VALIDATION OF ANAYTICAL METHODS

P.S.RAMANATHAN DIRECTOR—CORPORATE ANALYTICAL OPERATIONS GHARDA CHEMICALS LTD. B-29, MIDC(PHASE I), DOMBIVLI(E)421203 DIST. THANE, MAHARASHTRA STATE
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Chemical Analysis as an Integral Process Chemical analysis of whatever material system can be described as a chain of decisions, actions, and procedures. Figure 1 shows the cyclic nature of many chemical analytical processes.

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The last step (interpretation and evaluation of results of analysis) should eventually provide an answer to the starting problem, generally stated by a client of the laboratory. If the answer is not satisfactory, the analysis cycle can be followed again, after a change or adaptation of one or more steps. Sometimes this leads to a development of a new method or (part of a) procedure in order, for example, to achieve better separation of certain components, or to attain a lower detection limit for specific compounds.
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Chemical analysis as a cyclic process This is illustrated in Fig. 1. Like any chain, a chain of chemical analysis is only as strong as its weakest link.

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Figure 1. Chemical analysis as a cyclic process
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In general, the weakest links in an analytical process are not the ones usually being recognised as parts of chemical analysis, such as chromatographic separation or spectrometric detection, but rather the preceding steps, often taking place outside the analytical laboratory such as the selection of object(s) to be sampled, the design of the sampling plan, and the selection and the use of techniques and facilities for obtaining, transporting, and storing samples.
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When the analytical laboratory is not responsible for the sampling, the quality management system often does not even take account of these weak links in the analytical process. Furthermore, if the preparation (extraction, clean-up, etc.) of the samples has not carefully been carried out, even the most advanced and quality controlled analytical instruments and sophisticated computer techniques cannot prevent that the results of the analysis become questionable.
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Finally, unless the interpretation and evaluation of results have a solid statistical base, it is not clear how significant these results are, which in turn greatly undermines their merit. We, therefore, believe that quality control and quality assurance should involve all the steps of chemical analysis as an integral process, of which the validation of the analytical methods is only one, though important, step.
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In laboratory practice, quality criteria should concern the rationale of the sampling plan, the validation of methods, instruments and laboratory procedures, the reliability of identifications, the accuracy and precision of measured concentrations, and the comparability of laboratory results with relevant information produced earlier or elsewhere.
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In this presentation, we will discuss the  Validation concepts in detail.  Thereafter, we  will discuss some related aspects  like  Certified Reference Materials(CRM) and their  traceability, and a brief review of Proficiency  Testing(PT) Programs. 

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1)The Joke of the Japanese prime minister talking to President Clinton. 2)Condolence message to a neighbour in the village, after the death of his wife.

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The language police(Newspeak.com) have added more words to their politically correct lexicon—so it is no longer okay to call a housewife a “home maker”, an ugly woman, “a plain Jane” and a handicapped person “physically challenged”. According to the website that tracks modern speak and is dedicated and inspired by George Orwell, “domestic engineer” is the term for housewife.
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Visually challenging for the “ugly”. Handicapable for the physically impaired. Other examples are: Broken home: Dysfunctional family The website compares modern speak with “Newspeak”—the official language of the totalitarian state of Oceania in Orwell’s classic 184, which deleted all words that went againt party policy. The state’s propaganda department was called “The Ministry of Truth”.
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The rationaing dept.—The ministry of plenty. Words like “affirmtive action”, “homophobic”, “peace keepers”, “sexual harassment” and “the war on drugs”, were nonexistent before this century. They were fabricated by the government and special interest groups with the main aim of misleading the public and swaying public opinion. The website also lists words that it claims have lost their original meaning. “Patriot” –one who loves the country and culture—Now it 03/12/09 means “bigot, racist and a possible terrorist. 14

Family now means, “anyone or more people of any gender raising any one’s children anywhere”

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Validation: Element of Laboratory Quality Assurance Quality is a relative notion; never high or low, in an absolute sense. Rather, it is adequate or inadequate in terms of the extent to which a product, a process, or a service meets the requirements specified beforehand by an objective or a customer.

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The principal product of an analytical chemical laboratory is information about the chemical composition of material systems, usually in terms of the identity and/or quantity of one or more relevant components in samples taken from these materials. The quality of scientific information, in general, is evaluated by internationally accepted standards of objectivity, integrity, reproducibility, and traceability, in any case, prior to publication.
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Essential criteria for the quality of produced chemical information are the utility and the reliability, which are closely related to the margins of uncertainty in the measurement results regarding both the identity and the concentration of the target components.

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With respect to these correlated criteria, minimum requirements are generally set by the customer and usually deduced from a previously specified purpose. The quality of produced chemical information is therefore factually to be acknowledged by the customer as the end-user of this information.

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For chemical measurements, this could be a clinical chemist, who needs to know the identity of certain isolated compounds from a biological fluid, a polymer chemist who wishes to verify the molecular structure of a product of synthesis, or a health researcher who wants to know whether the concentration of a certain toxic compound in certain food is above certain concentration level.
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It is not hard to imagine the consequences in terms of costs, health risks, and so on , when, on closer examination or statistical evaluation of the measurement results, a positive finding turns out to be false, or the uncertainty margin of a measured concentration appears to be 100% and not the initially reported 10%. Evaluation and validation of analytical methods and laboratory procedures are, therefore, of paramount importance, prominent means being the use of adequate (preferably certified) reference materials and participation in interlaboratory 03/12/09 21 proficiency tests.

Quality demands made on the infrastructure, equipment, operating procedures, personnel, and organization of the laboratory are to be deduced from the quality requirements, that the produced chemical information should meet. A formal recognition of this type of quality can be achieved through accreditation or certification, based on international quality standards and guidelines, as issued by International Organization for Standardization (ISO),
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Organisation for Economic Co-operation and Development (OECD), and European Committee for Standardization (CEN). Validation of analytical methods is one, though an essential step in the integral process of quality assurance and quality control of chemical measurements in material systems.

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Validation Concepts in Analytical Chemistry —General Aspects “An Analytical Chemist is one, who applies  knowledge of the principles of the Physical,  Life and Engineering Sciences, to providing  and utilising the means, whereby the  constitution of substances is established,  and exercises professional judgement, in the  interpretation and use of the information  obtained."                             The Royal Society of  Chemistry 03/12/09 24

Besides other assignments undertaken by him, an analytical chemist must use his skill and judgement to: (a)Study the fundamental principles of the methods used for analysis, (b)Develop new analytical methods and instrumentation, (c) Develop and validate analytical techniques, (d) Assess the degree of reliability of the results, prior to reporting the results and (e) Supervising and training juniors.
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What is a Validated method? A good analytical result must have the potential to withstand the closest scrutiny. Often, reports are made on cases, where analytical data have been inadequate. Unknown interferences and biases all go towards unreliable data.

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Validation of a method ensures that the accuracy of the concentration reported is known and appropriate for the purpose. All known interferences and biases will have been investigated and evaluated and the method will have been tested against Certified Reference Materials, where available, and, if possible, by collaborative studies with peer laboratories.
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The Validation process in analysis A good analyst must, therefore, acquire complete understanding of the method used for analysis, and its potential applications. He has to ensure whether the method is capable of answering the questions put by the customer. He has to ascertain the limits to the range of applications. He must have full information about the factors which are liable to upset the method. All these efforts, cumulatively, constitute the method validation process, which is an essential activity if an analytical measurement is going to be fit for the 03/12/09 28 purpose for which it was intended.

Thus, generating quality analytical results involves fully understanding and evaluating the set of procedures that make up the method, knowing how the result is going to be used, and maintaining close collaboration with the customer for that analysis.

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During the last few decades analytical measurement has come under very close scrutiny. The indirectness of the analytical measurement presents the analyst with unique problems. In fact, every new sample poses a different set of challenges, even to the seasoned analytical chemist.

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The analytical chemist may use an in-house method, or a method reported in a standard (like the Bureau of Indian Standards or pharmacoepia) or the method may be set out as a standard method in some piece of legislation. The fundamental requirement of a good analytical chemist is to satisfy himself as to whether the methods he uses actually measure what they are supposed to measure. How much is the method affected by outside interference?
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Have all the factors affecting the reliability of the method been fully understood and investigated? Does the analysis produce results which are fit for the purpose to which it is put, and are the results comparable with those of other laboratories performing similar analyses? These are the problems faced by a seasoned analytical chemist, as a matter of routine.
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Any Validated analytical measurement program needs full support from adequate education and training programs. There must also be a well designed and organized program of international collaboration aimed at the harmonization of analytical measurement technology.

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The availability of well characterized reference materials(ideally with matrices which match the sample in question) is undoubtedly one of the corner stones of method validation and establishing traceability to primary measurement standards. The procedure adopted to check that the results are comparable with those of other laboratories(performing the same analysis) is termed “Proficiency testing program”.
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These schemes are run by authorized government agencies like the NABL(National Accreditation Board for Testing and Calibrating Laboratories) or by some wellestablished central co-ordinating laboratories. Internationally accepted standard protocols(ISO/IUPAC/AOAC) are available for operating proficiency testing programs.

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National and international cooperation are essential and, in fact, indispensable, for the harmonization of analytical methods and protocols, which would pave the way for increase in the national and international acceptance of analytical data. Achieving this acceptance is one of the key steps in the process of ensuring more freedom and flexibility for international trade, which will ultimately lead to greater economic growth and increased benefits to the customer.
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Formation of organizations like EURACHEM(a network of European National Laboratories) , CITAC(Cooperation on International Traceability in Analytical Chemistry), etc., are efforts to harmonise chemical testing procedures and supporting infrastructure. The aims and objectives of EURACHEM and CITAC have been described in reference.
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The VAM bulletin, published by the “Laboratory of the Government Chemist”, UK, is an excellent publication, which provides information and guidelines on various aspects connected with Valid Analytical Measurement. It has been claimed that this publication has a current circulation of more than 10,000 copies.

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It should be noted that “validation” does not guarantee that a method is free from error. It just confirms that the method is adequate to meet the specification prepared for the analysis. This confirmation will usually require the laboratory to carry out an experimental study but, in certain circumstances, the experience of the analyst, together with validation data obtained previously or elsewhere, may be sufficient.
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Whatever the approach used, it is important that the analyst notes in the laboratory’s records the agreed analytical specification, in terms of various performance characteristics of the method, and the evidence or other reasons for believing that the specification has been met. This should be done before any samples are analysed and, where the method is used routinely, checked periodically.
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It must be emphasized that reliable chemical analysis will always ultimately depend on the skill of the analyst. From the wealth of literature available for guidance, it is the duty of the analytical chemist to select from it a proper approach in order to provide a method which is fit for a specific purpose. Judged from this angle, it can be seen that the method validation for the everyday work of the laboratory is both essential and realistic.
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Checking the validity of procedures for environmental monitoring, food analysis, etc., has, now, become a matter of utmost importance because of the need to regulate environmental pollution and contamination of food articles. It is worth mentioning here that the validity of analytical measurements is of such key importance to many aspects of business and social activities that it should be high on the agenda of those who teach science in colleges and universities. 03/12/09 42

It may not be out of place to mention here that in an intercomparison of results from expert international laboratories, (available in the literature and provided in the bar chart form), the results of different laboratories showed significant variability.

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The average percentage of error varies from less than 1% to about 13%( out of the results provided by 16 laboratories). The results provided are for relatively straightforward analyses of inorganic elements in solution. Such practical problems must be kept in mind while understanding the subject of Valid analytical measurements and its practical importance.
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PT schemes provide laboratory analysts and their customers with valuable information about the way in which they carry out their analyses. A major development is the publication an International Harmonised Protocol on PT. This is the result of collaboration of scientists from many countries, under the joint auspices of ISO, IUPAC and AOACI.

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Valid Analytical Measurements A chemist can generate a number, and call it a result!! A standard analyst must be able to stand up in a court of law and say that, if the analysis is repeated, the result will always come within the expected range. Another analyst, applying the same or different method to the same sample, should be able to come up with a comparable result.
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Professional analysts do not generate numbers---they perform Valid analytical measurements.

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Globalisation Q: What is the truest definition of Globalisation? Answer: Princess Diana’s death. Q: How come? A: An English princes with an Egyptian boyfriend crashes in a French tunnel, driving a German car with a dutch engine, driven by a Belgian who was drunk on Scottish whisky, followed closely by Italian paparazzi, on Japanese motorcycles, treated by an American doctor, using Brazilian medicines…..
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……sent to you by an American, using Bill Gate’s technology and you are probably reading this on your computer, that uses Taiwanese chips and a Korean monitor, assembled by Bengaladeshi workers in a Singapore plant, transporeted by Indian lorry drivers, hijacked by Indonesians, unloaded by Sicillian longshoremen and trucked to you by Mexican illegals…. That, my good friends, is real globalisation!!
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The importance of validated analytical measurements Several billion analyses are carried out per year, in various countries. They form a vital part of ensuring the quality of goods and commodities. They assist the government in policy development, and the proper enforcement of regulations. Analytical measurements of proven validity pervades all aspects of the economy and everyday life.
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Obtaining the necessary accuracy and precision in analysis is important (a) in terms of the quality of industrial goods, (b) R&D, (c) the health, safety and welfare of the community, (d) protection of the environment and (e) economic well-being of the nation.

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Often analytical results do not get  reproduced within the same laboratory, and  other laboratories, as well.  If this is so, the  analytical community is failing to meet one  of its fundamental objectives­­to provide  customers with results of demonstrable  accuracy.

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The costs associated with wrong results are  usually enormous. Unnecessary actions  taken based on inaccurate results are not  acceptable from the economic point of view.  About 10% of the analyses carried out in the  world are estimated to need repetition(Costs  billions of dollars).

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Method validation process includes, among other things: (a)Ensuring that the method offers satisfactory answers to the needs of the customer, (b)Understanding the limits to the range of application and (c) Identifying the factors that may upset the results.

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The VAM(Valid Analytical Measurement) Initiative, is a programme funded by the U. K. Dept. of Trade and Industry. The VAM Initiative seeks to improve the quality of analytical data and to facilitate the mutual recognition of analytical results by promoting 6 key principles of good analytical practice.
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The Six VAM Principles The six VAM principles enable organisations to implement best practice, and make valid measurements. They are designed to control all factors that might affect the reliability of analytical results, thereby reducing the cost and risk of unreliable measurements.

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Principle 1:Analytical measurements should be made to satisfy an agreed requirement. Principle 2:Analytical measurements should be made using methods and equipment, which have been tested to ensure, they are fit for purpose. Principle 3:Staff, making analytical measurements, should be, both qualified and competent, to undertake the task.
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Principle 4:There should be a regular independent assessment of the technical performance of a laboratory. Principle 5:Analytical measurements made in one location should be consistent with those elsewhere. Principle 6:Organisations making analytical measurements should have well defined quality control and quality assurance procedures.
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In other words, key principles of valid analytical measurements include, (1) Measurements should be made using properly validated methods. (2) Reference materials should be used to ensure traceability of measurements. (3) Laboratories should seek independent assessment of their performance by participating in national and international proficiency testing schemes. (4) Labortories should seek independent approval of their quality system, preferably by accreditation or licensing, to a recognised quality standard. 59 03/12/09

When VAM are not realised, data of poor quality are reported by a lab. In such circumstances, the existing problems and costs for the end-user of the analytical data can be substantial and are associated with consequences such as : 1)the costs involved in repeat measurements to correct poor data; (2)the faulty decisions making that ensues when invalid results are acted upon; (3)damage to reputation and credibility that results when an end-user is associated with poor 03/12/09 60 data;

(4)possible loss of business where the endusers customer is compromised by poor data; and (5)any legal and financial liability incurred from the use of poor data. It is important that those who commission labs to undertake analytical measurements on their behalf appreciate the critical need to select only competent labs.
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To sum up, validation is "establishing documented evidence, that a system does what it purports to do". Validation master plan, in any organisation, includes, among other things, analytical methods for products and low level detections, standardisation of equipments and related facilities, and validated documentation.

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HR Manager’s loveletter Ever wonder how an HR manager would write a love letter to his girl friend? Dearest Juliet: I am very happy to inform you that I have fallen in love with you, since the October 14, 2005, (Saturday), 12 noon to be precise. With reference to the meeting held between us on the 13th of October at 15.00 hrs., in the HRD Conference room, I would like to present myself as a prospective lover of your good self.
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Our love affair would be kept confidential, and maintained on probation for a period of three months. Depending on compatibility, and your detailed medical examination by a lady doctor, and appropriate physical examinations by me, it would be made permanent. Of course, upon completion of probation, there will be continuous on-the-job training and performance appraisal schemes, leading upto promotion from lover to spouse. The expenses incurred for coffee and entertainment would initially be shared equally 03/12/09 64 between us.

Later, based on your performance and inherent skills I might take up a larger share of the expense. However, I am broad-minded enough to be taken care of, on your expense account as well. I request you to respond to this offer within 30 days of receiving this letter, failing which, this offer would be cancelled, without further notice, and I shall have no alternative other than considering some other suitable candidate for the position indicated above. If due to any reason, you do not wish to take up this offer, I request you to forward this letter to your sister, who is also under my consideration65for 03/12/09 the said post.

Please acknowledge receipt of this letter. As a token of your having received and accepted this offer, please sign the duplicate copy of this letter and send the same to the undersigned by courier service or Regd. Post, eventhough hand delivery, in the absence of others in my cabin, may be the most welcome approach. Wishing you all the best, and looking forward to get a favourable response, before the date stipulated above, and thanking you in anticipation, Yours sincerely 03/12/09 HRD Manager
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Method validation is a major challenge for analytical chemists in the present global scenario. The reasons include, (a) The increasing importance of accurate analytical measurements, (b) Concern about the existing deficiencies in the quality of data, (c) The onus to demonstrate the validity of its data is on the analytical community, (d) Sole dependence on the skill and experience of the analyst to ensure the quality of analytical results is no longer accepted, and (e) Evidence is needed, which can 67 be03/12/09 provided by systematic QA protocol.

The Food and Drug Administration of USA has defined Validation as, “The documented program providing high degree of assurance that, specific process or equipment, will consistently produce product, meeting predetermined specification and quality attributes”.

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The documentation process consists of the following steps. (1)A trained and knowledgeable person prepares a written document, (2)Review by a higher or more qualified person, (3) Approval by the Head of the Department and (4)Filing and control of the prepared document.
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Validation of results obtained by any analytical method is a process to confirm that, the analytical procedure employed for a specific test is suitable for its intended use. Methods need to be validated or revalidated, a) before their introduction into routine use, b) whenever the conditions change for which the method has been validated, e.g., instrument with different characteristics and c) whenever the method is changed, and the change is outside the original scope of the method.
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A laboratory, applying a specific method, should have documentary evidence that, the method has been appropriately validated. “The responsibility remains firmly with the user, to ensure that the validation documented in the method is sufficiently complete to meet his or her needs."

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This holds for standard methods, for example, from Environmental Protection Agency(EPA), American Society for Testing Materials(ASTM), International Organisation for Standardisation(ISO) or US Pharmacoepia(USP), as well as for methods developed in-house.

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If standard methods are used, it should be verified that, the scope of the method and validation data, for example, sample matrix, linearity, range and detection limits, etc., comply with the laboratory’s analyses requirements; otherwise, the validation of the standard method should be repeated, using the laboratory’s own criteria. The laboratory should demonstrate the validity of the method in the laboratory’s environment.
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It has been stated that “Doing a thorough  method validation can be tedious, but the  consequences of not doing it right are,  wasted time, money, and resources”. 

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General concepts of  “Single Laboratory  Method validation” and  “Full method  validation”    In many cases, it has proved impracticable  or uneconomical, for methods of routine  analysis to be collaboratively validated.  Single Laboratory (former In­House)  Validation of a method requires the  evaluation of a number of appropriately  selected performance characteristics. 
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For example, in the case of analytical methods for pesticide formulations, these characteristics are defined by CIPAC(Collaborative International Pesticides Analytical Council) and AOAC(Association of Official Analytical Community) protocols and EU(European Union) directives. Since an interlaboratory study is not conducted, estimation of the laboratory effect (bias) can be made, either by use of the Horwitz equation, or by the analysis of Certified Reference Material (CRM), where the combined laboratory and method bias are assessed. 03/12/09 76

Method Validation makes use of a set of tests that both 1) test any assumptions, on which the analytical method is based and 2) establish and document the performance characteristics of a method.

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The term “Method validation”, in all definitions given by various international organizations (Food and Agricultural Organisation(FAO), International Atomic Energy Agency(IAEA), Eurachem, International Union of Pure and Applied Chemists(IUPAC), AOAC), comprises of two key parts: 1) establishment of performance characteristics and 2) fitness for purpose.
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According to FAO/IAEA: “Method validation  may be described as the set of tests used to  establish and document, the performance  characteristics of a method and, thereby,  demonstrate that the method is fit for a  particular analytical purpose”,  while  according to the Eurachem Guide “Method  validation is 1) 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  03/12/09 79 extent. 

Which analytes can it determine, in which  matrices, in the presence of which  interferences? Within these conditions, what  levels of precision and accuracy can be  achieved?). 2) The process of verifying that,  a method is fit for purpose (i.e., for use for  solving a particular analytical problem.)” 

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Different levels of validation have occurred, due to the different needs for application of each particular method, and the different levels of quality required from the results. Methods, that are to be applied in a wide variety of instruments, in many different countries worldwide, under different climatic conditions, by analysts of different levels of knowledge and expertise, have to be “fully validated”.
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On the other hand, methods, that are to be used for a specific purpose in a specific laboratory by personnel of a particular degree of training, can be validated at a lower level, what is known as “in-house” validation, or more accurately “Single laboratory validation”.

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“Full” validation for an analytical method is usually taken to comprise an examination of the characteristics of the method in an interlaboratory method performance study (also known as collaborative study or collaborative trial). This study could be viewed as the co-ordinated “Single laboratory validation” study of the analytical method, in usually 10-15 laboratories.
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From the accumulated results of the interlaboratory performance of the method, some characteristics, that cannot be assessed within a single laboratory, are taken into account, and quantified.

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Depending on the filed of application of a method, the performance characteristics required for validation vary. For example, there is no need for determining the limit of detection for a method, that is used for quality control of pesticide formulations, while it is imperative for a method for the determination of residues of a pesticide in any matrix.

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In their excellent article Balayiannis et al have summarized the requirements for “Single laboratory validation” of analytical methods, for the determination of the chemical composition of pesticide formulations. Special attention have been drawn by them to the requirements demanded by the CIPAC and EU Directive 91/414 “Concerning the placing of plant protection products on the market”.
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Method validation is a key element in the establishment of reference methods and in the assessment of a laboratory’s competence in producing reliable analytical data. Hence, the scope of the term, “Method validation”. is wide, especially if one bears in mind the role of Quality Assurance/Quality Control (QA/QC). Validation has been put in the context of the process, generating chemical information. Basic performance parameters, included in the validation processes, have been well discussed in the literature, including evaluation of current 03/12/09 87 approaches to the problem.

Validation parameters Validation of results / Method validation has received considerable attention in the literature, and from industrial committees and regulatory agencies. “The Guidance on the Interpretation of the EN 45000 Series of Standards and ISO/IEC 17025 (2005)” includes information on the validation of methods, with a list of nine validation parameters.
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The International Conference on Harmonization (ICH) of Technical Requirements for the Registration of Pharmaceuticals for Human Use has developed a consensus text on the validation of analytical procedures. The document includes definitions for eight validation characteristics. The United States Food and Drug Administration (US FDA) has proposed guidelines on submitting samples and analytical data for methods validation. The United States Pharmacopoeia (USP) has published specific guidelines for method 03/12/09 89 validation for compound evaluation

Based on the various guidelines available, one can summarise that Validation of the analytical system includes, among other things, verification of the appropriateness of the test method(Assay validation), System suitability test, Hardware validation(Design stage, Design qualification, Installation, Inspection and Regular inspection) and Software validation. Validation and testing are not the same.
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According to the Directive 91/414/EEC [4 ], methods for the quantification of the active substance in formulated pesticide products are required to be robust, accurate and precise. Validation studies of quantitative analytical methods, according to CIPAC and EU guidelines, should determine the following performance characteristics:

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(i)Applicability (ii)Specificity (a definition of the species being determined) (iii)Selectivity (a demonstration of no interference from excipients). (iv)Linearity of response for the analyte in the method. (v)A demonstration of the accuracy of the procedure. (vi)Trueness (Bias) (vii)An estimation of the precision of the procedure (repeatability and reproducibility). 03/12/09 92

However, according to FAO/IAEA, some additional parameters have to be assessed, in accordance to AOAC International: (viii)Calibration (ix)Range (x)Sensitivity (xi)Ruggedness (xii)Practicability
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Brief explanations of the parameters, connected with validation, and the requirements for each individual parameter, taken from the available guidelines, are given below.

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Applicability According to the IUPAC after validation, the documentation should provide, in addition to any performance specification, information about the identity of the analyte (e.g., “fenthion”), specification of the range of matrices of the test material covered by the validation (e.g., “pesticide formulations”), concentration range (e.g., “0–50 ppm”).
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The intended application, and its critical uncertainty requirements, (e.g., “The analysis of pesticide formulations for screening purposes) and, finally, a protocol, describing the equipment, reagents, procedure (including permissible variation, unspecified instructions, e.g., “heat at 100± 5°C for 30±5 min”), calibration and quality procedures, and any special safety precautions required.
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Specificity According to CIPAC Guidelines [3], the specificity of the method is a definition of the species giving rise to the signal, used for quantification. It shows that the detected signal is due to the analyte, and not due to another compound. Specificity is a quantitative indication of the extent to which a method can distinguish between the analyte of interest and interfering substances on the basis of signals produced under actual experimental conditions. Random interferences should be determined using representative blank 03/12/09 97 samples.

Selectivity (interference) The capability of an analytical method, to reliably discriminate among chemically or physically related substances. It is sometimes quantified as cross sensitivity. According to IUPAC, selectivity is the degree to which a method can quantify the analyte accurately, in the presence of interferents.
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The terms specificity and selectivity are often used simultaneously, to describe the same phenomenon. However they have specific meaning. Specificity describes the performance of detection (rise of signal), while selectivity is used for characterising the chromatographic separation (discrimination between the signals of two substances).

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According to chromatographic theory [8 ] Rs = 1.18 (tR2 –t-R1)/(Wh1 + Wh2) where Rs = resolution for two consecutive peaks tR1 = retention time of first peak tR2 = retention time of second peak Wh1 = peak width at half peak height of the first peak Wh2 = peak width at half peak height of the second peak and the acceptable values are Rs>1.0 (and preferably >1.54 from the practical point of 03/12/09 100 view).

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.

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The USP monograph(REFERENCE) defines selectivity of an analytical method as its ability to measure accurately an analyte, in the presence of interference, such as synthetic precursors, excipients, enantiomers and known (or likely) degradation products, that may be expected to be present in the sample matrix. Selectivity in Gas chromatography is obtained by choosing optimal columns and setting chromatographic conditions, such as column temperature and detector.
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Linearity The linearity of a test procedure is its ability (within a given range), to obtain test results, proportional to the concentration (amount) of analyte in the sample. According to EU and CIPAC Guidelines, the linearity of response to the analyte should be demonstrated, at least over the range: nominal concentration ±20%. At least three concentrations should be measured, with duplicate measurements for each.
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Linearity can be tested informally, by examination of a plot of residuals, produced by linear regression of the responses, on the concentrations, in an appropriate calibration set. The linearity of an analytical method is its ability to elicit test results that are directly, or by means of well-defined mathematical transformations, proportional to the concentration of analytes in samples, within a given range.
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Linearity is determined by a series of three to six injections of five or more standards, whose concentrations span 80-120 percent of the expected concentration range. The response should be directly, or by means of a well defined mathematical calculation, proportional to the concentrations of the analytes. A linear regression equation, applied to the results, should have an intercept, not significantly different from zero. If a significant nonzero intercept is obtained, it should be demonstrated that, there is no effect on the accuracy of the 03/12/09 105 method.

Frequently, the linearity is evaluated graphically, in addition or alternatively to, mathematical evaluation. The evaluation is made by visual inspection of a plot of signal (height or peak area) as a function of analyte concentration. Because deviations from linearity are sometimes difficult to detect, two additional graphical procedures can be used. The first one is to plot the response against the concentration. For linear ranges, the deviations should be equally distributed between positive and negative values.
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Another approach is to divide signal data by their respective concentrations yielding the relative responses. A graph is plotted with the relative responses on the Y-axis, and the corresponding concentrations on the X-axis, on a log scale. The line obtained should be horizontal over the full linear range. At higher concentrations, there will typically be a negative deviation from linearity. Parallel horizontal lines are drawn in the graph corresponding to, for example, 95 percent and 105 percent of the horizontal line. 03/12/09 107

The method is linear up to the point, where the plotted relative response line intersects the 95 percent line. Fig. 3.1 shows a comparison of the two graphical evaluations, carried out in a typical GC evaluation.

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Figure 3.1. Graphical presentations of a typical GC estimation. Plotting the sensitivity (response/amount) gives clear indication of the linear range. Plotting the amount on a logarithmic scale has a significant advantage for wide linear ranges. Rc = Line of constant Response

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109

The linearity range for examination depends on the purpose of the test method Under most circumstances acceptable regression coefficient (r) is 0.999. Intercept and slope should be indicated.

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Range Range is defined as the difference between the largest and the smallest observed value of a quantitative characteristic. In practical terms, this means that the “range” is the interval of concentrations, within which the analytical procedure demonstrates a suitable level of precision and accuracy. The analytical range may result from an analytical curve, that is linear or not linear.
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The range of an analytical method is the interval between the upper and lower levels (including these levels) that have been demonstrated to be determined with precision, accuracy and linearity, using the method as written. The range is normally expressed in the same units as the test results (e.g. percentage, parts per million) obtained by the analytical method.
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The chemists normally employ two terminologies with respect to the analysis carried out. One is the working range and the second is the linear range. For any quantitative method, there is a range of analyte concentrations over which the method may be applied. At the lower end of the concentration range, the limiting factor is the value of the limit of detection and/or limit of quantification. At the upper end of the concentration range, limitations will be imposed by various effects, depending on the detection 113 03/12/09 mechanism.

Within this “working range”, there may exist a “linear range”, within which the detection response will have a sufficiently linear relation to analyte concentration. The working and linear range may differ in different sample types, according to the effect of interferences arising from the sample matrix.

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It is recommended that, in the first instance, the response relationship should be examined over the working range, by carrying out a single assessment of the response levels to at least six concentration levels. To determine the response relationship within the linear range, it is recommended that three replicates are carried out at each of at least six concentration levels.
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Accuracy Accuracy is the closeness of agreement between a test result, and the accepted reference or true value of the property being measured. Accuracy is a qualitative concept [13 ], and cannot be given a numerical value, but can be expressed on an ordinal scale such as “poor”, “fair” and “good”.

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Trueness (Bias) The closeness of agreement between the average value obtained from a large series of test results, and the accepted reference or true value of the property being measured. Trueness is a measure of systematic error. The measure of trueness is usually expressed in terms of bias. As ihe true value cannot be determined by chemical analysis, it has been replaced by the “accepted reference value” or “the most probable value”.
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Bias is the difference between the mean value (expectation) of the test results, and an accepted reference value. The bias is the total systematic error. The accuracy of an analytical method is the extent to which test results, generated by the method, and the true value agree. The true value for accuracy assessment can be obtained in several ways. One alternative is to compare results of the method, with results from an established reference method. This approach assumes that the uncertainty of the reference 03/12/09 118 method is known.

Secondly, accuracy can be assessed by analyzing a sample with known concentrations, for example, a certified reference material, and comparing the measured value with the true value, as supplied with the material. If such certified reference material is not available, blank sample matrix of interest can be spiked with a known concentration by weight or volume of the pure material and analysis carried to evaluate the recovery.
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Accuracy refers to closeness of agreement between the true value of the analyte concentration and the mean result obtained by applying experimental procedure to a large number of homogeneous samples. It is related to systematic error and analyte recovery. Systematic errors can be established by the use of appropriate certified reference materials (matrix-matched) or by applying alternative analytical techniques.
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Recovery is expressed as the amount/weight of the compound of interest, analyzed, as a percentage to the theoretical amount present in the medium. Accuracy is the measure of how close the experimental value is to the true value. Accuracy studies for drug substance and drug product are recommended to be performed at the 80, 100 and 120% levels of label claim.
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Recovery data, at least in triplicate, at each level (80, 100 and 120% of label claim) is recommended. The mean is an estimate of accuracy, and the RSD is an estimate of sample analysis precision. The concentration should cover the range of concern, and should, particularly, include one concentration close to the quantitation limit.
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The expected recovery depends on the sample matrix, the sample processing procedure, and on the analyte concentration. The AOAC Manual for the Peer Verified Methods program(REFERENCE) includes a table (Table 3.1), with estimated recovery data, as a function of analyte concentration.

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Table 3.1. Analyte recovery at different concentrations
Active Ingred. [ %] 100 >=10
>=1

Unit 100% 10% 1% 0.1 % 100 ppm 10 ppm 1 ppm 100 ppb 10 ppb 1 ppb

Mean recovery [%] 98-102 98-102 97-103 95-105 90-107 80-110 80-110 80-110 60-115 40-120
124

>=0.1 0.01 0.001 0.0001 0.00001 0.000001 0.0000001
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Limit of Detection and Quantitation The limit of detection is the point at which, a measured value is larger than the uncertainty associated with the measurement. It is the lowest concentration of analyte in a sample, that can be detected, but not necessarily quantified. In chromatography, the detection limit is the injected amount, that results in a peak, with a height at least three times as high as the baseline noise level.
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The limit of detection is usually expressed as the analyte concentration corresponding to the sample blank plus three sample standard deviations, based on 10 independent analyses of sample blanks.

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126

The limit of quantitation is the minimum injected amount, that gives precise measurements. If the required precision of the method at the limit of quantitation has been specified, then the following approach can be used. A number of samples with decreasing amounts of the analyte are injected six times. The calculated RSD% of the precision is plotted against the analyte amount. The amount, that corresponds to the previously defined required precision. is equal to the limit of quantitation.
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Figure 3.2. Limit of quantitation

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128

The limit of quantification is the lowest concentration of analyte that can be determined with an acceptable level of uncertainty or, alternatively, it is set by various conventions to be five, six, or ten standard deviations of the blank mean. It is also sometimes known as the limit of determination.

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Sensitivity Sensitivity is the measure of the change in instrument response, which corresponds to a change in analyte concentration. Where the response has been established as linear with respect to concentration, sensitivity corresponds to the gradient of the response curve.

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Sensitivity is the capability of an analytical procedure, to reliably discriminate between samples, having differing concentrations, or containing differing amounts of an analyte. The sensitivity of a method is correctly defined as the slope of the calibration curve.

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Precision (repeatability and reproducibility). Precision is the measure of how close the data values are to each other for a number of measurements under the same analytical conditions. ICH has defined precision to contain three components: repeatability, intermediate precision and reproducibility. The precision of a method is the extent to which the individual test results of multiple injections of a series of standards agree.
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The measured standard deviation can be subdivided into three categories: repeatability, intermediate precision and reproducibility. Repeatability is obtained when the analysis is carried out in one laboratory by one operator using one piece of equipment over a relatively short time span. At least 5 or 6 determinations of three different matrices at two or three different concentrations should be done and the relative standard deviation calculated. The acceptance criteria for precision depend very much on the type of analysis. 03/12/09 133

While for compound analysis in agrochemical / pharmaceutical quality control precision of better than 1 % RSD is easily achieved, for biological samples the precision is more like 15% at the concentration limits and 10% at other concentration levels. For environmental and food samples, the precision is very much dependent on the sample matrix, the concentration of the analyte and on the analysis technique. It can vary between 2% and more than 20%.
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Precision is the closeness of agreement between independent test results obtained under stipulated conditions. The measure of precision is usually expressed in terms of imprecision, and computed as a standard deviation or relative standard deviation of the test results. Precision is a measure of random errors, and may be expressed as repeatability and reproducibility. These terms are defined in ISO 5725-1986E:
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For single laboratory validation, two sets of conditions are relevant [7]: a) Precision under repeatability conditions, describing variations observed during a single run and b) precision under run-to-run conditions, describing variations in run bias. Usually, both of these sources of error are operating on individual analytical results. The two precision estimates can be obtained most simply by analysing the selected test material, in duplicate, in a number of successive runs.
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The combined precision can be estimated directly, by the analysis of the test material once, in successive runs, and estimating the standard deviation from the usual equation.

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Repeatability Repeatability is the closeness of agreement between mutually independent test results, obtained with the same method, on identical test material, in the same laboratory, by the same operator, using the same equipment, within short intervals of time. The measure of repeatability is usually expressed in terms of Relative Standard Deviation, RSDr.
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Reproducibility Reproducibility is the closeness of agreement between test results, obtained with the same method, on identical test material, in different laboratories, with different operators, using different equipment. The measure of reproducibility is usually expressed in terms of Relative Standard Deviation, RSDR.

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Theoretical repeatability and reproducibility values can be calculated from the Horwitz equation. Horwitz trumpet and equation is RSDR = 2(1-0.5logC) where C is the concentration of analyte in the sample, expressed as a decimal fraction.

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Horwitz has derived the equation, after studying the results from many (~3,000) collaborative trials. The equation is an approximation of the possible precision that can be achieved. The data points from all the “acceptable” collaborative trials lie within a range of one half to twice the values derived from the equation. Their graphical representation is an idealised smoothed curve, independent of the analyte, method, matrix, laboratory and time (state of the art).
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In general, the values taken from this curve are indicative of the precision that is achievable and acceptable of an analytical method, by different laboratories. Its use provides a satisfactory and simple means of assessing method precision acceptability. The assumption that RSDr = 0.67 RSDR was made by Horwitz, after he noted that values for RSDr were, usually, between half and two thirds of that of RSDR.
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It should be noted that the equation has been recalculated in the light of recent collaborative trials. This has now been published by Thompson, who recommends that, for values less than 120 μg/kg, a constant value for the relative standard deviation of 22% should be used. However, for many purposes, e.g., mycotoxins and pesticide residues, the original form is still applicable, in many cases.
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The AOAC manual for the Peer Verified Methods program includes a table (Table 3.2), with estimated precision data, as a function of analyte concentration.

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Table3.2. Analyte concentration versus precision within or between days
Analyte % 100 10 1 0.1 0.01 0.001 0.0001 0.00001
0.000001

Unit 100% 10% 1% 0.1 % 100 ppm 10 ppm 1 ppm 100 ppb 10 ppb 1 ppb

RSD (%) 1.3
2.3

2.7 3.7 5.3 7.3 11 15 21 30
145

0.0000001
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Intermediate precision was previously known as part of ruggedness. The attribute evaluates the reliability of the method in a different environment, other than that used during development of the method.

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The objective is to ensure that the method will provide the same results, when similar samples are analyzed once the method development phase is over. Depending on time and resources, the method can be tested on multiple days, analysts, instruments, etc. Intermediate precision in the test method can be partly assured by good system suitability specifications. Thus, it is important to set tight, but realistic, system suitability specifications.
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Reproducibility, as defined by ICH, represents the precision obtained between laboratories. The objective is to verify that the method will provide the same results in different laboratories. The reproducibility of an analytical method is determined by analyzing aliquots from homogeneous lots in different laboratories, with different analysts, and by using operational and environmental conditions that may differ from, but are still within the specified parameters of the method (inter laboratory 03/12/09 148 tests).

Validation of reproducibility is important, if the method will be used in different laboratories. Both repeatability and the reproducibility are expressed in terms of standard deviation, and are generally dependent on analyte concentration. It is, therefore, recommended that the repeatability, and within-laboratory reproducibility, are determined at different concentrations across the working range, by carrying out 10 repeated determinations, at each concentration level. 03/12/09 149

As stipulated by Horwitz and Albert (10), the variability among laboratories is the dominating error component in the world of practical ultratrace analysis. They conclude that a single laboratory cannot determine its own error structure, except in the context of certified reference materials or consensus results from other laboratories.

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Standard deviation The standard deviation is usually understood in terms of a hypothetical normal (Gaussian) distribution of the error.

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151

Ruggedness/robustness Ruggedness is the ability of a chemical measurement process, to resist changes in results, when subjected to minor changes in environmental and/or operating conditions, similar to those likely to arise in different test environments. It is a measure of how effectively the performance of the analytical methods stands up to, less than perfect implementation.
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In any method there will be certain parts, which will severely affect the method performance, unless they are carried out with sufficient care. These aspects should be identified and, if possible, their influence on the method performance should be evaluated, using the ruggedness tests, sometimes also called robustness tests.
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The ruggedness/robustness tests provide important information for the evaluation of the measurement uncertainty. The methodology for evaluating uncertainty given in the ISO Guide relies on identifying all parameters that may affect the result (that is, the potential sources of uncertainty), and on quantifying the uncertainty contribution from each source.
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This is very similar to procedures used in robustness tests, which identify all the parameters, likely to influence the result, and determine the acceptability of their influence through control. If carried out with this in mind, the robustness tests can provide information on the contribution to the overall uncertainty, from each of the parameters studied.
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Robustness is the ability of a chemical measurement process, to resist changes in results, when small but deliberate variations, similar to those likely to arise during normal usage, are applied to method parameters. Robustness test examines the effect of operational parameters on the analysis results.

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156

For the determination of a chromatographic method’s robustness, a number of chromatographic parameters, for example, flow rate, column temperature, injection volume, are varied, within a realistic range, and the quantitative influence of the variables is determined.

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157

If the influence of the parameter is within a previously specified tolerance, the parameter is said to be within the method’s robustness range. Obtaining data on these effects will allow to judge whether a method needs to be revalidated, when one or more of parameters are changed, for example, to compensate for column performance, over time.

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158

ICH defines robustness as a measure of the method's capability to remain unaffected by small, but deliberate variations in method parameters. Robustness can be partly assured by good system suitability specifications. Thus, it is important to set tight, but realistic, system suitability specifications.

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159

In chromatographic procedures one has to include System suitability tests(SST), which will consist of the following investigations. (a) Capacity factor, (b) Resolution, (c)Tailing factor and (d) Theoretical plate number. Details of these would be given in one of the following sections.

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Practicability The ease of operation (in terms of sample throughput and costs), the ability to achieve the required performance criteria and, thereby, meet the specified purpose. Conclusions In conclusion it can be stated that, in modern laboratories, methods can be “single laboratory” validated and, thus, produce reliable results, without necessarily undergoing wide interlaboratory trials. 03/12/09 161

Single laboratory method validation is appropriate in several circumstances. One case is the initial estimation of the performance of a method, prior to a costly interlaboratory trial. Another case is, when the correct use of “off the shelf” validated methods is ensured. Finally, when evidence of reliability is provided in the case of methods, with no collaborative studies available, or where the conduct of such studies is not possible.
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Single laboratory validation of methods, developed for the determination of active ingredients’ and impurities’ content of pesticide formulated products, requires the determination of specific performance characteristics, defined specifically by CIPAC and EU official guidelines. FAO/IAEA have defined some additional parameters to be assessed, in accordance with the general specifications of AOAC International.
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Methods should not be validated as a one-time situation, but methods should be validated by the developer or user, to ensure ruggedness or robustness throughout the life of the method. The variations, due to the laboratory sample preparation procedure and the instrument performance, contribute to the accuracy of the data obtained for a particular analysis.
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With proper validation and tight chromatographic performance (system suitability) criteria, an improvement in the reliability of the data can be obtained. Only with good reliable validated methods, can data that are generated for purity, stability, and pharmacokinetics be trust-worthy.

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165

Control charts Validation studies often demonstrate the performance of an analytical method, before its routine application. The validity of the assessed performance for the routine measurements can be controlled by repeated analyses of control samples. The results are monitored in a control chart, with warning and action limits. Application of a stable control sample also provides necessary information for the interpretation of long term, trend studies. 03/12/09 166

Method Validation approach in a typical Chromatographic procedure An outline of the validation approach in a typical chromatographic analytical procedure is given below. Chromatographic methods are to be validated before first routine use. Variables to be considered are: Sampling procedure, sample preparation, chromatographic separation, detection and data evaluation, using the same matrix as that of the intended sample. 03/12/09 167

The proposed procedure must go through a rigorous process, verified by laboratory studies. All validation data must be documented. There are no general rules for validation--Only guidelines are available. What is appropriate for one application, in one laboratory, may not be appropriate for another application in the same or a different laboratory. One has to demonstrate that, the performance characteristics of the method are fit for the intended purpose, and the evidence for this is 03/12/09 168 statistically valid.

System Suitability Specifications and Tests for chromatographic methods The accuracy and precision of data collected begin with a well-behaved chromatographic system. The system suitability specifications and tests are parameters that provide assistance in achieving this purpose.

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1.Capacity factor Capacity factor (K’) is a measure of where the peak of interest is located with respect to the void volume, i.e., elution time of the nonretained components. The peak should be well-resolved from other peaks. Generally the value of k' is > 2.

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170

2. Resolution (R) R is a measure of how well two peaks are separated. For reliable quantitation, wellseparated peaks are essential for quantitation. This is a very useful parameter, if potential interference peak(s) may be of concern. The closest potential eluting peak to the analyte should be selected. R is minimally influenced by the ratio of the two compounds being measured. R of > 2, between the peak of interest and the closest potential interfering peak (impurity, excipient, degradation product, internal standard, 03/12/09 171 etc.), is desirable.

3. Tailing factor (T) The accuracy of quantitation decreases with increase in peak tailing, because of the difficulties encountered by the integrator in determining where/when the peak ends, and, hence, the calculation of the area under the peak. Integrator variables are preset by the analyst for optimum calculation of the area for the peak of interest. If the integrator is unable to determine exactly, when an upslope or down slope occurs, accuracy drops.
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4. Theoretical plate number (N) Theoretical plate number (N) is a measure of column efficiency. N is fairly constant for each peak on a chromatogram with a fixed set of operating conditions. H, or HETP, the height equivalent of a theoretical plate, measures the column efficiency per unit length (L) of the column. The theoretical plate number, in general, should be > 2000.
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System suitability testing is essential for the assurance of the quality performance of the chromatographic system. The amount of testing required will depend on the purpose of the test method. For acceptance, release, stability, or impurities/degradation methods using external or internal standards, k' (capacity factor), T (Tailing factor), R (Resolution) and RSD are recommended as minimum system suitability testing parameters. 03/12/09 174

In practice, each method submitted for validation should include an appropriate number of system suitability tests, defining the necessary characteristics of that system. The validation procedure should have data demonstrating (a) Suitable accuracy, precision and linearity over the range of interest (b) Specificity of the method and the determination of the limits of detection/quantitation for all the components including degradation products/impurities 175 03/12/09

(c) Recovery from the sample matrix (d) That neither fresh nor degraded placebo interferes with the method (e) Reproduction of chromatograms and instrumental recordings (f) Ruggedness of data: Characterising day-today, lab.-to-lab, analyst-to-analyst and column-to-column variability (g) System suitability tests for chromatographic assays.
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The evaluation of robustness should be considered during the development phase, and depends on the procedure under study. It should show the reliability of an analysis with respect to deliberate variations in the method parameters. If measurements are susceptible to analytical conditions, they should be suitably controlled or a precautionary statement should be included in the procedure.
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Typical variations are: Stability of analytical solutions, Extraction time, pH variations in HPLC, Mobile phase composition, temperature, flow rate, etc. Modern chromatographic systems are provided with GLP support softwares, which help in method validation, and system suitability evaluation. The parameters evaluated include those already specified.
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The System suitability test is performed, every time, daily, when analysis is carried out. For SST, in chromatographic assays, analysis is made, using the standard sample for calibration, control sample for repeatability or accuracy check, unknown sample, and again the control sample/standard sample. System suitability standarsises the integrated system for a given method.
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Failing SS requirement invalidates the use of the integrated system for the intended method application. When all the results of evaluation are satisfied, the results of quantitative analysis of the unknown are approved as valid data. Any partial data obtained cannot be handled separately
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The case of some aspects of validation, carried out for Isoproturon analysis in our laboratory, will be illustrated. They include (a) Linearity of response (b) System precision and (c) Assay accuracy and precision. Similar experiments for the impurities are to be carried out. One, then, carries out the five batch analysis, getting a closure as close to 100 as possible. Structure of active ingredient and impurities are to be given. Methods followed are to be given. 03/12/09 181

Linearity should be evaluated by visual inspection of signal vs concentration. If there is a linear relationship, test results should be evaluated by appropriate statistical methods(e.g., by calculation of a regression line, by the method of least squares, correlation coefficient, Y-intercept, slope of the regression line, etc.) To establish linearity, a minimum of five concentrations are required.
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The range depends on the intended application of the procedure. For assay of the major component: 80-120% of the concentration. For content uniformity: 70-130% For precision a minimum of 6 determinaltions at 100% of the test concentration. Accuracy should be assessed using a minimum of 9 determinations over a minimum of 3 concentration levels covering the specified range. Accuracy should be reported as percent 03/12/09 183 recovery.

ICH(International Conference on Harmonisation) Guidelines: Minimum nine determinations over a minimum of three concentration levels covering specified range(for e.g., 3 concns. With 3 replicates each AOAC Guidelines given in Table 3.1 provide Estimated Recovery at various concentration levels.
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Precision: ICH Guidelines: Minimum of 9 determinations for 3 concentration levels covering specified range(3 levels with 3 replicates each) AOAC Guidelines given in Table 3.2 give estimated precision at various concentration levels. The data related to HPLC method validation of isoproturon are given in Tables 3.3 to 3.9.
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Table 3.3: Linearity of response for the determination of isoproturon S.No. 1) 2) 3) 4) 5) 6) 7) Nominal wt. 36.3 (mg/ 50 ml) 40.2 45.4 50.5 55.5 60.6 65.6 Concn. nominal % 72.6 80.4 90.8 101.0 111.0 121.2 131.2 Peak areas* 1734511 1923978 2238330 1947210 1750430 2165342 2437310 2683268 2891407 2423821 3120371 2640086 2856872
186

* Normalised with respect to IS
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3124240

Table 3.4 System precision for the determination of Isoproturon
S.No. Determined peak areas* Mean Concn. (mg/ml) Coeff. of Variation (%)

1) 2) 3) 4) 5) 6) 7)

2406344 2407694 2405475 2405442 2400166 2392064 2409267 2403779 1.01 0.24

*Normalised with respect to IS
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Table 3.5 Assay accuracy and precision of Isoproturon active estimation
Nominal concn. (mg/50ml) Determined concn. (mg/50ml) 40.6 40.5 40.2 100.8 40.5 45.9 45.9 45.9 50.9 50.9 50.9 % recovery Mean recovery (%) 100.7 Coeff. of variation

1)

101.0 100.7 100.7 101.1 101.1 101.1 100.8 100.8 100.8 100.5 100.8 101.1

0.17 (%)

2)

45.4

0.0

3)

50.5

0.0

4)

0.63

55.5 55.4 100.9 100.9 99.2 99.2 99.2 55.9 60.1 55.9 60.1 60.1

99.8

5)

60.6

99.2

0.0

Overall assay accuracy: 100.5%; 03/12/09 Overall assay precision:0.16%

188

Table 3.6 Linearity response for impurity 1
S.No. Nominal wt. mg/ml 1) 0.0019 0.09 22410, 22023 Concn. nominal% Determined peak areas Coeff. Of correlation

2)

0.0040

0.20

49652, 49836

3)

0.0060

0.30

76170, 79460

0.9965

4)

0.0080

0.40

100814, 100716

5) 6)

0.0100 0.0119

0.50 0.60

116455, 118623 139046, 138684

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Table 3.7 System precision for the determination of impurity 1

S. No.

Determined peak areas

Mean

Concn. (mg/ml)

Coeff. of variation

1)

137835

2)

139955

3)

139046

4)

138684

138091

0.0119

1.6

5)

133854

6)

140420

7)

136846

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190

Table 3.8 Assay accuracy and precision of impurity 1
Nominal concn. (mg/ml) 0.0019 Determined concn. (mg/ml) 0.0018 0.0018 0.0017 0.0040 0.0040 0.0040 0.0061 0.0064 0.0058 0.0080 0.0081 0.0081 0.0080 0.0093 0.0100 0.0119 0.0095 0.0101 0.0111 0.0111 0.0110 % recovery Mean recovery 93.0 Coeff. Of variation % 3.2

94.7 94.7 89.5 100.0 100.0 100.0 100.0 104.9 95.1 101.3 101.3 100.0 93.0 95.0 101.0 93.3 93.3 93.0

0.0040

100.0

1.5

0.0061

100.0

4.9

100.9

0.74

96.3

4.3

93.3

0.56

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Overall assay accuracy % : 97.1 Overall assay precision %: 2.5

191

Table 3.9 Isoproturon 5 batch analysis Batch no. 1) 2) Component A,%(w/w) 99.1 98.8 98.6 99.1 98.9 Component D,%(w/w) 0.58 0.64 0.70 0.70 0.64 Total 99.7 99.4 99.4 99.8 99.5

3)
4) 5)

Impurities B, C, E and F are less than 0.05% The structres of the active ingredient and impurities are to be presented. A description of each method used in the study is to be presented.
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GLP COMPLIANCE OF GC and HPLC The following parameters are to be specified by the user of GC/HPLC. (a) Limit of detection (b) Limit of quantitation, (c) Selectivity, (d) Detector sensitivity and linearity and (e) Accuracy.

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Automatic performance measurement generally includes, plate number of a column, capacity factor, peak width at half height, tailing factor, resolution between two peaks, selectivity relative to preceding peaks, precision of peak retention time, peak areas, heights or amounts, baseline noise and S/N, baseline drift and linearity.

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The quality of the analytical data can be supported by keeping records of the actual instrument conditions at the time of measurement. Precolumn pressure and temperature of the column compartment are recorded before, during and after the run and are stored together with chromatographic raw data.

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The system suitability test includes testing of sample injection, chromatographic separation and associated data handling. A system suitability test should be designed so that it proves whether the method achieves the same or better accuracy/precision on the current system as shown during validation.

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Substituted benzenes have peaks at 205 nm for low wavelength checks. Anthracene shows absorbance peaks at 250 and 360 nm for calibration checks at mid and near UV wavelengths. Far UV transparent solvents like acetonitrile or water must be used for wavelength calibration checks in the far UV, mid UV and near UV regions.

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The universal test mixture used consists of 4 alkyl benzenes(Toluene, ethyl benzene, isopropyl benzene and 1-butyl benzene) and anthracene. The relative standard deviations in area and retention time can be arrived at by overlaying 6 replicate injections of known amounts(say 5 microlitres). Flow rate sensitivity can be evaluated by carrying out HPLC with 0.02-0.03 ml/min. increments(In all the five cases of analytes). 198 03/12/09

Gradient precision sensitivity can be  evaluated by recording 1.0, 1.2, 1.4 gradient  curves in all the five cases.     Quantitative linearity is evaluated by  injecting various amounts of each analyte.   Linearity plots are constructed in each case.  S/N calculations are made in each case. Injection sequence for holistic validation procedure
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Automatic sequence of 13 injections  standard: Toluene, ethyl benzene, Isopropyl   benzene, 1­butyl benzene(0.1%);  Anthracene(5 ppm).  All dissolved in 35%  acetonitrile /65% water.   The 13 injections suggested are given for the  following purposes. Reproducibility –6 Carry over­­­2(Blank) Linearity, Wave Length calibration­­­­ 3, 5, 20,  30,50 microlitres—5 injections    03/12/09 200

CURRENT APPROACHES TO VALIDATION In a recent review, van der Voet and co-workers discuss current approaches to validation of analytical chemical methods, identifying some shortcomings of existing validation schemes, such as insufficient coverage of variability in space or time and mismatches between validation criteria and intended use of the method, giving an example of regulatory control.
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The authors make an attempt to link validation concepts used in different fields, such as measurement uncertainty, and the prediction error. They recommend general statistical modelling approach for combining different aspects of validation, and illustrate it with an example. This type of modelling should be the basis for the development of new statistically underpinned validation schemes, which integrate current validation and quality assurance activities.
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It is stated that validation includes the initial assessment of performance characteristics, several types of inter-laboratory testing, and quality control. Validation is thus concerned with assuring that a measurement process produces valid measurements; this has also been called measurement assurance.

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The validation of an analytical method as a concept may be understood in (at least) three senses. In the narrow and traditional sense, the term denotes validation of a chemical method, as described in a standard operating procedure (SOP). In a wider sense, validation may be concerned with a method of analysis (e.g., in an ISO standard) which explicitly leaves freedom to adapt the procedure to the infrastructure, in a specific situation. In this case, there are more SOPs, all in conformity with the master method.
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Finally, in a still wider and perhaps unconventional sense, validation of analytical methods may be considered from the perspective of those, who use analytical results for other purposes. The method of analysis for end-users of analytical results amounts to the specification of an analytical result (e.g., clenbuterol in liver.), with the implied statement: analysed by any reasonable method.
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Accordingly, a specific method of analysis in the analytical chemical sense can be considered as just one realisation of the class of all methods, currently applied to measure component X in matrix Y.

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In principle, each modification of the  protocol invalidates an existing validation, according to ISO 5725. Much work on  validation has been performed in joint efforts of International Union of Pure and Applied  Chemistry (IUPAC), ISO, and Association of  Official Analytical Chemists (AOAC  International). Results appear as a series of  harmonised protocols. 
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The second edition of the ISO 5725 standard has much in common with the IUPAC/ISO/AOAC Protocol for design, conduct and interpretation of collaborative studies. Important contributions to some of the problems mentioned above were made in two other protocols on proficiency testing (PT), and on internal quality control (IQC).

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The harmonised international protocol for the proficiency testing of (chemical) analytical laboratories considers laboratory performance studies, in which each laboratory uses its own analytical method, as opposed to the methodperformance studies of ISO 5725.

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Although the primary purpose of proficiency testing is often the evaluation or improvement of laboratory performance, it is also reasonable to consider it as a methodperformance validation in the wider sense of the definition. This would solve problems of different laboratories having different SOPs, and of SOPs changing every now and then in each laboratory.
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The prescribed repetition in proficiency testing schemes considers reasonable, the frequencies of once every two weeks to once every four months. This solves the problem with the static nature of ISO 5725 validation, and, by varying the test materials, the problem of not assessing matrix variability.

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Despite all the advantages, proficiency testing according to the IUPAC guidelines (16) cannot be considered as a complete validation methodology on its own. First of all, it does not provide for SOP-specific validation. More importantly, the scheme requires repeated interlaboratory studies, which severely restricts the amount and variety of samples that can be analysed.
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Therefore, proficiency testing is an extensive validation methodology. Finally, the current protocol has limited consideration of performance to laboratory bias, most often in the form of a z-score. This information alone may be insufficient to evaluate a method’s fitness for the purpose.

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It has been shown that an effective measurement assurance requires validation at different scales. Newly developed or implemented methods are usually first validated through in-house validation. This type of validation should be supplemented by ongoing internal quality control validation in each laboratory, and by participation of the laboratory in interlaboratory schemes.
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Considering the complex nature of many modern methods of analysis, proficiency testing schemes, allowing laboratory-specific SOPs, are more to the point than the methodevaluating schemes like ISO 5725.

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Currently, the three validation schemes, inhouse validation, internal quality control, and proficiency testing, are not sufficiently linked. The development of integrated validation approaches is possible from the various guidelines available.

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A recent Joint FAO/IAEA Expert Consultation on validation of analytical methods for food control defined the .ideal validated method. as follows: The ideal validated method is one that has progressed fully through a collaborative study in accordance with internationally harmonised protocols for the design, conduct and interpretation of method performance studies.
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This usually requires a study design involving a minimum of 5 test materials, the participation of 8 laboratories reporting valid data, and most often includes blind replicates or split levels, to assess within-laboratory repeatability parameters. Limiting factors for completing ideal multilaboratory validation studies include high cost, lack of expert laboratories available and willing to participate in such studies, and overall time constraints. 03/12/09 218

Validation by using different analytical methods. Above all, the described validation strategies assume that methods are applied on a routine basis in various laboratories. In a research environment, a rather unique method might be developed and validated for the use in only one or a few studies. Then, of course, one would like to establish the same amount of information on the validity of the method.
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However, some of the usual tools, like participation in proficiency schemes and the use of reference materials are probably not possible. Hogendoorn and co-workers discuss a number of practical examples such asscreening and analysis of polar pesticides in environmental monitoring programmes by coupled-column liquid chromatography and gas chromatography-mass spectrometry (GC-MS).
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One example is a study on the levels of ethylenethiourea (ETU) in groundwater. In the validation of both methods, the calibration procedure is very important, and provides information for several of the criteria. The calibration is based on spiked samples, which are comparable to samples of groundwater to be analysed which, by comparison with the analysis of standards, in itself gives the recovery data.

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The calibration samples are analysed, in time, around and between the real samples. Possible influence of the conditions during the analysis (ruggedness) of the samples is automatically captured in the calibration sequence. To determine the working range of both methods, the calibration data are evaluated by the Calwer spreadsheet application.
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The application enables an extensive evaluation of the calibration curve with respect to the appropriate calibration model , showing that the assumption of equal variance over the working range is nearly always severely violated. The strategy of variance models, weighted regression in combination with the maximum likelihood criterion, is formalised for method comparison calibration/validation in ISO.
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The selectivity of the method is checked by comparing the shape and the retention of real samples and calibration samples with the chromatogram of standard solutions. More details are available in the paper cited.

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Interlaboratory method validation Another approach to validation is by interlaboratory tests. An example of the validation of GC determination of chlorophenols in water has been summarized. Details can be found in reports of Hoogerbrugge and co-workers. The results obtained complied with the general variation in interlaboratory studies as found by Horwitz.
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It has been stated that “Global decisions  made on the basis of chemical analytical  results need global comparability. The  concept of traceability is important and  necessary, but not sufficient to achieve  global comparability, especially for routine  analysis in environmental protection, health  care and public safety. Sometimes,  comparability is only determined by the  method. In this case, validated methods have  to be known, and recognized internationally. 
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In addition, a minimum level of proficiency of  the personnel, producing analytical results,  has to be guaranteed. Finally, a system of  measures, designed to build up trust, is  needed for worldwide acceptance of  analytical results. The method of self­ declaration by the supplier of chemical  analytical services has the advantage of  presenting specific responsibility and  gaining a good reputation. Additional  methods should be employed. The  evaluation of reference materials by specific  quality criteria is mentioned as an example”. 03/12/09 227

Accreditation Accreditation of test laboratories, and adoption of certified Quality systems by manufacturing industries, can eliminate repeated checks on quality of the same product. Mutual recognition of test results between organisations would automatically emerge, if both adopt quality systems. Accreditation provides independent endorsement of a laboratory’s quality systems. In the field of chemical testing, it is recognised as an essential ingredient in achieving valid analytical measurements.
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OTHER USEFUL ELEMENTS IN INHOUSE VALIDATION PROCEDURE A Comprehensive Discussion of the Role of Certified Reference Materials(CRM) in Chemical Analysis At this point, it would be worthwhile to provide a comprehensive coverage of CRMs used in chemical analysis.
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It must be emphasized that standard samples, which have been analysed by a number of skilled analysts, are commercially available. These include, certain primary standards(sodium oxalate, potassium hydrogen phthalate, arsenic(III) oxide and benzoic acid) and ores, ceramic materials, irons, steels, steel –making alloys and nonferrous alloys.
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Reference materials play an important role in analytical chemistry, where they are used by analysts for a variety of purposes, including: checking and calibrating instruments; validating methods and estimating the uncertainty of analytical measurements; checking laboratory and analyst performance; and internal quality control.
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Literature provides adequate guidance and information for the users of Certified reference materials (CRMs), explaining how they can best be used to achieve valid analytical measurements, and improve quality in the analytical laboratory. General information on CRMs, and how they are produced, have been well documented. The main uses of CRMs, and statistics relating to CRM use, are also available.
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If relevant reference materials are available, they are a powerful tool in the assessment and control of the accuracy of the performance of the applied analytical method. In practical laboratory application, one should, however, realise that, additional control experiments are often necessary since, reference materials usually do not reflect the complete range of application of the method with respect to concentration, matrix effects, and possible inhomogeneity of samples.
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All chemical measurements, whether qualitative or quantitative, depend upon and are, ultimately, traceable to a CRM, or standard material of some sort. Qualitative measurements of identity based(e.g., retention time measurement in gas chromatography or spectroscopic properties), require a reliable, authentic, reference material to calibrate the particular instrument or test used.
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Quantitative determination have the additional requirement, that the instrument is calibrated with an accurately known amount of the reference material concerned. This is because, an instrument generates only a signal , and it is the responsibility of the analytical chemist to convert the signal into concentration.
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An instrument converts a chemical information into an observable form. (i) It generates a signal. (ii) Transduction, (iii) Amplification, and (iv) Display of results. Developments in electronics have influenced chemical instrumentation in a bewildering manner(Fig. 3.3).

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Fig.3.3. Parts of a typical instrument
SIGNAL GENERATOR ANALYTICAL SIGNAL DETECTOR ELECTRICAL/ MECHANICAL INPUT SIGNAL SIGNAL PROCESSOR

METER (ANALOGUE SIGNAL)
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RECORDER PRINTER-PLOTTER/ INTEGRATOR

DIGITAL READ OUT
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Emphasis on CRM in the Six VAM Principles The key role of CRMs in analytical chemistry is recognized in the six Valid Analytical Measurements (VAM) principles indicated earlier. The testing of methods and equipment, referred to in VAM Principle 2, is most effectively accomplished by the use of appropriate CRMs.
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For example, the accuracy of the wavelength scale of UV-Visible HPLC detector can be verified by the use of a CRM, comprising a solution, that has absorption peaks, with well characterised reference values for the wavelengths of maximum absorbance. Likewise, the accuracy of an entire analytical method can be checked by the use of a CRM.

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VAM Principle 5 emphasises the importance of comparability between analytical data, produced in different laboratories and locations. One effective means of achieving this is, to ensure that all analytical data are traceable to reliable CRMs. A laboratory can check the repeatability of its data, by setting up Internal Quality Control(IQC) procedures, to provide evidence of day-to-day consistency in its results, but a laboratory relying exclusively on IQC procedures could conceivably be producing 03/12/09 240 consistent, but biased results.

The use of CRMs, as measurement bench marks, can provide the essential reference or anchor points, against which analysts can achieve comparability of their measurements. When several laboratories can achieve the same analytical result(within the uncertainty specified), for a given CRM, they will have demonstrated the comparability of their measurements.
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The areas in which Reference materials would find use are depicted in Fig. 3.4.
Instrument Performance Verification Instrument Calibration Laboratory Performance Verification

Internal Quality Control

REFERENCE MATERIALS

Method Validation

Uncertainty Estimation

Analyst Performance Verification

Figure 3. 4. Areas in which Reference materials would find use

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The Reference Material as a Transfer Standard in the Traceability Chain Most results of chemical analytical measurements are directly related to reference materials, which are used for the calibration of the measurement process. Two decisive preconditions must be fulfilled in order to obtain a result, that is traceable to the SI (Système International d’Unités). First, the reference material itself must carry an SItraceable value, and an attached uncertainty.
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Second, the whole measurement procedure – from sampling to calculation of the result – must be fully validated, and the uncertainty must be evaluated according to common rules While the analyst has to validate his procedure, and to evaluate his measurement uncertainty by himself, he may take for granted, that the value declared on the label of the reference material is traceable to the SI. Thus, the reference material plays a most important role, as it serves as a transfer standard in the traceability chain. 03/12/09 244

One of the key factors affecting laboratories’ capabilities to produce reliable test data is the availability of standard materials, with property values, that can be relied upon by their users. The quality of a result is dependent on the quality of standards used to validate a method, or calibrate an analytical instrument. Every day, standards are used to calibrate instruments, or validate test methods, e.g., pH meters, Conductivity systems, Auto-titrators, Karl-Fisher titrators, Chromatographs, etc. If the standard is not accurate, this inaccuracy will be reflected in the test result. 03/12/09 245

“No matter how skilled the analysts or how sophisticated the analytical technique used, if the calibration of the system is in error, the analytical result will always be wrong”. The definitions of various kinds of Reference materials used by analysts, and some related aspects, published by NIST, are summarized below.
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Certified Reference Material (CRM) Reference material, accompanied by a certificate, one or more of whose property values are certified by a procedure, which establishes traceability to an accurate realization of the unit in which the property values are expressed, and for which, each certified value is accompanied by an uncertainty at a stated level of confidence.
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In other words, a CRM is a reference material, one or more of the property values of which are certified by a technically valid procedure, accompanied by a certificate(See Annexure III), or other documentation, which is issued by a certifying body. This certificate will provide detailed information on the analyte values, their associated uncertainties, methods of analysis, and traceability. Their production and certification is expensive. Therefore, they are not used for routine analysis.
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Reference Material (RM) - Material or substance, one or more of whose property values are sufficiently homogeneous and well established, to be used for the calibration of an apparatus, the assessment of a measurement method, or for assigning values to materials. Reference Material Certificate - Document accompanying a certified reference material, stating one or more property values and their uncertainties, and confirming that the necessary procedures have been carried out to ensure their validity and traceability. 03/12/09 249

NIST Standard Reference Material® (SRM) A CRM, issued by NIST, that also meets additional NIST-specific certification criteria, and is issued with a certificate or certificate of analysis, that reports the results of its characterizations, and provides information regarding the appropriate use(s) of the material (NIST SP 260-136).

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Note: An SRM is prepared and used for three main purposes: (1) to help develop accurate methods of analysis; (2) to calibrate measurement systems used to facilitate exchange of goods, institute quality control, determine performance characteristics, or measure a property at the state-of-the-art limit; and (3) to ensure the long-term adequacy and integrity of measurement quality assurance programs.
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NIST Reference Material - Material issued by NIST, with a report of investigation, instead of a certificate to: (1) further scientific or technical research; (2) determine the efficacy of a prototype reference material; (3) provide a homogeneous and stable material so that investigators in different laboratories can be ensured that they are investigating the same material;
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and (4) ensure availability, when a material produced and certified by an organization other than NIST, is defined to be in the public interest, or when an alternate means of national distribution does not exist. A NIST RM meets the ISO definition for a RM, and may meet the ISO definition for a CRM (depending on the organization, that produced it).
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NIST Traceable Reference MaterialTM (NTRMTM) - A commercially-produced reference material with a well-defined traceability linkage to existing NIST standards for chemical measurements. This traceability linkage is established via criteria and protocols defined by NIST to meet the needs of the metrological community to be served (NIST SP 260-136). Reference materials producers, adhering to these requirements, are allowed use of the NTRM trademark. A NIST NTRM may be recognized by a regulatory authority, 254 as 03/12/09 equivalent to a CRM. being

NIST Certified Value - A value reported on an SRM certificate or certificate of analysis, for which NIST has the highest confidence in its accuracy in that, all known or suspected sources of bias have been fully investigated or accounted for by NIST. (NIST SP 260-136) NIST Reference Value - A best estimate of the true value provided on a NIST certificate, certificate of analysis, or report of investigation, where all known or suspected sources of bias have not been fully investigated by03/12/09 NIST. (NIST SP 260-136) 255

NIST SRM Certificate or Certificate of Analysis - In accordance with ISO Guide 31: 2000, a NIST SRM certificate is a document containing the name, description, and intended purpose of the material, the logo of the U.S. Dept. of Commerce, the name of NIST as a certifying body, instructions for proper use and storage of the material, certified property value(s) with associated uncertainty(ies), method(s) used to obtain property values, the period of validity, if appropriate, and any other technical information deemed necessary 03/12/09 256 for its proper use.

A Certificate is issued for a SRM, certified for one or more specific physical or engineering performance properties, and may contain NIST reference, information, or both values, in addition to certified values. A Certificate of Analysis is issued for a SRM, certified for one or more specific chemical properties. Note: ISO Guide 31 is updated periodically; ISO provides the updated version.
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NIST Certificate of Traceability - Document stating the purpose, protocols, and measurement pathways, that support claims by an NTRM to specific NIST standards or stated references. No NIST certified values are provided, but rather the document references a specific NIST report of analysis, bears the logo of the U.S. Department of Commerce, the name of NIST as a certifying body, and the name and title of the NIST officer, authorized to accept responsibility for its contents.
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NIST RM Report of Investigation - Document issued with a NIST RM, that contains all the technical information necessary for proper use of the material, the logo of the U.S. Department of Commerce, and the name and title of the NIST officer, authorized to issue it. There are no NIST certified values provided, and authorship of a report's contents may be by an organization, other than NIST.
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NIST Report of Analysis (ROA) - Document containing the certification of the material, and including such information as the base material used, how the SRM was manufactured, the certification method(s) and description of procedures, outside collaborators, instructions for use, special instructions for packaging, handling, and storage, and plan for stability testing. The ROA is intended for internal NIST use only.
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The goal of any analytical measurement is to get accurate, reliable and consistent data. Prerequisites for achieving accurate results in analytical laboratories are correct sampling, correct weighing of the sample and standards, use of well-maintained and calibrated equipment, qualified operators, validated methods and procedures for data validation. Most important is the use of accurate standards or Certified Reference Materials.
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Eventhough the chief role of reference material is to ensure accuracy for a specific method, there is another, equally important use of such materials: they enable the laboratory and a specific user to verify the performance of equipment, procedures and operators at any time.

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Agreement in analysis results with the certified value proves that, not only the method is right, but also the equipment and the chemicals used for sample preparation are right, and that the operator did a good job. The laboratory can conclude that, the data generated for this particular procedure are correct.

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All laboratories obtaining the same results are 'intercalibrated', and in line with the technically competent organization, which certified the material. Any disagreement between the certified value and the value determined by the laboratory indicates a problem with the analysis, which requires a thorough follow-up.

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Commercially available Reference Materials Reference materials are available from a large number of producers. Among them are private companies, federal research laboratories, and metrology institutes. ISO has set up several rules to assure a suitable quality of reference materials which should be clearly indicated in a certificate(See Annexure III).
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Producers of CRM’s include ¨ National Institute of Standards and Technology NIST (USA)* ¨ Commission of the European Communities BCR (Belgium) ¨ Laboratory of the Government Chemist LGC (UK) ¨ National Institute for Environmental Studies NIES (Japan) ¨ Laboratoire National d’Essais LNE, (France) and many more. A comprehensive list of ref. standard suppliers is given in Annexure I. 266 03/12/09

Criteria for all Standard Materials Stability The stability of a standard material is defined as its ability to maintain its property / value over a specified period of time. The aim of stability testing is to determine whether the standard maintains its reference value from the time of production to time of use. The frequency of stability testing depends on the risk of any change in reference value with time.
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Resulting from a well defined stability trial, one should be in a position to (i) Identify suitable packaging materials, Determine shelf life of the product, (ii) Determine optimal storage conditions, for the standard material being investigated.

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The ideal standard material should be physically and chemically stable. Most materials change due to evaporation, or chemical reaction initiated by temperature, light, air, or humidity. Precipitation, bacteriological activity, and interaction with storage container may lead to instability. The producer of the standard should carry out extensive stability studies, and assign expiry dates, based on these studies.
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Homogeneity The Standard must be homogeneous; i.e., the difference between representative sample measurements must be less than the overall uncertainty limits of the measurement. A material can only be said to be homogeneous at or above the weight / volume of the representative samples analysed. Accuracy of Reference Value Accuracy of the value / property of a standard material may be defined as the closeness of the agreement between the reported value and the 03/12/09 270 true value of the analyte.

Uncertainty of Reference Value The reference value of the material must be determined with rigorous estimates of uncertainty, the value being as close as possible to the true value. Uncertainty of Measurement is defined as the parameter associated with the result of a measurement that characterizes the dispersion of the values, that could reasonably be attributed to the measurand.
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Application of Standards Standards provide vital ingredients in every day lab. analysis; these include, Instrument calibration, ensuring the analytical device is giving a correct result over the analyte range of interest. It is always advisable to check operator’s manual for the manufacturer’s requirements for frequency of "actual calibration" of an instrument.
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The broad areas of application of standards include, Validation of test methods, Analytical QC, Production of working standards, Establish traceability, Check equivalence of methods and Comparability. By the use of Standards, the analyst can demonstrate the validity of an analysis, the concept of measurement traceability can be realized and the comparability of analytical data between laboratories can be improved.
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Selection of a Standard When deciding on the most appropriate standards for a particular use, one must first decide on the accuracy of result required. The use of Primary Standards / CRMs are appropriate to work of highest accuracy, or when a specific procedure requires their use.

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Secondary standards are more appropriate to routine analysis. They provide known accuracy, traceability, certification, and economy. When selecting a Standard, the analyst must consider, the accuracy of standard required, stability of standard, matrix of standard and concentration of analyte (value of standard).
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General Guidelines for Handling Standard Materials. Standards, when supplied, are accurate, homogeneous, and stable. The guidelines to handle and store standards are given below. Store in original container, Store in accordance with accompanying instructions, Check the Expiry Date before use, Replace cap when standard is not in use, Avoid contact with fumes, Avoid the possibility of contamination during removal of sample.
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Dispose of any ‘unused’ sample of Standard material; do not return to the original container. Standards are chemicals, in some cases they may be classed as hazardous substances. One must handle, and dispose of, in accordance to local safety regulations. Most importantly they must be accompanied by appropriate certification.

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Standard samples can be obtained from various sources. Annexures 1 and II give Sources of reference materials, and important guidelines available for Certification of reference standards, respectively. In Annexure III a typical Certificate issued by NIST(for Hydrogen sulfide) is reproduced, just to illustrate the various aspects covered in such a certificate.
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Assigning the traceable value to the CRMs There have been several discussions on how the traceable value has to be assigned to the reference material, and who is responsible for the assignment. However, there is still no generally accepted guideline at present. In fact, one has to face a contradictory situation today. On the one hand, there is a growing demand for reference materials, which carry traceable values. On the other hand, assigning a traceable value means putting much effort into a primary measurement. 03/12/09 279

For a private company, this may not be economically feasible. Thus, not all the produced reference materials will carry traceable values. Several studies at LNE (Laboratoire National d’Essais, France) and EMPA (Swiss Federal Laboratories for Materials Testing and Research) have shown that even the values declared for monoelemental standard solutions, which are used for calibration and which are relatively simple materials with respect to their matrices, are often not traceable. 03/12/09 280

An Effective Solution As the need for reference materials will keep growing, a division of tasks may be the most effective solution. Federal institutes and private companies will focus on their unique skills and core activities. While a private company may be able to produce a reference material very efficiently on a large scale, they may not be equipped for the assignment of a traceable value. 03/12/09 281

In many cases, the declared value has been regarded as a traceable value by most analysts. Federal institutes, especially the metrological institutes, are well-equipped to assign a traceable value, because, normally, it is one of their core tasks to provide the link to the SI, by performing a primary measurement. However, they often may not be equipped well enough for an efficient production.
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Regarding the large number of reference materials that are already available, one could state, that there is already enough production capacity. There is, however, a lack of capacity for a traceable value assignment. As a consequence, it would be more effective to combine the different skills in a complementary way.

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Concept for the Value Assignment of Reference Materials The way, described above, has been chosen by several well reputed organizations. A suitable quality assurance system is required from each partner. An ISO DIS 9001:2000 certification is in place at EMPA, and FLUKA. The production and certification of a monoelemental standard solution follows an acceptable flow chart, with different well defined steps. 03/12/09 284

If several fundamentally different methods of analysis for a given constituent are available, e.g., gravimetric, titrimetric, spectrophotometric or spectrographic, the agreement between atleast two methods of essentially different character can, usually, be accepted, as indicating the absence of an appreciable systematic error in either( a systematic error is one which can be evaluated experimentally or theoretically).
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Primary Standards(Primary reference materials) A Primary Reference Material is one, whose value is accepted without reference to other standards of the same quality. The standard must be pure, stable, have high equivalent, be soluble under the conditions in which it is to be used, react with the standard solution instantaneously and stoichiometrically. Primary Standards are expensive, but offer the highest accuracy.
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Many other relevant information regarding primary standards given in Ref. (36A) and are summarized in several literature sources. A primary standard is a compound of sufficient purity from which a standard solution can be prepared by direct weighing of quantity of it, followed by dilution to give a defined volume of solution. The solution produced is then primary standard solution. The substances commonly employed as primary standards are given below.
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Acid - Base reactions: Sodium carbonate Na2CO3, Sodium tetraborate Na2B4O7, Potassium Hydrogen phthalate KH (C8H4O4), Potassium Hydrogen Iodate KH (IO3)2. Complex formation reactions: Pure metals (e.g. Zinc, magnesium, copper and manganese) and salts, depending upon the reaction used. Precipitation reactions: Silver, Silver nitrate, sodium chloride, potassium chloride and potassium bromide.
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Oxidation - Reduction titrations: K2Cr2O7, KBrO3, KIO3, KH (IO3)2 , Potassium Oxalate, Na2C2O4, arsenic (III) oxide As2O3 and pure iron. Hydrated salts, as a rule, do not make good standards; this is because it is difficult to dry them efficiently. However, those salts which do not effloresce, such as sodium tetraborate Na2B4O7. 10 H2O and copper sulfate CuSO4. 5H2O are found by experiment to be 03/12/09 289 satisfactory secondary standards.

Secondary Standards A secondary standard is a substance, which may be used for standardization, and whose content of the active substance has been found by comparison against a primary standard. It follows that, a secondary standard solution is solution in which the concentration of dissolved solute has not been determined from the weight of the compound dissolved but by reaction (titration) of a volume of the solution against a measured volume of primary standard solution. A secondary standard is traceable to a primary 03/12/09 290 standard, or a CRM.

Working Standard A Working Standard is used as an alternative to a secondary standard. It may be characterized by, either primary or secondary methods. Typical applications would include internal quality control, and instrument performance checks. Typically it might be obtained by dilution of a Secondary Standard.

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Certification of Secondary Standards As the reported test results forms the basis for the ‘Certificate of analysis’ of the standard, the testing procedure is critical to the reported value. All tests must be carried out using Validated methods. Such methods must be technically valid, i.e., i) have a valid and well described practical foundation, (ii) have been experimentally evaluated so that reported results have negligible systematic errors, (iii) have a high level of precision and (iv) have high reliability. 03/12/09 292

Where possible, a primary method should be used, i.e., titrimetry, gravimetry, coulmetry and depression of freezing point. The term traceability is used to describe the reliability of measurements, but it is not always clear exactly what that means(36H). In the science of chemical measurements comparability of results is a primary requirement, and traceability is a tool to help achieve comparability.
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Definition of traceability provided in ISO 8402 is “Traceability is the ability to trace the history, application or location of an entity by means of recorded identifications”. Nowadays, there is great emphasis, not only on the precision of the results obtained using a specified method, but also on their traceability to a defined standard or SI unit. Traceability refers to the completeness of the information about every step in a process chain.
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The formal definition of Traceability is the “ability to chronologically interrelate the uniquely identifiable entities in a way that matters”. The term is, for example, used to refer to an unbroken chain of measurements, relating an instrument’s measurements to a known standard. Traceability can be used to certify an instrument's accuracy, relative to a known standard.
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