- Email: [email protected]
Recent developments in analytical toxicology: for better or for worse
Toxicology Letters 102]103 Ž1998. 103]108
Recent developments in analytical toxicology: for better or for worse Rokus A. de ZeeuwU Department of Analytical Chemistry and Toxicology, Uni¨ ersity Centre for Pharmacy Deusinglaan 1, NL-9713 AV Groningen, The Netherlands Received 6 July 1998; received in revised form 4 August 1998; accepted 10 August 1998
Abstract When considering the state of the art in toxicology from an analytical perspective, the key developments relate to three major areas. Ž1. Forensic horizon: Today forensic analysis has broadened its scope dramatically, to include workplace toxicology, drug abuse testing, drugs and driving, doping, environmental and veterinary toxicology, etc. Ž2. Basic analytical issues, focusing on a strongly widening array of relevant substances and metabolites at ever decreasing levels in a large variety of matrices. Ž3. Validation and interpretation: Because forensic analyses may have severe, punitive consequences, validation of methods and approaches plus proficiency testing are of utmost importance. Also, interpretation of the analytical results must be done with prudence in view of chemical and biological diversity in society. This review discusses the various pros and cons in these developments. Q 1998 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Analytical toxicology; Screening; Identification; Drug abuse testing
1. Introduction Analytical toxicology comprises the detection, identification and Žoften. the quantitation of potentially harmful substances in relevant matrices. In the past, it was mainly limited to casework in living persons Žclinical toxicology. or in persons that had died under suspicious circumstances Žforensic toxicology..
U
Tel.: q31 50 3633336; fax: q31 50 3637582; e-mail: [email protected]
The first two issues, detection and identification, relate to qualitative analysis, and these are by far the most demanding and challenging. A proper analysis should be able to detect all substances of toxicological relevance, regardless of their structure or chemical properties, followed by their identification beyond reasonable doubt. In recent years, analytical toxicologists have tried to cope with the many demands in their field. Undoubtedly, this has led to a variety of important achievements. Yet, not all developments can be considered positive ones; some give reason for considerable concern, whereas some
0378-4274r98r$ - see front matter Q 1998 Elsevier Science Ireland Ltd. All rights reserved. PII S0378-4274Ž98.00293-8
104
R.A. de Zeeuw r Toxicology Letters 102]103 (1998) 103]108
key issues still remain unresolved. In this review, the pros and cons of the state of the art will be discussed and related to the perspectives for the future. 2. Forensic horizon Not so long ago, forensic toxicology was mainly restricted to postmortem investigations in suspicious cases of poisoning. The number of substances involved was relatively small and the levels encountered were relatively high. Also, the number of cases was small, although it remains unknown how forensic toxicologists performed during those days. Only detected cases were reported ŽCurry, 1998.. However, during the last 15 years, the forensic horizon has broadened dramatically ŽTable 1.. From Table 1 it is obvious that the scope of forensic toxicology has changed substantially. The majority of the work is now being done in living subjects, the levels to be analyzed are often much lower Žin the ppb or even ppt range., and the number of relevant substances has expanded. Moreover, the demand for forensic analysis has grown exponentially. In general, forensic toxicologists have been able to respond relatively well to these new challenges, making ingenious use of advances in basic analytical techniques and instrumentation. To give some examples, morphine, its 3- and 6-glucuronides and 6-monoacetylmorphine could be analyzed in body fluids at the low ppb level by using solid phase extraction ŽSPE. and liquid chromatography-mass spectrometry ŽBogusz et al., 1997.. Cocaine and benzoylecgonine can be analyzed in hair segments down to the low ppb range using Table 1 New application areas of forensic toxicology Classical forensic toxicology Urine drug testing Human performance testing Occupational toxicology Food toxicology Animal toxicology Ecotoxicology Warfare toxicology
Postmortem cases Drugs of abuse, doping Workplace, drugs and driving Exposure at work Residues in food products Cattle, wildlife, fish, pets Environmental pollution, exposure Gases, defoliation
GC-MS or LC-MS techniques ŽCone, 1995.. Flunitrazepam and its major metabolites could be determined in biofluids at the low ppb level using SPE and HPLC with UV-detection ŽDeinl et al., 1998.. Yet, it should be noted that the majority of the advances dealt with directed analyses, i.e. it focused on a particular set of drugs or on a drug class. In these cases one can optimize the procedures, taking into account the physico-chemical properties of the substance Žs. of interest. When undirected analyses are to be carried out Ži.e. the search for substances whose presence are uncertain and whose identities are unknown., the analytical approach is much more complex and hinges very much on qualitative issues. Is something present that should not be there, and } if so } what is its identity. This is also called general unknown analysis or systematic toxicological analysis ŽSTA. and is discussed below in more detail. 3. Basic analytical issues The undirected search, or STA, usually consists of three major steps: 1. Sample work up, isolation and concentration. This may include hydrolysis of conjugates, digestion of tissues, removal of proteins, lyophilization, etc. to be followed by SPE, liquid]liquid extraction ŽLLE., supercritical fluid extraction ŽSFE. or immunoaffinity chromatography ŽIAC.. 2. Differentiation and detection. The most common techniques for these purposes are: immunoassays ŽIA. and receptor assays ŽRA.; thin layer chromatography ŽTLC. with color reactions; gas chromatography ŽGC. combined with flame ionization detection ŽFID., nitrogen]phosphorus detection ŽNPD., electron capture detection ŽECD. or mass spectrometry ŽMS.; high performance liquid chromatography ŽHPLC. combined with ultraviolet ŽUV., diode array detection ŽDAD., electrochemical detection ŽECD. or MS detection. 3. Identification, which is usually done by comparing the data found for the unknown substance Žs. with data bases of reference substances and by finding proper matches.
R.A. de Zeeuw r Toxicology Letters 102]103 (1998) 103]108
In order for STA to be effective, the following prerequisites must be met. Step 1 must retain all relevant substances but should remove all matrix interferences and other non-relevant compounds. Step 2 must detect with optimum sensitivity and universality and should yield maximum differentiation in a minimum amount of time. Step 3 relies on comprehensive and up-to-date data bases. Unfortunately, it is still a widely held belief that STA is only needed in general unknown cases. This is wrong, however. Also in cases in which the toxic agent is known Žor suspected. a systematic analysis must be applied to check the presence of additional toxic agents. It will be clear that the latter is even more important now that multi-substance intoxications are presently the rule rather than the exception. Thus, the ultimate aim of STA can be defined as: Ž1. to detect all toxicologically relevant agents present and to identify them beyond reasonable doubt; and Ž2. to exclude the presence of all other relevant agents. Notwithstanding its importance, STA is becoming more and more neglected in various areas, because it is considered too time-consuming and too costly. As a result, clinical intoxications are being treated only symptomatically, whereas in forensic casework only a limited number of directed tests are carried out. Needless to say that this development is deplorable, scientifically, legally and socially. The latter is further illustrated in the area of drug abuse testing. Here, the Mandatory Guidelines issued by the National Institute for Drug Abuse ŽNIDA. in the USA ŽMandatory Guidelines for Federal Work Place Drug Testing Programs, 1988. played a pivotal role. It singled out the so-called NIDA-Five as the substances to be tested for: Ž1. amphetamine and methamphetamine; Ž2. morphine and codeine; Ž3. cocaine, determined as benzoylecgonine; Ž4. cannabis, determined as 11-nor-D9 -THC-9carboxylic acid; and Ž5. phencyclidine. In addition, it set rules for the analytical approach, namely screening by immunoassays and confirmation by GC-MS. This has led to a strong fixa-
105
Table 2 Other abused amphetamine-type drugs MDMA ŽXTC. MDEA ŽEve. MDA ŽLove. MMDA MBDB PMA DMA TMA DOB DOM ŽSTP. DOET Benzphetamine Ephedrine
Fencamfamine Fenproporex Clobenzorex Mefenorex Methoxyphenamine Methylphenidate Norephedrine Amphepramone Fenfluramine Norfenfluaramine Phenmetrazine Phentermine Phenylpropanolamine
tion on these substances and on the analytical approach to the extent that other issues and developments have been ignored. It is now well realized that, particularly in Europe, the number of narcotics and amphetamines used at the drug scene is much larger Žsee for example the list of other abused amphetaminetype drugs in Table 2.. Also, benzodiazepines and barbiturates are now recognized as abused drug classes. Moreover, we see occasional appearances of exotic drugs such as mescaline, LSD, psilocibine, cathine, g-hydroxybutyrate, bromathan, ketamine, etc. However, suitable guidelines and approaches to test for this much broader spectrum of abused drugs are not available, also because of political disunity and inaptitude. As regards the analytical approach in the NIDA-guidelines, it seems that screening by IA, followed by GC-MS confirmation is being adopted, but wrongly so, as the gold standard in forensic toxicology as a whole. Of course, IAs may offer the advantage of simplicity, speed and cost-effectiveness, but it must be remembered that: Ža. antibodies have to be raised; and Žb. that the efficacy to test for a broad spectrum of analogs is entirely dependent on the cross-reactivity of the available antibody. Receptor assays ŽRA. may be a very elegant and powerful alternative to IAs, at least for all substances that exert their activity in the body through receptor mediation Ž˘ Smisterova ´ et al., 1994.. Though there still remain some difficulties to be resolved, such as matrix interfer-
106
R.A. de Zeeuw r Toxicology Letters 102]103 (1998) 103]108
ence and receptor purification and stability, RAs may have a lot of potential in analytical toxicology, also because non-radioactive RAs are being developed ŽJanssen, 1997.. Also, it should be realized that GC-MS has its limitations and that it is not infallible. Obviously, an inherent disadvantage of STA is the fact that research and method development is so time- and effort-consuming because of the large number Žand variety. of substances one must take into account. Nevertheless, various research groups have been actively engaged in this area, also by joining forces. This has led to a reasonable understanding of how STA should be approached and which analytical techniques are most suitable for this purpose, at least for organic substances ŽDe Zeeuw, 1997.. For sample work up, SPE on mixed-mode columns has emerged as the technique of choice, giving clean extracts, good recoveries and good reproducibilities for a wide variety of drugs ŽChen et al., 1992; Lai et al., 1997.. An additional advantage of SPE, also in view of the increased workload, is its amenity to automation and miniaturization ŽBlevins and Hall, 1998.. Supercritical fluid extraction seems more suitable for directed analysis than to extract a broad spectrum of drugs. For the differentiation]detection phase, GC on capillary columns ŽPolettini, 1996. and HPLC on reversed phase columns ŽBogusz and Erkens, 1994. remain the workhorses, with mass spectrometry being the most powerful detection mode. More recently, reasonably priced LC-MS instruments have appeared on the market. This combination is much more universally applicable than GC-MS, also allowing the analysis of thermolabile substances and non-polar and non-volatile substances, such as metabolites, conjugates and peptides. The combination of LC-DAD has also proven to be a very useful and cost-effective one, but requires that the analytes absorb UV- or visible light. For some purposes, ion mobility spectrometry can be very valuable Že.g. for explosives and for detecting contamination of objects with drugs of abuse., but the technique is also very sensitive to various types of interferences ŽKoole et al., 1998.. Last but not least, TLC should be mentioned. Though it offers limited separation efficiency and repro-
ducibility, the technique remains a very versatile tool because it offers speed and the possibility to run a number of plates in parallel. Moreover, when detection is done by means of a series of consecutive color reactions on the plate, satisfactory identification power can be obtained ŽDe Zeeuw, 1997.. This is important to know in developing countries and laboratories that cannot afford sophisticated instrumentation. In addition, it has been shown that TLC can be used successfully under extreme climatic conditions, such as in tropical countries, provided that proper R f-correction procedures are applied ŽDe Zeeuw et al., 1992, 1994.. The utility of capillary electrophoresis ŽCE. and related techniques such as micellar electrokinetic chromatography ŽMEKC. to analytical toxicology appears promising at the moment ŽTagliaro et al., 1996; Von Heeren and Thomassen, 1997., but its potentials for STA need to be investigated. The third step in STA, the identification, has also seen reasonable progress, although many scientists are still not fully aware of the key factors involved. Important contributions were written by Spiehler et al. Ž1988. and Ferrara et al. Ž1994.. Recently, Hartstra developed a probabilistic model to carry out computerized substance identification by comparing analytical data for the unknown substance Žs. with those present in data bases of known substances ŽHartstra, 1997.. For the latter, extensive data compilations exist for TLC R f-values with color reactions, GC Retention Indices with molecular weights and HPLC Retention Indices with diode array detection ŽHartstra et al., 1995.. Yet, despite considerable advances in our analytical abilities and knowledge, there remain many areas that need further attention. To name a few: the analysis of polar substances, including metabolites, conjugates, peptides, nucleotides, etc.; inorganic cations and anions; agrochemicals; toxins of biological origin; the ability to deal with ‘dirtier’ matrices such as Žputrefied. tissues, animal specimens, soil specimens; and the creation of suitable data bases and their updating, etc. Concerted research efforts and interlaboratory cooperation are highly needed.
R.A. de Zeeuw r Toxicology Letters 102]103 (1998) 103]108
4. Validation and interpretation In recent years, much emphasis has been paced on the quality and reliability of the results obtained in toxicological analysis. Given the fact that the analytical results may have a great impact on decisions made in criminal or civil justice, the former must be as error-free as possible. This requires the implementation of Good Laboratory Practice rules, including the use of Standard Operating Procedures and of properly validated analytical methodologies. In addition, the overall performance of the laboratory should be monitored by both internal and external proficiency testing ŽPT. programs ŽFerrara, 1998.. Fortunately, various PT programs are now available that are suitable for analytical toxicology laboratories. In North America, there is a clear trend in requesting successful participation in these type of programs as the basis for an official Laboratory Accreditation. Suitable PT programs for analytical toxicology are also available in Europe but participation in these programs is still almost entirely on voluntary basis. Mandatory EU-rules are urgently needed, but this seems to be hampered by political disunity. So far, the experience with external PT programs is very good in that regular participation in these programs considerably improves proficiency when dealing with the more prevalent toxic substances. However, the PT programs have also indicated that many laboratories have great difficulties with substances that are less often seen in daily practice. Thus, apart from the need for regular PT programs, preferably on an international basis, there is also a need to expand the spectrum of substances to be included. Finally, in a nutshell, some words on interpretation. With our knowledge advancing continuously, we have come to appreciate that the interpretation of analytical data must be done with utmost care for a variety of reasons. To name a few: often, the data relate to only one time point; site-dependent differences may exist for biofluids like blood; concentration ratio’s between blood and other compartments in the body are usually unknown Že.g. blood ] brain; blood ] saliva; blood]tissue.; drugs may redistribute after death
107
or may be prone to decomposition; and effects of drugs or combinations of drugs may depend on gender, race, age, illness, tolerance. One of the reasons for intersubject variation that is presently drawing much attention is metabolic polymorphism, the genetically determined ability to perform metabolic reactions in the body, particularly the polymorphic enzymes of the cytochrome P-450 systems ŽWest et al., 1997.. Up till now our knowledge on the potential impact of enzymatic polymorphism in causing intoxications is virtually non-existent. Yet, an intoxication with dextromethorphan, a relatively non-toxic drug, in a victim with a metabolic deficiency has been described recently ŽBauman et al., 1997.. Thus, also in the field of interpretation, a lot remains to be learned and investigated. References Bauman, P., Vlatkovic, D., Macciardi, F., 1997. Dextromethorphan intoxication in an adolescent with a genetic CYP2D6 deficiency. Therapie 52, 607]608. ´ Blevins, D.D., Hall, D.O., 1998. Recent Advances in Disk Format Solid Phase Extraction. LC-GC Current Trends and Developments in Sample preparation, May, S16]S21. Bogusz, M.J., Erkens, M., 1994. Reversed phase high performance liquid chromatographic database of retention indices and UV spectra of toxicologically relevant substances and its interlaboratory use. J. Chromatogr. A 674, 97]126. Bogusz, M.J., Maier, R.D., Erkens, M., Driessen, S., 1997. Determination of morphine and its 3- and 6-glucuronides, codeine, codeine]glucuronide and 6-monoacetylmorphine in body fluids by liquid chromatography]atmospheric pressure chemical ionization mass spectrometry. J. Chromatogr. B 703, 115]126. Chen, X.H., Wijsbeek, J., Franke, J.P., De Zeeuw, R.A., 1992. A single column procedure on Bond Elut Certify for systematic toxicological analysis of drugs in plasma and urine. J. Forensic Sci. 37, 61]71. Cone, E.J., 1995. Testing for codeine in hair. In: De Zeeuw, R.A., Hosani, I., Munthiri, S., Magbool, A. ŽEds.., Hair Analysis in Forensic Toxicology. Abu Dhabi Police Dept., Abu Dhabi, pp. 136]160. Curry, A.S., 1998. Murder. Bull. Int. Assoc. Forensic Toxicol. 28, 9]10. Deinl, I., Mahr, G., Von Mayer, L., 1998. Determination of flunitrazepam and its main metabolites in serum by HPLC after mixed-mode solid phase extraction. J. Anal. Toxicol. 22, 197]202. De Zeeuw, R.A., 1997. Drug screening in biological fluids. The need for a systematic toxicological approach. J. Chromatogr. B 689, 71]79.
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De Zeeuw, R.A., Franke, J.P., Dik, E., Ten Dolle, W., Kam, B.L., 1992. Impact of tropical conditions on TLC in analytical toxicology: high temperatures and moderate humidities. J. Forensic Sci. 37, 984]990. De Zeeuw, R.A., Franke, J.P., Van Halem, M., Schaapman, S., Logawa, E., Siregar, C.J.P., 1994. TLC under tropical conditions: impact of high temperatures and high humidities on screening systems for analytical toxicology. J. Chromatogr. 664, 263]270. Ferrara, S.D., 1998. Quality control in toxicological analysis. J. Chromatogr. B 713, 227]243. Ferrara, S.D., Tedeschi, L., Frison, G., et al., 1994. Drugs of abuse testing in urine, statistical approach and experimental comparison of immunochemical and chromatographic techniques. J. Anal. Toxicol. 18, 278]291. Hartstra, J., 1997. Computer Aided Identification of Toxicologically Relevant Substances by Means of Multiple Analytical Methods. Ph.D. Thesis, University of Groningen. Hartstra, J., Franke, J.P., De Zeeuw, R.A., 1995. Computerized identification of toxic substances and their metabolites. GIT Labor-Med. 18, 272]279. Janssen, M.J., 1997. Development and Perspectives of Fluorescent Receptor Assays. Ph.D. Thesis, University of Groningen. Koole, A., Luo, Y., Franke, J.P., De Zeeuw, R.A., 1998. Dramatic signal reduction in ion mobility spectrometry by residues of solvents. J. Anal. Toxicol. 22, 191]196. Lai, C.K., Lee, T., Au, K.M., Yan-Wo Chan, A., 1997. Uni-
form solid phase extraction procedure for toxicological drug screening in serum and urine by HPLC-DAD. Clin. Chem. 43, 312]325. Mandatory Guidelines for Federal Work Place Drug Testing Programs, 1988. Federal Register 53, 11970]11989. US Government Printing Office, Washington, DC. Polettini, A., 1996. A simple automated procedure for the detection and identification of peaks in gas chromatography-continuous scan mass spectrometry. J. Anal. Toxicol. 20, 579]586. ˘Smisterova, ´ J., Ensing, K., De Zeeuw, R.A., 1994. Methodological aspects of quantitative receptor assay. J. Pharm. Biomed. Anal. 12, 723]745. Spiehler, V.R., O’Donnell, M., Gokhale, D.V., 1988. Confirmation and certainty in toxicology screening. Clin. Chem. 34, 1535]1539. Tagliaro, F., Smith, F.P., Turina, S., Equisetto, V., Marigo, M., 1996. Complementary use of capillary electrophoresis and micellar electrokinetic chromatography for mutual confirmation of results in forensic drug analysis. J. Chromatogr. A 375, 227]235. Von Heeren, F., Thomassen, W., 1997. Capillary electrophoresis in clinical and forensic analysis. Electrophoresis 18, 2145]2154. West, W.L., Knight, E.M., Pradhan, S., Hinds, T.S., 1997. Interpatient variability: genetic predisposition and other genetic factors. J. Clin. Pharmacol. 37, 635]648.
Recent developments in analytical toxicology: for better or for worse Rokus A. de ZeeuwU Department of Analytical Chemistry and Toxicology, Uni¨ ersity Centre for Pharmacy Deusinglaan 1, NL-9713 AV Groningen, The Netherlands Received 6 July 1998; received in revised form 4 August 1998; accepted 10 August 1998
Abstract When considering the state of the art in toxicology from an analytical perspective, the key developments relate to three major areas. Ž1. Forensic horizon: Today forensic analysis has broadened its scope dramatically, to include workplace toxicology, drug abuse testing, drugs and driving, doping, environmental and veterinary toxicology, etc. Ž2. Basic analytical issues, focusing on a strongly widening array of relevant substances and metabolites at ever decreasing levels in a large variety of matrices. Ž3. Validation and interpretation: Because forensic analyses may have severe, punitive consequences, validation of methods and approaches plus proficiency testing are of utmost importance. Also, interpretation of the analytical results must be done with prudence in view of chemical and biological diversity in society. This review discusses the various pros and cons in these developments. Q 1998 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Analytical toxicology; Screening; Identification; Drug abuse testing
1. Introduction Analytical toxicology comprises the detection, identification and Žoften. the quantitation of potentially harmful substances in relevant matrices. In the past, it was mainly limited to casework in living persons Žclinical toxicology. or in persons that had died under suspicious circumstances Žforensic toxicology..
U
Tel.: q31 50 3633336; fax: q31 50 3637582; e-mail: [email protected]
The first two issues, detection and identification, relate to qualitative analysis, and these are by far the most demanding and challenging. A proper analysis should be able to detect all substances of toxicological relevance, regardless of their structure or chemical properties, followed by their identification beyond reasonable doubt. In recent years, analytical toxicologists have tried to cope with the many demands in their field. Undoubtedly, this has led to a variety of important achievements. Yet, not all developments can be considered positive ones; some give reason for considerable concern, whereas some
0378-4274r98r$ - see front matter Q 1998 Elsevier Science Ireland Ltd. All rights reserved. PII S0378-4274Ž98.00293-8
104
R.A. de Zeeuw r Toxicology Letters 102]103 (1998) 103]108
key issues still remain unresolved. In this review, the pros and cons of the state of the art will be discussed and related to the perspectives for the future. 2. Forensic horizon Not so long ago, forensic toxicology was mainly restricted to postmortem investigations in suspicious cases of poisoning. The number of substances involved was relatively small and the levels encountered were relatively high. Also, the number of cases was small, although it remains unknown how forensic toxicologists performed during those days. Only detected cases were reported ŽCurry, 1998.. However, during the last 15 years, the forensic horizon has broadened dramatically ŽTable 1.. From Table 1 it is obvious that the scope of forensic toxicology has changed substantially. The majority of the work is now being done in living subjects, the levels to be analyzed are often much lower Žin the ppb or even ppt range., and the number of relevant substances has expanded. Moreover, the demand for forensic analysis has grown exponentially. In general, forensic toxicologists have been able to respond relatively well to these new challenges, making ingenious use of advances in basic analytical techniques and instrumentation. To give some examples, morphine, its 3- and 6-glucuronides and 6-monoacetylmorphine could be analyzed in body fluids at the low ppb level by using solid phase extraction ŽSPE. and liquid chromatography-mass spectrometry ŽBogusz et al., 1997.. Cocaine and benzoylecgonine can be analyzed in hair segments down to the low ppb range using Table 1 New application areas of forensic toxicology Classical forensic toxicology Urine drug testing Human performance testing Occupational toxicology Food toxicology Animal toxicology Ecotoxicology Warfare toxicology
Postmortem cases Drugs of abuse, doping Workplace, drugs and driving Exposure at work Residues in food products Cattle, wildlife, fish, pets Environmental pollution, exposure Gases, defoliation
GC-MS or LC-MS techniques ŽCone, 1995.. Flunitrazepam and its major metabolites could be determined in biofluids at the low ppb level using SPE and HPLC with UV-detection ŽDeinl et al., 1998.. Yet, it should be noted that the majority of the advances dealt with directed analyses, i.e. it focused on a particular set of drugs or on a drug class. In these cases one can optimize the procedures, taking into account the physico-chemical properties of the substance Žs. of interest. When undirected analyses are to be carried out Ži.e. the search for substances whose presence are uncertain and whose identities are unknown., the analytical approach is much more complex and hinges very much on qualitative issues. Is something present that should not be there, and } if so } what is its identity. This is also called general unknown analysis or systematic toxicological analysis ŽSTA. and is discussed below in more detail. 3. Basic analytical issues The undirected search, or STA, usually consists of three major steps: 1. Sample work up, isolation and concentration. This may include hydrolysis of conjugates, digestion of tissues, removal of proteins, lyophilization, etc. to be followed by SPE, liquid]liquid extraction ŽLLE., supercritical fluid extraction ŽSFE. or immunoaffinity chromatography ŽIAC.. 2. Differentiation and detection. The most common techniques for these purposes are: immunoassays ŽIA. and receptor assays ŽRA.; thin layer chromatography ŽTLC. with color reactions; gas chromatography ŽGC. combined with flame ionization detection ŽFID., nitrogen]phosphorus detection ŽNPD., electron capture detection ŽECD. or mass spectrometry ŽMS.; high performance liquid chromatography ŽHPLC. combined with ultraviolet ŽUV., diode array detection ŽDAD., electrochemical detection ŽECD. or MS detection. 3. Identification, which is usually done by comparing the data found for the unknown substance Žs. with data bases of reference substances and by finding proper matches.
R.A. de Zeeuw r Toxicology Letters 102]103 (1998) 103]108
In order for STA to be effective, the following prerequisites must be met. Step 1 must retain all relevant substances but should remove all matrix interferences and other non-relevant compounds. Step 2 must detect with optimum sensitivity and universality and should yield maximum differentiation in a minimum amount of time. Step 3 relies on comprehensive and up-to-date data bases. Unfortunately, it is still a widely held belief that STA is only needed in general unknown cases. This is wrong, however. Also in cases in which the toxic agent is known Žor suspected. a systematic analysis must be applied to check the presence of additional toxic agents. It will be clear that the latter is even more important now that multi-substance intoxications are presently the rule rather than the exception. Thus, the ultimate aim of STA can be defined as: Ž1. to detect all toxicologically relevant agents present and to identify them beyond reasonable doubt; and Ž2. to exclude the presence of all other relevant agents. Notwithstanding its importance, STA is becoming more and more neglected in various areas, because it is considered too time-consuming and too costly. As a result, clinical intoxications are being treated only symptomatically, whereas in forensic casework only a limited number of directed tests are carried out. Needless to say that this development is deplorable, scientifically, legally and socially. The latter is further illustrated in the area of drug abuse testing. Here, the Mandatory Guidelines issued by the National Institute for Drug Abuse ŽNIDA. in the USA ŽMandatory Guidelines for Federal Work Place Drug Testing Programs, 1988. played a pivotal role. It singled out the so-called NIDA-Five as the substances to be tested for: Ž1. amphetamine and methamphetamine; Ž2. morphine and codeine; Ž3. cocaine, determined as benzoylecgonine; Ž4. cannabis, determined as 11-nor-D9 -THC-9carboxylic acid; and Ž5. phencyclidine. In addition, it set rules for the analytical approach, namely screening by immunoassays and confirmation by GC-MS. This has led to a strong fixa-
105
Table 2 Other abused amphetamine-type drugs MDMA ŽXTC. MDEA ŽEve. MDA ŽLove. MMDA MBDB PMA DMA TMA DOB DOM ŽSTP. DOET Benzphetamine Ephedrine
Fencamfamine Fenproporex Clobenzorex Mefenorex Methoxyphenamine Methylphenidate Norephedrine Amphepramone Fenfluramine Norfenfluaramine Phenmetrazine Phentermine Phenylpropanolamine
tion on these substances and on the analytical approach to the extent that other issues and developments have been ignored. It is now well realized that, particularly in Europe, the number of narcotics and amphetamines used at the drug scene is much larger Žsee for example the list of other abused amphetaminetype drugs in Table 2.. Also, benzodiazepines and barbiturates are now recognized as abused drug classes. Moreover, we see occasional appearances of exotic drugs such as mescaline, LSD, psilocibine, cathine, g-hydroxybutyrate, bromathan, ketamine, etc. However, suitable guidelines and approaches to test for this much broader spectrum of abused drugs are not available, also because of political disunity and inaptitude. As regards the analytical approach in the NIDA-guidelines, it seems that screening by IA, followed by GC-MS confirmation is being adopted, but wrongly so, as the gold standard in forensic toxicology as a whole. Of course, IAs may offer the advantage of simplicity, speed and cost-effectiveness, but it must be remembered that: Ža. antibodies have to be raised; and Žb. that the efficacy to test for a broad spectrum of analogs is entirely dependent on the cross-reactivity of the available antibody. Receptor assays ŽRA. may be a very elegant and powerful alternative to IAs, at least for all substances that exert their activity in the body through receptor mediation Ž˘ Smisterova ´ et al., 1994.. Though there still remain some difficulties to be resolved, such as matrix interfer-
106
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ence and receptor purification and stability, RAs may have a lot of potential in analytical toxicology, also because non-radioactive RAs are being developed ŽJanssen, 1997.. Also, it should be realized that GC-MS has its limitations and that it is not infallible. Obviously, an inherent disadvantage of STA is the fact that research and method development is so time- and effort-consuming because of the large number Žand variety. of substances one must take into account. Nevertheless, various research groups have been actively engaged in this area, also by joining forces. This has led to a reasonable understanding of how STA should be approached and which analytical techniques are most suitable for this purpose, at least for organic substances ŽDe Zeeuw, 1997.. For sample work up, SPE on mixed-mode columns has emerged as the technique of choice, giving clean extracts, good recoveries and good reproducibilities for a wide variety of drugs ŽChen et al., 1992; Lai et al., 1997.. An additional advantage of SPE, also in view of the increased workload, is its amenity to automation and miniaturization ŽBlevins and Hall, 1998.. Supercritical fluid extraction seems more suitable for directed analysis than to extract a broad spectrum of drugs. For the differentiation]detection phase, GC on capillary columns ŽPolettini, 1996. and HPLC on reversed phase columns ŽBogusz and Erkens, 1994. remain the workhorses, with mass spectrometry being the most powerful detection mode. More recently, reasonably priced LC-MS instruments have appeared on the market. This combination is much more universally applicable than GC-MS, also allowing the analysis of thermolabile substances and non-polar and non-volatile substances, such as metabolites, conjugates and peptides. The combination of LC-DAD has also proven to be a very useful and cost-effective one, but requires that the analytes absorb UV- or visible light. For some purposes, ion mobility spectrometry can be very valuable Že.g. for explosives and for detecting contamination of objects with drugs of abuse., but the technique is also very sensitive to various types of interferences ŽKoole et al., 1998.. Last but not least, TLC should be mentioned. Though it offers limited separation efficiency and repro-
ducibility, the technique remains a very versatile tool because it offers speed and the possibility to run a number of plates in parallel. Moreover, when detection is done by means of a series of consecutive color reactions on the plate, satisfactory identification power can be obtained ŽDe Zeeuw, 1997.. This is important to know in developing countries and laboratories that cannot afford sophisticated instrumentation. In addition, it has been shown that TLC can be used successfully under extreme climatic conditions, such as in tropical countries, provided that proper R f-correction procedures are applied ŽDe Zeeuw et al., 1992, 1994.. The utility of capillary electrophoresis ŽCE. and related techniques such as micellar electrokinetic chromatography ŽMEKC. to analytical toxicology appears promising at the moment ŽTagliaro et al., 1996; Von Heeren and Thomassen, 1997., but its potentials for STA need to be investigated. The third step in STA, the identification, has also seen reasonable progress, although many scientists are still not fully aware of the key factors involved. Important contributions were written by Spiehler et al. Ž1988. and Ferrara et al. Ž1994.. Recently, Hartstra developed a probabilistic model to carry out computerized substance identification by comparing analytical data for the unknown substance Žs. with those present in data bases of known substances ŽHartstra, 1997.. For the latter, extensive data compilations exist for TLC R f-values with color reactions, GC Retention Indices with molecular weights and HPLC Retention Indices with diode array detection ŽHartstra et al., 1995.. Yet, despite considerable advances in our analytical abilities and knowledge, there remain many areas that need further attention. To name a few: the analysis of polar substances, including metabolites, conjugates, peptides, nucleotides, etc.; inorganic cations and anions; agrochemicals; toxins of biological origin; the ability to deal with ‘dirtier’ matrices such as Žputrefied. tissues, animal specimens, soil specimens; and the creation of suitable data bases and their updating, etc. Concerted research efforts and interlaboratory cooperation are highly needed.
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4. Validation and interpretation In recent years, much emphasis has been paced on the quality and reliability of the results obtained in toxicological analysis. Given the fact that the analytical results may have a great impact on decisions made in criminal or civil justice, the former must be as error-free as possible. This requires the implementation of Good Laboratory Practice rules, including the use of Standard Operating Procedures and of properly validated analytical methodologies. In addition, the overall performance of the laboratory should be monitored by both internal and external proficiency testing ŽPT. programs ŽFerrara, 1998.. Fortunately, various PT programs are now available that are suitable for analytical toxicology laboratories. In North America, there is a clear trend in requesting successful participation in these type of programs as the basis for an official Laboratory Accreditation. Suitable PT programs for analytical toxicology are also available in Europe but participation in these programs is still almost entirely on voluntary basis. Mandatory EU-rules are urgently needed, but this seems to be hampered by political disunity. So far, the experience with external PT programs is very good in that regular participation in these programs considerably improves proficiency when dealing with the more prevalent toxic substances. However, the PT programs have also indicated that many laboratories have great difficulties with substances that are less often seen in daily practice. Thus, apart from the need for regular PT programs, preferably on an international basis, there is also a need to expand the spectrum of substances to be included. Finally, in a nutshell, some words on interpretation. With our knowledge advancing continuously, we have come to appreciate that the interpretation of analytical data must be done with utmost care for a variety of reasons. To name a few: often, the data relate to only one time point; site-dependent differences may exist for biofluids like blood; concentration ratio’s between blood and other compartments in the body are usually unknown Že.g. blood ] brain; blood ] saliva; blood]tissue.; drugs may redistribute after death
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or may be prone to decomposition; and effects of drugs or combinations of drugs may depend on gender, race, age, illness, tolerance. One of the reasons for intersubject variation that is presently drawing much attention is metabolic polymorphism, the genetically determined ability to perform metabolic reactions in the body, particularly the polymorphic enzymes of the cytochrome P-450 systems ŽWest et al., 1997.. Up till now our knowledge on the potential impact of enzymatic polymorphism in causing intoxications is virtually non-existent. Yet, an intoxication with dextromethorphan, a relatively non-toxic drug, in a victim with a metabolic deficiency has been described recently ŽBauman et al., 1997.. Thus, also in the field of interpretation, a lot remains to be learned and investigated. References Bauman, P., Vlatkovic, D., Macciardi, F., 1997. Dextromethorphan intoxication in an adolescent with a genetic CYP2D6 deficiency. Therapie 52, 607]608. ´ Blevins, D.D., Hall, D.O., 1998. Recent Advances in Disk Format Solid Phase Extraction. LC-GC Current Trends and Developments in Sample preparation, May, S16]S21. Bogusz, M.J., Erkens, M., 1994. Reversed phase high performance liquid chromatographic database of retention indices and UV spectra of toxicologically relevant substances and its interlaboratory use. J. Chromatogr. A 674, 97]126. Bogusz, M.J., Maier, R.D., Erkens, M., Driessen, S., 1997. Determination of morphine and its 3- and 6-glucuronides, codeine, codeine]glucuronide and 6-monoacetylmorphine in body fluids by liquid chromatography]atmospheric pressure chemical ionization mass spectrometry. J. Chromatogr. B 703, 115]126. Chen, X.H., Wijsbeek, J., Franke, J.P., De Zeeuw, R.A., 1992. A single column procedure on Bond Elut Certify for systematic toxicological analysis of drugs in plasma and urine. J. Forensic Sci. 37, 61]71. Cone, E.J., 1995. Testing for codeine in hair. In: De Zeeuw, R.A., Hosani, I., Munthiri, S., Magbool, A. ŽEds.., Hair Analysis in Forensic Toxicology. Abu Dhabi Police Dept., Abu Dhabi, pp. 136]160. Curry, A.S., 1998. Murder. Bull. Int. Assoc. Forensic Toxicol. 28, 9]10. Deinl, I., Mahr, G., Von Mayer, L., 1998. Determination of flunitrazepam and its main metabolites in serum by HPLC after mixed-mode solid phase extraction. J. Anal. Toxicol. 22, 197]202. De Zeeuw, R.A., 1997. Drug screening in biological fluids. The need for a systematic toxicological approach. J. Chromatogr. B 689, 71]79.
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De Zeeuw, R.A., Franke, J.P., Dik, E., Ten Dolle, W., Kam, B.L., 1992. Impact of tropical conditions on TLC in analytical toxicology: high temperatures and moderate humidities. J. Forensic Sci. 37, 984]990. De Zeeuw, R.A., Franke, J.P., Van Halem, M., Schaapman, S., Logawa, E., Siregar, C.J.P., 1994. TLC under tropical conditions: impact of high temperatures and high humidities on screening systems for analytical toxicology. J. Chromatogr. 664, 263]270. Ferrara, S.D., 1998. Quality control in toxicological analysis. J. Chromatogr. B 713, 227]243. Ferrara, S.D., Tedeschi, L., Frison, G., et al., 1994. Drugs of abuse testing in urine, statistical approach and experimental comparison of immunochemical and chromatographic techniques. J. Anal. Toxicol. 18, 278]291. Hartstra, J., 1997. Computer Aided Identification of Toxicologically Relevant Substances by Means of Multiple Analytical Methods. Ph.D. Thesis, University of Groningen. Hartstra, J., Franke, J.P., De Zeeuw, R.A., 1995. Computerized identification of toxic substances and their metabolites. GIT Labor-Med. 18, 272]279. Janssen, M.J., 1997. Development and Perspectives of Fluorescent Receptor Assays. Ph.D. Thesis, University of Groningen. Koole, A., Luo, Y., Franke, J.P., De Zeeuw, R.A., 1998. Dramatic signal reduction in ion mobility spectrometry by residues of solvents. J. Anal. Toxicol. 22, 191]196. Lai, C.K., Lee, T., Au, K.M., Yan-Wo Chan, A., 1997. Uni-
form solid phase extraction procedure for toxicological drug screening in serum and urine by HPLC-DAD. Clin. Chem. 43, 312]325. Mandatory Guidelines for Federal Work Place Drug Testing Programs, 1988. Federal Register 53, 11970]11989. US Government Printing Office, Washington, DC. Polettini, A., 1996. A simple automated procedure for the detection and identification of peaks in gas chromatography-continuous scan mass spectrometry. J. Anal. Toxicol. 20, 579]586. ˘Smisterova, ´ J., Ensing, K., De Zeeuw, R.A., 1994. Methodological aspects of quantitative receptor assay. J. Pharm. Biomed. Anal. 12, 723]745. Spiehler, V.R., O’Donnell, M., Gokhale, D.V., 1988. Confirmation and certainty in toxicology screening. Clin. Chem. 34, 1535]1539. Tagliaro, F., Smith, F.P., Turina, S., Equisetto, V., Marigo, M., 1996. Complementary use of capillary electrophoresis and micellar electrokinetic chromatography for mutual confirmation of results in forensic drug analysis. J. Chromatogr. A 375, 227]235. Von Heeren, F., Thomassen, W., 1997. Capillary electrophoresis in clinical and forensic analysis. Electrophoresis 18, 2145]2154. West, W.L., Knight, E.M., Pradhan, S., Hinds, T.S., 1997. Interpatient variability: genetic predisposition and other genetic factors. J. Clin. Pharmacol. 37, 635]648.