Method Validation of Analytical Procedures

Transcription

1 32 Revew Artcle Method Valdaton of Analytcal Procedures Prakash Chanda Gupta QC Executve, Natonal Healthcare Pvt. Ltd., Nepal ABSTRACT After the development of an analytcal procedure, t s must mportant to assure that the procedure wll consstently produce the ntended a precse result wth hgh degree of accuracy. The method should gve a specfc result that may not be affected by external matters. Ths creates a requrement to valdate the analytcal procedures. The valdaton procedures conssts of some characterstcs parameters that makes the method acceptable wth addton of statstcal tools. Keywords: Valdaton, Analytcal Procedure, Accuracy, Precson, Robustness INTRODUCTION Valdaton of an analytcal procedure s the process by whch t s establshed, by laboratory studes, that the performance characterstcs of the procedure meet the requrements for the ntended analytcal applcatons. [1] Method valdaton provdes an assurance of relablty durng normal use, and s sometme referred to as the process for provdng documented evdence that the method does what t s ntended to do. The man objectve of the valdaton s to demonstrate that the analytcal method s sutable for ts ntended purpose, s accurate, specfc and precse over the specfed range that an analyte wll be analyzed. Analytcal Method Valdaton s to be performed for new analyss methods or for current methods when any changes are made to the procedure, composton of the drug product and synthess of the drugs substances. Common types of analytcal procedure that can be valdated [2] Identfcaton tests; Quanttatve tests for mpurtes content; Lmt tests for the control of mpurtes; Quanttatve tests of the actve moety n samples of drug substance or drug product or other selected component(s) n the drug product. Typcal valdaton characterstcs whch should be consdered are lsted below: [1] Accuracy Precson Specfcty Detecton Lmt Quanttaton Lmt Lnearty Range Robustness The valdaton characterstcs are to be evaluated on the bass of the type of analytcal procedures. Table 1: Evaluaton of Valdaton Characterstcs Characterstcs Type of Analytcal Procedures Identfcaton Impurtes Quanttatve Tests Quanttatve Lmt How to cte ths artcle: PC Gupta; Method Valdaton of Analytcal Procedures; PharmaTutor; 2015; 3(1); 32-39

2 33 Accuracy Not evaluated Evaluated Not evaluated Evaluated Precson Not evaluated Evaluated Not evaluated Evaluated Specfcty Evaluated Evaluated Evaluated Evaluated Detecton Lmt Not evaluated Not evaluated Evaluated Not evaluated Quanttaton Lmt Not evaluated Evaluated Not evaluated Not evaluated Lnearty Not evaluated Evaluated Not evaluated Evaluated Range Not evaluated Evaluated Not evaluated Evaluated METHODS AND TERMINOLOGY Accuracy The accuracy of an analytcal method s the closeness of the test results obtaned by that method to the true value. [3] Ths s sometmes termed trueness. It s recommended that accuracy should be determned usng a mnmum of nne determnatons over a mnmum of the three concentraton levels, coverng the specfed range (3 concentratons/3 replcates each of total analytcal procedures). [4] It s measured as the percent of analyte recovered by assay. The recovery can be determned by the equaton: Recovery = Analytcal Result x 100% True Value The recovery should be n the range of Control lmt. The followng method can be appled for calculatng the Upper Control Lmt (UCL) and Lower Control Lmt (LCL). The method nvolves the movng range, whch s defned as the absolute dfference between two consecutve measurements ( x x 1 ). These movng range are averaged ( MR ) and used n the followng formulae: [5] UCL and MR x 3 d 2 LCL MR x 3 d 2 Where, x s the ndvdual analytcal result, x s the sample mean, and 2 d s a constant commonly used for ths type of chart and s based on the number of observatons assocated wth the movng range calculaton. Where n = 2 (two consecutve measurements), as here, d 2 = Precson The precson of the analytcal method descrbes the closeness of repeated ndvdual measures of analyte. [6] The precson of an analytcal procedure s usually expressed as the standard devaton or relatve standard devaton (coeffcent of varaton) of a seres of measurements. It s ndcated by Relatve Standard Devaton, RSD, whch s determned by the equaton: 1/ 2 n 2 ( ) 100 x x 1 (%) n RSD x n 1 Where x s an ndvdual measurement n a set of n measurement and x s the arthmetc mean of the set. Generally, the RSD should not be more than 2%.

3 34 Repeatablty Repeatablty refers to the use of the analytcal procedure wthn a laboratory over a short perod of tme usng the same analyst wth the same equpment. [3] Repeatablty should be assessed usng a mnmum of nne determnatons coverng the specfed range for the procedure (.e., three concentratons and three replcates of each concentraton or usng a mnmum of sx determnatons at 100% of the test concentraton). [4] assay and mpurty tests by chromatographc procedures, specfcty can be demonstrated by the resoluton of the two components whch elute closest to each other. [9] It s not always possble to demonstrate that an analytcal procedure s specfc for a partcular analyte (complete dscrmnaton). In ths case a combnaton of two or more analytcal procedures s recommended to acheve the necessary level of dscrmnaton. Reproducblty Reproducblty expresses the precson between laboratores (collaboratve studes, usually appled to standardsaton of methodology). Reproducblty s usually demonstrated by means of an nter-laboratory tral. [7] Intermedate Precson Intermedate precson s the results from wthn lab varatons due to random events such as dfferent days, dfferent analysts, dfferent equpment, etc. [8] The standard devaton, relatve standard devaton (coeffcent of varaton) and confdence nterval should be reported for each type of precson nvestgated. Specfcty Specfcty s the ablty to measure accurately and specfcally the analyte of nterest n the presence of other components that may be expected to be present n the sample matrx such as mpurtes, degradaton products and matrx components. It must be demonstrated that the analytcal method s unaffected by the presence of spked materals (mpurtes and/or excpents). In case of dentfcaton tests, the method should be able to dscrmnate between compounds of closely related structures whch are lkely to be present. Smlarly, n case of Lnearty Lnearty s the ablty of the method to elct test results that are drectly, or by a welldefned mathematcal transformaton, proportonal to analyte concentraton wthn a gven range. [10] It should be establshed ntally by vsual examnaton of a plot of sgnals as a functon of analyte concentraton of content. If there appears to be a lnear relatonshp, test results should be establshed by approprate statstcal methods. Data from the regresson lne provde mathematcal estmates of the degree of lnearty. The correlaton coeffcent, y-ntercept, and the slope of the regresson lne should be submtted. It s recommended to have a mnmum of fve concentraton levels, along wth certan mnmum specfed ranges. For assay, the mnmum specfed range s from 80% -120% of the target concentraton. [11] Regresson lne, y ax b Where, a s the slope of regresson lne and b s the y - ntercept. Here, x may represent analyte concentraton and y may represent the sgnal responses. Correlaton Coeffcent, r n ( x x)( y x) ( x y)( y y) 1/ 2

4 35 Where x s an ndvdual measurement n a set of n measurement and x s the arthmetc mean y of the set, s an ndvdual measurement n a set of n measurement and y s the arthmetc mean of the set. DETECTION LIMIT AND QUANTITATION LIMIT The Detecton Lmt s defned as the lowest concentraton of an analyte n a sample that can be detected, not quantfed. The Quanttaton Lmt s the lowest concentraton of an analyte n a sample that can be determned wth acceptable precson and accuracy under the stated operatonal condtons of the analytcal procedures. [12] Some of the approaches to determne the Detecton Lmt and Quanttaton Lmt are: [13] the determnaton of Detecton Lmt and relably quantfed for the determnaton of Quanttaton Lmt. A sgnal-to-nose rato between 3 or 2:1 s generally consdered acceptable for estmatng the detecton lmt and A typcal sgnal-to-nose rato s 10:1 s consdered for establshng the quanttaton lmt. c. Standard Devaton of the response and the Slope. The Detecton Lmt may be expressed as: DL 3.3 / s The Quanttaton Lmt may be expressed as: QL 10 / s Where, s standard devaton of the response and s s slope of the lnearty curve. a. Vsual Evaluaton Vsual evaluaton may be used for nonnstrumental methods. For non-nstrumental procedures, the detecton lmt s generally determned by the analyss of samples wth known concentratons of analyte and by establshng the mnmum level at whch the analyte can be relably detected. And the quanttaton lmt s generally determned by the analyss of samples wth known concentratons of analyte and by establshng the mnmum level at whch the analyte can be determned wth acceptable accuracy and precson. Vsual Evaluaton approach may also be used wth nstrumental methods. b. Sgnal to Nose Ths approach can only be appled to analytcal procedures that exhbt baselne nose. Determnaton of the sgnal-to-nose rato s performed by comparng measured sgnals from samples wth known low concentratons of analyte wth those of blank samples and establshng the mnmum concentraton at whch the analyte can be relably detected for The method used for determnng the detecton lmt and the quanttaton lmt should be presented. If DL and QL are determned based on vsual evaluaton or based on sgnal to nose rato, the presentaton of the relevant chromatograms s consdered acceptable for justfcaton. Range The range of an analytcal procedure s the nterval between the upper and lower levels of analyte (ncludng these levels) that have been demonstrated to be determned wth a sutable level of precson, accuracy, and lnearty usng the procedure as wrtten. The range s normally expressed n the same unts as test results (e.g., percent) obtaned by the analytcal procedure. [10] The followng mnmum specfed ranges should be consdered: [14] For Assay of a Drug Substance (or a drug product) the range should be from 80% to 120% of the test concentraton.

5 36 For Determnaton of an Impurty: from 50% to 120% of the acceptance crteron. For Content Unformty: a mnmum of 70% to 130% of the test concentraton, unless a wder or more approprate range based on the nature of the dosage form (e.g., metered-dose nhalers) s justfed. For Dssoluton Testng: ±20% over the specfed range (e.g., f the acceptance crtera for a controlledrelease product cover a regon from 20%, after 1 hour, and up to 90%, after 24 hours, the valdated range would be 0% to 110% of the label clam). Robustness The robustness of an analytcal procedure s a measure of ts capacty to reman unaffected by small but delberate varatons n procedural parameters lsted n the procedure documentaton and provdes and ndcaton of ts sutablty durng normal usage. Robustness may be determned durng development of the analytcal procedure. [15] If measurements are susceptble to varatons n analytcal condtons, the analytcal condtons should be sutably controlled or a precautonary statement should be ncluded n the procedure. One consequence of the evaluaton of robustness should be that a seres of system sutablty parameters (e.g., resoluton test) s establshed to ensure that the valdty of the analytcal procedure s mantaned whenever used. [16] Examples of typcal varatons are: Stablty of analytcal solutons; Extracton tme. Dfferent columns (dfferent lots and/or supplers); Temperature; Flow rate. In the case of gas-chromatography, examples of typcal varatons are: Dfferent columns (dfferent lots and/or supplers); Temperature; Flow rate. System Sutablty Testng System sutablty testng s an ntegral part of many analytcal procedures. The tests are based on the concept that the equpment, electroncs, analytcal operatons and samples to be analyzed consttute an ntegral system that can be evaluated as such. System sutablty test parameters to be establshed for a partcular procedure depend on the type of procedure beng valdated. They are especally mportant n the case of chromatographc procedures. [16] INTERPRETATION AND TREATMENT OF VARIATION OF ANALYTICAL DATA Analytcal procedures are developed and valdated to ensure the qualty of drug products. The analytcal data can be treated and nterpreted for the scentfc acceptance. The statstcal tools that may be helpful n the nterpretaton of analytcal data are descrbed. Many descrptve statstcs, such as the mean and standard devaton, are n common use. Other statstcal tools, such as calculatng confdence nterval, outler tests, etc. can be performed usng several dfferent, scentfcally vald approaches. In the case of lqud chromatography, examples of typcal varatons are: Influence of varatons of ph n a moble phase; Influence of varatons n moble phase composton; 1. Confdence Interval: A confdence nterval for the mean may be consdered n the nterpretaton of data. Such ntervals are calculated from several data ponts usng the sample mean (x) and sample

6 37 standard devaton (s) accordng to the be performed on the same sample, f possble, formula: [17] or on a new sample. [17] x t s x t / 2, n1, / 2, n1 n s n n whch t / 2, n1 s a statstcal number dependent upon the sample sze (n), the number of degrees of freedom ( n 1), and the desred confdence level ( 1 ). Its values are obtaned from publshed tables of the Student t-dstrbuton. A confdence nterval provdes lmts around the expermentally determned value of the mean wthn whch the true value les wth a gven value of probablty, usually 95%. 2. Outlyng Results: Occasonally, observed analytcal results are very dfferent from those expected. Aberrant, anomalous, contamnated, dscordant, spurous, suspcous or wld observatons; and flyers, rogues, and mavercks are properly called outlyng results. Lke all laboratory results, these outlers must be documented, nterpreted, and managed. Such results may be accurate measurements of the entty beng measured, but are very dfferent from what s expected. Alternatvely, due to an error n the analytcal system, the results may not be typcal, even though the entty beng measured s typcal. When an outlyng result s obtaned, systematc laboratory and process nvestgatons of the result are conducted to determne f an assgnable cause for the result can be establshed. Factors to be consdered when nvestgatng an outlyng result nclude but are not lmted to human error, nstrumentaton error, calculaton error, and product or component defcency. If an assgnable cause that s not related to a product or component defcency can be dentfed, then retestng may When used approprately, outler tests are valuable tools for pharmaceutcal laboratores. Several tests exst for detectng outlers such as the Extreme Studentzed Devate (ESD) Test, Dxon's Test, and Hampel's Rule. Choosng the approprate outler test wll depend on the sample sze and dstrbutonal assumptons. Many of these tests (e.g., the ESD Test) requre the assumpton that the data generated by the laboratory on the test results can be thought of as a random sample from a populaton that s normally dstrbuted, possbly after transformaton. 3. Generalzed Extreme Studentzed Devate (ESD) Test Ths s a modfed verson of the ESD Test that allows for testng up to a prevously specfed number, r, of outlers from a normally dstrbuted populaton. Let r equal 1, and n equal 10. Normalze each result by subtractng the mean from each value and dvdng ths dfference by the standard devaton. Take the absolute value of these results, select the maxmum value R, and compare t to a 1 prevously specfed tabled crtcal value 1 based on the selected sgnfcance level (for example, 5%). If the the maxmum value s larger than the tabled crtcal value, t s dentfed as beng nconsstent wth the remanng data. If the maxmum value s less than the tabled crtcal value, there s not an outler. Sources for -values are ncluded n many statstcal textbooks. CONCLUSION Method Valdaton s an mportant analytcal tool to ensure the accuracy and specfcty of

7 38 the analytcal procedures wth a precse agreement. Ths process determnes the detecton and quanttaton lmt for the estmaton of drug components. The valdaton procedures are performed along wth the system sutablty. Some statstcal tools are also used to nterpret the analytcal results of the valdaton characterstcs. The valdaton of analytcal methods not only requres the performance of characterstcs parameter but also the statstcal treatments of the analytcal data. The acceptance of the varaton of the analytcal data s determned by these treatments. REFERENCES 1. Valdaton of Compendal Procedures <1225>, The Unted States Pharmacopea, 32th Rev., and The Natonal Formulary, 27th Rev., Rockvlle, MD: The Unted States Pharmacopeal Conventon Inc., 2009; I: Valdaton of Analytcal Procedures: Text and Methodology Q2(R1), ICH Harmonsed Trpartte Pharmaceutcals For Human Use, 2005; Valdaton of Compendal Procedures <1225>, The Unted States Pharmacopea, 32th Rev., and The Natonal Formulary, 27th Rev., Rockvlle, MD: The Unted States Pharmacopeal Conventon Inc., 2009; I: Valdaton of Analytcal Procedures: Text and Methodology Q2(R1), ICH Harmonsed Trpartte Pharmaceutcals For Human Use, 2005; Analytcal Data-Interpretaton and Treatment <1010>, The Unted States Pharmacopea, 32th Rev., and The Natonal Formulary, 27th Rev., Rockvlle, MD: The Unted States Pharmacopeal Conventon Inc., 2009; I: Gudelne on boanalytcal method valdaton, European Medcnes Agency, London, UK, 2011; I: Valdaton of Analytcal Procedures SC III F, Brtsh Pharmacopea, Brtsh Pharmacopea Commsson, Valdaton of Analytcal Procedures: Text and Methodology Q2(R1), ICH Harmonsed Trpartte Pharmaceutcals For Human Use, 2005; Valdaton of Analytcal Procedures: Text and Methodology Q2(R1), ICH Harmonsed Trpartte Pharmaceutcals For Human Use, 2005; Valdaton of Compendal Procedures <1225>, The Unted States Pharmacopea, 32th Rev., and The Natonal Formulary, 27th Rev., Rockvlle, MD: The Unted States Pharmacopeal Conventon Inc., 2009; I: Valdaton of Analytcal Procedures: Text and Methodology Q2(R1), ICH Harmonsed Trpartte Pharmaceutcals For Human Use, 2005; Valdaton of Compendal Procedures <1225>, The Unted States Pharmacopea, 32th Rev., and The Natonal Formulary, 27th Rev., Rockvlle, MD: The Unted States Pharmacopeal Conventon Inc., 2009; I: Valdaton of Analytcal Procedures: Text and Methodology Q2(R1), ICH Harmonsed Trpartte

8 39 Pharmaceutcals For Human Use, 2005; Valdaton of Analytcal Procedures: Text and Methodology Q2(R1), ICH Harmonsed Trpartte Pharmaceutcals For Human Use, 2005; Valdaton of Compendal Procedures <1225>, The Unted States Pharmacopea, 32th Rev., and The Natonal Formulary, 27th Rev., Rockvlle, MD: The Unted States Pharmacopeal Conventon Inc., 2009; I: Valdaton of Analytcal Procedures: Text and Methodology Q2(R1), ICH Harmonsed Trpartte Pharmaceutcals For Human Use, 2005; Analytcal Data-Interpretaton and Treatment <1010>, The Unted States Pharmacopea, 32th Rev., and The Natonal Formulary, 27th Rev., Rockvlle, MD: The Unted States Pharmacopeal Conventon Inc., 2009; I: 399.