12. Methoden der Drogenanalyse ~ ~ O ~

% Cannabis resin (hashish) 25 Cannabis (plant material) 6 Cannabis extract 0.2 False cannabis (henna etc) 4 Amphetamine and related compounds 38 Heroin 3 Cocain 0.2 Prescribed drugs used illegally 18 Negative results 5

A11in all, over 130 compounds are included on the drug list in Sweden. Similar numbers are found in most industrialized countries.

The analysis proceeds in the following steps: 1. Registration and labelling 2. Weighing of each item 3. Sorting into groups according to physical appearance 4. Screening and possible further grouping 5. Identification 6. Quantitative analysis (with certain exceptions) 7. On request: Determination of the impurity profile of one-

component drugs (cf. below) 8. Report writing 9. Feeding of the results into the central police computer.

Some of these procedures have been reported in a previous publication [1].

Screening is usually done by thin-layer chromatography. Ultraviolet spectrophotometry often gives information on the class of compounds present.

The most important step is the unequivocal identification of the drug in question. It would obviously be inexcusable to send an innocent person to prison for up to 12 years on the basis of a faulty analytical result. At our laboratory drug identifications are based on four different chromatographic criteria from high performance GLC and TLC [2, 3] or, alternatively, 3 analytical methods, whereof at least one must be highly selective, such as IR or GCMS.

Quantitative analyses are often needed for determining the seriousness of drug offences. Powders containing 2 % or 95 % of amphetamine are judged differently by many courts of law. Additives and adulterants can give valuable hints for discovering drug trafficking.

Our laboratory has been a pioneer in the field of impurity profiles, also called chemical signatures, esp. by the work of

Strtmberg. The technique was first used for comparative analysis of hashish seizures. Since this material is of natural origin it contains hundreds of individual components which, upon gas chromatographic analysis, give a rich "fingerprint" pattern [4].

Later, profiles of amphetamines and related compounds were investigated [5-9]. In these cases, the main component is removed by extraction and the remaining impurities concen- trated and analyzed by gas chromatography. Work on heroin is in progress and will extend this kind of investigation. The purpose of these techniques is to establish connections between different seizures. This in turn can prove drug trafficking over a large area. It further serves as an important tool for drug intelligence work. Furthermore, we are considering the establish- ment of connections between active constituents of drug prepara- tions. The comparative analysis will then be based on individual components, i.e. parts of the profiles. The requested chromatog- raphic resolution for this purpose is fullfilled by the latest generation of capillary gas chromatographs. Since these instru- ments are computerized and automated, the number of analyses may be increased at a moderate cost.

References

1. Str6mberg L, Maehly AC (1978) J Anal Toxicol 2 : 7 - 1 2 2. Aim S, Jonson S, Karlsson H, Sundholm EG (1983)

J Chromatogr 254:179-186 3. Sundholm EG (1983) J Chromatogr 265:285-291 4. Str6mberg L (1972) J Chromatogr 68 : 253- 258 5. Strtmberg L, Maehly AC (1978) Advances in chemical

signature analysis of drugs. Proc Intern Symposium on Instrumental Applications in Forensic Drug Chemistry, Washington DC, USA, May 29-30 , pp 202-209

6. Strtmberg L (1975) J Chromatogr 106: 335- 342 7. Strtmberg L, Maehly AC (1975) J Chromatogr 109: 6 7 - 72 8. Bergkvist H, Str6mberg L (1980) Comparative gas chroma-

tographic analysis of amphetamine sulphate adulterated with sugars. Proc 1st Scand Conf Forens Sci, Link6ping (Sweden), June 11-13, pp 153-156

9. Strtmberg L, Bergkvist H, Edirisinghe EAMK (1983) J Chromatogr 258 : 6 5 - 72

Fresenius Z Anal Chem (1984) 317 : 636 - 637 �9 Springer-Verlag 1984

Immunochemical Analysis of Body Fluids

R. N. Smith

Metropolitan Police Forensic Science Laboratory, 109 Lambeth Road, London SE1 7LP, UK

Immunehemische Analyse von K/Jrperfliissigkeiten

The analysis of drugs and their metabolites in body fluids involves the detection and identification of a few substances of interest in the presence of numerous other materials, some of which may have physical or chemical properties similar to those of the target compounds. Chromatographic methods are widely used for drug analysis but they become more demanding as the analyte concentration decreases. Simpler and less expensive alternatives are therefore desirable and immunochemical meth-

ods have much to offer in this respect since they are sensitive and relatively unaffected by endogenous constituents of the sample.

Radioisotopes have traditionally been used as the labels in immunoassays but numerous alternatives have been proposed in recent years. Each alternative has its advantages and disadvan- tages but, as far as sensitivity is concerned, alternative labels have no advantage over radioisotopes in a conventional labelled- antigen immunoassay. In theory, radioimmunoassay (RIA) using a high-avidity antiserum can detect a concentration of a few femtogrammes/ml which is well below therapeutic drug levels. The choice of assay for a particular application therefore depends solely on methodological considerations.

The two most important components of a conventional immunoassay are the labelled antigen and the antiserum. Numerous methods of preparing labelled antigens have been described ranging from the introduction of a radioisotope directly into the molecule to the covalent linkage of a labelled

637

S ~ r 12. Methods of Drug Analysis

"tag" to the antigen via a suitable functional group. It is not necessary for the antiserum to bind the labelled antigen with the same avidity as the unlabelled antigen; the only requirement is that neither compound should be excessively bound under the conditions of the assay. It is therefore advisable for the linkage between a labelled "tag" and an antigen to differ from that in the anfigen-protein conjugate that was used to raise the antiserum in order to avoid excessive binding of the labelled antigen by antibodies recognizing the linkage.

There are general guide-lines rather than definitive rules for the production of polyclonal anti-drug sera and the control of specificity. Bleeds from different animals of the same species can vary enormously in the titre and pattern of cross-reactivity displayed, and even consecutive bleeds from the same animal can show marked differences. Some properties of an antiserum may be due to in vivo modification of the drug-protein conjugate used to immunize the animals or to free drug that is physically rather than covalently bound to the protein. An antiserum can often be used without any pre-treatment but, if necessary, it can be fractionated by precipitation methods or affinity chromatog- raphy. The latter process is of limited use for isolating antibodies against small molecules since extreme conditions are required to dissociate the bound antibodies from the affinity matrix. Monoclonal antibody technology is a more effective means of obtaining pure antibodies in quantity, but there is as yet no evidence that monoclonal antibodies have any marked advan- tage over polyclonal antisera in drug immunoassay.

Receptor preparations are an alternative to antisera in immunoassays. They display cross-reactivity patterns towards

related drugs that reflect the biological effectiveness of the drugs, but this useful property is offset by the limited availability and stability of receptor preparations and the laborious procedures required to isolate them.

The evaluation of an immunoassay is a lengthy procedure. Antiserum and label concentrations must be matched to give the required dose-response curve; the specificity of the antiserum must be determined by assaying all available drugs and meta- bolites that might be expected to cross-react; blank and "spiked" samples should be assayed to determine sensitivity, accuracy and precision, and, if possible, the performance of the assay should be compared with that of an established and totally unrelated method. Erroneously low results may occasionally be due to endogenous anti-drug antibodies in blood samples, while falsely elevated results may sometimes be caused by, for instance, anti- rabbit antibodies in a blood sample interfering with a second- antibody separation of the bound and free fractions by a solid- phase sheep anti-rabbit serum.

Current developments in drug immunoassay are more technical than fundamental. There is a noticeable bias, partic- ularly in the case of diagnostic reagent and instrument manu- facturers, towards automated, high-throughput, non-isotopic, homogeneous assays. Such assays are suitable for routine thera- peutic drug monitoring but, for many other applications and in the development of new assays for drugs, RIA is likely to remain the immunoassay method of choice.

Fresenius Z Anal Chem (1984) 317:637-638 �9 Springer-Verlag 1984

Chromatographic Techniques in Drug Analysis

A. C. Moffat

Drugs and Toxicology Division, Home Office Central Research Establishment, Aldermaston, Reading, Berkshire RG7 4PN, UK

Chromatographische Techniken bei der Analyse von Drogen

Manuscript not submitted

Manuskript nicht eingegangen

Toxicological Drug Analysis by Spectrometric and Combined Techniques

R. A. A. Maes

Center of Human Toxicology, State University, Vondellaan 14, NL-3251 GE Utrecht, The Netherlands

Toxikologische Drogenanalyse durch spektrometrische und kombinierte Verfahren

Although spectrophotometric analysis of drugs in biological material is still widely used the technique might be hampered by the lengthy purification and extraction procedures needed to

638

reduce interference. The major rule of UV-spectrophotometry in toxicology is the analysis of weak acids, neutral compounds and certain weak bases in biological specimens. The advantages of classical UV-spectrophotometry are ease of use, short setup time for instruments, rapid analysis time after sample preparation, qualitative and quantitative data and with a few exceptions, the non-destructive nature of the procedure and the relatively low cost. Some disadvantages are that drugs which do not have a sufficiently strong UV-spectrum cannot be analyzed, and that structurally related compounds such as amphetamine, phenetyl- amine and other amine derivatives exhibit similar absorption characteristics, making specific identification very difficult or even impossible.

On the other hand UV-VIS derivative spectrophotometry is a relatively new analytical technique that offers enhancement of