Chapter 23 Aspects of quality assurance in forensic toxicology

M.J. Bogusz (Ed.). Forensic Science Handbook of Analytical Separations, Vol. 6 r 2008 Published by Elsevier B.V.

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Aspects of quality assurance in forensic toxicology Rolf E. Aderjan Institute of Legal Medicine and Traffic Medicine, University Hospital – Ruprecht-Karls University of Heidelberg 69115 Heidelberg, VoX str. 2, Germany

23.1 INTRODUCTION Aristotle (384–322 B.C.) distinguished 10 basic categories of human being and thinking, one of which is ‘‘quality.’’ Its elements are taken to be either objective distinguishing marks or stipulated by human perception. John Locke (1632–1704) drew a distinction between (external) sensation and (internal) reflection. He assigned their quality to (objective) primary elements, such as dimensions in space or time, and to secondary subjective ones, such as color or olfaction (due to imagination). In other words, in addition to measurable ones, various subjective components of quality exist whose weighting may entail affirmative, negative, and limiting utterance, respectively. Correspondingly, any decision is subject to either intentional or unconscious considerations of the quality of a matter, an object, or an activity, in order to approve or refuse it. If several alternatives are available for choice, they need to be compared and evaluated by ranking their quality elements. Depending on the individual matter and the way of decision, more or less weighted subjective and objective factors may contribute to quality ranking. After all, in contrast to prevailing objective quality signs, any decision may be governed by subjective factors. Such consideration is relevant for the understanding of quality aspects. In the view of customers and manufacturers, quality has to do primarily with the relation between cost and benefit. Goods may be preferred according to their being ‘‘fit for use.’’ From his observation of nature, Darwin deduced the principle of evolution, ‘‘survival of the fittest.’’ Based on environmentally supportive and selective conditions, both replication (related to replicability, repeatability, and reproducibility) and mutation (as a base of accommodation) are genetic quality-raising processes by which present information, abilities, and knowledge are passed into the future. In humans, as in other organizational structures in nature, quality generally is a moving

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target demanding a change. Therefore, an aphorism, attributed to Lichtenberg (1742–1799), is still true: I cannot say whether things will get better if we change; what I can say is they must change if they are to get better.

Clearly, all quality assurance aspects in forensic toxicology should refer to the primary ‘‘quality product’’ expected from a forensic toxicologist (FT). This product is to provide objective analytical chemical results, including the bare analytical values, and their interpretation as within definite limits obtained also by appropriately trained staff in other forensic analytical laboratories. To serve as a means of evidence, results must correspond to legal requirements and be capable of being conclusively testified to in court. Even if it should be admitted that courts have limited capacity to review the FT’s work, that work is always open to cross-examination. FTs have always been concerned about the quality of their results, most likely more so than other laboratory organizations. Therefore, forensic science laboratories have probably developed quality assurance and quality control activities as long as they have existed. While accreditation and certification was requested in calibration and testing laboratories for several reasons, such as safety, improvement, or equal conditions, in forensic science laboratories quality assurance and control were always thought of as good laboratory practice (GLP). The development towards worldwide quality terms and the standards of today, such as GLP and the ISO standards, were initiated in order to eliminate the bad reputation of national products using quality badges, such as ‘‘made in y,’’ and also because of complaints about inadequate drug toxicology studies performed by contract laboratories for big manufacturing companies. New regulations, which formulated the expectations and requirements for an organization engaged in nonclinical laboratory studies aimed at the safety of regulated products, were established in GLP by the American Food and Drug Administration (FDA) in the U.S. Federal Register in 1978 [1–3]. The U.S. Environmental Protection Agency (EPA), too, had issued partly more specific GLP standards for toxic substances and pesticides in parallel with those of the FDA. Similar regulations entitled GLP in the Testing of Chemicals were adopted by the Organization of Economical Cooperation and Development (OECD) in 1982 and also by the European Community (CE) in 1987. Since 1967, U.S. Clinical Laboratories have been subjected to regulation by the U.S. Clinical Laboratories Improvement Act (CLIA). Any laboratory handling human specimens for analysis must hold a license issued by the U.S. Secretary of Health and Human Services. Since 1989, the general requirements for the accreditation of analytical chemical laboratories in Europe have been established in the standard called the General Criteria for the Operation of Testing Laboratories (EN 45001), which has also become the national standard of individual European countries. In addition, at an international level, General Requirements for the Competence of Calibration and Testing Laboratories (ISO/IEC Guide 25) have been created [4]. These requirements are designed to be applied to the performance of all objective measurements. According to the requirements, in cases of dispute, the accreditation bodies should

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adjudicate unresolved matters. With reference to EN 45001 and ISO/IEC Guide 25, an interpretation of the accreditation requirements was produced by a joint working group of the European Cooperation of Accreditation of Laboratories (EAL/CEOC). It was aimed at specific guidance on accreditation regarding staff, equipment and calibration, test procedures, handling of items and components, measurement uncertainty, proficiency testing, as well as at the subcontracting of tests. Guidelines according to ISO 9000, ISO 17025, and ISO 15189 in their updated versions are now authoritative documents for laboratory accreditation [5], which in 2005 were subjected to amendments concerning the effectiveness of the management system and its correction or improvement. However, in order to fit with forensic demands, additional requirements were being introduced and developed in the ILAC – Guidelines for Forensic Laboratories and for dealing with management and technical requirements of objective testing, as practiced in laboratories involved in forensic analysis and examination [6,8]. The International Laboratory Accreditation Conference (ILAC) was initiated by the United States in 1979 and holds meetings biannually. ILAC’s efforts are concerned with coordinating the use, competence, and reliability of national testing bodies in more than 40 countries, as follows: 1. legal aspects of international reciprocity of test data, 2. the format of an international directory of laboratory accreditation systems, and other schemes for assessment of laboratories, and 3. definitions and common criteria for national accreditation systems. In order to give guidance in forensic quality terms, professional organizations such as The International Association of Forensic Toxicologists (TIAFT) [7],1 the Gesellschaft fu¨r Toxikologische und Forensische Chemie (GTFCh) [8], or Society of Forensic Toxicologists (SOFT) [9] and Swiss experts [10] were formulating and updating national and international guidelines for (objective testing in) forensic or analytical toxicological laboratories, based on both scientific and quality aspects, even before the need for accreditation was disseminated. Necessary additional guidelines had to be developed, introduced, and complemented, taking national legal regulations and jurisdictions into account, for instance, the specific detection of substances referred to in legislation covering driving under the influence in several European countries.2 Due to the widely recognized need in many western industrial countries, private, professional, and governmental organizations promote the evaluation, upgrading, and periodic checking of the performance of laboratories and also examine their testing ability for conformance to standards. Quality assurance principles of testing laboratories are now generally accepted. Accreditation bodies now (at a cost) certify 1

TIAFT-Guidelines: a still valid version was adopted in 1993 and republished in 2006 [7].

2

For instance, Section 24 a.2 of the German Traffic Act and corresponding guidelines referring to the problems of detecting definite compounds in blood after the usage of heroin, cocaine, amphetamines, and cannabis.

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766 Quality management & planning

management of strategic activities

management of operating process activities

structures & means

processes

staff facilities

management of normative activities outcome

forensic outcome preexamination examination postexamination

equipment, supplies

scientific outcome business outcome

processing phases

Fig. 23.1. Important quality areas that need to be managed in forensic-toxicological laboratories with respect to quality.

laboratories, providing assurance of their testing ability and adherence to procedures on inspection. Besides formal safety regulations, quality concepts were developed from a more basic point of view. While earlier in the 1960s, quality control predominantly focused on the product being manufactured, it is now clear that quality skills themselves need to be produced. Quality experts have described the important essentials of producing quality. In 1980, Donabedian [11] analyzed the elements of quality for health care services. He proposed a concept in which three sections of quality were distinguished: (1) structural quality: the availability and organization of human, physical, and financial resources, such as facilities, space, staff, equipment, and consumables; (2) processing quality: the activities necessary to deliver a product or service including examination, quality assurance, reporting, interpretation, and consultation; and (3) the quality of the outcome with respect to patients’ health. As shown in Fig. 23.1, these elements can also be applied to create a comprehensive quality concept in analytical toxicology.

23.1.1 Organizing quality and quality concepts To introduce such new principles to manage quality, it may be necessary to overcome a high activation energy in a laboratory. First, the generally accepted guidance documents need to be studied. For the realization of a quality management system (QMS), one member of the staff needs to be enabled and nominated as a quality supervisor. Step by step all the members of the staff need to be informed and involved in the coming activities. In addition to the creation and implementation of standard operating procedures (SOP), all regulations, including internal audits, have to be explicitly written in a quality management manual. The SOPs refer to all logistical

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activities, examinations, and equipment as well as quality control procedures. In order to comply with actual practical procedures, SOPs and quality documents should first and must preferably be written by the corresponding coworkers, supported by experienced external accreditation experts. Then all documents need to be examined and released by the previously trained superior ‘‘in-house experts.’’ Implementation of a QMS can only be achieved by continuously performing all the required steps. Besides its necessity in a high-throughput/workload laboratory, a well-designed computerized electronic laboratory information and management system (LIMS), which models the in-house organization principles, is an indispensable quality management tool, after whose implementation a step-by-step introduction of the quality concept can be effectively transferred into action. Finally, accreditation and certification may be required, by more or less voluntary applications for external audits organized by authorized national accreditation bodies. Usually, certification is possible after introduction of a QMS according to the ISO EN DIN 9000 series. In contrast, accreditation means a quality proof being based on the EN 45000 series and now on the ISO/IEC 17025 standard. These require suitable internal quality controls and successful participation in external quality controls as well as an approval of the laboratory’s conformity with its QMS. In its 2005 Edition, ISO/IEC 17025 introduces some changes referring to QMS qualification and developing appropriate QMS monitoring and improvement strategies, including customers’ satisfaction. For further developing quality and QMS issues and to eliminate any deficits, which are identified, Deming [12] (according to Malorny [13]) has recognized the PDCA cycle (a continuous sequence of planning, doing, checking, and adjusting). In contrast to industry, where high quality should usually delight the customer and consumers, in forensic science the toxicological expert may have several ‘‘customers,’’ some of whom may not be pleased by his product Prosecutors, judges, defenders, and last, but not least, the accused have different and even conflicting interests in the FT’s reporting. Therefore, in case of doubt, analytical results may be questioned from diametrically opposed points of view. This fact has a major influence on quality management in forensic toxicology. Forensic toxicologists must anticipate potentially relevant facts. Do they keep in mind all known difficulties or interference of the methods that are used when they start to work on a case? Reasonable doubts must accompany the toxicologist’s daily casework. Is he or she always aware of possibly working on a case in which afflicted people seriously distrust results or conclusions? Have all of the substances been determined, which will be necessary for conclusive case-based reasoning? The multitude of such questions makes it understandable that educational ring tests as well as true proficiency testing are accepted as a highly important means to increase the degree of certainty of analyses aiming at the detection of previously unknown substances and their quantitative determination. However, along with progress in science and increasing knowledge about metabolism, human formation, and elimination kinetics of an increasing number of toxicologically relevant parent compounds and their metabolites, new concepts and methods will be developed, entailing a permanent involvement in continuing advanced education. That is why the GTFCh has introduced an References pp. 814–819

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acceptance procedure including the appointment as ‘‘Forensic Toxicologist GTFCh,’’ attesting to a qualifying knowledge according to updated guidelines [14] and requiring a continuing advanced education in the future. According to German accreditation regulations, forensic accreditation requires the acquisition of this appointment for laboratories and their responsible toxicological directors [8]. Limited laboratory services may be selected because of large case numbers and economic pressures. Also, the pressure of the high responsibility required for forensically demanding and time-consuming cases may stimulate a wish to limit laboratory services more towards the analysis of definite analytical parameters. Sometimes in automated laboratory analysis indicators for questionable results are carried along with the analytical method, including sample consumption. In contrast to clinical samples, however, forensic samples must be carefully treated as unique specimens, and analytical pitfalls should be anticipated by experienced staff as much as possible. In laboratories performing a selected number of standardized clinical analytical methods, services, such as blood alcohol determination and drugs of abuse in urine, are being offered. The analyst may then obtain objective, precise, and accurate results. In contrast, in forensic toxicology laboratories the analyst often must first solve the fundamental task of poison detection in human organs and body fluids, at times in very complicated cases, before being able to apply or, if necessary, to work out and validate a new analytical method. As justice should remain affordable and because the economical selection of analytes entails a loss of analytical experience, it needs to be stressed that time-consuming and less cost-effective tasks must not be excluded from forensic-toxicological standard methods and moved to limited-service laboratories due to economics, including some risks of quality. Forensic analysts are sometimes confronted with unexpected or difficult cases including decomposed bodies. According to the results of a general unknown analysis or to a question, new or problem substances need to be determined, requiring methods previously not tried and tested. Consequently, the repertoire of methods needs to be continuously increased by developing appropriate approaches including, at times, the costly validation of methods applied only a few times, or even once. Running parallel to activities since the 1960s aimed at defining standards, scientific societies continuously work out their recommendations and guidelines for forensic analytical work and laboratories.3 However, merely acting according to working guidelines or formally fulfilling standards does not guarantee that the top quality levels needed in forensic toxicology are permanently achieved and in specific cases. Forensic toxicology is a scientific discipline in which ongoing efforts to improve methods of poison detection point out the close relationship between quality and scientific development. This becomes obvious regarding frequently occurring problems and deficits in interpretations experienced during casework. Besides the formal and theoretical principles of quality assurance, there is one more facultative requirement regarding the FT’s management activities. This requirement

3

Newly structured Guidelines of the GTFCh are presently being prepared (2006) including appendices for specific analytes, method validation,, minimum limits of detection and quantification.

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refers to goal finding in science and corresponding organizing activities in a forensic toxicological laboratory. At universities, for example, scientific work usually needs to be carried out in addition to casework. Approved methods are often developed by one’s own efforts. However, analysts trying to run ‘‘solutions’’ offered in the literature often experience the need to vary or improve them. Therefore, the quality concept must include the FT’s maintaining competence in qualification and expertise, and a need for continuous self-education by updating and improving his or her scientific knowledge, especially by doing his or her own research in analytical and human toxicology.

23.2 A QUALITY CONCEPT FOR ANALYTICAL TOXICOLOGICAL LABORATORIES Donabedian understood quality care to aim at a successful change in patients’ present and future health status. In order to reach such an outcome, the appropriate quality of both the structures and the processes of health care services are required. For a toxicology laboratory, similar needs can be seen (Fig. 23.1).

23.2.1 Quality of the structure 23.2.1.1 The staff-management and basic requirements One key to maintaining quality and suitable performance of any chemical analytical laboratory is successful interaction of all staff members. This is achieved by the performance of the laboratory director as well as by the selection of qualified supervisors and trained non-supervisory staff. The laboratory director must have appropriate education and experience regarding proper planning, organizing, staffing, leading, and controlling. Most of these activities involve abilities in personnel management. In order to be able to apply management concepts and techniques, it is recommended to the FT that his postgraduate education should ideally include a sequence of training courses in current personnel management skills. As all work needs to be organized into individual jobs, an optimal level of laboratory performance is only achieved if appropriate personnel are recruited and placed into the correct positions. In addition, realistic goals and fair pay policies in combination with a bi-directional flow of information within a laboratory are important quality requirements. The staff must be aware of the meaning and importance of their tasks and know how to contribute to the goals of the management system. Appropriate communication pathways need to be implemented and communication about the effectiveness of the management system must take place. Regular evaluation of the employees and a positive attitude to their work is crucial to improve motivation. The management needs to create an appropriate environment to encourage the staff members to participate in decisions including the perception of their vital interests. As mistakes inseparably belong to daily work, an References pp. 814–819

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effective strategy of improvement of the individual is probably more important than trusting in the usefulness of rewards or discipline as a situation appears to demand. 23.2.1.2 Core competencies of a forensic toxicologist as a laboratory director Because they are the most important aspects for quality assurance, an organization’s attention must mainly be directed towards personnel qualifications. Besides the primary laboratory management skills, the FT basically needs scientific skills, technical skills such as observation of, knowledge of, and the introduction of new technologies, continuous self-education, and participation in and sharing of knowledge in the scientific society. Guidelines have been developed by the GTFCh for the fundamental skills required for certification as an FT [14]. To be certified, an applicant must demonstrate that he or she fulfills a series of minimum requirements. They include:
an academic degree in a subject of natural science related to forensic toxicology, such as chemistry or pharmaceutical science;
seven years of professional toxicological work including testifying in courts (presently updated to only five years of FT-coached in-house training including examination by GTFCh experts.);
participation in scientific symposia and in workshops of the GTFCh as well as appropriate postgraduate education;
the ability to suitably understand and discuss analytical procedures;
the ability to correctly interpret forensic toxicological testing results; and
sufficient forensic testifying experience by presentation of advanced written expert opinions taken from his or her professional work. The size and organization of a laboratory influences management structures. Therefore, the qualification and experience of superiors and coworkers depend on the requirements and responsibilities of each position. As a minimum, the laboratory director must overlook all analytical activities undertaken and have a comprehensive scientific analytical and methodological knowledge and responsibility. According to the full analytical scope of forensic toxicology, he needs to be supported by at least two coworkers in supervisory positions. Except for the maintenance of complex analytical instruments, most of the basic analytical activities presuppose coworkers with a sound technical-analytical but not necessarily academic education. As a consequence of the scientific development of a laboratory, highly evolved analytical instrumentation purchased for scientific reasons has become or will possibly be commonly used in the future. For this reason, a laboratory’s list of supervisory positions must include specialists who can take care of one or more of such complex instruments, analytical methods, and the tasks to be carried out by them. Unfortunately, forensic toxicology laboratories of some university institutes of legal medicine have become relatively small. In these, because of the lack of appropriate positions, the working scientist at the bench may belong to the supervisory staff without a clear-cut definition of his or her responsibility. This understanding of responsibility is critical

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with respect to science, quality, and accreditation requirements as, on the one hand, relevant scientific research interests may easily be obstructed by casework overload and, on the other hand, a non-analytically educated head of the department takes responsibility for reports, but transfers analytical responsibility. In order to revise such deficient structures, some universities have started to establish positions for a fully responsible FT. Developing more of such well-tailored positions will be a major challenge in these times of increasing knowledge requirements and decreasing financial resources. The rapid scientific development of forensic toxicology and the use of more and more advanced technical analytical instrumentation is another reason why it seems advisable to exclusively employ a scientifically qualified FT for the position of a fully responsible laboratory director. Along with a rating according to scientific evaluation requirements, the level of performance and quality is becoming decisive for both the existence of a forensic toxicology laboratory and its structural conditions. 23.2.1.3 Quality aspects regarding the role of an expert witness Independent of the national legal system, a scientist’s unique role as an expert witness (EW) is to be unbiased in helping a judge (and the jury in the case of the United States and United Kingdom as well as other related adversary legal systems) to understand the scientific truth. This duty, however, may not always be simple. In specific cases, a thorough preparation and updating of current scientific knowledge is required. Interestingly, there is little information available about the professional training of an EW, which is particularly important for FTs testifying in an adversary system. Recently, a condensed overview has been presented in order to summarize the training requirements for an EW in the United States [15]. Eight ‘‘qualities’’ needed to be developed [16]. The expert must:









perform a thorough investigation; be personable, genuine, and natural; have an ability to teach; be generally competent; be believable; persuade without advocacy; be prepared; and demonstrate enthusiasm.

In Ref. 17, the ‘‘expert witness (EW)’’ has been defined as follows: ‘‘... a witness having special knowledge of the subject about which he is to testify. That knowledge must generally be such as it is not normally possessed by the average person. This expertise may derive from either study and education or from experience and observation. An expert witness must be qualified by the court to testify as such.’’ Compared to this definition, some of the qualities refer to behavioral and technical skills, which, however, need to be based on scientific knowledge. Among some advice regarding pre-trial procedures, such as discovery of evidence and deposition or trial procedures such as cross-examination, useful hints are given in order to keep current References pp. 814–819

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and competent, improve personal presentation or avoid taking sides, and help an attorney win the case. As the opposing attorney in the adversary system may have several goals during cross-examination, the EW needs to focus on this part of the trial. According to Refs. 15 and 18, such goals may consist of:






discreditation of the EW by attacking conduct and character; attacking qualifications by establishing gaps in the EW’s professional resume; showing inconsistencies in the EW’s statements; exposing the EW’s bias; giving reasons why testimony is slanted; attacking the witness’s fact basis; tests conducted were inadequate, inappropriate, or incorrect; and
discrediting the EW’s conclusions by showing that it is confused and therefore can be wrong (changing the hypothetical used on direct). In contrast to the adversary system, e.g., in the German legal system, one or more expert witnesses may be accepted or appointed by the judge and need to be present in the courtroom in order to observe the entire taking of evidence and then give his or their expert opinions in order to support the judge in his or her assessment of evidence. Such a procedure has major advantages. First, the opposing attorney’s experts can directly be examined by each other and also by the judge regarding the scientific quality of their statements. Second, the judge can appoint an expert of his or her own choice to obtain a superior scientific expertise including examination of written opinions and the excerpts of the procedure. 23.2.1.4 Non-supervisory staff The selection of assistant and technical staff working under the supervision of the scientific staff is very important for a laboratory. Appropriate professional, chemical, and analytical education of technicians, selection according to an appropriate pre-employment interview, a knowledge of the principles of the methods used in a laboratory and the ability to apply them, handling of equipment, as well as understanding the importance of quality assurance are major requirements to be expected at this level. The recognition and reporting of potential sources of error and of situations where quality standards may be missed are other expectations addressed by technical staff. As such qualifications are usually based on experience, repeated teaching and training of staff members is indispensable. 23.2.1.5 Facilities, equipment, supplies All guidelines demand suitable facilities, equipment, and supplies belonging to the important structural presuppositions. All of them need to be managed skillfully. The availability of a sufficient number of clean vented laboratory rooms suitable for trace analysis and the prevention of contamination during all analytical processes is a fundamental requirement. Additionally, in forensic toxicology, all laboratory rooms need to be protected in order to exclude access by unauthorized persons.

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The equipment and supplies depend on laboratory functions, which need to be described in a QMS. In high-throughput laboratories, running a suitable laboratory information system is obligatory for properly monitoring the state of progress of individual casework, which may consist of only one, several, or even multiple analytical tasks, including tasks given to subcontractors. However, even if the decision to purchase the right equipment and supplies suitable for chemical toxicological analysis clearly belongs to quality aspects, focusing on details or the criteria of the corresponding considerations would be beyond the scope of this chapter. In a fitting and appropriately developed QMS, all procedures are clearly described and updated analytical method SOPs are used, which include their validation data. QMS introduction will be an investment whose benefit may not immediately be obvious in terms of the costs. However, clear and completely written information and easy access to contact persons,, manufacturers, providers, and supplies will help to optimize organizational procedures by clarified responsibilities, and a rapid understanding of the pathways will save the staff ’s time in favor of performing all quality assurance procedures necessary, including instrument checks, monitoring of control charts, giving helpful comments, and undertaking corrections when parameters have become out of range or whenever necessary, and by avoiding errors. Therefore, regarding visible and hidden costs, an effective quality management supported by all staff member may turn out to be an economical necessity. 23.2.2 Quality of the processes 23.2.2.1 Preanalytical phase procedures Regardless of the objectives of a chemical analysis, suitable collection of the samples is the most important preanalytical activity. Only in very few cases, this action, regrettably, is carried out by FTs themselves. Therefore, it is recommended that detailed advisory preanalytical information is provided to all personnel involved in the collection, storing and transportation of samples [19,20]. In Germany, for example, taking of not only urine samples or hair strands but also venous blood samples regularly serves as a means of evidence in criminal cases. As decreed according to the code of criminal procedures, the taking of blood samples by physicians ordered by the police has proved a powerful means to clarify both the individual state of influence as well as the respective situation in which the afflicted person has been. However, the instability of drugs such as heroin and cocaine as well as irreversible binding of cannabinoids to matrix constituents and other factors, such as temperature, oxygen, or UV-radiation sensitivity of analytes, are commonly encountered pre-analytical problems. All physicians and police officers responsible for sampling supplies, taking of samples, and their transportation to laboratories must be informed about such details in order to understand and to comply with procedures as issued by the governmental agency. Besides this, the stability or degradation and decomposition of analytes by various influences is an important matter of interest to the FT, easily being neglected in routine analysis. References pp. 814–819

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Post-mortem sampling procedures need to be strongly distinguished from collecting samples from living subjects. The proper documentation of both the origin and quality of a sample is particularly important to the analyst. Clear-cut sampling and its documentation are both obligatory with respect to the interpretation of analytical results and the conclusions drawn. Site dependence, post-mortem changes, and redistribution of drugs and their metabolites are important phenomena [21–23] with which medical examiners need to be familiar when they collect the specimens. The publication of literature reporting on post-mortem redistribution, including animal experiments, continues [24–34]. In particular, substances with high distribution volume and specific tissue binding, such as cardiac glycosides or cannabinoids [35], and many other drugs are subject to redistribution after death. The mechanisms are diffusion, breakdown of membranes or decomposition of tissue, changes of water content, and agglutination of blood constituents, etc. Therefore, blood concentration measured is often unpredictably site dependent. Finally, the compounds themselves will decay with time after death. At autopsy, the case history may not necessarily be known in detail. Therefore, welldocumented sites, comprehensive specimen collection, and the taking of relevant samples must be carried out particularly in cases of possibly previously unknown homicidal poisoning. Hence, the early exchange of information at autopsy between the medical examiner and the laboratory needs to be stressed. There is still a great need for studies aimed at detailed knowledge about substance stability or the occurrences and causes of concentration changes in samples. As cannabinoid analysis in blood and urine has become an analytical task whose frequency nearly equals that of ethanol analysis in blood, relevant reporting on the stability of the major oxidative THC metabolite is taken as one example among many others [36]. In addition, more knowledge of specific non-metabolic post-mortem breakdown products needs to be gathered and analyzed. In a forensic laboratory, clearly defined and well-documented procedures are major points of specific care such as:
advising customers in proper sample storage and transport;
treatment, starting at the arrival in the laboratory;
specimen registration, sample portioning, including sample treatment, and how to avoid mistakes;
sample storage, including the decision about the necessity of freezing or refrigerating only;
registration and documentation of samples with respect to the chain of custody; and
security checks and storage of samples for additional examination and further investigations. Proper sampling of post-mortem specimens has recently being reviewed and, accordingly, GTFCh recommendations for post-mortem sampling and handling of samples have been published [37].4 4

Versions in English are being prepared.

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23.2.2.2 Examination and methods Forensic toxicologists need to perform poison detection in human organs and body fluids. Therefore, a careful selection of appropriate methods and their suitable application is a major point regarding laboratory performance in forensic toxicology. Two kinds of validated methods and corresponding equipment need to be maintained regarding the tasks to be carried out. First, approved general unknown analysis and selective screening methods need to be deployed. Second, various quantitative methods need to be applied according to specific requests or in cases in which particular results were previously obtained by general unknown analysis. For the introduction of a new method into regular casework, following a logical sequence of the PDCA cycle, several steps need to be taken:







elaboration of a new analytical method; testing of the method using spiked samples; measures of internal quality control; participation in external quality control; development of a plan for corrective actions; and steps to define a problem, investigate and determine its causes, initiate corrective actions to eliminate the problem, and check if the problem has been eliminated.

23.2.2.3 Screening methods and general unknown toxic substances Depending on the scope of substances that need to be detected or identified, so-called general unknown analysis and screening methods are used. As it is most important to the analyst to have indicators of substances present in a material, a screening procedure should but must not necessarily include substance identification. Based on the purpose of screening procedures, their diagnostic selectivity and sensitivity need to be high enough to avoid too many negative confirmation results. A universal screening purpose and ideal general unknown analytical method would include all toxic substances. Such a claim has remained an ideal comparable to the medieval legend of the ‘‘Holy Grail.’’ In contrast, usually a sequence of screening procedures is carried out using a combination of separation and detection methods to find the toxic substances present. The term ‘‘screening’’ refers to a limited number of substances related to each other by chemical, physical, and/or structural properties. Immunoassays for drugs or GC-head space analysis for screening of volatile compounds are examples of different areas of screening. Analytical screening is more or less extensive and its extent can be regarded as a quality. In general, complete information about the practical performance of screening methods for qualitative poison detection in practical cases is difficult to obtain. Some of the methods propagated for use in systematic toxicological analysis (STA) are based on the parent compound but not on their metabolites. In addition, the testing of practical detection limits in difficult matrices is lacking. The identification of unknown substances and the amounts that need to be determined are more critical in cases under suspicion of homicidal poisoning than in suicidal or accidental poisonings because of higher amounts of substances usually References pp. 814–819

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present in such cases. At present, for the general unknown analysis of toxic lowmolecular-weight substances, their possible detection is based on suitable sample preparation methods and the extraction of as many substances as possible. Upon chromatographic separation, any unknown substances need to be identified by consequent application of a spectroscopic method. Based on sufficient chromatographic separation prior to recording, the identification power of spectra mainly depends on their informational specificity. GC and HPLC are now the most common separation methods which, depending on the chromatographic principle, compete with each other effectively regarding the palette of accessible substances. The additional use of GC [38,39] or HPLC and retention indices [40] of reference substance mixtures helps to characterize the chromatographic column and improve the discrimination power of the analytical procedure needed to identify unknown substances. However, in contrast to substance quantization, for screening purposes, the baseline resolution of chromatographic peaks is not required under any circumstances. Gas chromatographic separation in combination with mass spectroscopy (GC/MS) using several libraries for comparison of standardized 70 eV mass spectra is generally available in forensic-toxicological laboratories [41]. A dual-column chromatography using MS and nitrogen-sensitive detection can be a useful extension of GC/MS screening. Matrix interference plays an important role in substance discovery and identification issues. However, knowing the limits of identification of individual substances in screening procedures used for various materials is more a matter of experience [42] than a result of circumstantial and nearly impossible method validation. Recently, in a consensus approach, the U.S. American Society of Forensic Toxicologists (SOFT) defined the maximum detection limit for drugs recognized to be used in sexual assault. (Table 23.1, [43]). Obviously, such minimum detection limits need to refer to specific search runs using single ion monitoring and also to general unknown screening procedures. Similar recommendations for minimum detection limits have been published for analytes listed referring to diagnosis and confirmation of brain death (Table 23.2). Clearly, the use of recovery-sensitive internal standards is recommended. In general unknown analyses, acidic and basic deuterium-labeled compounds can also be used (e.g. barbiturates or tricyclic antidepressants). It is recommended, as well to check an instrument’s performance and calibration with special attention to amphoteric substances with critical chromatographic peak shapes, such as benzoylecgonine or morphine [44]. The analyst’s attention needs to be drawn to suitable recording of mass spectra or ion monitoring of xenobiotics as both may be disturbed in the presence of high amounts of substances co-eluting in GC (e.g., polyethylene glycols of different chain lengths liberated from pharmaceutical formulations). Due to interference within the ion source, the extraction efficiency of charged particles may be reduced, as was observed for parent drug substances and their deuterated analogues [45,46] and for environmental compounds [47]. Other problems of mass spectra generation may arise by the uncritical use of an ion trap (and other types of) MS that needs careful evaluation of data in order to generally achieve

Target Analytes Ethanol Ethanol GHB and analogs Gamma-hydroxybutyrate

Parent Drug

Trade Names/Street Names!

Recommended Maximum Detection Limit

Ethanol

Alcohol, ethyl alcohol, ‘‘booze’’

Gamma-hydroxybutyrate

Xyrem, ‘‘GHB,’’ Easy Lay,’’ ‘‘G,’’ ‘‘Georgia Home Boy,’’ 10 mg/mL ‘‘Grievous Bodily Harm,’’ ‘‘Liquid Ecstasy,’’ ‘‘Liquid E,’’ ‘‘Liquid G,’’ ‘‘Liquid X,’’ ‘‘Salty Water,’’ ‘‘Scoop,’’ ‘‘Soap’’ ‘‘1,4-BD,’’ ‘‘Enliven,’’ ‘‘Inner G,’’ ‘‘Revitalize Plus,’’ ‘‘Serenity,’’ ‘‘SomatoPro,’’ ‘‘Sucol B,’’ ‘‘Thunder Nectar,’’ ‘‘Weight Belt Cleaner,’’ ‘‘White Magic’’ ‘‘GBL,’’ ‘‘Blue Nitro,’’ ‘‘G3,’’ ‘‘Gamma G,’’ ‘‘G.H. Revitalizer,’’ ‘‘Insom-X,’’ ‘‘Invigorate,’’ ‘‘Remforce,’’ ‘‘Renewtrient,’’‘‘Verve’’

1,4-Butanediol

Gamma-butyrolactone

10 mg/dL

777

Benzodiazepines Many benzodiazepines are biotransformed into glucuronide-conjugated metabolites. To improve detection limits and times, it is recommended that laboratories use instrumental techniques that will detect the glucuronide metabolites or hydrolyze urine specimens to free the conjugate before extraction. Alprazolam Alprazolam Xanax, Niravam 10 ng/mL a-Hydroxy-alprazolam Chlordiazepoxide Chlordiazepoxide Librium, Libritabs 10 ng/mL

Aspects of quality assurance in forensic toxicology

References pp. 814–819

TABLE 23.1 INTERNET PRESENTATION (ONLY FIRST PAGE) OF RECOMMENDED URINARY MAXIMUM DETECTION LIMITS REFERRING TO COMMON DRUG FACILITATED SEXUAL ASSAULT (DFSA) DRUGS AND METABOLITES (HTTP://WWW.SOFT-TOX.ORG/DOCS/SOFT%20DFSA%20LIST.PDF). SOCIETY OF FORENSIC TOXICOLOGISTS (SOFT). DRUG-FACILITATED SEXUAL ASSAULT COMMITTEE. RECOMMENDED MAXIMUM DETECTION LIMITS FOR COMMON DFSA DRUGS AND METABOLITES IN URINE SAMPLES

778

TABLE 23.1 CONTINUED

Target Analytes Clonazepam 7-Aminoclonazepam Diazepam Flunitrazepam 7-Aminoflunitazepam Lorazepam Nordiazepam Oxazepam Temazepam Triazolam 4-Hydroxy-triazolam

Parent Drug

Trade Names/Street Names!

Recommended Maximum Detection Limit

Clonazepam

Clonapin, Klonopin, Rivotril

5 ng/mL

Diazepam Flunitrazepam

Valium, Diastat, Dizac Rohypnol

10 ng/mL 5 ng/mL

Lorazepam Diazepam, Chlrodiazepoxide Oxazepam, Diazepam, Chlrodiazepoxide, Nordiazepam, Temazepam Temazepam, Diazepam Triazolam

Ativan Serax

10 ng/mL 10 ng/mL 10 ng/mL

Normison, Restoril Halcion

10 ng/mL 5 ng/mL

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TABLE 23.2 RECOMMENDED MINIMUM DETECTION LIMITS TO SUPPORT BRAIN DEATH DIAGNOSIS ACCORDING TO HALLBACH ET AL. [2002] TOXICHEM & KRIMTECH 69 (3) 124–127

Wirkstoff Thiopental Pentobarbital (Thiopental-Metabolite) Phenobarbital Methohexital Midazolam Diazepam Nordazepam (Diazepam-Metabolite)

Untere Grenze Destherapeutischen Empfohlene Untere Bereiches (mg/L) Messbereichsgrenze (mg/L) 1.0 1.0 10.0 0.5 0.04 0.2 0.2

0.5 0.5 5.0 0.25 0.02 0.1 0.1

acceptance [48]. Recently introduced mass spectrometric techniques, such as timeof-flight (TOF) spectrometers, need to be evaluated as useful new tools for screening analysis operating with full mass spectra even in trace analysis (see below). As a consequence of a laboratory’s need to assess the performance of the screening methods used, and due to the lack of simple validation criteria for generally unknown analytical methods, the participation in external quality assessment schemes including case report-oriented analysis of general unknowns is strongly recommended [49]. Among substances often overlooked in screening strategies may likely be the highly water-soluble compounds, such as glycols [50], and low-chain aliphatic acids such as glycolic acid and beta- or gamma-hydroxybutyric acid. There is increasing use in general unknown analysis of MS in combination with HPLC [51]. Depending on individual substances, suitable ionization modes, such as electrospray and atmospheric pressure chemical ionization systems [52–54], are now increasingly used and are affordable. Besides costs, however, the comparability of mass spectra recorded under specific conditions is still a limiting factor with respect to the widespread use of HPLC/MS and data collection for libraries. As spectra may vary between instruments of different manufacturers, more work needs to be carried out in this area. The use of such techniques for the detection of common drugs of abuse in serum or urine has been published [55,56]. Progress in the detection and quantitation of toxic plant [57] or animal peptides and proteins [58] is expected, including increasing forensic use of HPLC/MS. In spite of far less discrimination and identification power, HPLC and UV spectra are commonly used in combination with retention time or retention index concepts and library search techniques [59]. Usually using specific flame ionization, but increasingly the mass-selective detectors, head space GC is successfully applied to sensitively detect and quantitatively determine gases and volatiles in body fluids or organ tissues. For other groups of chemical substances, such as organic and inorganic toxic anions, practically no comprehensive systematic methods for poison detection have been proposed and approved. Separate testing of compounds under suspicion seems to be usual. Even if high-performance ion liquid chromatography has become an approved method for analysis of drinking water, up to now no satisfactory application of this method has References pp. 814–819

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been described for use in post-mortem samples. As molecular weight information is obtained from mass spectra, HPLC/MS/MS is, as expected, becoming a powerful tool for substance identification and confirmation of results of screening methods for toxic cations, quaternary ammonium compounds and various anions, and for toxic proteins. Chemical diagnosis of metal poisoning is another area of toxicological analysis in which quality assurance is urgently needed. Instead of separate testing for the presence of an individual metal, inductively coupled plasma mass spectroscopy (ICP-MS) seems appropriate for use in screening procedures and the identification of metals. As acute metal poisoning is rare, such expensive instrumentation may be used in collaboration with specialized laboratories for economic reasons. In conclusion, quality results of a general unknown analysis will strongly depend not only on a shrewd analytical strategy as conducted by an experienced toxicologist but also on the availability of appropriate equipment, the use of approved methods, and a regularly updated reference data collection. Progress in general unknown analysis continues. A laboratory’s participation in external quality control (EQC) and an educational ring test is the best means to examine performance and to compare and improve methods of performance in general unknown screening. New approaches in general unknown analysis include using GC 70 eV ionization and LC ionization techniques and time of flight mass spectrometry (GCMS-TOF, LC/MS-TOF) in which the drift velocity of ions in an electric field is measured. Precise molecular weight, peak recognition, and deconvolution software is applied. By such aids, all ions of mass scans can be recorded for substance identification in one run [60–64]. 23.2.2.4 Quantitative determination of toxic substances and their metabolites Quantification of parent compounds and, as often required, their metabolites presumes:
qualified and trained staff, appropriate equipment, supplies, and reagents;
the use of approved methods validated regarding their purpose including calibration; and
corresponding stocks of certified pure reference substances including proper storage conditions (or at least easy access to substances possibly found in general unknown analysis). GC/MS is routinely used for quantification of licit and illicit drugs, synthetic chemicals, and poisons. In order to achieve highly precise and accurate determinations, the analysis needs to be conducted using internal standards with chemical properties similar to the analyte and, if available, the deuterated analogues of the analytes. For an acceptable precision of a method, the recovery of spiked substances should be not less than 50%. Depending on the selectivity of the chromatographic separation, selected ion monitoring (SIM) based on four or at least three ions is sufficient for identification and quantification of trace amounts. The ion ratio is acceptable if varying within 720% of the theoretical value. This criterion seems to be

Aspects of quality assurance in forensic toxicology

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derived from a practical approach rather than from a systematic investigation serving as a decisive base. In qualitative trace analysis, however, little above the limit of detection and below the limit of quantitation (LOQ) small signal intensities will be detected. As statistical errors in the reading of less intensive ions become more pronounced, enlarging deviations must be considered. Some details regarding substance identification will be discussed below. Tandem-MS-MS and high-resolution MS (HRMS) may help to diminish the time needed for quantitative batch analysis and chromatographic separation using detection techniques (e.g., selected reaction monitoring, neutral loss scan, and precursor ion scan after collision gas-induced decomposition in triple-stage quadrupole instruments). The use of such techniques, far-reaching elimination of the background, and high discrimination power are other advantages that allow substance identification by specific monitoring of a single ion transition instead of several correlated ion intensities in single low-resolution quadrupole MS instruments. With increasing use of LC/MS/MS instruments, however, pitfalls [65] have recently become obvious, which should lead to increased validation efforts in HPLC/MS including more individual real samples to check for matrix-dependent effects of cross-talk [66] and ion suppression [67]. 23.2.2.5 Validation of methods Regardless of the use of approved isolation, purification, or separation steps, any new analytical method needs to be validated before its use in order to ensure that it is capable of yielding acceptable results. Validation is a procedure that determines whether an analytical instrument or method as applied in a laboratory provides the analyst and customer with results fitting their intended purpose. In general, validation activities refer to sampling, stability of substances or samples, performing analytical methods, calibration of instruments and methods, suitability of reference materials, calibrators, reagents, data acquisition, data documentation, and data statistics. Usually, the term ‘‘validation’’ is not used for elements of quality assurance in a laboratory other than analytical elements.. Primarily, validation seems necessary where new elements are taken into a procedure, such as new products, new analytical methods, new analytical instruments, or new quality testing criteria. That is why chemical analysts generally need to know three major validation aspects: 1. the performance of the instruments used; 2. the performance characteristics of the method; and 3. by whom and for which purpose will the measurements be used? Suitable forensic validation should include interlaboratory comparability and traceability as needed for objective testing in forensic toxicology. If correct procedures have been followed by different laboratories at different times, the same chemical analysis performed with the material under investigation should produce results agreeing with each other within definite limits. Traceability means that this comparability of results References pp. 814–819

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can be traced back to appropriate national or international measurement standards via an unbroken chain of comparisons. Typically, validation characteristics can be classified with respect to identity, pureness, and content of a substance. Data obtained during method validation should cover the performance characteristics as described in the following sections. They are some of the key ingredients that contribute to the analyst being in a position to understand and control the uncertainties that will affect the measurement. Where generally accepted validation data do not exist, e.g., in the development of a new standard method for a regulatory authority or for legal decisions, the proper course of action is to validate a method through a collaborative study involving at least eight laboratories [68]. In order to support scientific development, the definition of analytical standards, which need to be met by a scientist’s own method, has proved more useful in forensic toxicology than establishing standard methods. Regarding such standards, the introduction of other improved methods may be delayed and circumstantial. In addition, in forensic toxicology, many scientists are strongly convinced of their own method. It may become difficult to find a corresponding number of laboratories using identical methods for purposes of necessary collaborative studies. Under urgent circumstances, quantitative analytical methods previously not applied and not appropriately tested may sometimes be applied for rapid information for customers, e.g., an intensive care unit or the police. If such results are later used for legal decisions, for obvious reasons the methods should be validated as soon as possible. As in complex cases, new or unusual questions may arise later in the course of criminal procedures, and it is recommended to update validation and internal quality control data frequently with respect to detailed examination. Practical method validation requires investigation of performance characteristics that depend on the method’s purpose. As shown in Table 23.3, several performance levels of analytical methods in relation to validation can be distinguished. As has been demonstrated for polychlorinated biphenyls [69], the critical points of control in environmental quantitative trace analysis procedures were the following:
It must not miss any substance present at or above the limit of detection, and possibly at or above a selected threshold limit concentration.
It must not report false positives.
The recovery must be 60–100%, with a coefficient of variation of 4 20%.
Values found, corrected for recovery, must agree within 20% with the recoverycorrected values obtained by the official method.
There must be a documented validation that the extraction procedure removes compounds of interest from the sample matrix. Both the progress and the practice of method validation were recently reviewed [70]. Based on this survey, a generally accepted approach to method validation was given as an appendix to the GTFCh guidelines [71].5 Accordingly, a practical solution and scheme to calculate corresponding validation parameters by reporting 5

English version given in: Bulletin of the TIAFT XXXII(1), 16–23 (2003).

Action/ Level Measurement of physical parameters Identification Measurement of physical-chemical parameters Qualitative (trace) analysis (one substance or more)

Content, quantitative trace determination

Propositions Needed for Proper Working

Deviations Due to Instruments and Methods

Testing Method Acquaintance Performance in with Resolution/ Relation to Definite Separation Power Requirements

Interlaboratory Comparison

Reaction to Adjusting, Necessary Changes

Calibration of the instrument Measurement precision Measurement precision and method precision Measurement precision and method precision Measurement precision and method precision

Selectivity

Selectivity

Robustness

Linearity, precision accuracy

Accuracy

Robustness

Selectivity robustness

Limit of detection, limit of identification

Robustness

Selectivity robustness

Possible range of measurements, linearity, precision, accuracy, limit of detection, limit of identification, limit of quantification

Selectivity completeness of detection and identification Reproducibility, accuracy

Aspects of quality assurance in forensic toxicology

References pp. 814–819

TABLE 23.3 PERFORMANCE LEVELS OF ANALYTICAL METHODS IN RELATION TO VALIDATION

Robustness

783

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the appropriate analytical data into an easily taught Excels-based computer program with fill-in sheets has been developed [72]. Another tool useful in forensic toxicology is regular participation in external quality control trials as well as use of certified reference material (CRM), both of which are now available for illicit drugs in serum as a result of round robin testing and external quality control trials organized by the GTFCh. 23.2.2.6 Extraction recovery The correct use of deuterated internal standards is an excellent method to check the recovery. In order to yield acceptable precision and detection certainty, the recovery should be as high as possible. Currently deuterated standards are used, and an overall recovery of better than 50% with variability of 710% may be sufficient. 23.2.2.7 Selectivity and specificity The investigation of the method’s selectivity aims at low-interference frequency possibly resulting from normal and atypical tissue constituents such as xenobiotics and contamination, particularly at low concentration levels. No definite procedure exists for completely investigating the selectivity with respect to economical validation. First, it is recommended to analyze around 100 previously tested blank samples for typical metabolites in order to find co-eluting peaks. Second, in order to find the origin of and kind of co-eluting material in samples containing the analyte, the analyst may check the whole mass spectrometric background at the retention time of a poorly resolved peak and change the chromatographic conditions if necessary. 23.2.2.8 Calibration linearity and analytical sensitivity In order to calibrate properly, a linear relationship is expected between signal intensity and amount measured. The slope of the calibration line is a measure of the power of discrimination between concentration levels (analytical sensitivity). High recovery and narrowing of the chromatographic peak shape will increase sensitivity and yield better measurement precision and fit of the calibration line. The calibrators used should cover the whole expected concentration range. In human and other biological samples ranges depend on substance toxicity, dosage, body weight, species, individual tissues, and pharmacokinetic or toxicokinetic properties. As concerns calibration and analytical limits, the statistically based German DIN 32645 [73] states that the midpoint of the calibration should be near the statistical mean of the concentrations to be tested. Five ideally equidistant calibrator levels should be chosen. With respect to both measurement precision and detection limit, for trace analysis in forensic toxicology the calibrator concentration should be shifted towards the lowest levels. With use of a computer program and statistical functions as described in the DIN, the calculation of GC/MS-SIM calibration data gained by peak by integration during frequent trace determinations of tetrahydrocannabinol in human serum in actual cases has shown that four of these calibrator levels may

Aspects of quality assurance in forensic toxicology

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be chosen at 15, 35, 50, and 75% of a maximum concentration of 10 mg/L [74]. Additionally, higher concentrations in a control calibrator can be analyzed separately. 23.2.2.9 Limits of detection, quantitation, and identification limits Interrelated critical limits of a quantitative analytical method need to be determined during validation. The limit of detection as well as the LOQ are fundamental data to document assay performance. In forensic toxicology, the detection of a substance often must include its identification, especially when the presence of a substance is in question. Regarding the determination of the limit of detection, empirical and statistical approaches exist. Empirically, the limit of detection of a method was assessed by measuring decreasing concentrations of a single analyte in order to establish the lowest concentration that can be detected with an acceptable response. According to the definition of the International Federation of Clinical Chemistry [75], the detection limit is ‘‘the smallest single result which, with a stated probability of (commonly) 95%, can be distinguished from a suitable blank.’’ A similar definition was given by the International Union of Pure and Applied Chemistry [76]. For practical reasons and in order to certify new GC/MS instruments for analysis of illicit drugs in urine, the limits of detection (LOD) were defined as ‘‘the lowest concentration of drug that a laboratory can detect in a specimen with forensic certainty at a minimum of 85% of the time.’’ Overall batch acceptance criteria were as follows: acceptable quantitation of control material within 20% of the target; chromatography (peak shape, symmetry, integration, peak, and baseline resolution); retention time within 0.1% of the extracted reference compound; mass ion ratio within 720% of the extracted item; and individual specimen. The acceptance criteria excluded quantitation requirements in the individual specimen. According to the German DIN 32365 standard, the limit of detection of an analytical method can be calculated daily using calibration data. This approach assumes a normal (‘‘Gaussian’’) frequency distribution of readings at a definite concentration. The terms a and b represent the error probability levels to (falsely) classify an analyte as present or not present, respectively. Each quantitative method has a critical value representing a concentration at which the probability of an a error reading in a sample not containing the analyte is 0.01. In contrast to the above approaches, DIN defines two LOD. The lower is defined as a concentration at which the analyte is classified as present or not present with equal probability. Consequently, this concentration is calculated from calibration data taking b ¼ 0.5 and a ¼ 0.01. Depending on the analytical problem, other values of b and a can be chosen. That is why the statistical solution provides a second and higher LOD. As defined by the German DIN 32365, this limit refers to the smallest quantity that can be detected at a probability level of 1-b (detection certainty of 95% or 99% if b ¼ 0.05 or 0.01, respectively). This concentration threshold has higher coverage. The certainty to detect an analyte is 99%, meaning that the analyte is regularly detected when present at this concentration. References pp. 814–819

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23.2.2.10 GC/MS substance identification using selected ion monitoring The LOQ refers to the minimum quantity being determined with both a defined probability level (e.g., bp0.01) and an acceptable relative uncertainty. In the DIN approach, both the lower LOD and the critical value are of theoretical interest and no values are suitable for the customer’s use: Only the LOD has high detection certainty. However, it refers to a single signal as detected using MS/ MS-techniques or high-resolution MS. For methods using low-quadrupole mass resolution, LOD is not a suitable parameter to guarantee substance identification. The appropriate detection of three or even four characteristic ions is required to identify an analyte in such quantitative GC/MS analysis. For GC/MS-SIM quantitation, usually, the most intensive signal is chosen as the target ion. Regarding substances with less than four intensive ions, SIM may not be sufficient for identification. The corresponding identification power depends on the availability of qualifiers with suitable abundance. In order to identify an analyte, the least intensive of three qualifiers must be recorded at least at a 90% detection certainty level, including suitable mass ion ratios (within 720% deviation of theoretical ratio). Regarding the calculation of the LOD (i.e., certainty to detect and identify6), the calibration data (peak areas) of least intense ion being monitored need to be used. In contrast, the calculation of the LOQ can be based on the most intensive ion. In pharmacokinetic studies, for instance, the detection and even quantitation of a known substance may be needed in terminal elimination at concentrations at which the substance can no longer be identified in a forensic sense, e.g., assuming monitoring of the 100% intensive target ion (and far less intensive qualifiers). Consequently, for forensic purposes, the LOQ must not be chosen lower than the limit at which identification is possible. 23.2.2.11 Cut-offs In contrast to clear characterization of LOD or quantitation of qualitative and quantitative methods, the term ‘‘cut-off’’ is used more or less arbitrarily in screening procedures using immunoassays. The meaning of a cut-off is mainly a decision limit defined by a nominally given concentration level of the analyte used for calibration. The analyte, however, may not necessarily be present in real samples (e.g., cannabinoids and benzodiazepines in urine, where instead of parent compounds their glucuronides represent the target analyte). However, the analytical meaning of a cut-off is different from a diagnostic meaning. It may be useful to avoid false-positive readings in screening tests at both a concentration level as low as possible (optimum diagnostic sensitivity) and at a high probability level of analytical trueness (commonly at least of 95%). An analytical cut-off often includes possible readings of cross-reactions within the substance group and should exclude responses of material not representing any analytical target. Any false sources of response in a test result usually cannot be recognized or 6

There is semantic difficulty in translating the limit terms as used in the German DIN standard.

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distinguished from correct ones. By using cut-offs, however, testing should exclude analytical false-positives as far as possible. In contrast, diagnostic cut-offs may be needed to: 1. separate collectives, e.g., healthy and ill persons; 2. avoid misleading interpretation of results, e.g., regarding the origin of illicit drug metabolite excretions in urine; 3. allow a corresponding application of appropriate confirmation methods; 4. have comparable testing preconditions regarding regulatory, authority, and legal decisions; and 5. have comparable testing preconditions in proficiency testing. Regarding the above topics for workplace testing, national and international proposals for cut-offs exist for immunoassay screening and for confirmation of illicit drugs and their metabolites in urine, as shown in Tables 23.2 and 23.3. NIDA cut-offs were the first ones used for abuse testing of drugs in urine [77]. The U.S. Department of Health and Human Services and its Substance Abuse and Mental Health Administration (SAMHSA) have defined federal standards for urine drug testing. On the Internet [78], SAMHSA’s Workplace Resource Centre gives access to cut-off concentrations as specified in the Mandatory Guidelines for Federal Workplace Drug Testing Programs that include immunoassays and GC/MS confirmation. These cut-offs (2001 version) for immunoassays and GC/MS confirmation are presented in Table 23.4. In order to yield comparable results as well as unavoidable measurement uncertainty, no information is presented about minimum accuracy and precision performance needed at a level of 15 ng/mL, as these were defined for cannabinoids earlier. In Germany, no mandatory regulations have been enacted or accepted to date. Nevertheless, for abstinence testing of drugs of abuse, the cut-off regulation is useful TABLE 23.4 CONFIRMATORY TEST LEVELS FOR WORKPLACE TESTING OF SUBSTANCE ABUSE AND QUANTITATIVE CONFIRMATION AS PUBLISHED BY THE US DEPARTMENT OF HEALTH AND HUMAN SERVICES Initial IA Test Levels ng/mL (Equivalents of Reference Substance) Marijuana metabolites

50

Opiate metabolites

300

Cocaine metabolites Phencyclidine Amphetamines

300 25 1000

GC/MS Confirmatory Test Delta-9-tetrahydro-canna-binol-9carboxylic acid Morphine Codeine Benzoylecgonine Phencyclidine Amphetamine Methamphetamine, and coincidend with amphetamine

Test Levels (ng/mL) 15 300 300 150 25 500 500 200

References pp. 814–819

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788

TABLE 23.5 URINE THRESHOLD CONCENTRATIONS AS PROPOSED IN CLINICAL AND EU WORKPLACE TESTING ACCORDING TO [81] AND [105]

Analyte or Group

KKGT Dutch Health Care Cut-off

UKNEAS – Clinical Thresholds (mg/L)

EU – Workplace Testing Threshold (mg/L)

1000

300

500 300

– 50

500 500

– 300

1000 1000 1000 1000 1000 500 15 3000 300 300 500 500

200 200 200 200 200 – 15 150

Screening tests Amphetamine group Barbiturate group Cannabinoide group Benzodiazepine group Opiate group Methadone Single analytes Amphetamine Methamphetamine Methylendioxyamphetamine Methylendioxymethamphetamine Methylendioxyethylamphetamine Specific barbiturate Delta-9-THC-carboxylic acid Cocaine Benzoylecgonine Specific benzodiazepine Methadone Morphine Dihydrocodeine Buprenorphine Phencyclidine Lysergic acid diethylamide

(Reference substance 4) 1000 (Methylamphetamine) 300 (Secobarbital) 100 (11-nor-THC delta 9 COOH) 300 (Morphine) 300 (Oxazapam) 300 (Methadone)

1000 1 25 5

200 after splitting of conjugates – – –

and recommended; however, it should include monitoring of the performance of both test kits and testing Tables 23.4 and 23.5. In 1998, a regulation in Germany within the Road Traffic Act has been promulgated. According to an appendix to y24a II of the Road Traffic Act, the presence of illicit drugs in the blood of drivers is regarded as a violation. Therefore, analytical threshold limit values for GC/MS analysis of illicit drugs (parent compounds) were proposed by an expert group in order to combine the analytical and minimum risk assessment aspects in regard to drivers and drug abuse [79]. These concentrations were halved in 2001 [80]. Accordingly, in 2004, the German Federal Constitutional Court judged a limit of 1 ng THC/mL serum to be the minimum concentration at which a driver can operate a vehicle in spite of being impaired. This limit is said to include a safety margin of 100% relative to a limit of detection of 0.5 ng THC/mL serum. Some questions are still open regarding the introduction, size, and role of measurement

Aspects of quality assurance in forensic toxicology

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uncertainty at this concentration and about a single value measured with a correspondingly validated high-precision method. As clinical testing aims at appropriate therapeutic treatment, performance criteria other than forensic testing may be suitable. In emergency toxicology and cases of drug overdose, rapid, low-cost, and possibly less sensitive methods may be chosen. In specific cases, the identification of substances and trace analysis may be less important than the exclusion of their presence. The usefulness of higher clinical cut-offs than those for forensic or EU workplace testing has been reported [81] (Table 23.5). Clinical thresholds reflect the use of less sensitive methods for the confirmation of immunoassay results by clinical laboratories. For clinical testing, some single analyte thresholds for parent compounds were chosen that were higher than screening thresholds, indicating possibly nonrealistic testing conditions, e.g., for unchanged cocaine in high concentration in relation to its major metabolites. Immunoassays usually show cross-reaction to several known and possibly unknown metabolites. Some tests are not capable of detecting the parent compound when applied to human body fluids or tissues, which is why confirmatory testing in urine must usually detect concentrations lower than the cut-offs of the immunoassays used. As a consequence, forensic or workplace testing laboratories must not refer to clinical quality assessment. As some sites may provide both clinical and workplace testing, an optional selection of cut-offs would be in contrast to appropriate proficiency testing. As a consequence of the lowering of detection limits by more and more powerful analytic instrumentation, future forensic work concerning cut-offs must concentrate on the definition of thresholds for substance traces or substance metabolites in order to discriminate exogenous substances that are taken or given from those that originate from natural or systemic sources or from small substance doses taken up unknowingly. One known example is the systemic excretion of g-hydroxy butyric acid and its discrimination from traces excreted after criminal poisoning [82–85]. Another example illustrates the problem: While painting, a painter who is under alcohol abstinence control may inhale ethyl acetate. Does this compound produce ethyl glucuronide, which tests as an alcohol marker throughout the world? If excreted in urine, can ethyl glucuronide be present in measurable concentration and, if so, at which maximum level? 23.2.2.12 Immunochromatographic tests: lateral flow assay 23.2.2.12.1 Easy Use of LFA? For many trained and untrained users immunochromatographic lateral flow assay (LFA) technology is a welcomed alternative to collection, transport of urine samples, and subsequent quality-controlled laboratory analysis. Lateral flow immunochromatographic testing and targeting of the more commonly abused substances became a multimillion-dollar business; however, as a designated qualitative test LFA may entail some losses of quality. LFA formats range from dipsticks or pipette strip tests to multiple test cards or plastic cassettes to cups. The amount of sample needed for testing ranges from a few drops of urine to a minimum amount of 20–30 mL as dictated by sample collection vessels or cup dimensions. To screen for the presence of References pp. 814–819

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a single drug or drugs of an abuse class or to detect several groups of drugs such as amphetamines, benzodiazepines, cannabinoids, cocaine, and opiates, all known devices use the immunoassay principle. The devices utilize several antibody reaction principles such as agglutination reactions, fluorescence, or chromogenic (gold) labeling of antibodies and labeled drug conjugates. It is common to distinguish them according to their visible response, usually a test line, which is generated by a drug present at or above the designated nominal concentration threshold. In laboratory testing, the chosen nominal cut-off (numerical reading) combines the exclusion of minimum (potentially unspecific) test reactions with diagnostic group differentiation needs (e.g., passive consumption of cannabis). In contrast, lateral flow test cut-offs are numbers greatly differing from laboratory test cut-offs with calibrated readings. In reality, in corresponding tests, LFA cut-off numbers mean a generally precisely reached threshold saturation of a labeled antibody by present drug molecules fully suppressing the antibody’s binding at the test line. (Fig. 23.2) Similar to drug radioimmunoassay with low-molecular-weight compounds (haptens) in which the loss of competitive antibody binding of a radioactive tracer can be counted in relation to the concentration of the hapten present, the absence of a line or color means the test result is positive. At the lateral flow cut-off, the test reaction on the strip should have developed close to its maximum. Therefore, even the development of a less intensive line or color indicates that the drug is present (below the threshold). According to saturation analysis principles related to an antibody’s affinity, the lateral flow immune response consists of two steps: a primary (pre)incubation of the

signal intensity%

binding inhibition or colour intenstity of LFA test line 100 90 80 70 60 50 40 30 20 10 0

specify cut off

treshold "cutoff"

1

10

100

1000

concentration [ng/mL] Fig. 23.2. Difference between cut-off numbers in machine-based liquid-phase immunoassays and lateral flow immunochromatography tests: While the conventional understanding of cut-off principles refers to cross-reactivity and specificity issues (shown by +), the lateral flow ‘‘cut-off’’ means test line visibility (disappearance, open circle) at a (technically adjustable) threshold. As can be seen from the flattening shape of the dose–response curve at high binding inhibition, lateral flow visual readings at thresholds (‘‘cut-offs’’) must have relatively high imprecision due to the inborn variability of both binding kinetics of the antibodies and antibody desorption from the analyte contact zone into the chromatographic flow.

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antibody with the target analyte, and a secondary competitive reaction between the unbound (and also the target analyte bound) antibody sites and the haptenprotein conjugate at the test line. Because of their in-line combination, these two steps may merge into each other. This disadvantage may also depend on the still incomplete release of antibody sites from the sampling pad, and the incomplete saturation of antibody binding sites when reaching the hapten-conjugate at the test line. Both the inborn variability of the antibody binding kinetics and the lateral flow kinetics at a relatively high target analyte concentration may give rise to a relatively imprecise calibration curve, hence, visual readings at the designated cut-off. Unlike other qualitative applications, most LFA devices for drugs of abuse give a negative visual sign when the drug of interest is at or above the defined threshold. To avoid quantitative measures, such testing was called qualitative testing. At present, there are only a few devices that indicate the presence of the designated drug with the appearance of a line. The term ‘‘cut-off’’ needs to be considered further in selecting a device, as these devices will impact the number of samples requiring confirmation. Because of the above disadvantage, the statistical likelihood of obtaining a negative result for a sample containing the drug or its metabolites near the cut-off should be defined by the manufacturer. Validation studies during selection and implementation should include testing of the defined cut-off. According to an antibody’s specificity, as with laboratory-based methods, most tests detect drug metabolites instead of the parent drug and often detect other metabolites than those present in the sample. One example is, again, the calibration using 11-nor-delta-9-carboxytetrahydrocannnabinol (which in practice is not present) instead of its acylic glucuronide (the usual major cannabinoid metabolite present in urine). Although urine samples may need to be transferred, most of the steps for lateral flow testing require an operator’s intervention such as a sample application, possible transfer of the sample to another portion of the device, timing of the immune reaction, reading or interpreting the visual endpoint, and recording and documenting the result. Visual endpoint readings clearly depend on the chosen technological approach. Turnaround times seen from the initial sample application to reading of the result are up to 15 min, but they usually take much less than this time. The operator is responsible for all potential manipulations of the sample, the reagent application, timing, interpretation, and recording of the results, including quality control indicators. At times there may be little control of the sample volume applied, depending on the principle: Some dipstick devices use dipping time; cassette tests use small plastic pipettes to introduce the sample. This technique greatly differs from automated immunoassay-based laboratory methods for serum/plasma or urine with primary tube sampling. There may well be a potential need for regulations for the application of immunochromatographic lateral flow (LFA) testing in different forensic, clinical, and non-clinical situations (e.g., at the workplace or for abstinence testing, for testing of drug users in road traffic, for testing of former drug users whose driving licenses have been withdrawn, or for rapid drug testing in emergency situations). References pp. 814–819

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One problem may be that some commercial providers of LFA devices may have a limited background and limited information to correctly train purchasers or users on function, application, and especially careful interpretation of results. Such training should include quality issues as well as well-known device limitations. All users of LFA devices should understand the devices’ limitations, including statistical and analytical sensitivity, specificity, and nomenclature used. Users need to be aware of commonly experienced interferences resulting from the presence of substance-related or unrelated drugs or metabolites that could have an impact on readings of results as well as the interpretation of results. In practice, however, such necessary information is sometimes not available or is incomplete and therefore is rarely or inadequately given. Aimed at a relevant diagnosed population, a careful evaluation needs to be conducted by well-educated test users and needs to take place in those environments where the tests will be used. For instance, roadside testers may check their tests at various low and high outdoor temperatures and also after storage at various automobile interior temperatures. Controlled studies on population and the purposes of testing are rarely published, perhaps because test providers and distributors have limited information about the background knowledge of their customers, the test applicants, and the environments in which the purchasers want to use the tests. Testers need to be aware of any known interferences from reagents, chemicals, and other methods of adulteration or manipulation that could influence the results and interpretation. Procedures need to be adopted within a protocol framework, which should ensure that specimens remain tamper-free. If required, the type of testing chosen should enable the tester to detect manipulation and adulteration of the sample by the donor. According to accreditation principles, all analyses must be subject to quality control and quality assurance, which should encompass a quality system that includes effective training, record keeping, and review. As costs come into play, a legally defensible approach and the recording of data need to be considered, and insufficient evidence for or against specimen stability as a justification for the testing location must be taken into account. Although generally reliable in comparison to automated screening methods for drugs of abuse, immunochromatographic devices do not have sufficient specificity to be used for legal or forensic applications. Results may be subject to legal challenge unless positive results are confirmed by a generally accepted additional method. LFA for drugs of abuse may, under some circumstances, provide limited but adequate information for clinical purposes or intervention. In case of doubt, when a definitive penal or legal action is to be feared, laboratory confirmation is unavoidable. Screening using LFA can only be effective when issues of quality and data recording are adequately addressed. When used by trained laboratory personnel, currently available immunochromatographic test strip devices for drug screening in urine may produce results comparable to liquid-phase and machine-based laboratory screening methods, according to some evidence.

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When used by trained non-laboratory personnel, as is becoming more and more common, outcome and results are much poorer. Increases in testing by experienced laymen run into the risk of a serious loss of quality in drugs of abuse testing. 23.2.2.12.2 Lateral flow tests and alternative matrices For LFA, urine is presently the best established matrix. Cut-off thresholds, interferences, and interactions have been established and studied more in urine than in other matrices. If alternate matrices are to be used for LFA, the antibodies and cutoffs must be optimized with respect to the most abundant parent drug or metabolite in that matrix. [One example is the possible presence of tetrahydrocannabinol (THC) in saliva or oral fluid resulting from contamination by cannabis smoke, but without the presence of original substance or metabolites, which antibodies for urine should target.] Unsatisfactory results for certain highly serum protein bound drugs, especially for THC, benzodiazepines, or opiates detection, are reported using oral fluids for drug screening by LFA. There is a lack of evidence regarding the obvious quantitative and qualitative limitations of oral fluid testing. As routines for sampling at specific sites and different collection methods for oral fluids or sweat definitely exist, procedures need to be standardized. 23.2.2.13 Substance identification and confirmatory tests In forensic toxicology, one important principle is substance identification including confirmation of qualitative and quantitative results by the application of at least two independent methods or specific detection techniques. Identification of a substance can be achieved applying a step-by-step isolation procedure to the material, including extraction and chromatographic or other separation, which allows safe discrimination of the substance of interest and possible interference. After chromatographic separation and, preferably, calculation of retention indices, substance identification is usually safely accomplished by recording appropriate spectroscopic information, such as mass spectral or infrared spectral data, and their comparison to data of a pure reference substance. As the suitable combination of two or more quantitative determination methods and an agreement of results is regarded as a useful tool of objective testing, a valid result cannot be obtained by multiple applications of screening or re-screening tests. There is no gain in validity if specimens initially tested for drug screening using immunoassay techniques are forwarded for a drug immunoassay with different specificity in order to eliminate false-positive or false-negative measurements. A specimen found positive for a substance or class of substances in an initial screening test needs to be confirmed by using a method that ensures substance identification, such as chromatographic or other separation techniques in combination with MS, selected four-ion monitoring, or high-resolution MS and MS/MS techniques. The usefulness of UV-spectra for substance identification depends on the specificity of an individual spectrum. A UV spectrum shows less characteristic

References pp. 814–819

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information of a chemical substance than its IR spectrum (e.g., in the fingerprint range). As the discrimination power in HPLC is less than that in GC [86,87], a careful use of HPLC and more or less nonspecific detection and further confirmation may be required. 23.2.2.14 Accuracy and precision Both accuracy and precision are terms related to error. Strictly speaking, the extent of inaccuracy and imprecision needs to be determined. Assuming that a set of observations is made under the same testing conditions, the arithmetic mean and the standard deviation will provide the information required. While the standard deviation refers to the precision and the agreement of results of a set of replicate measurements among themselves, accuracy means the closeness of an individual result as well as of the arithmetic mean of several results to the true, expected, or accepted value. Two classes of error – systematic error and random error – contribute to the total error of a result. While random error reflects fluctuations in the use of the method, which are unpredictable and unavoidable, systematic errors result from a multiplicity of causes due to poor analytical practice or failure in the application of methods and instruments. Internal quality control measures using control charts as described in the literature [88] is one of the means to identify sources of error. Especially for analyses of large numbers of the same type of samples, and if they cover a sufficient period of time, control charts may provide useful information on accuracy and precision as well as on the occurrence of unexpected analytical trends and a lack of randomness. 23.2.2.15 Internal quality control Internal quality control (IQC) in a forensic toxicology laboratory includes all activities by which conditions and procedures can be controlled in order to warrant reliable and suitable analytical results. IQC needs to concentrate on the quality of processing. Any successful analytical work is based on using approved or validated methods. IQC is to check whether in casework the individual analytical runs met the performance characteristics as determined by method validation. IQC is also to inform about changes. In order to provide appropriate analytical preconditions the analyst must be sure that:
analytical instruments are regularly checked, well maintained, properly calibrated, and adjusted;
instruments for sample preparation or subsampling are clean and trouble-free;
refrigerators and freezers for sample storage or reagents are in acceptable condition;
suitable materials, reagents, and solutions are available and prepared for use;
appropriate control samples, blank samples, and reference material are run in the same way as the case samples;

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procedures are implemented in order to exclude carryover of the analytes and contamination of an individual testing sample; and
staffs comply with operation procedures such as subsampling, sample preparation, analytical methods, and documentation. Regarding IQC, different areas of attention can be distinguished in qualitative and quantitative analysis. 23.2.2.16 Internal quality control of qualitative testing First, the purpose of forensic chemical analysis is to give an answer to the question of whether there is one or more analytes of interest present in a specimen or a dead human body. Besides the sample being representative, in order to be found, the analyte must be present in a sample in a concentration above the limit of detection of the method applied. That is why any quantitative analytical method is basically a quantitative one. Second, the concentration of the analytes needs to be determined in order to know at which level they have been present and to draw plausible conclusions regarding their origin, toxicological action, etc. Accordingly, the performance of a qualitative method can be checked by analyzing one or more samples that do not contain the analytes in order to compare the results to a control sample containing the analytes in a concentration at and little above the limit of detection. Additionally, the application of control samples with two different higher concentration levels is recommended. Control samples containing a highly immunoreactive reference substance are also used for immunoassays (IA) and other related tests. However, IA need to be distinguished from other methods used as qualitative screening tests. IA are indirect methods using a competitive reaction principle which is usually sensitive to more than one substance present if their chemical structures are very closely related. Thus, in a way, IA are specific but not selective methods. IA readings may be subject to possible reaction failure or specific, unspecified cross-reactions and matrix effects. Therefore, especially near the theoretical limit of detection, IA readings usually show relevant imprecision. Consequently, as mentioned, several specific reasons exist as to why cut-offs need to be used (e.g., a 95% confidence limit to avoid a very high rate of false-positive results). In forensic terms and also in general, because of limited specificity, IA cannot be used to identify a single compound and cannot even safely indicate the presence of chemicals structurally related to the reference compound. As for screening tests based on chromatography, like general unknown analysis, both the analytical instrument’s performance of substance detection and the efficiency of the extraction method need to be checked. In GC/MS, usually the limit of detection should refer to the full scan mode and ten most intensive diagnostic ions, respectively, including a signal-to-noise ratio of 3. However, as stated in a review [42], most of the papers referring to chromatographic toxicological analysis and detection limit only contain data on pure substances. Detection limits are only useful if they are measured using spiked biosamples and if the drug itself is the predominant compound in the sample. In many instances, compared to the parent References pp. 814–819

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compounds, the metabolites can be detected at higher concentration and for a longer time. Additionally, commercial chemical analytical companies or the manufacturers of the medical drugs do not necessarily provide many of the drug metabolites. Therefore, a the usual methods of validation can be difficult. The analyst expects that by using his or her method, the presence of various compounds and their metabolites should be indicated or excluded. However, especially regarding different sample quality and sample sites in post-mortem analysis, it seems desirable (1) to validate a method regarding the corresponding LOD in various different body tissues and fluids as well as (2) to provide corresponding control samples covering the numerous toxic drugs and chemicals. Four approaches are possible in order to achieve effective IQC when using an approved method for general unknown analysis: 1. Samples should preferably contain a minimum concentration of both a polar and a less polar water-soluble compound with unfavorable chromatographic peak shape and relatively low total extraction recovery. 2. Frequent participation in a proficiency-testing program including ring tests designed for educational quality assessment (e.g., as it is offered by the GTFCh [89]). 3. As performed for a relevant systematic data collection [41], the duration of elimination of therapeutic doses of pharmaceutical drugs and their metabolites in urine of humans or other species can be monitored in order to investigate the analytical power of the method proposed [90]. 4. The use of blind samples. The final guidelines notice of the Mandatory Guidelines for Federal Workplace Testing in the United States [1] dictates agency responsibilities for the submission of a quantity of blind samples equal to 10% of their regular specimens. These blind samples should include 80% negative samples and 20% positive samples spiked with various drugs of the agency protocol. As was reported for testing laboratories used by U.S. customs, analyte concentration ratios for blind quality assurance specimens were found in acceptable agreement to those found by the reference laboratories of the provider. Except for ‘‘THC,’’ 35 laboratories had routinely achieved a level of performance on complete blind samples, which approached the levelss expected for an open proficiency specimen [91]. 23.2.2.17 Internal quality control of quantitative testing Internal quality control of quantitative testing is effectively performed by using reference samples of a known concentration. For monitoring of the precision of a method there is no need to know exactly the true concentration in a sample. Accuracy and precision can only be checked by the use of a CRM, the true concentration of the analyte of which must be both known and stable. For blood, serum, or urine, such material is difficult to obtain. Testing of spiked lyophilized serum samples in ring tests and consequent testing of their long-term stability has

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been started by the GTFCh ring test scheme in 1998 in order to provide reference sera for illicit drugs and their metabolites for internal quality control. Originally, Shewhart [92] had proposed charts for quality testing of manufactured products. Similar charts were used in clinical chemistry in 1950 [93]. The use of control charts, as recommended and explained in the relevant literature on analytical quality assurance [88,94], is now generally accepted. In order to monitor accuracy and precision of quantitative methods and to discover typical errors that may come up in an analytical laboratory with time, both control samples and CRM should be used, if available. 23.2.2.18 Instrument performance checks Different frequencies of instrument checks are recommended and mainly depend on an instrument’s inherent tendency towards decreasing performance. Proposals for instruments and their checking frequency are given in the literature [88,94]. Important recommendations for the use of GC/MS instruments were first given by Cody et al. [95]. 23.2.2.19 Post-examination phase Usually a laboratory provides a form on which the customer has specified the incoming order according to the laboratory services. Both the order and the samples need to show unequivocal data referring to the customer, date of order, origin and type of sample (clear personal and sampling data), and the investigation requested. The order should include a short case report if necessary. Consequently, the forensic toxicological report must unmistakably refer to the sample, personal, and case data mentioned in the order and give an answer to the questions asked. Only qualified staff members must report. Competent reporting of analytical results gives information about the tests used and their analytical performance characteristics and certainty. The interpretation consists of an analytical and a toxicological part. The analytical part refers to the verification of analytical data with respect to preanalytical conditions, chain of custody documents, methods specification, calibration, analytical limits, decision limits, analytical certainty, quality assurance data, outliers, results other than primarily requested, etc. The toxicological interpretation refers to biological plausibility of results and explains what the results mean with respect to the case history, to time dependence of analytical results with respect to metabolism, taking personal data such as.body weight, age, sex, individual constitution, possible illness, prescribed medication, time and ways of substance incorporation, inter-individual variability of toxic actions, tolerance phenomena, substance interactions, pharmacogenetics, and pharmacokinetics into account. The information must be given in a way that analytical laymen and experts can understand. It must be stressed that the limitations of the tests used , e.g., lack of identification power of immune tests, need to be mentioned to avoid a wrong understanding of the results. For instance, the reporting of positive immune tests for opiates References pp. 814–819

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or cannabinoids in serum or urine without comment may lead to a misunderstanding by analytical laymen, as this result is easily changed by heroin consumption and recent marijuana intoxication, respectively. Nevertheless, the reporting of immunoassay results without any forensic-toxicological or clinical explanation of their meaning is not a rare event. Casework in forensic analysis more than in clinical toxicology includes appropriate documentation of analytical results. Clearly, documentation must provide the original analytical data. The data need to be stored in such a form, which allows duplication of qualitative and quantitative information and conclusions when an analytical expert is asked to examine the data. 23.2.3 External quality control (EQC) External quality control has a bi-directional purpose. Laboratories participating in external quality assessment schemes and proficiency testing need to know and also to show whether their methods are sufficiently applicable to detect a wide range of analytes in toxicological analysis. The need for quality assurance and control was not just a quest of the last decades of the twentieth century. More than 150 years ago, the German chemist Remigius Fresenius complained about the lack of guidelines for quality control: ‘‘Nevertheless, it seems to me as if in this part of the science still much is left to be carried out, both in respect of the assessment of the most secure methods for poison detection,’’ y (it would be helpful) ‘‘if the state authorities would – in form of a standard – stipulate well-tested methods which have been approved for poison detection, and each chemist performing toxicological analysis would be asked to follow these’’ [96]. In the United States, since the late 1940s, the College of American Pathologists (CAP) has conducted interlaboratory comparisons designed to assess the state of the art in clinical laboratory practice [97,98]. Some reports on proficiency testing in forensic toxicology were published in 1976 and 1977 [99–102]. However, in 1985, the need for adequate external quality control over laboratory performance was dramatized in an article referring to studies conducted in the 1970s on laboratories providing drug screen services to federally funded drug treatment programs [103]. Furthermore, the study had focused attention on the general lack of quality control programs. Consequently, several such testing programs for drugs of abuse in urine were initiated. Programs were conducted by the U.S. Department of Defense, Armed Forces Institute of Pathology (both open proficiency-testing samples), and, starting in 1984, by the College of American Pathologists. Since 1980, a urine toxicology program became available from the American Association of Bioanalysts. State programs as conducted by the states of California, Pennsylvania, and New York are providing blind sample testing and open proficiency testing. Other individual and commercial programs also exist [97]. In a feasibility study on proficiency testing in forensic toxicology [104], it was shown that the methods used by the participants satisfied requirements of accuracy, although they were tested in Europe [105].

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23.2.3.1 The relation between clinical drug of abuse testing and forensic or clinical– toxicological proficiency testing There may be a need to harmonize both the criteria of successful participation in ring tests and the corresponding substance panels tested. Harmonization, however, may easily be upset by the goal of testing and comparing as many European drug testing laboratories as possible. For comprehensive proficiency testing as needed in forensic toxicology, neither limitation to commercial IA-based drug testing with confirmation analysis nor a European centralized survey of the most frequent drug tests (including the corresponding laboratories) will help warrant an effective screening of abused substances. Regarding national legal requirements, different drug-taking behavior in individual countries, and the subsequent necessity for national quality control schemes to exist, it seems advisable not to mix the goals of clinical chemical, workplace, or forensic toxicological proficiency testing. In comparing the purpose of proficiency tests, it must be stated that there are relevant differences between forensic and clinical testing. Diagnostically sensitive, rapid, and cost-effective drug testing in clinical chemistry differs from the need for substance identification in forensic testing and for legal issues. As the detection window is longer than in blood and the concentrations are higher, clinical testing by using commercially available IA test kits for frequently abused drugs is preferably performed in urine. Other clinical proficiency testing is focused on therapeutic drug monitoring (TDM) in serum. Clinical chemical proficiency testing therefore demands a selection of analytical parameters that need to be performed rapidly and often with respect to a definite concentration range. However, ring tests restricted to a set of drugs of abuse will focus more on the performance of commonly used specific tests than on general screening performance. Analytical results often need to be interpreted in order to make it possible for public authorities to make decisions and to judge if further expert knowledge is required. For this reason, forensic proficiency testing needs to focus on laboratories being able to provide reliable screening and identification as well as suitably interpreted results. As an example, the toxic effects of benzodiazepines, their different potencies and dosages, their concentration in blood, the need for substance identification, and the qualitative and quantitative testing of various benzodiazepines, as well as a lack of suitable external quality control in both forensic and clinical toxicology [106] have been obvious for a long time. Only recently have further details of the problem been characterized [107,108]. Forensic testing has focused on the detection and identification of poisons and intoxicants whose presence was previously unknown. In addition to urinalysis, in forensic toxicological analysis quantitative substance determination in blood (and also other tissues) is frequently performed. Forensic toxicological PT schemes should conduct the corresponding ring tests. Since IA is used for screening of substance groups only, but not for substance identification, in forensic toxicological PT no IA results need to be reported.

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In addition, forensic PT schemes should organize the collaborative studies necessary in order to:
investigate performance criteria for various methods and standards fitting with the requirements as needed in qualitative and quantitative analysis [109] for objective testing including trace determination; and
test the use of reference material. Besides the question of which method used by laboratories is best for identifying a particular chemical compound, both forensic toxicological analysis and proficiency testing must refer to frequently and also rarely observed analytes. The panel of substance needs to be open to newly discovered illicit and licit drugs which must first be qualitatively detected and, if necessary, quantitatively determined to pass the ring test. Additionally, the identification of metabolites rather than of parent compounds may cause identification difficulties. The presence of a new drug, such as N-methyl-3,4-methylendioxyphenyl2-butanamine (MBDB,) in a urine ring test has caused disastrous analytical results including numerous false-positive ‘‘identifications’’ of MBDB when amphetamines were present [110]. In a clinical context, the question posed is generally not whether the subject has abused drugs at some unspecified time in the past, but more typically whether a non-prescribed compound was taken within the past 24–72 h? Therefore, diagnostic sensitivity and specificity of available analytical techniques are factors that need to be taken into account in a setting of the desirable standards of the performance of illicit and licit drug assays. In regard to both these definitions, the techniques used for the detection of drug abuse in urine, external quality assessment in the UK has shown in 1991 [111] that as far as clinical understanding of sensitivity is concerned, the use of confirmation methods such as chromatography had inadequate diagnostic sensitivity. In a report on the same scheme, gas chromatography with mass spectroscopy as used in the participating laboratories, was reported to be significantly less sensitive than other techniques for the detection of 0.5 mg/L of benzoylecgonine (71%) and 1.5 mg/L of morphine (88%). Interestingly, in contrast, high-performance liquid chromatography turned out to be the most (100%) sensitive for amphetamine. Regarding diagnostic sensitivity, commercially available immunoassays for drugs of abuse performed well when operating above their specified cut-off concentrations. The authors state that threshold concentrations are therefore greater than those of the field of employee testing [112], though the consequences of analytical errors are no less serious in the damage they may cause to the patient. The data demonstrated that the laboratories did not always achieve the goals of easily confirming the presence of an analyte by the use of two techniques based on different physical chemical principles. 23.2.3.2 Choice of proficiency-testing schemes As laboratories may change to the most convenient scheme, proficiency testing should cover the services offered. However, due to increasing competition and for

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economic reasons, various laboratories may offer their toxicological analytical services, primarily designed for clinical purposes, to non-professional customers with the belief that the results will be sufficient for forensic evidence. Laboratories without suitable (expensive) confirmation facilities probably frequently report drug abuse positives exclusively based on the use of IA techniques. Consequently, IA confirmation is performed by more qualified laboratories and on request only. Thus, voluntary choice of the scheme and participation of laboratories with respect to favorable performance criteria and well-tailored testing schemes may create problems regarding quality and use of results and forensic expertise. As a distinction is drawn between routine clinical drug abuse testing and forensictoxicological analytical work, the same discrimination should also be made for proficiency testing. In a forensic as well as in a clinical toxicological context, a clear distinction needs to be made between educational ring tests helping laboratories to improve their methods and other tests aimed at laboratory certification. As mentioned above, approved case-based proficiency testing for clinical toxicology laboratories was successfully introduced in the Netherlands. In this scheme, reasonable toxicological testing was supported by a case history. 23.2.3.3 GTFCh’s forensic toxicological proficiency-testing scheme In the Federal Republic of Germany, method comparison and toxicological ring tests for various drugs were introduced in 1969 [113]. Consequently, ring tests were performed in 1970–1975 [114–118] and continued after a gap of several years [119]. Parallel to those, ring tests for the detection of both lead and thallium were organized [120,121]. Since 1985, ten ring tests were organized by the committee ‘‘Qualita¨tskontrolle’’ of the GTFCh, which referred to opiates, splitting of their glucuronides, benzodiazepines, and hypnotics in solution, urine, and serum [122]. Since 1982, ring tests for blood alcohol determination have been introduced to the German Society of Clinical Chemistry and Biochemistry (DGKC). As no other PT scheme was available, forensic laboratories also participated in these ring tests. Unfortunately, they did not conform to forensic requirements. According to (recently updated) guidelines for forensic blood alcohol determination [123], the mean of four independent values must be reported by forensic laboratories to which the blood samples are submitted. Both the values of two separate determinations using, e.g., head-space gas chromatography and two separate determinations using enzymatic ADH-method need to be within a precision range of 0.1 g/kg below a BAC of 1 g/kg and within 75% of the mean (above 1 g/kg). As the scheme is designed for clinical alcohol determination, it refers to the accuracy of a single value but not to the precision of four values as required in forensic blood alcohol determination. As a consequence, based on internationally harmonized protocol [124], the GTFCh organized a forensic blood alcohol proficiency-testing program in 1995 and, at the same time, qualitative and quantitative proficiency testing of drugs of abuse in blood and serum. References pp. 814–819

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The introduction of proficiency testing of drugs of abuse in blood and serum became necessary because of a new paragraph in the German Road Traffic Act (y24a II StVG), at present referring to illicit drugs and driving. The GTFCh proficiencytesting program is organized according to the above reflections and offers ring tests in the following areas:







illicit drugs in blood (serum); benzodiazepines in blood (serum); ethanol in serum and whole blood; substance identification in urine; markers of alcoholism; and qualitative general unknown analysis (in combination with a brief case history).

Before the program was started, collaborative studies on THC trace determinations were performed [125,126] followed by the introduction of quantitative ring tests [127]. Supported by the German Ministry of Transportation and the Bundesanstalt fu¨r StraXenwesen (BASt), the program has given rise to a significant increase of performance with respect to interlaboratory comparability of quantitative results which are just above the detection limit. In Fig. 23.3, the results of the first round-robin testing and the 10th testing round are compared. Since then, a regularly used external analytical quality control scheme has been established, as scientifically conducted by the GTFCh and technically achieved by Arvecon GmbH [128]. Figure 23.4 shows the year 2005 performance of GCMS determination for cannabinoids in serum using deuterated standards of 64 participating laboratories seen in the ring tests of the GTFCh. The data show that:
the continued use in daily casework and proficiency testing led to a change of the methods used in the beginning;
the variability of the concentration measurements has improved; and
using GC/MS and deuterated internal standards, the coefficients of variation can nowadays be less than in 1982 [129]. This underlines both the necessity and the effectiveness of proficiency testing. Clearly such proficiency testing must refer to the threshold limit value concept as preferred by the German legislation and jurisdiction and to acceptable practical

Fig. 23.3. The comparison of (A) the 1st ring test (1995) and (B) the 11th ring test of GTFCh-EQA-scheme of D-9-tetrahydrocannabinol in serum (1998) shows impressive improvement of the participating laboratories due to the introduction of external quality control. 1st ring test: reference labs mean ¼ 3.77 7 1.04 (SD), coefficient of variation ¼ 0.27, participants mean ¼ 9.21 mg THC/L serum 7 9.13 (SD). 11th ring test: consensus mean ¼ 2.6 mg THC/L serum 7 0.5 (SD), coefficient of variation ¼ 0.19. The upper bar charts show the values determined; the lower bar charts refer to the z-score ( ¼ (measured value  mean)/target standard deviation). A z-score of two or less means an acceptable or better result. The preliminary ‘‘educational’’ testing did show not only some false high IA results due to the metabolite concentration present, but also some questionable GC/MS results. (No use of a standard method requested in both ring tests.)

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A) THC ring test 1/95 (57 labs, 44 of which used GC/MS, and 13 IA) 25 Meßwerte der Teilnehmer

µg THC / L

20 GC/MS

IA

15 10 5 0 8 Abweichung der Meßwerte vom Sollwert (Z-Score)

Z-Score [σ]

6 4 2 0 -2 -4 B) THC ring test 3/98 (40 participants using GC/MS) 5 Meßwerte der Teilnehmer

µg THC / L

4

GC/MS

3 2 1 0 4 Abweichung der Meßwerte vom Sollwert (Z-Score)

Z-Score [σH]

2

0

-2

-4

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Fig. 23.4. Ring test results and performance of MS determination of THC and cannabinoids in serum in the year 2005.

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identification criteria (Appendix to the guidelines of the GTFCh, in preparation [130]). 23.3 QUALITY OF THE OUTCOME For a toxicology laboratory, outcome is measured not only by successful casework and testifying in lawsuits to discover the scientific truth, but also by progress in scientific research and development of the scientific discipline. Scientific outcome of an organization has substantial relevance with respect to the scientific reputation of an institution. Cooperation between institutions, their influence on legal procedures, or even legislation on a national level need to be considered. Besides a scientist’s efforts, the working processes chosen, and research areas, scientific outcomes also depends on policies and on given internal and external structures of a laboratory’s organization. The evaluation of the outcome may become difficult due to the elements of quality which can be a mixture of various objective but also some subjective elements. More simply, outcome can be regarded in terms of business results. 23.3.1 Toxicological analytical services and business results Business results have an important influence on the satisfaction of the customer. The quality of a product is usually judged by the customer in terms of its performance and cost. In order to have proper business results, short-term and long-term business strategies need to be developed, which are not within the scope of this article. Nevertheless, business strategies need to involve cost-effective organizing of both analytical work and quality. Quality management brings about visible and invisible costs. Therefore, administrative instruments will be needed by which quality improvement can be observed and the costs of quality deficits and the cost-effectiveness of their prevention can be determined and lowered to a minimum. If quality-oriented formal procedures are installed in small laboratories in order to apply for future accreditation, additional personnel and increased paperwork and possibly the help of a consulting company need to be considered. Accreditation costs are relatively high; however, working without being accredited may become a major disadvantage with respect to business competition with potentially equivalent providers. Therefore, economical accreditation and quality costs are important for an acceptable business result. Besides internal efforts to achieve objective analytical quality, it must be remembered that customers compare the service of different providers. Regarding a forensic analytical laboratory in terms of a service company, not only the internal procedures must run satisfactorily. Additional factors play an important role about which individual staff members may be unaware. The nature of individual coworkers often determines whether they can be motivated, are interested in participating in the goals of their organization, and properly understand the decisions of the management. Some key factors are considered essential with respect to the satisfaction of the customer (Table 23.6 and 23.7). References pp. 814–819

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TABLE 23.6 CUT-OFF CONCENTRATIONS AS SPECIFIED IN THE MANDATORY GUIDELINES FOR FEDERAL WORKPLACE DRUG TESTING PROGRAMS Agency: Substance Abuse and Mental Health Services Administration, Date: November 1, 2001 The following cut-off concentrations are used by certified laboratories to test urine specimens collected by Federal agencies and by employers regulated by the Department of Transportation Initial test cut-off concentration (ng/mL) Marijuana metabolites 50 Cocaine metabolites 300 Opiate metabolites 2000 Phencyclidine 25 Amphetamines 1000 Confirmatory test cut-off concentration (ng/mL) Marijuana metabolitea 15 Cocaine metaboliteb 150 Opiates Morphine 2000 Codeine 2000 6-Acetylmorphined 10 Phencyclidine 25 Amphetamines Amphetamine 500 Methamphetaminec 500 a

Delta-9-tetrahydrocannabinol-9-carboxylic acid.

b

Benzoylecgonine. Specimen must also contain amphetamine at a concentration and gt; ¼ 200 ng/mL. d Test for 6-AM when morphine concentration exceeds 2000 ng/mL. c

TABLE 23.7 IMPORTANT KEY FACTORS IN RESPECT OF THE CUSTOMER’S SATISFACTION Key Factor Access Comprehension Communication Competence Politeness Credibility Responsiveness Reliability Keeping of time-limits, meeting of deadlines Security Material environment

Example Easy contact by phone or fax is possible Individual awareness Ready for and capable of correct information Technical ability shown during contact Respectfulness and kindness of the staff Reputation of a lab, personal attitude of coworkers Rapid reaction of the staff in time Accuracy and precision of measurements and values Physical and financial security Facilities, equipment, technical aids

23.3.2 Scientific outcome in forensic toxicology 23.3.2.1 Scientific work Practicing forensic toxicology and performing toxicological analysis is in fact not possible without a strong scientific background. Sensitivity to new views and

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questions arising during casework must be developed. Forensic and clinical casework are both sources and destinations of new solutions tried for in scientific work. Bringing in new approaches and publishing specific casework is a must for the FTs, who need to share their knowledge. That is why a research vision and goal-finding as well as planning and performing relevant studies oriented towards the future needs of forensic toxicology are most important concerning qualified outcome. Like other researchers in natural sciences, FT must focus on important abilities such as:







knowledge of the state of the art; inquiring and knowing scientifically relevant needs of research in his or her subject; developing a working hypothesis; assessing its feasibility; proper and timely applications for grants; developing new analytical methods or tools (of poison or drug detection) and their application in order to improve (forensic-toxicological) data acquisition; and
applying validated and approved analytical methods in order to solve recognized (forensic-toxicological interpretation) problems. Purchasing of expensive analytical tools usually depends on the scientific evaluation of previous work and on peer-reviewed application for grants. Especially when a scientist applies for grants, the fulfillment of both formal and intrinsic scientific quality aspects is indispensable. When independent experts of scientific commissions finally decide about research grants, planned projects will usually be rated by a peer group of leading scientists regarding:


















scientific relevance; innovation; originality; feasibility; scientific qualification of the applicant(s); and previous grants. Criteria for a clear-cut design the study are: the objective(s) of the study are precisely stated; actual state of research in relation to the study; state of the previous investigations carried out; all working steps necessary; details of the way to achieve them; available staff, material, and technical (analytical) devices; allowance to use the basic equipment of the institution; realistic requirement of additional staff materials and devices; realistic schedule; and acceptable relation between objectives, costs, and outcome.

Forensic toxicology units are often associated with institutes of forensic medicine. In concurrence with other scientific subjects, scientific activities in forensic toxicology are evaluated not only in regard to casework, but also in relation to their scientific impact. Researchers, suspicious that scientific work in forensic toxicology is not References pp. 814–819

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financially supported as much as other more currently appreciated scientific subjects, need to remember that the instruments for scientific evaluation are at least congruent with the criteria of good quality. To be supported with equal priority and because of the undisputed need for proper forensic-toxicological investigations, not only the scientific community but also society in general needs to be well informed. With respect to public health and jurisdiction, society should strongly be convinced about our scientific progress and the importance of the goals of forensic toxicology. 23.3.2.2 Impact of scientific work within the subject and in relation to other subjects Along with an increasing reduction of national research funds and scientists competing harder for positions, nowadays finding the means to move forward becomes increasingly difficult. One of the criteria by which the work of scientists eventually can be measured may be found using the ‘‘Science Citation Index’’ (SCI). It has been brought up by one of the pioneers of bibliometric methods for evaluation research performance [131]. Scrutinizing the reference lists for all articles published in thousands of scientific journals led to a huge database. By storing and indexing the bibliographical information it became possible to calculate, document, and compare: 1. the number of times the work of a certain first author of an article is cited; and 2. the number of times a certain journal is cited. By monitoring the productivity of individual scientists (articles per author) over a period of time it is also possible to determine the bibliographical impact of: 1. a particular article (citations per paper); and 2. a first author (citations per author). As a measure of frequency with which the average journal article has been cited, the corresponding Impact Factor (IF) can be regarded as a reflection of a journal’s importance and influence within the scientific community. As a consequence, it seems possible that important work may be overlooked if it is published in a less favored journal. As other modes of scientific impact may exist, this specific kind of interaction in the literature must not be identified with the true influence of the work of a scientist. According to SCI journal citation reports, the IF of a journal is determined by dividing the number of all current citations of source items published in the journal during the previous two years by the number of articles that the journal publishes in these two years. When publishing articles that become highly cited, a journal will get a high IF. Publication of articles rarely or never cited will result in a low IF. In fact, however, most scientific articles are cited only once (possibly self-citations) or are not cited at all. Some scientists complained that in spite of some obvious deficiency, the IF has been unintelligibly picked as a scientometric measure at German universities [132]. Others drew a negative picture regarding the IF and exposed problems of the IF including its role in possibly marking a gloomy future for young scientists in less broad subjects

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[133]. As the IF is extracted from journal citation reports, the base of calculation was thought to be inappropriate. The uncritical uses of citation analysis to compare the importance of scientific journals or to judge research significance have been frowned upon by those who prefer traditional peer assessment [134]. The use and misuse of journal impact factors for assessing the work of individual scientists and their professional standing and esteem among their peers has been much debated in popular journals and weekly magazines [135–137]. One point of criticism was that it is the editorial board of a private Institute of Scientific Information that, without entering into criteria, decides on journals to be taken as source journals, the citations of which are used for calculation purposes [132]. However, it seems that most peer-reviewed journals are included in the database. Other problems seen are outlined below:
Possible frequent self-citation or alleged citation circles in source journals as well as shallow or limited literature search might have decisive influence on the data used for the index.
Favouring of frequent negative citations.
Non-English languages and non-Latin alphabets having relevant disadvantage, and consequently, possibly rapid scientific development in such countries may not be recognized in Anglo-American countries.
Articles aiming at professional education are necessarily published in a national language; however, they will not be cited as those to whom they are addressed will not publish in their part.
Specific subjects like forensic toxicology will not reach IF in their journals as high as those of the basic and substantial scientific subjects that lead the hit list.
The time during which citations are registered is two years. Thus, short half-lives of information and predominantly the most favorite and broad publication organs are supported. (It was complained that in 1995 out of 10 of the mostly cited relevant medical journals, only two were among those with the highest IF. In contrast, the impact winner had a very low number of citations.)
The quality of a reference list may be a problem of the IF. (How many authors do really read the original publications and how many of them read results of literature searches only or copy the reference lists of others to save time and efforts? How many of them bring in a complete reference list after a comprehensive studying of the literature?) In the final analysis [138], impact was shown to reflect the ability of journals and editors to attract the best papers available. However, the IF gives limited information about the relevance of a journal in its own discipline. Forensic science and forensic toxicology are applied sciences. In general, interest in forensic toxicology is attracted only by a relatively small number of scientists. Compared to basic research subjects, there are only a few corresponding forensic toxicology departments at European universities with appropriate permanent research positions including undergraduate and postgraduate education. This results in fewer papers and lower frequency of citation. Furthermore, forensic scientists are usually employed at government laboratories that have different qualification rules and, to some extent, References pp. 814–819

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these organizations tend to discourage open publication research. This is another reason why forensic science journals have low impact factors [139]. The present climate of performance evaluation, quality assessment, and competitiveness will hopefully help to make scientists in government and university laboratories more inclined not only to publish their work but also to publish in peer-reviewed journals. However, prolific authorship obviously does not carry as much weight for recognition and esteem in all fields of forensic science as it does in academic medicine, where with similar effort leading academic positions can be reached. In order to judge the relative importance of different scientific journals, misleading information will be obtained when simply comparing the size of their IF. As shown in [140], by information also gleaned from SCI, a scope-adjusted impact factor can be obtained by dividing the conventional IF for a particular journal by the number of citing journals and multiplying by 1000. A calculation including original and new ranking of the scope-adjusted impact factor of 11 journals of interest to forensic scientists is given in [139]. Some heads of departments encouraged young scientists to publish their work in impact journals above a threshold of 2.0 in order to virtually boost its impact. Interestingly, none of the forensic journals met this demand [141]. However, some authors may have experienced that the IF of forensic science journals may change unexpectedly according to publication and consequent citation fluctuations [139,141]. For assessing the track records and the quality of published work, instead of counting the sum and average of IF of journals in which an author has published, the number of citations to individual articles should be investigated. This exercise, however, would entail much more effort [142,143]. In addition to this, for adjusting IF and citation counts, there will be the problematical handling of names and numbers of coauthors contributing to articles. Perhaps the ordering of the names of multiauthor articles should be considered as well [144,145]. Whatever the pros and cons of citation counting may be, there is no need to discredit an objective method of evaluating the scientific literature that can be one important pillar of scientific quality assessment [139,141,146]. The use of the IF in this respect presumes the knowledge of its rules, the limit, and the limitations. Uncritically using the IF like scientific currency should not be accepted, as it would be a non-scientific onset leading to ignoring of the competence at universities in favour of a pseudo-objective measure. All in all, a standardization is needed, particularly within a scientific subject, considering peer-reviewed publications in national journals as well. Some research results may need a long time until their significance is fully understood. As the interest in a particular work may increase if its usefulness has been recognized from a changing point of view, the relevance of scientific work may be disregarded at first. Therefore, the IF should not be used exclusively as a unique parameter indicating the research activities of scientists, their institution, and their quality. In contrast, the SCI may by helpful as a contribution to any careful individual technical examination. Besides being always justified, evaluation of forensic toxicology facilities needs to be rated from a distinguished point of view when compared to other medical subjects. Like legal medicine, forensic toxicology has many elements of an interdisciplinary

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subject. Forensic toxicology is related to a sequence of other subjects such as forensic pathology, clinical and emergency toxicology, analytical chemistry, pharmacology, clinical chemistry, clinical pharmacology, pharmaceutical science, environmental chemistry, and also criminal and behavioral science. Therefore, the impact of forensic toxicological scientific work within these subjects needs to be increased and weighted. The effort of scientists to strengthen their positions needs to focus on sound projects attracting the attention of the scientific community. 23.3.2.3 The impact of forensic toxicology on society The improvement of analytical instruments and laboratory methods as a means of evidence in cases of violation or in criminal cases and procedures has important relevance in a society. In this context, forensic toxicology services contribute to public health and to risk prevention with respect to crime fighting, crime investigation, traffic accidents, drug addiction, etc. Some national regulations, specific problems of legislation, jurisdiction, and police activities may be based on the availability and proper use of valid chemical-analytical testing methods. Costs and benefit always need to be considered. In Germany, for example, according to the code of criminal procedures, drawing and analyzing a blood sample in addition to urine analysis is generally accepted. Consequently, results of blood analyses serve as a regular means of evidence in criminal procedures, e.g., concerning the various time-dependent states of drunkenness and inebriation evoked by drugs, as are euphoria, loss of inhibition and self-control, intoxication, withdrawal, symptoms, dysphoria, and aggressiveness. Assuming a definite blood alcohol concentration–effect relationship and road accident risk assessment, in 1990 the German Supreme Court proclaimed (lowered) the scientifically based threshold limit value of the BAC as 1.1 (g/kg). As absolute driving inability is assumed [147] at or above this concentration, drivers are penalized including a time-limited withdrawal of their driver’s license. In contrast, driving and BAC values above 0.5 g/kg is a violation which entails fines, and – above 0.8 g/kg – a limited driving prohibition. Following requests from the police, in spite of obvious biological disagreement between blood alcohol and breath alcohol values [148], in 1998 the German government ignored these differences and introduced a regulation in which the measurements of blood alcohol or breath alcohol concentrations entails identical legal consequences [149]. In spite of this, the regulation was consequently adopted in the jurisdiction. In 2007, a ‘‘zero’’ alcohol tolerance regulation was introduced for young drivers. Accordingly, a zero BAC of 0.2 % has been proposed including a basic level of 0.1% and a safety margin of 100%. As can be seen from preliminary ring test results (Fig. 23.5), this safety margin can be regarded sufficient: When a spike value of 0.1 g ethanol/liter serum was distributed, 94 participating laboratories clearly confirmed the spike value with both the GC-head space method and the enzymatic ADH-method (consensus value of 0.1 g/L), and did not report any level above 0.15 g ethanol/liter serum. For illicit drugs, up to now, no concentration–effect relationship comparable to ethanol can be deduced. Satisfactory toxicological models aiming at such a References pp. 814–819

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Fig. 23.5. Results of the 4th blood alcohol ring test in 2006 using serum spiked with 0.1 g ethanol/liter serum.

relationship could not be developed. Consequently, no similar threshold values could be determined. Alternatively, for blood ethanol concentration in drivers above 0.8 g/kg, the mere presence of illicit drugs in the blood of drivers is now a violation entailing fines and limited driving prohibition; however, no immediate withdrawal of the driving license takes place. Analytical Threshold Limit Values were proposed by relevant scientific societies, reflecting both the analytical identification of the parent compounds and acute risks caused by the consumption of illicit drugs [79]. An appropriate metabolite, e.g., benzoylecgonine (cocaine) or morphine (heroin), needs to be determined if the parent compound is not stable enough in an unpreserved blood sample. As the determination of an inactive metabolite like benzoylecgonine would not be consistent with intentions of the y24a StVG, an Analytical Threshold Limit Value considerably higher than an acceptable limit of detection was proposed. With respect to the biological plausibility of the analytical result, however, relevant metabolites generally need to be determined in addition to the parent or primary compound, respectively. As threshold limit values may have some cut-off function and are not identical with analytical detection limits, it remains to be seen if they will be accepted in jurisdiction. Therefore, analytical methods for the determination of

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alcohol, illicit drugs, as well as markers of alcoholism in blood (serum) need to be validated according to specific standards [150] to meet scientific and legal requirements. Improving evidence and the interpretation of results presumes that the human metabolic, toxicokinetic, and toxicodynamic background must be investigated and sometimes reinvestigated for new and old substances. Forensic toxicological laboratories help to prepare both scientifically based legislation and jurisdiction by successful implementation of new substance identification techniques and investigation of the metabolic fate of toxic chemicals and intoxicants in humans under various consuming conditions, drugging, and poisoning. This has considerable relevance with respect to public health.

23.4 CONCLUSIONS Forensic toxicology is a science in which – like other sciences – providing quality is more than developing or adopting procedures and analytical methods that merely need to be performed according to international standards and guidelines or technical requirements of objective testing. Accreditation is important. It requires a great deal of energy and expense but does not, however, guarantee all of the quality levels needed. The relation of costs to benefits of laboratory accreditation needs to be carefully considered. Nevertheless, the conformity of a laboratory with accepted quality and management structures will be required in future. Forensic toxicologists take care of the science of poison detection in human organs and body fluids. Our scientific research means acquiring knowledge to develop and improve forensic toxicological tools and better evidence. What scientists think today will be the reality of the future. Therefore, a careful development and selection of new appropriate methods and instrumentation as well as their suitable application is one major crucial point regarding the claims and laboratory performance in forensic toxicology. Two kinds of approaches, methods, and corresponding equipment need to be further developed, validated, if possible, and maintained regarding open tasks to be carried out: 1. Approved general unknown analysis and selective screening methods. 2. Various quantitative and confirmation methods need to be applied according to specific requests and in cases in which specific qualitative results were previously obtained by general unknown analysis. There are a practically unlimited number of poisons that may be present in individual cases and under particular circumstances. Therefore, forensic toxicology is a scientific discipline in which permanent efforts to complete and improve the methods of poison detection show its close relation to raising quality. How can the analyst effectively test the power of the methods used in his or her laboratory? The most important means to increase the degree of certainty of analyses aimed at the detection of previously unknown substances and their quantitative determination consist of a References pp. 814–819

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combination of educational ring tests and true proficiency testing. External quality control and ring tests in forensic toxicology need specific guidance because of the multiplicity of the possible analytes and matrices presenting considerable difficulty compared to other areas of testing. Clinical chemical testing and external quality control prefers a selection of analytical parameters which need to be performed rapidly and often with respect to a definite concentration range. Ring tests restricted to a set of drugs of abuse will focus more on the performance of commonly used specific tests than on general substance screening performance. Differences between post-mortem sampling and sample taking from living persons need to be considered as well as the corresponding interpretation of results. Forensic toxicological analytical results therefore need to be carefully interpreted to make it possible for public authorities to make decisions and investigate whether further expert knowledge is needed. The difficult, often time-consuming scientific analytical tasks in forensic toxicology and casework should not be separated from drug of abuse testing with respect to large-scale testing of a few analytes and some kind of outsourcing for economic reasons. Improving evidence and the interpretation of results presumes that human metabolic, toxicokinetic, and toxicodynamic background must be investigated and sometimes reinvestigated for new and old substances in both case samples and scientific experiments. Forensic toxicological laboratories and qualified research help to prepare both scientifically based legislation and jurisdiction by the successful implementation of new substance identification techniques and investigation of the metabolic fate of toxic chemicals and intoxicants in humans under various circumstances of drug abuse and poisoning. This has considerable social importance. By being financially supported with appropriate priority and because of the undisputed need of proper forensic toxicological investigations, not only the scientific community but all of society need to be permanently informed about the importance of scientific goals and progress in forensic toxicology. 23.5 REFERENCES 1 2

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