10 Assess Validation

8/12/2019 10 Assess Validation

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Medicines Compendium General Information / 10Assessing Validation Parameters 1

PPAC10Assessing Validation Resolution: NLT 1.5 for each impurity

Parameters for Reference andLinearity and Range

Acceptable ProceduresGuidelineSamples: NLT 6 independent replicate Standard solutionsfor Donors/Instructions for Staff over the range the procedure is intended to be used, butNLT at 80%, 90%, 100%, 110%, and 120%Analysis: Analyze the results of each data series of solu-tions as described in Precision and Accuracy.PPAC: NLT 0.95 for each concentration point

PURPOSE

This document (Document 5) provides information to de- ORGANIC IMPURITY LIMIT PROCEDUREtermine whether Reference Procedures or Acceptable Proce-dures for the Assay and Impurity tests in the USP MedicinesCompendium (MC)are acceptable. The approach is based ona defined set of Procedure Performance Measures (PPMs) Precision (Repeatability)and associated Procedure Performance Acceptance Criteria(PPACs) and experiments. Further information is provided Test samples: Six independent samples of the materialin the Appendix. While the concepts in the Guideline are under test, spiked with reference materials for the specifiedgeneral, it is directed to chromatographic methods for impurities at the indicated limitchemical ingredients and products. Adjustments may be PPACneeded for non-separation methods and ingredients/

Relative standard deviation: NMT 2C0.1505%. Cis theproducts.concentration fraction of the impurity [and is calculated bydividing the impurity concentration by the major compo-nent concentration. A spiked impurity present at 0.1% ofASSAYthe major peak would have a concentration fraction, C, of(0.1%/100%) = 103, which would lead to an acceptableRSD of 5.7%. Therefore, an alternative impurity procedure

Precision and Accuracy would present acceptable precision if it yielded arepeatability of 5.7%]. [NOTEThe Horwitz equation isbased on the comparison of a procedures reproducibility asSamples: Six replicate Standard solutionscompared to the concentration factor. The repeatability hasAnalysis: Determine the Precision valueand Accuracy value.been reported to be 1/2of that of the reproducibility. There-

Precision value: Determine the % RSD of the Standard so- fore, the equation based on the repeatability analysis wouldlutions. lead to the equation: Acceptable % RSD = C0.1505. However,Accuracy value: Mean of the percentage content of the for the evaluation of a limit test, the broader reproducibilityStandard solutionscorrected for the stated content of the based equation is used because the precision is less impor-Reference material. tant in this type of test.]

Analyze results using MS Excel:Result = NORMDIST(UpperCert, Mean, SD, TRUE) Limit of Detection

NORMDIST(Lower+ Cert, Mean, SD, TRUE)

Control sample: A preparation of reference materials forthe impurity(ies) of interest at the target concentration(s)Test sample 1: A sample of material under test, spiked

Upper = upper limit of the Acceptance Range (e.g., 102% with reference materials for the specified impurities at thein the default case) target concentration, prepared in triplicate

Cert = measurement uncertainty of the reference stan- Test sample 2: A sample of material under test, spikeddard used (= 0 unless CRM or SRM used) with reference materials for the specified impurities 100

(2C0.1505)% of the target concentration for the specified im-Mean = Accuracy Valuepurity(ies), prepared in triplicateSD = Precision ValuePPAC: Each Test sample 1provides a peak response equiva-TRUE = logical operator lent to or greater than that of the Control sample. Test sam-Lower = lower limit of the Acceptance Range (e.g., 98%ple 2must provide a peak response that is less than that ofin the default case)

the Control sample. [NOTE

The signal from each sample ob-tained must show a change from the value obtained com-PPAC: NLT 0.95pared to a blank determination.]Specificity: The procedure must be able to unequivocally

Specificity assess, with acceptable Accuracy and Precision(see previoussections), each specified impurity in the presence of compo-nents that may be expected to be present, including otherSpiked standard solution(s): Contains the measurand atspecified impurities, the parent compound counter-ions, sol-the same concentration as the Standard solutionand eachvent fronts, and matrix components.specified impurity at 10x the specified value and unspecified

Where specified impurities are separated by a resolutionimpurities at 1.0%. [NOTEExclude all impurities that con-of greater than 1.5, then the procedure has acceptabletribute an acceptable level of assignable bias.]specificitySamples: Standard solutionand Spiked standard solution(s)

Where specified impurities are not well-resolved, then aAnalysis: Analyze results, and determine the Resolutionof spike and recovery study of the closely eluting peaks iseach impurity from the primary peak. completed. If the specified impurity of interest meets


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2 10Assessing Validation Parameters/ General Information Medicines Compendium

the previous Precision requirementswhile in the presence APPENDIX: THEORETICAL CONSIDERATIONSof interfering peak, then the procedure is acceptable.

Organic Impurity Quantitative Procedures Introduction

The principles of validation are provided in United StatesPharmacopeiaNational Formulary (USPNF)general chapter

ACCURACY Validation of Compendial Procedures 1225. Principles ofequivalence are presented in the following publication: Ac-

Standard solutions: Prepare solutions of the specified im- ceptable, Equivalent, or Better: Approaches for Alternatives topurities at concentrations ranging from 50% to 150% of the Official Compendial Procedures. One form of equivalence in-limit value for each impurity, using appropriate reference volves demonstrating that a procedure meets pre-set criteriamaterials or other reliable impurity source. that indicate acceptability of either a Reference Procedure orSample solutions: Prepare solutions of the material under Acceptable Procedure in the USP Medicines Compendiumtest spiked with appropriate reference materials at concen- (MC). The pre-set criteria consist of the Procedure Perfor-trations ranging from 50% to 150% of the indicated limit mance Measures (PPM) and the Procedure Performance Ac-value for each specified impurity. ceptance Criteria (PPAC) for a specific application. Often

the PPM and the PPAC are presented together in a mono-PPACgraph or method-specific general chapter (see Elemental Im-Spike recovery: = C0.1505%for the mean of three repli-puritiesProcedures 233). The PPAC may also be found incate preparations at each concentration. [NOTEThe datamethod-specific general chapters (such as Chromatographyobtained for this validation parameter are used for Limit of621). This Appendix provides a general discussion ofQuantitation (LOQ), Range, and Linearityin the followingPPMs in determining whether Reference Procedures or Ac-section.]ceptable Procedures meet predetermined criteria for accept-

able PPACs.PRECISION

Procedure Performance MeasuresRepeatability

Test samples: Six independent samples of material General chapter Validation of Compendial Proceduresunder test, spiked with appropriate reference materials for 1225defines the critical validation parameters (CVP) nec-the specified impurities at the indicated levels essary to validate a procedure. The CVPs can also be consid-

ered as the PPMs for the determination of the acceptabilityPPACof procedures. The methods (chromatographic, spectro-Relative standard deviation: NMT C0.1505%; where Cisscopic, gravimetric, etc) used in a monographs test proce-the concentration fraction of the impuritydures each have terms, measures, and assumptions that areused to describe critical information about acceptability ofthe procedure itself. These method-specific terms and meas-RUGGEDNESSures may not align well with the PPMs necessary to definean acceptable procedure. Additional approaches are thusThe effect of random events on the analytical precision ofneeded to determine acceptability of a monographs proce-the method must be established. Acceptable experiments

dure. These approaches are termed PPMs.for establishing intermediate precision include performingthe Repeatabilityanalysis

On different daysChromatographic Assay With different instrumentation, or

With different analysts.The validation parameters for a chromatographic assay areNote that executing only one of the three experiments

defined as; accuracy, precision, specificity, linearity, andlisted is required in order to demonstrate intermediaterange. There are thousands of experts practicing chroma-precision.tography throughout the pharmaceutical industry and these

PPAC individuals have all come to a general, unwritten agreementRelative standard deviation: NMT 2C0.1505%; where C as to what an adequate procedure would entail. Most of

is the concentration fraction of the impurity these individuals would describe the resolution, peak shape,co-elution, baseline drift, dead volume and other chromato-graphically relevant terms. However, all of these terms are

SPECIFICITY specific measures that describe the features of a separation,but do not parallel the CVPs defined above. For example,

The procedure must be able to unequivocally assess each the specificity of a procedure can be described in part byspecified impurity in the presence of components that may

the resolution of critical pairs of peaks, and partially by thebe expected to be present, including other specified impuri- likelihood of co-elution and also by the number and heightties, the parent compound counter-ions, solvent fronts, and of theoretical plates, and several others. Each of thesematrix components. measures help an analyst describe the goodness of their

Where specified impurities are separated by a resolution procedure. However, these measures cannot adequately de-of greater than 1.5, then the procedure has acceptable scribe an acceptable procedure in isolation. What is neededspecificity. is an approach that allows an analyst to quickly and deci-

Where specified impurities are not well-resolved, then a sively determine that their procedure is acceptable. The ac-spike and recovery study of the closely eluting peaks is com- ceptable values have been included in general chapter Chro-pleted. If the specified impurity of interest meets the previ- matography 621using the rationale that follows.ous Precisionrequirements while in the presence of interfer-ing peak, then the procedure is acceptable.

PRECISIONANDACCURACYLimit of Quantitation, Range, and Linearity The precision of a procedure affects the acceptability of all

of the CVPs. As the precision of a procedure increases, anyDemonstrated by meeting the Accuracyrequirement.


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Figure 1. Bias%CV Tradeoff, 98%102% Limits, True Value = 100, Probability Passing 0.95.

bias inherent in the system becomes more apparent, the must provide flexibility to the user, while clearly defining thelinearity and range become better defined, the ability to de- boundary condition between acceptable and unacceptabletermine an error caused by a lack of specificity becomes chromatography. This boundary is defined statistically as amore apparent, and the likelihood of a material meeting the simple normal distribution that describes the likelihood (at aAssay acceptance criteria receiving a passing result is im- 95% confidence level) of a passing value to demonstrateproved. Of these relationships, Precision and Accuracy are compliance to an Assay acceptance criterion of 98%102%the most closely linked. (see Figure 1).

For the 98%120% case, a user could simply plot theAccuracy: Accuracy of a liquid chromatographic procedurevalues of Biasand %CV. If the point described by a proce-is linked to the accuracy of the detector, detector samplingdures accuracy and precision falls in the shaded area of Fig-rate, and the consistency and quality of the separation. Be-ure 1, then the procedure will (with a 95% probability) pro-cause chromatography is a relative technique, the results ofvide a passing value when the true value passes. Becausean unknown must be compared to those from a knownthis is a statistical evaluation and not a chemical determina-standard to obtain a quantitative result. The true value ortion, the results are conclusive.concentration of the known (Standard) solution must be de-

Although the Assay acceptance criteria of 98%102% istermined using an orthogonal procedure or through the usethe most prevalent in USPNF, there are many other criteriaof a standardized material. To empirically determine the

in use. For the CVP approach to determining the accepta-accuracy of a CVP, multiple (6) weighings of a standard bility of a procedures accuracy and precision to be appliedmaterial would be used to produce independent solutionsto cases other than the default, it is necessary to calculate ahaving the same concentration. Each of the solutions wouldnew boundary line for each case. The development of abe chromatographed, and corrected for weighing differ-new boundary line is not difficult, but is time consuming.ences. The difference between the average of the calcu-However, there is another approach. Because the boundarylated content of Standard solutions (obtained by using theline is based on the statistical evaluation of a normal distri-calibration curve developed during the validation of the pro-bution, it is possible to solve for the probability of the preci-cedure) and the known content of the Standard solutionsion and accuracy values providing the correct answer.would represent the procedure bias (accuracy).When the calculated probability is less than 0.95, then thePrecision: Like accuracy, the precision of a quantitativeprecision and accuracy are acceptable. One way to speedchromatographic procedure is directly linked to the preci-the calculation is to use a program such as Microsoft Excel.sion of the detector used. The precision of a measurementWhen using Excel, the probability would be calculated usingis also dependent upon the quality of the separation. Preci-the following equations:sion can be determined empirically using the data obtained1. Where the acceptance criteria are symmetric aroundfor the Accuracyevaluation. The data obtained and cor-100%, then calculate:rected are then used to calculate the relative standard devia-

tion or %CV.Result = NORMDIST(UpperCert, Mean, SD, TRUE) NORMDIST(Lower+ Cert, Mean, SD, TRUE)

RELATIONSHIPBETWEENACCURACYANDPRECISIONWhen making decisions about the best method for a par-

ticular analysis, there are a number of factors aside from the Upper = upper limit of the Acceptance RangeCVPs. These factors include: length of analysis, cost of sol-

Cert = measurement uncertainty of the Standard usedvent use and disposal, column life, solvent-column compati-(= 0 for Standards with unknown measurementbility, gradient lengths, equilibration times, pump capacity,uncertainty)etc. Therefore, it is often necessary to weigh Precision and

Mean = accuracy value (e.g., 100% in the default case)Accuracy requirements with these outside concerns. Obvi-SD = standard deviationously a method with 0 RSD and bias would be the best

available technique, but where it requires solvents and col- TRUE = logical operator umn to be maintained at 50C or that requires 2 hours to Lower = lower limit of the Acceptance Rangeequilibrate between each analysis, it is unlikely to be se-lected. Therefore, the definition of an acceptable procedure


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Figure 2. Guard Band for a CRM Labeled as 100% 0.06% Applied to Figure 1.

2. Where the acceptance criteria are asymmetric around sionwill need to be adjusted. This adjustment takes the100%, but the entirety of the range is between 97% and form of a guard band in the distribution. The guard band103% (includes about 85%* of monographs in USPNF) effectively tightens the acceptance criteria by the stated un-then: certainty of the standard. An example of a guard band for

The same equation is used, but the Lowerand/or Upper a certified reference material labeled as 100% 0.06% isvalue would be changed to describe the smallest symmetric illustrated in Figure 2. This offset is accomplished by a smallrange having a mean of 100%. change in the equation used to calculate acceptance

probability. The change is accomplished by adjusting the3. Where the acceptance criteria are asymmetric meanboundary conditions of the acceptance criteria using theother than 100%, and the value of 100% is not included inmeasurement uncertainty (Cert) in cases 13in the previousthe range, then calculate:subsection.

Result = NORMDIST(UpperCert, Mean, SD, TRUE) NORMDIST(Lower+ Cert, Mean, SD, TRUE)

SPECIFICITY

The ability to describe an adequate separation of a criticalanalyte from interferences is currently measured using sev-

Upper = upper limit of the Acceptance Range eral different approaches including: resolution, separationCert = measurement uncertainty of the Standard used factors, capacity factors, peak shape (tailing and fronting),

(= 0 for Standards with unknown measurement height and number of theoretical plates, retention times,uncertainty) and several others. All of these separation measures assume

that the detection system being employed is capable ofMean = target content = (Upper+ Lower)/2measuring the analyte and other materials likely to be pres-SD = standard deviationent. Resolution is the most effective measure of a separa-

TRUE = logical operator tion. Generally speaking, a resolution value of 2 would be

Lower = lower limit of the Acceptance Range described as being baseline separated and a resolution of 0would indicate no separation. Complete (baseline) separa-

4. 4.Where the range is larger than 97%103% and is not tion is preferred, but may represent much longer chromato-centered on 100%, the determination of the intended mean graphic run times, difficult chromatographic conditions, orand range need to be considered prior to calculating the may be unattainable. Therefore, it is necessary to determineacceptable value. The details of these very rare cases are what resolution is necessary to provide an adequate separa-still being considered. The problem of intent can be illus- tion of two close eluting peaks. One way to quantify thetrated using an example acceptance criteria of 85%103%. effect of resolution upon a chromatographic procedure is byThis criteria could be considered in two different ways: the evaluation of the error caused by a co-eluting or over-

A symmetric range around 94% or lapping peak.An asymmetric range around 100%

Assuming an SDof 4, case 1 would yield a probability ofSPECIFICITYERROR0.97 and would be considered acceptable, while case 2

would yield a probability of 0.54 which would not be ac-The signal (peak height or peak area) used to measureceptable. Further discussion on this topic is being

chromatographic response is susceptible to error due toconsidered.peak height or area shifts caused by co-elution of analytes.Most detectors do not allow the differentiation of chemicalentities present in a measured peak. The exceptions areGUARDBANDSmulti-dimensional detectors with deconvolution algorithms.Where peaks overlap, there are many different ways to esti-When a reference standard is used that includes a meas-mate the approximate individual component concentrationsurement uncertainty value, such as those found in CRMs orand each has a related error.SRMs, the results of the determination of Accuracy and Preci-


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Figure 3. Relationship Between Error and Resolution.

From a purely theoretical perspective, it is possible to cal- ASSIGNABLEBIASculate the percentage error caused by overlapping peaks byevaluating the effect of secondary Gaussian peak with differ- 1. If an impurity that co-elutes with the principal peak ising sizes on a primary peak. The error is directly related to considered a component of the Bias(assignable bias) in thethe size of the secondary peak, the width of the peaks, and analysis, then the likelihood of the presence of that impuritythe distance between their peak values (retention time). Be- impeding the quantification of the primary component cancause peak width and retention times are the critical vari- be calculated. The assignable bias is the known concentra-ables in calculation of resolution, then resolution and error tion of the impurity presented as a percentage of the totalare also directly linked. That relationship is described in primary component concentration. The calculation is theFigure 3. The values in Figure 3are the area counts ob- same as given previously, but with the assignable biastained by the traditional extending the sides of the peak to added to the accuracy value.baseline approach described in general chapter Chromatog-raphy 621with and without overlapping peaks. The size Result = NORMDIST(UpperCert, Mean, SD, TRUE) of the minor overlapping peaks range from 0.1% to 5% of NORMDIST(Lower+ Cert, Mean, SD, TRUE)the primary peak.

The generally accepted rule of thumb for chro-matographers is that a resolution of 1.25 is sufficient to ade-quately quantify overlapping peaks but that 1.5 is preferred.

Upper = upper limit of the Acceptance RangeBased on Figure 3, the rule of thumb appears to be correct.A resolution of 1.25 contributes significantly more error to a Cert = measurement uncertainty of the Standard useddetermination than a resolution of 1.5, but with more so- (= 0 for Standards with unknown measurementphisticated analytics (i.e., better peak area approximations) uncertainty)it is possible to reduce that error further. It is also clear that Mean = accuracy value + assignable bias (%)below a certain level the presence of a small impurity will SD = standard deviationcontribute an insignificant error in the quantification of the

TRUE = logical operator primary component. The size of an insignificant impurity is

Lower = lower limit of the Acceptance Rangedirectly linked to the Precision and Accuracyevaluation dis-cussed in the previous section. Please note that the pres-

2. If the Resultis less than 0.95, then the impurity is incon-ence of impurities in a drug substance is not desirable, butsequential to the Assay. Where the Resultis less than 0.95,for the purposes of assaying the content of the active ingre-then additional separation is necessary to assure the abilitydient, small impurities or undetectable impurities may notof the procedure to assay the primary component.need consideration. The determination of these impurities

Figure 3also provides insight into both the source of vari-will be considered separately.ability in the analysis and the level of separation necessaryto achieve an acceptable procedure. If an analyst knows the


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Figure 4. Effect of Random Error on CVP Linearity.

level of error (accuracy value + assignable bias) it would be sponse curve, but instead to evaluate the calculated concen-

possible to approximate the necessary resolution using Fig- tration-known concentration curve. This approach providesure 2. However, due to the significant convergence of the a means to assess the entire system including sample prepa-curves, it is safe to say that a resolution of 1.5 will provide ration, injection, separation, detection, and post-processing.sufficient separation for all but very large impurities. The calculated versus know concentration curve should be

very linear, and pass through the origin. However, as ran-dom error is introduced into the system, there is a direct

LINEARITYANDRANGE effect on the R2value of the linear regression. This effect isillustrated in Figure 4. This plot represents the results of a

The linearity and range of a liquid chromatographic pro- linear regression on data that ranged from 80% to 120% atcedure are largely controlled by the detector sensitivity and known values with a random noise of a known percentageby column capacity and retention time. Both the linearity added. As the amount of noise is increased, the variabilityand range can be empirically determined by evaluating of the R2value increases and the centers of the ranges de-Standard solutions ranging in concentration from 80% to crease.120% of the target concentration of the analyte. There Viewed another way, the error captured as the % RSD ofshould be five separate concentrations with each of the so- the data when compared to the square of the residuals indi-lutions being prepared in triplicate. The results are then cates a strong relationship (see Figure 5). This plot suggestsevaluated for adequate precision and accuracy as well as the that a R2value of greater than 0.994 and 0.990 would de-

function of a linear regression of the calculated concentra- scribe an acceptable procedure for 98%102% andtion versus known concentration plot. 97%103% acceptance ranges, respectively.The measurement of the linearity and range are coupled In summary, it is clear that the precision and accuracy of

for this discussion because, like precision and accuracy, the an analytical procedure are pivotal to defining the accepta-experiments and outcomes are directly linked. Up to this bility of a procedure. The specificity, linearity, and rangepoint, all of the CVPs have been focused on the ability of are also necessary that ensure the results of a procedurethe procedure to determine the true value of a sample having acceptable precision and accuracy will provide awhen present at a target concentration. However, not all meaningful approximation of the true value of an un-samples will be present at 100.0%. Therefore, it is neces- known sample. When taken together, it is possible to de-sary to ensure that a given procedure will yield acceptable scribe an acceptable procedure using measures that are in-results over a range of likely concentrations. This range is dependent of chromatographic figures of merit typicallydescribed as 80%120% of the target concentration in the used to describe a procedure.previous CVP section.Range: The acceptability of a range is evaluated by calcu-

Chromatographic Analysis of Impuritieslating the precision and accuracy values for each of the pre-pared solution concentrations described previously. Al-

Organic impurities in drug substances are chemical spe-though these solutions are prepared in triplicate and the

cies that should not be present. Their presence may pose aacceptance values calculated in the Precision and Accuracy health risk or may simply indicate a lack of quality. Thesection are based on 6 measurements, the calculations pro-acceptance criteria for individual impurities are evaluated byvide an adequate level of confidence in the acceptability ofthe regulatory agencies prior to the acceptance of a drugthe measurements. If all of the five solution triplicates yieldproduct for use in humans. Impurities are evaluated fora value of 0.95 or greater, then the range of the instrumentsafety and those that show toxicity or have the potential tois adequate for the measurement.be a health risk are termed toxic impurities and are specifi-Linearity: Typically linearity is evaluated by comparing thecally limited to low levels by the regulatory agency. Thosesignal to the concentration. However, the slope and inter-organic impurities that are not toxic at expected levels rep-cept of the linear regression of that line is of little conse-resent quality issues alone. International standard settingquence as long as it is not too vertical or too horizontal. Ingroups have determined that non-toxic impurities present ataddition, there are a number of detection systems in devel-less than 0.10% need not be quantified and those of lessopment and use that provide unique capabilities but requirethan 0.05% may be ignored. Individual regulatory agen-non-linear relationships between signal and concentrationcies may lower these limits to better represent the drug sub-that can be modeled. Therefore, the best manner to assessstance being approved, but for a public standard, the levelsthe linearity of a system is not to evaluate the signal-re-selected by the international body form an acceptable impu-


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Figure 5. Relationship Between % RSD and R2.

rity level. The acceptability of a procedure to evaluate non- body of data, he developed an empirical relationship fortoxic impurities in a drug substance is directly linked to the laboratory-to-laboratory reproducibility. The most commonCVPs defined by general chapter Validation of Compendial form of that relationship is:Procedures 1225. The validation chapter subdivides impu-

Predicted % RSD = 2 (10.5 log C)rity procedures into those used in a semi-quantitative limit

test and those intended to be quantitative. The require-where Cis the concentration as a dimensionless fractionments for each are significantly different; however, the re-

(see Table 1).quirements described for a limit test are insufficient for theWhen this equation is extended to concentrations of 100determination of acceptable limit tests. The means to

ppb (100 ng/g) or lower, the relationship becomes con-demonstrate the acceptable limit test are included in thestant. This observation was published by Thompson, et.al.following Chromatographic Limit Testssubsection. Fortu-, leading to the Horowitz-Thompson equation used by thenately, chromatography is typically used in a quantitativeAssociation of Official Analytical Chemists (AOAC) and bymode, and the validation parameters described in the vali-the Codex Alimentarius to describe acceptable procedures.dation chapter are a good indicator of those necessary toThis relationship is typically called the Horowitz ratio ordetermine an acceptable quantitative impurity procedure.HORRAT in these publications. Subsequently, MassartThe parameters and the performance characteristics neces-showed that the Horowitz ratio appears to be approximatelysary to demonstrate acceptability are also included in thetwice that found in the evaluation of repeatability. Thefollowing subsection.Horowitz relationship is plotted in Figure 6for reproducibilityand repeatability.

CHROMATOGRAPHICLIMITTESTS The recommended procedure for specificity is the evalua-tion of replicates of spiked standards. This is considered a

The CVP necessary to validate a limit test procedure in- repeatability study, but for the purpose of defining the PPACclude precision, specificity, and limit of detection (LOD). for a limit test, the use of the Horowitz-Thompson Repro-The PPM for LOD also provides information about that ac- ducibility factor is more appropriate as it allows a larger de-ceptability of the accuracy of the procedure for a limit test. gree of variability. For ease of use, Table 1includes com-The CVP for a limit test must answer the following ques- mon concentrations, associated fractions, the Horowitz-tions: (1) Will this procedure consistently provide the cor- Massart repeatability value, and the Horowitz-Thompson re-rect pass/fail decision for the specific impurity being evalu- producibility value.ated? and (2) What is the smallest change in Specificity: The specificity of a limit procedure is bestconcentration between a passing and a failing result? The stated in general chapter Validation of Compendial Proceduresanswer to the first question requires knowledge of the preci- 1225as the ability to assess unequivocally the analyte insion and specificity. The answer to the second question the presence of components that may be expected to berequires knowledge of the LOD and accuracy of the meas- present, including other specified impurities, the parenturement. However, the accuracy of a limit test and the compound, counter ions, solvent fronts, and matrix compo-LOD can be determined using the same PPM and therefore nents. For chromatographic procedures, such as for assays,is only described as the LOD for the purposes of this the resolution provides the best approach PPM for specific-discussion. ity. The rationale for the specificity PPAC is described in thePrecision: Unlike the analysis of an analyte that is a major Assay section. However, the basis for the PPAC is illustratedconstituent in a drug substance, the evaluation of minor in Figure 3, but the figure presents the error in the calcula-components of indeterminate concentration creates compli- tion of major component caused by co-elution with a minorcations for determining the acceptability of a procedure. component. In the case of Impurities testing, co-elutingThe major complication concerns the ability of the detection species will often be of equal or greater concentration thansystems to differentiate a measurand from background the impurity of interest (measurand), therefore causing annoise. The ability to differentiate is determined through unresolved peak to be over-estimated. However, from anmeasurement of the LOD, but the conclusion that a proce- acceptability perspective a false-positive result is preferabledure with sufficient LOD has the differentiating power capa- to a false negative. Therefore, the PPAC of a resolution of 1.ble of presenting a true result is unfounded. Instead, it is 5 is acceptable. However, there are times that a resolutionimportant to examine the precision of the measurement. of 1.5 cannot be achieved. In these cases, a complete spikeThe relationship between the analytical concentration and and recovery study or an orthogonal procedure will need tothe acceptable level of variability was extensively explored be completed or used, respectively. The PPAC for theseby William Horowitz in the 1980s. He found that as the studies is the requirement to meet the previously describedcontent of the measurand decreases, the amount of variabil- precision requirements of each of the specified impurities.ity from measurement to measurement increases, regardlessof the procedure used. Through the examination of a large


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Figure 6.

Table 1. Horowitz-Thompson Values

Horowitz-Horowitz-Massart Thompson

Concentration of Concentration of Repeatability ReproducibilityAnalyte Analyte Concentration Concentration Value Value

(%) (ppm) with w/w Units Fraction (RSD) (RSD)

100% 1000 g/g 1.0 1% 2%

1% 10,000 ppm 10 mg/g 0.01 2% 4%

0.1% 1000 ppm 1 mg/g 0.001 2.8% 5.7%

0.05% 500 ppm 500 g/g 0.0005 3% 6%

0.01% 100 ppm 100 g/g 0.0001 4% 8%

0.001% 10 ppm 10 g/g 0.00001 5.7% 11%

0.0001% 1 ppm 1 g/g 0.000001 8% 16%

0.00001% 100 ppb 0.1 g/g 0.0000001 11% 22%


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an acceptable procedure, negligible can be defined as ble of determining an accurate and precise quantity for thehaving a bias that is smaller than the acceptable precision of measurand over the range that a typical unknown measure-a measurement. Once again, the Horowitz/Massart ment will be made. The accuracy and precision require-repeatability value describes the acceptable precision relative ments described for a quantitative impurity test also pro-to the relative concentration of the measurand. vides this assurance. By determining the precision at several

points across the analytical concentration range appropriatePrecision: The precision of a measurement can be splitto the measurand, the need for a separate linearity require-into three different types: repeatability, intermediate preci-ment is eliminated.sion, and reproducibility. The first evaluates instrumental

and sample preparation sources of variability, the second ex- Limit of quantification and range: The limit of quantifica-amines variability caused by deliberate changes to the meas- tion and the range are values that are evaluated to ensureurement environment, and the third examines the variability that the analytical procedure is appropriate for the evalua-typically found through interlaboratory studies. In the de- tion of a measurand at an unknown value. In the case of atermination of an acceptable procedure an understanding of compendial procedure, the value of the measurand isall sources of variability does not need to be individually known before a procedure is validated. Typically, an impu-determined. Instead it is possible to model the types of rity procedure will define the maximum concentration thatvariability likely to be encountered in reproducibility through an impurity may be present in a sample. By completing thethe deliberate changes from the intermediate precision stud- accuracy requirement, the analyst knows that the procedureies. is capable of quantifying the measurand at half of the maxi-

mum acceptable value for the impurity. The accuracy re-RepeatabilityThe repeatability portion of a quantitativequirement will also ensure that a procedure will provide ac-procedure validation is conducted by preparing six spikedceptable results at twice the maximum acceptablesample solutions, each containing one or more of the impu-concentration of the impurity. Successful completion of therities of interest at the analytical significant concentration.accuracy requirements will provide sufficient confidence inThe relative standard deviation of these measurements isthe procedures adequacy, and therefore no additional eval-then compared to the Horowitz-Massart value appropriateuation of these characteristics is required to define an ac-

for the concentration of the measurand(s). ceptable procedure.Where it is not possible to spike the analyte with a stan-dard for the measurand, then the use of a retention timeand relative response factor can be used. Although this The Meaning of Acceptabilityapproach is not optimal, it may be the only way to evaluateunstable or transient degradation products. Alternatively, a

Up to this point, this document has focused on describingreference standard with the critical impurities already pres-an acceptable procedure from a purely empirical and ap-ent of developed in situcould be used. The knowledge ofproachable manner. To that end, the CVPs have been de-the actual concentration of these impurities is impaired, butfined and the performance criteria for each of these CVPsthe materials could be used to complete the validation ofhas been described. However, the fundamental question ofthe procedure. Wherever possible, a pure standard of thethe adequacy of the CDP has yet to be discussed. Compre-degradation product or process impurity is preferred.hensive descriptions of the types of equivalence should be

Intermediate precisionThe evaluation of the intermediate considered when comparing an analytical procedure with aprecision may be determined by repeating the Repeatability compendial procedure. The first, and in many ways mostmeasurements previously described after a controlled delib- appropriate, approach is that of an acceptable procedure.erate change is made to the testing environment. The This is a procedure that with pre-defined performance crite-types of changes that are most useful for the determination ria that describe a procedure adequate to achieve an ap-

of an acceptable procedure include different days, different proximation of the true value of an unknown without previ-instrumentation, different analysts, and using different in- ously defining the procedure itself.strumental conditions. The first three are particularly useful Looking at the criteria previously described, it should bebecause they link the variability to the Horowitz-Thompson clear to a chromatographer that any procedure meeting theequation. The evaluation of variability caused by all four PC described herein will provide adequate assurance thatspecified conditions is not necessary because the informa- the procedure under question is appropriate. Therefore, anytion provided by more than one condition does not gener- procedure that meets these criteria should be considered toally provide any additional information. Therefore, the ana- be equivalent or better than the Standard procedure.lyst should determine which conditions to be pursued. The Where a procedure is equivalent to the Standard procedureresult of the two sets of data (this should be 12 individual (or acceptable), then according to the General Noticesofmeasurements) should be compared to the Horowitz ratio USPNF, that procedure may be used as an alternative pro-appropriate for the concentration of the measurement(s). cedure without the need to actively compare theseSpecificity: The requirements of the specificity in a quanti- procedures.tative procedure are the same as those described above fora limit test.Linearity: The linearity of the procedure is intended to as-sure the analyst that the procedure being validated is capa-

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10 Assess Validation