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Analytical Letters
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Analytical Method Development and Validation for the Academic Researcher
Ira S. Krull; Michael Swartz

Online Publication Date: 01 January 1999

To cite this Article Krull, Ira S. and Swartz, Michael(1999)'Analytical Method Development and Validation for the Academic

Researcher',Analytical Letters,32:6,1067 — 1080
To link to this Article: DOI: 10.1080/00032719908542878 URL:

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360 Huntington Avenue. Northeastern University. 1067 Copyright © 1999 by Marcel Dekker. Krull* Department of Chemistry. and after reading numerous original research papers and reviews submitted for publication to various journals or books. . MA 02115. INTRODUCTION During the course of our scientific careers. 1067-1080 (1999) MINI-REVIEW ANALYTICAL METHOD DEVELOPMENT AND VALIDATION FOR THE ACADEMIC RESEARCHER Downloaded By: [UNAM Direccion General de Bibliotecas] At: 18:12 8 August 2008 Ira S. 32(6). academic researchers have little use for method validation studies or demonstrations. it has become apparent to us that many. Now. www. MA 01757. that may sound a bit harsh or a strong way to start this mini-review. USA. Boston. but this * Author to whom correspondence and reprint requests should be addressed.ANALYTICAL LETTERS. 102 Hurtig Building. and Michael Swartz Pharmaceutical Market Development R&D. 34 Maple Street. if not most.dekker. Inc. USA. Waters Corporation.

such as limits of detection or quantitation. mass spectrometry Downloaded By: [UNAM Direccion General de Bibliotecas] At: 18:12 8 August 2008 (MS) or other analytical techniques. and reproducibility. be that in high performance liquid chromatography (HPLC). to provide the readers with some idea of the already vast literature in the areas of analytical method validation. 4. ?). there is a glaring omission of analytical figures of merit.1068 KRULL AND SWARTZ situation has been our collective impression after carefully reading and commenting on hundreds of submitted journal manuscripts over perhaps the past 25 years. nor any application to simulated or real samples for an attempt at method validation. validity. will indeed provide some degree of method validation and assurance of applicability and transference of the reported methods? That is really the gist of this mini-review. and most importantly. robustness. There is also often an omission of repeat measurements on the very same sample extract. no statistical treatment of the data. How much more work will be needed on the part of those readers in order to learn whether or not the described methods will really work on their own samples in their own labs with their own instrumentation and skills? This unfortunate state of affairs in submitted and published manuscripts has resulted in a huge literature of methods that are of really questionable utility. accuracy and precision. How can we change this sorry state-of-affairs. other than to the authors. especially those analysts that might actually wish to employ the reported (purported) methods in the very near future. most papers from academia do not usually include attempts to partially or fully validate methods. Unfortunately. Many papers that are submitted for publication involve some type of method development. applicability. whether or not the method can indeed be routinely applied by other analysts in other laboratories with the same or similar instrumentation and skills. so that journals and authors of future submitted manuscripts. for academics . Such papers become of questionable and dubious value to the general reader. because the reader is not apprised as to the status of validation of the method being described. no indication of the number of such repeats (n = 3. or value. 5. high performance capillary electrophoresis (HPCE). calibration plots. This is very unfortunate. In many manuscripts.

It is. let us again summarize what is being routinely asked by the US FDA of pharmaceutical and biotechnology firms when . Investigational New Drug) or actual drug release (NDA.S. That appears incredulous and astounding today. That is not our intent. left to the discretion (if that is the proper word) of the reviewers. Academics do not. at the same time. with the editors rarely requesting additional validation evidence. There are no guidelines that we have ever seen in the directions to authors of any (!) analytical journals requiring or even suggesting that some amount of method validation data be routinely supplied when a manuscript is first submitted. need to provide the same degree of method validation as a pharmaceutical or biotechnology firm trying to get a new drug to market. What then is really needed on the part of these authors in order to provide some evidence of validation and applicability to future users? We are not. degree of method validation to be demonstrated by academic (or industrial) submitters of papers dealing with new analytical methods. too often. but it seems very clear in reading any number of journal descriptors to authors for papers to be submitted dealing with method development and optimization (validation as a word almost never appears).METHOD DEVELOPMENT AND VALIDATION 1069 as well as those making submittals to the regulatory agencies. and to try to encourage future authors of analytical manuscripts to routinely include some degree of method validation in the final manuscripts. at the very outset. such as U. New Drug Application). by and large. suggesting that all academics should in the Downloaded By: [UNAM Direccion General de Bibliotecas] At: 18:12 8 August 2008 future meet all USP/ICH (United States Pharmacopeia/International Conference on Harmonization) guidelines required of FDA (Food and Drug Administration) regulated firms that are making new drug submittals of methods for clinical studies (IND. But. Food and Drug Administration? Perhaps as a point of reference. What is Required Today of Industrial Firms Submitting New Analytical Methods to Regulatory Agencies. it would appear that most journals are not requiring any. nor do we feel that this would be warranted or accepted by the analytical community or analytical journal editors.

final FDA approval of an NDA. Figure 1 summarizes the ICH method validation parameters. and we will just summarize the more essential features in these1'15. The amount of method validation studies and number of validation parameters that must be met changes as a drug proceeds from discovery to Phase III and then. There are numerous reviews and overviews in the area of analytical method validation and USP/ICH requirements. This has all been sorted out by the USP and is discussed elsewhere1'14*16. Various analytical textbooks may also be scoured for more in-depth. they are submitting an IND or NDA for a new drug substance. academic . then not all of the validation parameters in Figure 1 need to be demonstrated. we need to critically and correctly define these terms. If the drug does not make it through all stages of Phases I-III clinical trials. 1: ICH method validation parameters. it is hoped. Since this mini-review is really being aimed at academics. too many academic analysts and those that develop newer analytical approaches are not always as familiar with these terms. Whatever the stage of a drug's progress towards commercialization and introduction. and what the FDA expects to see for drug method submittals1. Unfortunately.1070 1 *•[ KRULL AND SWARTZ Precision 1 [ Limit of Detection 1 Method Validation Downloaded By: [UNAM Direccion General de Bibliotecas] At: 18:12 8 August 2008 * • ^- Specificity Linearity ] 1 ) 1 * ^- Ranqe Robustness System Suitability 1 Fig. any pharmaceutical analyst must be familiar with the fundamental terms of Figure 1. soon to be adopted by the USP.

any validated method must demonstrate. Accuracy and precision therefore become the cornerstones of any quantitative analytical method. The closer the agreement amongst the different determinations done on the very same sample. Thus. and these must be very clearly demonstrated and reproducible. by and large. then the better the precision of that method. small deviations from the true value are usually found. the accuracy of levels determined in real samples. The method of demonstrating accuracy and precision can involve a variety of practices. is to demonstrate both high accuracy and precision. within one laboratory and one analyst. in this context relates to the agreement (or lack thereof) between the values determined for the drug of interest and the true or known level actually Downloaded By: [UNAM Direccion General de Bibliotecas] At: 18:12 8 August 2008 present. with each workup injected at least three times. in a reproducible manner. double-blind. and then between analysts in different laboratories. such as single-blind. no method is 100% accurate. then it is possible to determine precision. and others1'9"10. especially involving FDA submittals. interlaboratory collaborative study. of course. and the number of repeat injections from each aliquot is also greater than three. Analytical methods. are both qualitative and quantitative. from start-to-finish. this is so commonplace and so accepted that it is never questioned. . Accuracy. final utility and applicability of that method with real samples. It is recognized that no validated analytical method can possibly be done or repeated on the same sample less than three times. so that the number of samples is greater than three. That means one must have samples of known composition and known levels of the analytes of interest. but the closeness of method accuracy is an important marker of the overall. The goal. In a regulated industry.METHOD DEVELOPMENT AND VALIDATION 1071 discussions of these terms17'22. When the analytical method is repeated on different aliquots of the same sample several times. especially with regard to the levels of drugs and/or metabolites in any given sample. Only in academia does this question of the number of repeats come into the discussion and be questioned. Naturally.

which produces a certain signal-tonoise ratio. Limit of detection is a concentration point where accurate and precise (!) quantitation is not usually possible. LOD and LOQ. and reproducibility (ruggedness). Finally. from day-to-day. in a quantitative sense (LOQ). depending on the particular reference source or regulatory guidelines.1072 KRULL AND SWARTZ There are various parts to precision. This is different than selectivity. as well as other similar compounds . a number of these can be routinely used. Intermediate precision refers to how the method performs. It refers to how well the method qualitatively separates and then identifies that analyte of interest. Limit of quantitation or quantification. So long as one defines their terms. in a real sample. but the qualitative identification of the analyte as being present is. ICH guidelines and equations are routinely followed. within a given day. Specificity is a term that relates to how well a given method is able to uniquely (specifically) determine a given analyte in a sample to the exclusion of other possible interferents. as one attempts to quantitate an analyte at lower and lower concentration levels. using the currently accepted ICH equations and definitions2 . Repeatability means how the method performs in one lab and on one instrument. where it now starts being possible to accurately and precisely determine the absolute levels of the analyte present in a given sample. such as repeatability. to the perhaps 100% exclusion of all other substances present in the same sample that might interfere in the determination. There are numerous definitions of these terms. intermediate precision. from analyst-to-analyst. reproducibility refers to how that method performs from lab-toDownloaded By: [UNAM Direccion General de Bibliotecas] At: 18:12 8 August 2008 lab. both qualitatively and quantitatively. on the other hand. and from instrument-to-instrument. but now from instrument-to-instrument and dayto-day. Limit of detection (LOD) and quantitation (LOQ) relate to the concentration of the analyte. which is less specific. These validation parameters need to be demonstrated in order to indicate how the method functions. but for regulatory submittals. is a higher level. again in both qualitative and quantitative terms. in that it may determine the analyte of interest. within one lab.

it is not necessary according to ICH to demonstrate a range of 10 orders of magnitude. Ideally. but rather to limit that range to the samples of interest and their expected concentrations. Linearity relates to how well detector response (peak height or peak area or peak intensity) relates to or correlates with increasing concentrations of analyte present. and surely one is not required by ICH to demonstrate linearity of as many orders of magnitude as possible. including the concentration levels expected to be found in the final samples. standard additions. The ICH defines the linear concentration range depending on the nature of the sample and the type of method1. matrix-matched calibration plots)24. internal standard. That is. by . with or without the sample Downloaded By: [UNAM Direccion General de Bibliotecas] At: 18:12 8 August 2008 matrix present (e. starting from the LOD or LOQ. This concentration magnitude or spectrum is then defined as the range of the method.METHOD DEVELOPMENT AND VALIDATION 1073 in the same sample that are not the analyte itself. Linearity refers to the calibration plot and its slope of detector response vs.g. Again. but one that ICH and FDA have taken very seriously. There are very practical reasons for demonstrating a rather limited linearity range for real samples. Robustness is a term that academicians rarely utilize in their papers. as above. the calibration plot should be linear of several orders of magnitude. There can be external standard calibration plots. rather than the perhaps academic notion of demonstrating linearity over 5-7 orders of magnitude. It refers to how well the method performs over small. It is not necessary to demonstrate linearity beyond the expected concentration ranges. and that the peak of interest is the analyte of interest. Range relates to the linear portion of the calibration plot and its linearity. Robustness is actually demonstrated by experimentation. wherein accurate and precise quantitation should be best realized. intentional changes in the method's operational parameters.. concentrations injected. and nothing else. a calibration plot will be linear over a certain range of concentrations. True quantitation cannot be performed until the method is first shown to be specific. and others. with some extension at both ends.

It is used to show that the HPLC column. Robustness is very different from ruggedness or precision. if these chromatographic figures of merit do change greatly with small changes in these parameters. always before and always after the actual batch of samples. standard reference materials. A system suitability sample is run any day that real samples are being analyzed. robust is better than not. and instrument-toinstrument. such as pH. There is little use in promoting or promulgating a method in the literature that has never been shown robust. quality assurance samples. etc. Robustness is measured by noting changes in peak shape. and is really used just to show that the analytical instrumentation and overall system is performing properly . flow rates.1074 KRULL AND SWARTZ intentionally changing various operating parameters of the method. analyst-to-analyst. temperature. plate counts. often within that run of batches. Obviously. real samples. and accuracy of quantitation. for one then never knows how the method will perform in the hands of other analysts. and symmetry already demonstrated when the method was first validated. then the method is said to be robust. and the analyst should strive to demonstrate that such is the case in validating a method for future usage. plate counts. in order to demonstrate that the instrumental system is performing properly. is yielding the desired peak shapes. If the chromatographic peak shapes. ICH introduces something termed system suitability. retention time. mobile phase composition. gradient mixing. resolutions. retention times. resolutions. for example. and symmetry do not change much (and that must be defined) Downloaded By: [UNAM Direccion General de Bibliotecas] At: 18:12 8 August 2008 with perturbations in the operational parameters. which is really used after the method has been fully validated. as these operational parameters are modified. for it really describes how a given method performs when there may be slight. then the method is said not to be robust. when the validated method is being routinely used to actually analyze real samples25. It is different from quality control samples. On the other hand. unintentional changes in the operational parameters within a day or from day-to-day. laboratory-to-laboratory. plate counts. resolutions. Finally.

METHOD DEVELOPMENT AND VALIDATION 1075 before real samples are then run. System suitability samples are run to ensure the proper operation of the instrumental system itself. in replicate) as expected. from reviewer-to-reviewer. Our opinions here are just that. it seems to us that there are some basic requirements. We have now defined those USP method validation parameters that any FDA filing must meet in order to pass muster and avoid rejection of the application. and they may change in the future. However. Some authors and reviewers ask for less. others for more. and the reader is urged to approach more intense books and review articles that discuss this topic in much greater depth. we need to move forward. It is necessary to demonstrate the ability to fully resolve the analyte peak of interest from other compounds present in a real sample. not necessarily the entire method including sample preparation. they are not grounded in accepted facts or regulatory requirements. since this mini-review is really aimed at the academic researcher/analyst and their approaches to method validation. in order to have their method considered validated? In the past. There is more to regulatory method validation than just the above. It is necessary to . must the non-regulatory analyst include in their publications on a new or improved analytical method. opinions. validated methods of analysis. better. and some for none. this has varied from journal-to-journal. There are no firm (stated) requirements in writing. improved. make-believe. If the system suitability sample does not yield chromatograms (injected several times. What is Required Today of Academics Submitting New Analytical Methods to Journals for Eventual Publication? How much. partial sample (usually without the entire sample matrix present). even for academic analytical papers that describe new. and from editor-to-editor. and thus such a sample does not constitute a real sample as much as Downloaded By: [UNAM Direccion General de Bibliotecas] At: 18:12 8 August 2008 a made-up. then. then there is every reason not to proceed with real samples until the problem(s) is defined and corrected. However.

It is also necessary to demonstrate method optimization. in a true quantitative manner? Why is it that so little quantitation is demonstrated with real samples using more than a single analyst? r i . It is also necessary to demonstrate the time and effort involved in performing routine sample analyses with the newer method. the nature of that linearity (correlation coefficient.1076 KRULL AND SWARTZ demonstrate that peak is pure. whether one uses single. and the correct analyte (specificity). nor to demonstrate system suitability methods. slope.). etc. It is not necessary to demonstrate complete ruggedness from lab-to-lab or country-to-country. as above. It is necessary to demonstrate the ranges of linearity of the calibration plots. with actual sample matrices. whatever the method of quantitation utilized. or as a part of validation. in order to reach complete validation. double or zero blind methods of validation. It is necessary to demonstrate the precision of the overall method and how small changes in its operational parameters may affect the final results. since that is not really a part of method validation. prior to validation. with Downloaded By: [UNAM Direccion General de Bibliotecas] At: 18:12 8 August 2008 numerous. It is necessary to demonstrate percent recovery of the analyte of interest from several real sample matrices. It is necessary to demonstrate accuracy and precision of quantitation with real samples. or to^ndicate the number of repeats used per data point. just so that future users will have a better feel for where the method will and will not perform in an acceptable manner. repetitive injections of each and every sample preparation. It is also necessary to demonstrate acceptable (<2-4%) accuracy and precision. It is also necessary to demonstrate LOD and LOQ for the method. homogeneous. if not the actual costs so incurred. or how many times a given sample was assayed via different aliquots? Why do so many papers appear without any demonstration of true applicability or application to real world samples. y-intercept. in single or double blind manners. shortened validation approaches on a more routine basis? Why do they routinely avoid using statistics in their data. regardless of the method of quantitation being employed. Why then do academic analysts not utilize the above. It is necessary to demonstrate qualitative and quantitative repeatability and reproducibility.

Most readers may be much more interested in the science described. and journal owners or publishers. The reason that pharmaceutical analysts do perform such involved method validations is also quite simple. using some or all of the guidelines being suggested above. such as robustness. is not asked Downloaded By: [UNAM Direccion General de Bibliotecas] At: 18:12 8 August 2008 or expected by the journal. to satisfy both camps. and that most readers would not read that data if it were included. then it is much easier to just avoid so doing. or from the authors on request. with real samples. Glossary and List of Abbreviations HPLC = high performance liquid chromatography HPCE = high performance capillary electrophoresis ICH = International Conference on Harmonization IND = investigational new drug MS = mass spectrometry NDA = new drug application LOD = limit of detection LOQ = limit of quantitation USP = United States Pharmacopeia .these things are not being required by the reviewers. Otherwise. The fact of the matter is that the science of analytical chemistry will progress when any and all new methods of analysis are demonstrated valid at the time of first publication. but if is required by FDA in order to get a drug approved and remain in business. rather than in any method validation to demonstrate that the science really does work with real samples. Academics will do what is required from a purely scientific viewpoint or perspective for publication. editors. in a supplemental section of the journal available on-line or by writing. It has been argued that journals do not have the space needed to include full validation results.METHOD DEVELOPMENT AND VALIDATION 1077 Why indeed? The reason is really quite simple. it would always be possible to include the complete validation results via a website version of the paper. However. the reader will never know for certain that the method can indeed be taken from the literature and directly utilized.

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Validation Viewpoint: System suitability samples.1080 KRULL AND SWARTZ 24. 1999).E.1999 Accepted: January 25.S. Swartz. Downloaded By: [UNAM Direccion General de Bibliotecas] At: 18:12 8 August 2008 Received: January 15. 1999 .S. I. 16(12). Validation Viewpoint: Quantitation in method validation. Krull and M. Krull and M.E. and what they should do? LC/GC Magazine. I. LC/GC Magazine. 25. 1084(1998).what they are. why they are. Swartz. in press (January.

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