreement between independent test results obtained under stipulated conditions.Precision depends only on the distribution of random errors and does not relate to the true value or specified value. The measure of precision is usually expressed in terms of imprecision and computed as a standard deviation of the test results. A measure for the reproducibility of measurements within a set, that is, of the scatter or dispersion of a set about its central value.

EURACHEM Guide (1998) The Fitness for Purpose of Analytical Methods: A Laboratory Guide to Method Validation and Related Topics, p. 45; available at ttp://www.eurachem.ul.pt/guides/valid.pdf

23Sri Noegrohati, Fac of Pharmacy GMURANDOM ERROR Uncontrolled variables in the measurement Can be positive or negative Cannot be corrected for Random errors are independent of each otherRandom errors can be reduced by: Better experiments (equipment, methodology, training of analyst) Large number of replicate samples

Student's t-Tests"Student's t-test deals with the problems associated with inference based on "small" samples: the calculated mean (Xavg) and standard deviation () may by chance deviate from the "real" mean and standard deviation (i.e., what you'd measure if you had many more data items: a "large" sample).For example, it it likely that the true mean size of maple leaves is "close" to the mean calculated from a sample of N randomly collected leaves. At 95% of the time, the actual mean would be in the range:If N=5, Xavg 2.776 /N1/2; if N=10: Xavg 2.262 /N1/2 ; if N=20: Xavg 2.093 /N1/2 ; if N=40; Xavg 2.023 /N1/2 ; and for "large" N: Xavg 1.960 /N1/2 . (These "small-sample" corrections are included in the descriptive statics report of the 95% confidence interval.)Student" (real name: W. S. Gossett [1876-1937])26Sri Noegrohati, UGMCONFIDENCE INTERVALS

The confidence interval is given by:Where t is the value of students t taken from the tableThe confidence interval is the expression stating that the true mean, , is likely to lie within a certain distance from the measured mean, x. Students t test27Sri Noegrohati, UGMStudents t tabled.f.

90%95%99%99.9%23451020302.9202.3532.1322.0151.8121.7251.6974.3033.1822.7762.5712.2282.0862.0429.9255.8414.6044.0323.1692.8452.75031.59612.9418.6106.8694.5873.8503.646Worked example:Fluoride content of a sample determined potentiometrically in water is (mg/l) 4.50, 3.80, 3.90, 4.20, 5.00 and 4.80 for separate analyses.Mean = 4.37Standard deviation = 0.4890% confidence limits are: = 4.37 2.015 x (0.48/6) = 4.37 0.3999% confidence limits are: = 4.37 4.032 x (0.48/6) = 4.37 0.7928Sri Noegrohati, Fac of Pharmacy GMU

A t test is used to compare sets of measurements.Usually 95% probability is good enough.291) COMPARISON OF MEANS

Statistical tests are giving only probabilities. They do not relieve us of the responsibility of interpreting our results!Comparison of a measured result with a known (standard) valuetcalc > ttable at 95% confidence level results are considered to be different the difference is significant!APPLYING STUDENTS T:30Sri Noegrohati, Fac of Pharmacy GMUFor 2 sets of data with number of measurements n1 , n2 and means x1, x2 : Where Spooled = pooled std dev. from both sets of data

2) COMPARISON OF REPLICATE MEASUREMENTStcalc > ttable at 95% confidence level difference between results is significant.Degrees of freedom = (n1 + n2 2)A Comment on Minimum Numbers ofSamples

Impact of Number of Experiments on Capturing Variability in a Population of DataNumber of Samples NeededRelationship between a sample and a population of data

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Q TEST FOR BAD DATA

The range is the total spread of the data.The gap is the difference between the bad point and the nearest value. Example: 12.2 12.4 12.5 12.6 12.9

GapRangeIf Qcalc > Qtable discarded questionable pointThe VAM PrinciplesVAM = Valid Analytical Measurement1. Analytical measurements should be made to satisfy an agreed requirement.2. Analytical measurements should be made using methods and equipment that have been tested to ensure they are fit for their purpose.3. Staff making analytical measurements should be both qualified and competent to undertake the task.4. There should be a regular and independent assessment of the technical performance of a laboratory.5. Analytical measurements made in one location should be consistent with those made elsewhere.6. Organizations making analytical measurements should have well defined quality control and quality assurance procedures.

35Sri Noegrohati, UGMThe Analytical ApproachStatement of ProblemDefinition of ObjectiveSelection of ProcedureSampling, Sample Transport and StorageSample PreparationMeasurement/DeterminationData EvaluationConclusions and Report

Assumptions When Performing ValidationThe equipment on which the work is being done is broadly suited to the application. It is clean, well maintained and within calibration.The staff carrying out the validation are competent in the type of work involved.There are no unusual fluctuations in laboratory conditions and there is no work being carried out in the immediate vicinity that is likely to cause interferences.The samples being used in the validation study are known to be sufficiently stable.

System SuitabilityFitness for purpose is the degree to which data produced by a measurement process enables a user to make technically and administratively correct decisions for a stated purpose.39Sri Noegrohati, UGMCalibration of the instrumentBased on Lambert - Beer law linear correlation between absorbance and concentration calibration curve

Finding the Best Straight Line:a line that minimises the deviations in the y-direction using the sum of the square of these deviations)-method of least squares. Linearity and RangeLinearity defines the ability of the method to obtain test results proportional to the concentration of analyte.The Linear Range is by inference the range of analyte concentrations over which the method gives test results proportional to the concentration of the analyte. Working range is a set of values of measurands for which the error of a measuring instrument is intended to lie within specified limits.

41Sri Noegrohati, UGMMETHOD OF LEAST SQUARES Assumption:There is a linear relationship Errors in the y-values (measured values) are greater than the errors in the x-values minimise only the vertical deviations. Uncertainties for all y-values are the same minimise only the vertical deviationsEquation of a straight line: y = mx + cwhere m = slope and c = y-intercept

42Sri Noegrohati, UGMEstimation of the std. dev. for all y valuesThe vertical deviation can be calculated as follows: di = yi (mxi + c)Some deviations are positive (point lies above the curve) and some are negative (point lies below the curve) square the values so that the sign does not play a role di2 = (yi mxi - c)2Estimate the standard deviation for all y values

43Sri Noegrohati, UGM Standard deviation for the slope (m):

Standard deviation for the intercept (c):

DetectabilityIs the Analytical Signal distinguishable from the Blank? need to know the uncertainty of the measurements.scY detection limit = c+3sc LOD = (c+3sc)-c/m44Sri Noegrohati, UGM

lnear range, where the response of the analyte is linear with concentration. The dynamic range is the concentration where there is a measureable response to the analyte, even if it is not linear.

Sensitivity: calibration sensitivity = slope (m) of calibration curve.= ability to discriminate between small differences in analyte concentration analytical sensitivity (g) = slope (m)/standard deviation (Ss)45Sri Noegrohati, UGM

Linear Dynamic Range: linear region of calibration curve where the lower limit is ten times the standard deviation of the blank.LOQ - limit of quantitationLOL - limit of linearity

Concentration (mM)SensitivityCalibration of the fundamental Analytical Procedure Limit of detection (LOD) the concentraton that can be measured with reasonable statistical certainty of significantly different from blanc signal.Limit of quantitative measurement (LOQ) the lowest concentration of an analyte that can be determined with acceptable precision (repeatability) and accuracy under the stated conditions of the test using the chosen analytical procedure.

Limit of Linear Response (LOL)Point of saturation for an instrument detector, so that higher amounts of analyte do not produce a linear response in signal

How is this best done in practise?

ReproducibilityReproducible method the same or very similar results are obtained each time a sample is testedPrecision under reproducibility conditions, i.e. conditions where test results are obtained with the same method on identical test items in different laboratories with different operators using different equipment.Will you get the same result each time you test a sample?Different from repeatability, which is the precision under repeatability conditions, i.e. conditions where independent test results are obtained with the same method on identical test items in the same laboratory by the same operator using the same equipment within short intervals of time.Robustness (Ruggedness)Robust method successful results are obtained a high percentage of the time and few, if any, samples need to be repeatedThe 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 normal usage.You do not want the method to fail when you only have enough material for a single try.Specificity The ability of a method to measure only what it is intended to measure.Specificity is the ability to assess unequivocally the analyte in the presence of components which may be expected to be present. Typically these might include impurities, degradants, matrix, etc.

StabilityWill the method produce a result reliably over time?Control chart i.e. Shewhart chart, are an effective tool for monitoring stability and quality assurance over time

Development of Analytical Method

Funded by the CompanyPerformed by manufacturerPerformed by LabCommon Perceptions of Validation