The immuassay handbook parte94

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The immuassay handbook parte94

  1. 1. 963© 2013 David G. Wild. Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/B978-0-08-097037-0.00077-4 The nonmedical use of drugs such as heroin, cocaine, and amphetamines remains epidemic and affects all aspects of social and economic life. The prior establishment of a link between intravenous drug misuse and the spread of AIDS has fueled concern, particularly in Western countries. Legislative measures, no matter how punitive, have had marginal impact and it is now recognized that these must be accompanied by educa- tion, treatment, and rehabilitation programs if the prob- lem is to be brought under control. Diagnostic tests carried out on biological samples are an integral feature of these programs and those based on immunological principles are widely applied. Many of those who advocate a more radical approach to the problem, such as the decriminalization of cannabis use, often point to nicotine and alcohol as examples of sub- stances that are much more harmful and yet, in most coun- tries, are quite legal and widely available. Irrespective of the merits of this argument, there is no doubt that nicotine is highly addictive and that regular tobacco use can lead to a number of severe medical disorders. By the same token, chronic alcohol abuse has serious physical and neurologi- cal consequences as well as being a major cause of domestic upheaval and loss of livelihood. Smokers and alcohol abus- ers undergoing treatment are notorious, when faced with medical interrogation, for either claiming complete absti- nence from the habit or grossly under-reporting their con- sumption. Again, immunoassays can provide an objective measure of the true pattern of use. Self-administration of performance-enhancing drugs by high-profile athletes receives a great deal of media cover- age. It is known that the practice now extends to local com- petitions, e.g., school and county championships, and immunoassays have a role in detecting this type of drug misuse. Anabolic steroids taken in regular doses through- out an inter-competition training period help to build mus- cle and can boost athletic performance well beyond an individual’s natural capability. Apart from the fact that an unfair advantage may be gained, there is great concern about the subsequent physical and mental health of young people who embark on such a course. Anabolic steroid administration in order to increase the performance of race horses and other animals featured in sport is also prohib- ited, and testing of biological samples for this group of sub- stances is carried out on a regular basis using immunoassays. In competitive human sport, however, chromatographic and mass spectrometric techniques now take precedence. Applications DRUG DEPENDENCE TREATMENT CENTERS These centers may be either out-patient or in-patient units and are designed to deal with the more severely affected patients. New patients undergo a medical examination, are questioned on their drug abuse history, and a urine sample is requested for analysis. The history volunteered is often unreliable; many drug abusers are unaware of the compo- sition of “street” preparations; some will report heroin abuse but omit to mention regular intake of cannabis, ben- zodiazepines etc.; others may be drug free and attempting to obtain a prescription for drugs for subsequent illegal sale. The results of the urine analysis therefore provide the only objective evidence of the pattern of drug abuse and are crucial to the initial diagnosis. The ideal treatment aim is total and continued absti- nence from drug abuse. For some patients who are heavily dependent on opiate drugs such as heroin, this is an unreal- istic goal and it is common practice to prescribe one of the safer heroin substitutes methadone or buprenorphine. After a while, a sizeable proportion of these patients are unable to resist the craving for heroin and other psychoactive drugs and relapse into multiple drug abuse. The only reliable means of monitoring compliance with prescribed medica- tion in patients of this type is by regular urine analyses. Other patients may be successfully detoxified by con- trolled withdrawal of drugs and then embark on a course of rehabilitation. This invariably includes routine urine checks at appropriate intervals to ensure continued absti- nence from any form of nonmedical drug taking. PSYCHIATRIC CLINICS Abused drugs act on the central nervous system (CNS) and can provoke signs of mental illness. For example, drowsi- ness and slurred speech might suggest sedative or cannabis abuse. Stimulant drugs (amphetamines, cocaine) induce excessive agitation, and prolonged and heavy abuse can lead to psychosis. The hallucinogenic properties of lyser- gic acid diethylamide (LSD) and phencyclidine (PCP) are well documented. Urine tests for the presence of these drugs help to differentiate between endogenous and drug- induced mental disorders. MEDICAL–LEGAL APPLICATIONS Children of drug-abusing parents are often at high risk of mental and physical neglect. In certain circumstances, the child may be taken into care until such time that the mother or father can demonstrate prolonged abstinence from drug abuse. A decision to renew custody of the child may rest on Drugs of Abuse* Alun D. Hutchings1 (hutchingsad@cardiff.ac.uk) Brian Widdop2 C H A P T E R 9.23 1 This edition. 2 Previous editions. * Note: The product performance data reproduced here are likely to be from a previous version of the product. The data are for illustrative purposes only. Please contact the manufacturer or check the pack insert for the latest performance information.
  2. 2. 964 The Immunoassay Handbook the results of urine drug analyses on samples collected from the parent at weekly intervals for up to 3 months. Occasionally, drug abusers dose their own children either to gain respite from the child’s demands or out of malice. Analytical tests on samples from such children are crucial in establishing a diagnosis of poisoning and secur- ing their future safety by legal means. Drug abuse carries the danger of overdosage, particu- larly among intravenous users. This may be deliberate, as in a suicide attempt, or unintentional, for example when an illicit supply that is far more concentrated than that previ- ously tolerated is injected. The practice of smuggling packages of drugs across international boundaries by con- cealment in the rectum or vagina or by swallowing them (body packing) can cause severe and even fatal poisoning if the packages leak. Tests for drugs of abuse on biological samples form part of the clinical diagnosis. If the outcome is fatal, the results of these tests are used by the pathologist to establish the cause of death. Many countries legislate against driving under the influ- ence of drugs, and analyses of blood or urine specimens form part of the evidence put forward to secure a convic- tion. A more recent development is roadside testing by police officers equipped with hand-held devices that give presumptive results within 5min. Clearly, sample collec- tion has to be noninvasive and must not infringe privacy. For these reasons, saliva is the favored sample and the results of early trials are looking promising. Offenders with a history of drug abuse applying for renewal of a driv- ing license may be required to undergo periodic urine tests before this is granted. DRUG ABUSE IN THE WORKPLACE Employees who misuse drugs are more likely to make bad decisions, manufacture faulty goods, and cause accidents. In the USA, random urine testing for drug abuse is manda- tory in sensitive government posts, the armed forces, and the transport industries. Many other industries test job applicants before employment as a routine exercise. This approach has become widely practiced in several European countries and, because a high proportion of samples are negative, immunoassays, which are rapid and readily auto- mated, are much favored by the testing laboratories. It must be emphasized, however, that great care is needed to avoid false accusation of an innocent employee. A positive immunoassay result must always be substantiated by a sec- ond confirmatory analysis by gas chromatography/mass spectrometry (GC/MS) before any action is taken. Immunoassays for Drugs of Abuse Quantitative immunoassays for abused drugs in serum, plasma, whole blood, or saliva can be used to diagnose clinical overdose and fatal cases. Saliva tests for roadside testing after an accident are being seriously evaluated in Europe. Sweat patches that can soak up drugs excreted by the sweat glands over several days are now used in the USA to monitor prisoners for drug abstention during periods of parole. For the other situations described above, semi- quantitative tests on urine remain most prevalent. This chapter therefore deals mainly with immunoassays for detection of the drugs in urine. A distinction must be made between the sensitivity of the test, which can be defined as the lowest concentration of analyte that can be detected reliably in a given matrix, and the threshold (or “cutoff”) concentration. Urine is a complex and variable matrix that exacerbates the problem of distinguishing a signal due to the presence of a drug from that of background instrument noise. To overcome this, commercial assays are assigned a threshold concen- tration that exceeds the detection limit by several fold. The threshold adopted must also be practical in that it allows recent drug abuse to be detected. Once the threshold limit has been selected, test samples are assigned positive or negative status by comparison with the threshold (cutoff) response level. Selection of threshold levels is also gov- erned by the views of national bodies, for example in the USA the Substance Abuse and Mental Health Service Administration (SAMSHA) lays down its own guidelines on these for workplace testing (DHHS/SAMSHA, 1994). In the European Union, recommended threshold levels for workplace testing were put forward by an international panel in 1996 (Killander et al., 1997), but there is as yet no legislation to enforce them. Examples of common assay cutoff values are shown in Table 1. Note that for sub- stances which are extensively metabolized before reaching the urine (e.g., cocaine), cutoff values are assigned to the most prevalent urinary metabolite. Although radioimmunoassay (using 125I labeling) still has a place, particularly when testing for drugs present in very low concentrations, homogeneous non-isotopic imu- noassays such as the Enzyme Multiplied Immunoassay Technique (EMIT), the Cloned Enzyme Donor Immuno- assay (CEDIA), Fluorescence Polarization Immunoassay (FPIA), microparticle-based immunoassays such as OnLine, and heterogeneous assays such as ELISA (Enzyme-Linked Immunosorbent Assay) have proved the most popular in routine use. There has always been pressure on the manufacturers to develop very simple “dip-stick” tests which can be used with little or no train- ing by doctors, nurses, policemen, and the like to give on TABLE 1 Common Assay Cutoff Values for Drugs of Abuse Immunoassays Compound Cutoff Values (ng/mL) Amphetamines 300, 1000 Benzodiazepines* 300 Barbiturates 200, 300 Cannabis metabolite** 20, 50, 100 Cocaine metabolite† 300 Methadone 300 Opiates‡ 300 PCP 25, 75 *Usually as oxazepam. **11-nor-δ-9-tetrahydrocannabinol-9-carboxylic acid. †Benzoyl ecgonine. ‡Morphine.
  3. 3. 965CHAPTER 9.23 Drugs of Abuse the spot answers. The early devices were rather unreliable, but some of the recent products have to be taken seriously and will gain increasing favor over the next few years. The cross-reacting characteristics of the various antisera used are crucial in this area and these are presented in detail for some of the main kits commercially available under the appropriate sections. Space does not allow all products to be dealt with here in such detail, and the relevant informa- tion should be sought from the companies themselves. Although the manufacturers take great pains to evaluate the cross-reactivity of as many potentially interfering sub- stances as possible, their lists are not exhaustive. Although possible cross-reactivity can sometimes be forecast on the basis of a similarity in chemical structure, unrelated sub- stances can cause false-positive results. Moreover, lack of interference in the assays by parent compounds does not always rule out interference by their urinary metabolites. Sometimes a compound can affect the whole assay range as in the example of the antibacterial drug ciprofloxacin, which produces high absorbance readings in EMIT assays. Interference from natural products is very rare, although there have been reports of problems with fluorescence polarization immunoassays in subjects taking abnormally high quantities of riboflavin to treat migraine. It is most important for the analyst to be aware that adulteration of urine samples by donors wishing to avoid detection is a common practice. Immunoassays are vulner- able to changes in the sample matrix, pH, and ionic strength brought about by adding sodium chloride, sodium bicarbonate, bleach, and disinfectant. Other household products such as liquid detergent and hand soap also dis- rupt immunoassays. In theory, tightly-controlled sample collection procedures should eliminate this problem, but it is wise to examine samples carefully for abnormalities before analysis. Apart from visual abnormalities (odd col- ors, soap bubbles, undissolved solids), many laboratories check the pH, creatinine level, specific gravity, and osmo- lality for additional signs of tampering. Finally, the choice of assay system depends on several factors such as sensitivity required, specificity, sample numbers, equipment available, ease of use and, not least, cost. For most systems there are at least two suppliers and one may have the edge over the others when these factors are taken into account by an individual laboratory. In the sections that follow, an example of each system is included and one of the most important characteristics, i.e., speci- ficity, is considered in detail. However, inclusion of an assay here does not imply superiority over those produced by alternative diagnostic companies. AMPHETAMINE Structure See Fig. 1. Dose and Modes of Administration Orally or intravenously, 10–30mg; prolonged abuse leads to tolerance and dosage may exceed 200mg daily. Pharmacological Effects Amphetamine is a potent sympathomimetic amine with respect to stimulation of the CNS. Effective doses pro- duce elevation of mood, increased alertness, self-confidence, and ability to concentrate. Concomitant stimulation of the peripheral nervous system improves physical perfor- mance. The D-isomer (dextroamphetamine) is four times as potent as the L-isomer. Its use as an anorexiant in treat- ing obesity had little success and has long been abandoned. Toxic Effects Chronic abuse of high doses leads to weight loss, halluci- nations, and paranoid psychosis. Acute overdose causes agitation, hyperthermia, convulsions, coma, and respira- tory and/or cardiac failure. Assay Technology See METHYLENEDIOXYMETHAMPHETAMINE. METHAMPHETAMINE Structure See Fig. 2. Dose and Modes of Administration Orally, 2.5–15mg. The D-isomer is abused intravenously by addicts in doses of up to 200mg daily. Insufflation of the free base is also practiced. Pharmacological Effects These are identical to those of amphetamine. The L-isomer has weaker central, but greater peripheral sympathomimetic activity, and is used in some nonpre- scription inhalers as a decongestant. About 5% of a dose of methamphetamine is excreted as amphetamine in the urine. Toxic Effects These are similar to those of amphetamine. Assay Technology See METHYLENEDIOXYMETHAMPHETAMINE. FIGURE 1 Amphetamine. FIGURE 2 Methamphetamine.
  4. 4. 966 The Immunoassay Handbook METHYLENEDIOXYAMPHETAMINE Structure See Fig. 3. Dose and Modes of Administration Methylenedioxyamphetamine (MDA) is a ring-substituted derivative of amphetamine and is a member of the group of drugs popularly known as “Ecstacy” which also includes methylenedioxymethylamphetamine (MDMA) and meth- ylenedioxyethylamphetamine (MDEA). MDA is adminis- tered both orally and intravenously in doses of 50–250mg as an illicit drug. Pharmacological Effects This drug has, in the main, central stimulant properties and large doses induce hallucinations. Toxic Effects Overdosage causes agitation, tremor, tachycardia, hyper- thermia, muscular rigidity, hyperventilation, and coma. Assay Technology See METHYLENEDIOXYMETHAMPHETAMINE. METHYLENEDIOXYMETHAMPHETAMINE Structure See Fig. 4. Dose and Modes of Administration MDMA was previously used as an adjunct to psychother- apy. Drug abusers take oral doses of 100–150mg. It is metabolized to MDA. Pharmacological Effects MDMA has both central and peripheral sympathomimetic activity and doses of around 200mg can lead to visual, auditory, and tactile hallucinations. Toxic Effects MDMA abuse is associated with acid-house parties and deaths have been reported that were due to hyperthermia and to cardiovascular causes after “normal” dosage. Assay Technology (All Amphetamines) Commercial immunoassay kits for detecting amphet- amines in urine specimens have been available for many years. The antibodies used in these individual systems exhibit a wide range of cross-reactivities, particularly toward other drugs derived from phenylethylamine. Some of these, e.g., ephedrine and phenylpropanol- amine, are present in numerous over-the-counter medi- cines and normal use can trigger a positive response in certain amphetamine assays. Wide cross-reactivity can be an advantage if other related and abused drugs such as phentermine, mephentermine, MDA, MDMA, and other members of the Ecstacy group of drugs are to be detected. Some of the Ecstacy compounds, e.g., N-methyl-1-(3,4,-methylenedioxyphenyl)-2-butanamine (MBDB), are missed by the commercial immunoassays and if misuse of these substances spreads an effort will be needed to develop more sensitive tests for this group. The existence of isomeric forms of amphetamine and methamphetamine is a further complication. For exam- ple, L-methamphetamine, which is a much less potent CNS stimulant than D-methamphetamine, is used in non- prescription anticongestant inhalers. Some assays are designed to avoid significant cross-reactivity toward the L-species. The manufacturers are at pains to test antibody specific- ity toward a comprehensive range of common drugs, but their data are not exhaustive. On occasions they overlook the fact that a non-reactive parent drug will be metabo- lized to a reactive product, e.g., the anorectic drugs diethyl propion, clobenox, fenproporex have no cross-reactivity, but are metabolized to amphetamine. Cross-reactivity of other drugs, such as labetolol in the EMIT polyclonal assay, is unpredictable and any findings of this nature should be reported immediately to the manufacturer. Finally, amphetamine immunoassays are only prelimi- nary tests and positive results should always be confirmed by a chromatographic procedure such as GC or, for medical– legal purposes, GC/MS. Enzyme multiplied immunoassay technique EMIT technology was developed in the 1970s by the SYVA company and since that time several other manufac- turers have adapted the principle to produce tests which differ in cross-reactivity, sensitivity, and ease of use. Here the EMIT assays produced by the originators of the sys- tem are presented in detail. Similar data for alternative enzyme immunoassays can be obtained from the respec- tive companies. The EMIT polyclonal assay screens for the entire class of amphetamine compounds. Cross-reactivity data are shown in Table 2. The antibody cross-reactivity toward L-methamphet- amine has not been reported but is probably equivalent to FIGURE 3 Methylenedioxyamphetamine. FIGURE 4 Methylenedioxymethamphetamine.
  5. 5. 967CHAPTER 9.23 Drugs of Abuse that for D-methamphetamine. The L-isomer of metham- phetamine is 10 times less potent than the D-form and is used in the USA in common cold decongestants. To reduce the incidence of false positives, a monoclonal assay kit was developed with much less sensitivity toward the L-isomers of amphetamine and methamphetamine. This kit is also more sensitive toward MDA and MDMA (see Table 3). Both assays respond to phenethylamine compounds, some of which are present in proprietary cold cures, but the monoclonal kit is less prone to interference (see Table 4). An amphetamine confirmation kit is available which con- tains sodium periodate as an oxidant. Compounds with hydroxyl groups proximal to the amino group (e.g., ephedrine, phenylpropanolamine) undergo carbon–carbon bond cleavage at an aliphatic chain or oxidative deamina- tion. Compounds not susceptible to the reaction (e.g., isox- suprine, phentermine) still interfere. For medical–legal purposes, chromatographic confirmation procedures are essential. Other drugs known to cross-react in these assays after therapeutic dosage are listed in Table 5. Cloned enzyme donor immunoassay This technology has been around since 1986 but its commercial development is relatively recent. In the assay, enzyme donor units react with enzyme acceptor units to form a fully active tetrameric molecule of β-galactosidase, which then reacts with a substrate (galactopyranoside) to give a colored product. Competi- tive protein binding means that active enzyme formation and the amount of product depends on the concentra- tion of the analyte present in the same way as for EMIT. Cross-reactivity data for the Cloned enzyme donor immunoassay (CEDIA)™ amphetamine assay are given in Table 6. Although the CEDIA system detects fewer compounds from proprietary medicines, the number of false positives is still substantial. Producing antibodies that bind solely to amphetamine and methamphetamine is probably an unreal- istic goal and, as stated previously, wider specificity can be advantageous when related illicit compounds such as the those of the Ecstasy group (MDMA, MDA) are of concern. TABLE 2 Amphetamine Compounds Producing a Positive Result with the Syva EMIT Polyclonal Assay (Cutoff Level 300ng/mL D,L-amphetamine) Drug Concentration (ng/mL) D-amphetamine 300 D,L-amphetamine 300 D-methamphetamine 1000 MDA 10,000 MDMA 10,000 TABLE 3 Amphetamine Compounds Producing a Positive Result with the Syva EMIT Monoclonal Assay (cutoff Level 1000ng/mL D-Methamphetamine) Drug Concentration (ng/mL) D-amphetamine ≤400 D, L-amphetamine 1000 L-amphetamine 10,000 D-methamphetamine 1000 L-methamphetamine 12,000 MDA 1000 MDMA 3000 TABLE 4 Urine Concentrations (ng/mL) of Amphetamine-Like Compounds Above Which Positive Results May Occur Drug Polyclonal Assay Monoclonal Assay Ephedrine 1000 50,000 Fenfluramine —* 10,000 Mephentermine 500 10,000 Phendimetrazine —* 100,000 Phenethylamine —* 10,000 Phenmetrazine 1000 100,000 Phenylephrine —* 200,000 Phenylpropanol- amine 1000 75,000 *No data available. TABLE 5 Drugs Known to Produce False-Positive Results with Syva EMIT Amphetamine Assays After Therapeutic Dosage Polyclonal Assay Monoclonal Assay Labetalol Chlorpromazine N,N-dibenzylethylenediamine Chloroquine Phenelzine N-acetyl procainamide Procainamide Quinacrine Ranitidine TABLE 6 Cross-Reactivity of Amphetamine-Related Drugs in the CEDIA Amphetamine Assay Compound Cross-Reactivity (%) D,L-methamphetamine 67 L-ephedrine 0.5 D,L-amphetamine 52 MDA 2.2 MDMA 70 Phentermine 1.9 D-phenylpropanolamine <0.1 D-pseudoephedrine 0.6
  6. 6. 968 The Immunoassay Handbook The technology is designed for use on high-throughput clinical analyzers and various advantages in use are claimed. For example, reconstitution of the dried reagents in a volume of buffer is less critical compared to EMIT. One of the reagents (enzyme donor) is colored red and not likely to be confused with the non-colored enzyme acceptor reagent (both of the EMIT reagents are colorless). Abuscreen online In the OnLine series of assays drug–microparticle conju- gates react in solution with free antibodies and this causes aggregation, the rate of which can be measured by a change in light absorbance. In the presence of a test analyte, com- petitive antibody binding brings about a slowing down in the aggregation process that is proportional to the concen- tration of analyte present. Again, this type of technology is aimed for mass screening using automated clinical analyz- ers. Cross-reactivity data for the OnLine amphetamine assay are listed in Table 7. Other structurally-related substances such as ephedrine, mephentermine, and pseudoephedrine showed hardly any cross-reactivity with the assay, even at concentrations greater than 100,000ng/mL. No other drugs have been found to have any significant interference. This amphetamine assay has an extensive dynamic range and good precision at the cutoff, which improves the ability to discriminate between a negative sample and the cutoff and therefore provides a more specific test for amphetamine and methamphetamine. The OnLine assay also contains two monoclonal antibodies for amphet- amine and methamphetamine. Methamphetamine is metabolized to amphetamine and a positive urine sample should contain both compounds. At the 1000 ng/mL cut- off the methamphetamine antibody has very low response to methamphetamine, which means that there is little cross-reactivity toward over-the-counter (OTC) prepa- rations. If amphetamine is present, albeit in low concen- trations, the response is enhanced and the assay will report positive for any sample containing 200 ng/mL amphetamine and 500 ng/mL of methamphetamine. The objective of this design is to reduce the number of sam- ples requiring confirmatory analysis by GC–MS. Enzyme-linked immunosorbent assay Concateno has available a simple ELISA procedure that can be used on urine samples, serum and plasma, saliva, sweat, and also on more difficult matrices such as whole blood and hair extracts. The assay is based on horserad- ish peroxidase-labeled enzyme and antibody immobi- lized on the wall of a 96-well microplate. Sample and enzyme conjugates are incubated in the well for 30 min and after washing the plate the substrate (3,3′, 5,5′-tetra- methylbenzidine) is added. After a further 30 min of incubation, the reaction is stopped by adding sulfuric acid and the absorbance measured at 460 nm within half an hour. See Table 8. The specific amphetamine assay has far less cross- reactivity toward drugs commonly used in cold cures, but at the same time is less sensitive than the metham- phetamine kit toward MDEA and MDMA. However, TABLE 7 Cross-Reactivity of Amphetamine-Related Drugs in the Abuscreen OnLine Amphetamine Assay Compound Cross-Reactivity (%)* 500ng/mL Cutoff 1000ng/mL Cutoff D, L-amphetamine 62 56 p-hydroxyamphet- amine 25 9 L-amphetamine 5 7 D-methamphet- amine 98 0.5 D,L-methamphet- amine 47 0.2 MDA 35 35 MDMA 30 0.2 Phentermine 0.1 <0.2 D-phenylpropanol- amine 0.1 <0.2 β-phenethylamine 1.4 2.3 *Data derived by generating from inhibition curves for each compound and determining for each one the amount equivalent to the 500 and 1000ng/mL D-amphetamine cutoffs. TABLE 8 Cross-Reactivity of Amphetamine-Related Compounds in the Concateno Methamphetamine Microplate EIA Assay Compound Concentra- tion Added (ng/mL) Concentra- tion Found (ng/mL) Cross- Reactivity (%) D-Amphet- amine 1000 100 10 10,000 200 2.0 100,000 600 0.6 β-Phenyl- ethylamine 10,000 <25 — 100,000 79 0.08 250,000 170 0.07 L-Phenylala- nine 100,000 <25 <0.025 L-Ephedrine 10,000 290 2.9 100,000 >500 — Pseudoephed- rine 10,000 25 2.5 100,000 >500 — Phenylpropa- nolamine 10,000 80 0.8 100,000 190 0.19 Phentermine 10,000 60 0.6 100,000 100 0.1 50,000 311 0.62 MDEA 1000 10 1.0 5000 50 1.0 10,000 100 1.0 MDA 10,000 360 3.6 100,000 >500 — MDMA 10 125 1250 25 202 808 50 379 758 100 >500 —
  7. 7. 969CHAPTER 9.23 Drugs of Abuse the specific amphetamine test may still pick up MDMA use due to the strong affinity of the antibody for the MDA metabolite. See Table 9. BARBITURATES Structure See Fig. 5. Dose and Modes of Administration A variety of these barbituric acid derivatives, about 12 in all, are used as sedatives, hypnotics, anesthetics, and anti- epileptic drugs. Short-acting barbiturates, which have effects lasting up to 3 h, are the most commonly abused and include pentobarbitone and seconal (quinalbarbi- tone). These are taken mainly by the oral route in doses of up 200 mg with much larger doses taken by tolerant abusers. One of the long-acting barbiturates, phenobar- bitone, has on occasions been used as a heroin adulterant. Pharmacological Effects Barbiturates act as depressants on the CNS to produce drowsiness and sedation, which is often accompanied by a decrease in mental agility. With increasing dosage the speech becomes slurred and ataxia develops. Toxic Effects Overdose leads to dramatic falls in blood pressure and body temperature, depressed respiration and coma. The clinical syndrome resembles opiate poisoning. Continued use of barbiturates leads to marked tolerance and withdrawal can be hazardous; patients have suffered fatal grand mal sei- zures 2–3 days into withdrawal. Assay Technology The short-acting barbiturates are extensively metabolized by the liver to more polar and pharmacologically inactive hydroxylated compounds and only a very small proportion of the parent compound (<0.2%) appears in the 24h urine. However, with the large doses involved there is usually sufficient of the parent compound present to give an ade- quate response in the immunoassays and some of the hydroxylated metabolites will also cross-react. Secobarbi- tal (quinalbarbitone) is the most commonly used target analyte and calibrator. BENZODIAZEPINES Structure (oxazepam) See Fig. 6. Dose and Modes of Administration The benzodiazepines are the most widely prescribed sedative/hypnotic drugs and over 20 congeners are mar- keted. Because of the huge variation in potency, doses range from 1 to 200mg. Intravenous abuse of liquid encap- sulated forms (e.g., temazepam) occurs, but the usual route is oral. Pharmacological Effects The most prominent effects of benzodiazepines on the CNS are sedation, hypnosis, decreased anxiety, and anti- convulsant activity. There are virtually no effects on the peripheral tissues even in overdose. TABLE 9 Cross-Reactivity of Amphetamine-Related Drugs with the Concateno Amphetamine-Specific Microplate EIA Compound Concentra- tion Added (ng/mL) Concentra- tion Found (ng/mL) Cross- Reactivity (%) L-Phenylalanine 100,000 <25 <0.025 L-Ephedrine 100,000 <25 <0.025 L-Methamphet- amine 100,000 <25 <0.025 Pseudoephed- rine 100,000 <25 <0.025 Phenypropanol- amine 100,000 <25 <0.025 β-Phenethy- lamine 5000 33 0.66 10,000 134 1.3 100,000 442 0.44 Fenfluramine 100,000 <25 <0.025 Phentermine 1000 28 2.8 10,000 134 1.3 50,000 311 0.62 100,000 442 0.44 MDEA 100,000 160 160 MDA 10 21 213 MDMA 100,000 77 0.07 FIGURE 5 Secobarbital. FIGURE 6 Oxazepam.
  8. 8. 970 The Immunoassay Handbook Toxic Effects Chronic abuse leads to blurred vision, confusion, slow reflexes, slurred speech, and hypotension. Benzodiazepines are relatively safe drugs in overdose, and deaths are usually the result of concomitant ingestion of ethanol or other drugs. Assay Technology Benzodiazepines are extensively metabolized by the liver by processes of N-dealkylation and hydroxylation. Only trace amounts of the parent compounds appear in the urine. Hydroxylated metabolites of benzodiazepines and those with a hydroxyl group in the C3 position are conju- gated to glucuronic acid; these conjugates account for the major proportion of the dose eliminated in the urine. The diversity of ring substituents and metabolites is immense, and no immunoassay yet devised can claim to cover all members of the group. The compromise has been to raise antibodies toward the most common metabolites encountered (in particular, oxazepam and nordiazepam), and hope that there is sufficient cross-reactivity toward other products to widen the scope of the assay. Where the therapeutic dose is very low, as in the case of the so-called “date-rape” drug flunitrazepam, immunoassays often give false positives. Hydrolyzing samples with β-glucuronidase can increase detection rates, and some manufacturers incorporate this enzyme in the reagent. For some low- dose benzodiazepines such as alprazolam and triazolam, further improvement can be made by lowering the cutoff level. A comprehensive account of the problems of benzo- diazepine detection by immunoassays can be found in Fraser and Meatherall (1996). Enzyme multiplied immunoassay technique The EMIT d.a.u. benzodiazepine assay detects primarily those drugs that have oxazepam glucuronide as a major uri- nary metabolite. More recent benzodiazepine drugs such as alprazolam and midazolam are also readily detected by this assay. A positive result is based on a response greater than that of a 300ng/mL oxazepam calibrator (see Table 10). Cross-reactivity data relative to oxazepam are listed in Table 11. The EMIT assay is unlikely to detect the use of flu- razepam, flunitrazepam, or triazolam because of poor cross-reactivity and low concentrations of the urinary metabolites. False-positive results have occurred in samples contain- ing the nonsteroidal anti-inflammatory drug, oxaprozin, but no major interference from other non-benzodiazepine drugs has been reported. Cloned enzyme donor immunoassay The CEDIA assays have greater expected rate differences between the zero and cutoff calibrators than, say, EMIT II. This gives better discrimination between blank urine sam- ples and those with drugs at cutoff concentrations. The high sensitivity (HS) protocol benzodiazepine assay also improves the rate of positive detection by an enhancement of the sensitivity toward benzodiazepine glucuronides. Cross-reactivity data for the CEDIA benzodiazepine assay are listed in Table 12. False-positive results have been noted in samples containing metabolites of the antidepressant drug sertraline, but changing the antibodies has now elimi- nated the problem. In other reports, some interference was noted in samples containing the antihistamine embramine and more recently the nonsteroidal anti-inflammatory drug, oxaprozin, has been shown to cause false positives. Abuscreen online The Abuscreen OnLine benzodiazepine system is very sensi- tive and is claimed to detect at least 6ng/mL of nordiazepam. Manufacturers have been criticized in the past for publishing cross-reactivity data only for the parent drugs, but no such accusation can apply with this assay where a long list of ben- zodiazepines have been evaluated together with, in many cases, their metabolites. See Table 13. TABLE 10 Concentrations of Benzodiazepine Compounds Showing a Positive Response in the EMIT d.a.u. Benzodiazepine Assay. Cross-Reactivity Data Relative to Oxazepam Are Listed in Table 11 Compound Concentration (ng/mL) Chlordiazepoxide 3000 Clonazepam 2000 Demoxepam 2000 Desalkyflurazepam 2000 N-desmethyldizepam 2000 Diazepam 2000 Flunitrazepam 2000 Flurazepam 2000 Lorazepam 3000 Nitrazepam 2000 Oxazepam 300 TABLE 11 Relative Cross-Reactivity of Benzodiazepine Derivatives to Oxazepam in the EMIT d.a.u. Benzodiazepine Assay Compound Relative Cross-Reactivity* Chlordiazepoxide 0.03–0.33 Clidinium bromide 0.07 Clonazepam 0.15 Clorazepate 0.25 Demoxepam 0.15 N-desalkylflurazepam 0.14–1.00 Diazepam 0.15–0.63 Flurazepam >0.01–0.23 Hydroxyethylflurazepam >0.1 3-Hydroxydesalkyl-flurazepam 0.50 Lorazepam >0.01–0.23 Medazepam 0.06 Nitrazepam 0.15–0.35 Norchlordiazepoxide 0.17 Nordiazepam 0.15–1.11 Oxazepam 1.00 Temazepam (3-hydroxydiaz- epam) 0.45 *Relative cross-reactivity is defined as the test concentration of oxazepam divided by the concentration of cross-reacting compound required to give an equal response.
  9. 9. 971CHAPTER 9.23 Drugs of Abuse Enzyme-linked immunosorbent assay The Concateno benzodiazepines microplate EIA is designed for use on serum or whole blood and can give a semiquantitative result. The calibrators consist of a protein matrix with temazepam at concentrations of 0, 1, 10, and 100ng/mL (for a description of the kit see AMPHETAMINES). No major interferences have been reported and a list of benzodiazepine cross-reactivity data is given in Table 14. BUPRENORPHINE Structure See Fig. 7. Dose and Modes of Administration Buprenorphine is a synthetic thebaine derivative that has both analgesic and opioid antagonist properties. The usual dose is 0.3mg given parenterally, 0.2–0.4mg by the sublin- gual route, or the application of transdermal patches that deliver doses of 5–70µg/h. Pharmacological Effects The antagonist properties of buprenorphine are equiva- lent to naltrexone but as an analgesic it is significantly more potent than morphine. Although buprenorphine is used for the maintenance therapy of opiate addiction, it has abuse potential and may itself cause dependency. Buprenorphine is metabolized primarily by N-dealkyl- ation to form the pharmacologically active norbuprenor- phine, and by glucuronide conjugation of both parent drug and metabolite. Toxic Effects Symptoms of buprenorphine overdosage include nausea, vomiting, sedation, miosis (constriction of the pupil), hypotension, and respiratory depression. Assay Technology Immunoassay methods for measuring buprenorphine tend to measure the parent drug with the active metabo- lite norbuprenorphine exhibiting little cross-reactivity. A positive urine buprenorphine screen is generally con- sidered to mean concentrations of parent drug or metabolite being present at concentrations exceeding 5 ng/mL. Enzyme-linked immunosorbent assay Designed for use in urine, the Concateno microplate EIA has calibrators containing 0.5, 1, and 5 ng/mL. Cross-reactivity data for norbuprenorphine are shown in Table 15. TABLE 12 Cross-Reactivity of Benzodiazepines in the CEDIA Benzodiazepine Assay Compound Cross-Reactivity (%) Nitrazepam 100 Alprazolam 205 Bromazepam 110 Chlordiazepoxide 13 Clobazem 62 Clonazepam 140 Diazepam 247 Flunitrazepam 135 Flurazepam 190 Lorazepam 122 Medazepam 135 Oxazepam 107 Nordiazepam 210 Temazepam 144 Triazolam 191 TABLE 13 Cross-Reactivity of Benzodiazepine Compounds in the Abuscreen OnLine Benzodiazepine Assay Compound Concentration Equivalent to 100ng/mL Nordiazepam Cross- Reactivity (%) Alprazolam 112 89 α-Hydroxyprazolam 114 88 4-Hydroxyprazolam 116 86 Bromazepam 135 74 Chlordiazepoxide 172 58 Desmethylchlordiaz- epoxide 179 56 Clonazepam 167 60 Demoxepam 128 78 Diazepam 118 85 Oxazepam 139 72 N-Methyloxazepam 127 79 Flunitrazepam 182 55 Desmethylflunitraz- epam 169 59 3-Hydroxyflunitraz- epam 385 26 Flurazepam 164 61 Desalkylflurazepam 175 57 Didesethylflurazepam 125 80 Hydroxyethylfiuraz- epam 123 81 Lorazepam 169 59 Medazepam 345 29 Desmethylmedazepam 286 35 Midazolam 130 29 Nitrazepam 133 75 7-Acetamidonitrazepam 62.500 0.2 7-Aminonitrazepam 189 53 Pinazepam 127 79 Prazepam 139 72 Triazolam 127 79 α -Hydroxytriazolam 115 87 4-Hydroxytriazolam 196 51 No interference from other drugs has been reported.
  10. 10. 972 The Immunoassay Handbook Cloned enzyme donor immunoassay Designed for use in urine, the CEDIA assay has calibrators containing 0, 5, 20, 50, and 75ng/mL. Cross-reactivity data for norbuprenorphine are shown in Table 16. Cross-reactivity with other compounds is generally not significant but there are some exceptions which are shown in Table 17. While these cross-reactivities may seem small, urine samples collected from dihydrocodeine users will fre- quently exceed those concentrations required to produce a positive buprenorphine test. CANNABIS Structure (Tetrahydrocannabinol) See Fig. 8. Dose and Modes of Administration The Cannabis sativa plant produces a resinous substance containing various cannabinoids. The most potent prin- cipal is δ-9-tetrahydrocannabinol (THC). The resin (hashish) contains 3–6% THC and the flowering tops of the female plant (marijuana) 1–3%. Hashish oil is a much more potent form, which contains 30–50% THC. Syn- thetic THC (dronabinol) is marketed by Roxane as an TABLE 14 Cross-Reactivity of Benzodiazepines in the Concateno Microplate Benzodiazepine Assay Compound Concentration (ng/mL) Cross-Reactivity (%) Temazepam 1, 10, 100 100 Alprazolam 1 100 10 40 100 60 Nordiazepam 1, 10 10 100 5 1000 2 Oxazepam 10 5 100 0.5 500 1.0 Triazolam 100 2 1000 0.4 10,000 0.08 Nitrazepam 100 2 1000 0.4 10,000 0.7 Diazepam 1 1 10 10 100 100 Flunitrazepam 10, 100 5 1000 2 Clobazam 10 50 100 21 1000 6.5 TABLE 15 Cross-Reactivity of Norbuprenorphine in Concateno Buprenorphine EIA Cross- Reactant ng/mL Apparent Buprenorphine (ng/mL) % Reactivity Norbuprenor- phine 1000 >5.0 Unknown 500 3.5 0.7 100 1.0 1.0 50 0.8 1.6 10 <0.5 Unknown TABLE 16 Cross-Reactivity in Buprenorphine CEDIA Cross- Reactants ng/mL Apparent Buprenorphine (ng/mL) % Reactivity Norbu- prenorphine 1000 0.6 <0.015 Norbu- prenorphine glucuronide 1000 0.1 <0.015 TABLE 17 Cross-Reactivity of Norbuprenorphine in Concateno Buprenorphine EIA Cross- Reactants ng/mL Apparent Buprenorphine (ng/mL) % Reactivity Codeine 100,000 14.80 0.01 Dihydroco- deine 100,000 11.40 0.01 Hydrocodone 100,000 8.90 0.01 Nalorphine 100,000 86.70 0.09 Naltrexone 100,000 6.70 0.01 Norpropoxy- phene 100,000 5.50 0.01 FIGURE 8 Tetrahydrocannabinol. FIGURE 7 Buprenorphine.
  11. 11. 973CHAPTER 9.23 Drugs of Abuse anti-emetic under the trade name Marinol. Lilly pro- duces a synthetic cannabinoid (nabilone) with the trade name Cesamet that is also used as an anti-emetic. Can- nabis is usually smoked in cigarettes or pipes; oral use in cakes and confectionery is also popular. Although rare, intravenous injection of hashish oil has occurred, often with fatal consequences. An effective smoked dose is about 10mg of THC. Pharmacological Effects Crude plant extracts, synthetic THC, and other cannabi- noids have been used in the treatment of various medical conditions (glaucoma, asthma, multiple sclerosis) and as antinauseants in cancer chemotherapy. THC has sedative and euphoric properties and distorts the sense of space and time. Toxic Effects High and prolonged use has been linked to psychosis. Conjunctivitis is a frequent symptom and deleterious effects on the cardiovascular and respiratory systems can occur. Acute overdose can cause hallucinations, coma, and death. Assay Technology Only a small fraction of a dose of THC is excreted in the urine and therefore immunoassays are designed to detect a major inactive oxidation product, 11-nor-δ-9- tetrahydrocannabinol-9-carboxylic acid (11-COOH-THC). Enzyme multiplied immunoassay technique The EMIT d.a.u. is available in three kits with cutoff levels of 20, 50, and 100ng/mL. The assay detects the major metabolites of THC in urine (see Table 18 and Table 19). Cloned enzyme donor immunoassay The CEDIA d.a.u. multilevel THC system is very similar in performance to EMIT in terms of sensitivity and specificity. Like most non-isotopic assays, it detects about 10% fewer positives than the RIA techniques in compara- tive trials, but most of these false-negative samples are found to contain very low concentrations of the target metabolite, 11-nor-∆9-tetrahydrocannabinol-9-carboxylic acid (11-COOH-THC). Cross-reactivity data are listed in Table 20. The assay can also be extended to blood samples by first extracting with acetone, evaporating the extract, and then reconstituting the residue in a mixture of methanol and buffer. The same process can be used to adapt the FPIA system described below for blood analyses. Abuscreen online The Abuscreen OnLine THC system has been evalu- ated on more than one occasion against other methods. A false-negative rate of around 10% in samples contain- ing cannabinoids was put down to a change in the cali- brator material from a racemic mixture of the D- and L-isomers of 11-nor-∆9-tetrahydrocannabinol-9- carboxylic acid to the naturally occurring L-isomer. Be that as it may, the test is still adequate for screening purposes, and the false-negative samples were usually at the low end of the 11-THC-COOH concentration range. A list of cross-reacting cannabinoids is given in Table 21. More than 100 common drug compounds have been tested against the assay at concentrations of 100,000ng/ mL and none gave values in excess of the assay sensitivity level of 5ng/mL. TABLE 18 Concentrations of THC Metabolites Showing a Positive Response in the EMIT d.a.u. Cannabinoid 50ng Assay Compound Concentrations (ng/mL) 8-β-11-Dihydroxy-δ-9-THC 1000 8-β-Hydroxy-δ-9-THC 1000 11-Hydroxy-δ-8-THC 1000 11-Hydroxy-δ-9-THC 1000 11-Nor-δ-9-THC-9-carboxylic acid 50 TABLE 19 Compounds Shown to Give a Negative Response with the Syva EMIT d.a.u. Cannabinoid 50ng Assay Compound Concentration Tested (µg/mL) Acetylsalicylic acid 1000 Amitriptyline 1000 Amphetamine 100 Benzoylecgonine 400 Chlorpromazine 12* Meperidine 1000 Methaqualone 500 Morphine 200 Oxazepam 300 PCP 1000 Promethazine 125 Propoxyphene 100 Secobarbitone 1000 *Solubility limit for chlorpromazine under assay conditions. TABLE 20 Cross-Reactivity of Cannabinoids in the CEDIA THC Assay Compound Concentration (ng/mL) Cross- Reactivity (%) 11-Nor-∆9-THC-COOH 50 100 11-Nor-∆8-THC-COOH 40 125 ∆9-THC 500 10.4 11-OH-11-∆9-THC 125 43 8-β-OH-11-∆9-THC 1000 2.8 8,11-di-OH-11-∆9-THC 500 8.4 1-∆9-THC-glucronide 62 72 Cannabinol 1000 2.9 Cannabidiol 1000 <0.1
  12. 12. 974 The Immunoassay Handbook Enzyme-linked immunosorbent assay Designed for use in whole blood or serum, the Concateno microplate EIA has calibrators containing 0, 2, 10, and 50ng/mL of 11-nor-∆9-THC-COOH made up in a stabi- lized protein matrix. The cross-reactivity data for cannabi- noids are shown in Table 22. COCAINE Structure See Fig. 9. Dose and Modes of Administration Cocaine is rapidly hydrolyzed and inactivated in the stom- ach and is therefore abused intravenously or by nasal insuf- flation as the hydrochloride salt. Free-base forms (e.g., “crack”) are very potent and give an immediate “high” when smoked. Doses range from 10 to 120mg. Heavy abusers can consume up to 4g daily. Pharmacological Effects Cocaine stimulates the CNS, has local anesthetic proper- ties, and also increases blood pressure, heart rate, and body temperature. The euphoric effects show rapid onset but wear off within the hour, leaving anxiety, fatigue, and depression. Toxic Effects Cocaine has similar actions to amphetamine, and chronic abuse can lead to psychosis. Myocardial infarction, cardiac dysrrhythmias, and cerebrovascular accident can occur with both chronic abuse and overdose. Assay Technology Cocaine is rapidly hydrolyzed by blood cholinesterase to ecgonine methyl ester. Spontaneous chemical hydrolysis to benzoylecgonine also occurs and cocaine is therefore unstable in aqueous solution (and urine) at pH values above neutrality. Immunoassays are targeted at the polar benzoylecgo- nine breakdown product, which accounts for 30–40% of the dose eliminated in the urine. Enzyme multiplied immunoassay technique The EMIT d.a.u. cocaine metabolite kit is the most fre- quently used testing method and has a benzoylecgonine detection sensitivity of 300ng/mL. The assay also detects cocaine and ecgonine at levels greater than 25,000ng/mL and 5000ng/mL, respectively. Concentrations of compounds showing a negative response in the assay are shown in Table 23. No substances producing false-positive results with the assay have yet been reported. Cloned enzyme donor immunoassay This assay compares very favorably with both RIA and other non-isotopic methods such as EMIT in terms of reliability, sensitivity, and specificity. Like the other sys- tems, the target analyte is benzoylecgonine. Cross-reactiv- ity characteristics are listed in Table 24. Like most other immunoassays for cocaine detection the specificity is extremely high and no interfering compounds have been reported. Abuscreen online This system performs as well as any other immunoassay for cocaine detection and has very high specificity for the benzoylecgonine metabolite (Table 25). Enzyme-linked immunosorbent assay The Concateno microplate cocaine kit is designed for use in diluted serum or whole blood to detect the major cocaine metabolite benzoylecgonine. It also shows good cross-reactivity toward cocaine itself and reasonable cross-reactivity for the metabolic product formed by the simultaneous abuse of cocaine and alcohol, cocaethylene (Table 26). TABLE 22 Cross-Reactivity of Cannabinoids in the Concateno Microplate EIA Compound Concentration Added (ng/mL) Cross-Reactivity (%) Cannabinol 10 42 100 34.3 ∆8-THC 5 62 10 42 100 9.2 ∆9-THC 5 114 10 100 100 135 ∆9-THC- glucuronide 5 228 10 163 25 174 FIGURE 9 Cocaine. TABLE 21 Cross-Reactivity of Cannabinoids in the Abuscreen OnLine THC Assay Compound Equivalent Concentration (mg/mL)* Cross- Reactivity (%) 8-α-Hydroxy-∆9-THC 227 22 11-Hydroxy-∆9-THC 278 18 ∆9-THC 455 11 8-β-11-Dihydroxy-∆9- THC 500 10 11-Hydroxycannabinol 1000 5 Cannabinol 2500 2 *Represents the approximate concentration of each compound equivalent in assay reactivity to a 50ng/mL 11- ∆ 9-THC-COOH assay cutoff.
  13. 13. 975CHAPTER 9.23 Drugs of Abuse LYSERGIC ACID DIETHYLAMIDE Structure See Fig. 10. Dose and Modes of Administration LSD is taken orally as the tartrate salt. Doses ranging from 100 to 200µg were common for many years, but recently the tendency has been to take less (30–50µg). Pharmacological Effects The D-isomer of LSD is one of the most potent hallucino- genic agents known (the L-isomer is inactive). This drug belongs to the serotonin class of psychodelics, which dis- rupts the function of the brain 5-hydroxytryptamine sys- tems. LSD is structurally related to naturally occurring ergot alkaloids. Disorientation, euphoria, and hallucina- tion are the prevalent features that occur after a “trip” dose and these subside after 12h. Toxic Effects Recurrent symptoms without further dosage for several months (flashback) have been reported. Overdose can cause delirium and coma, but no deaths have been reported. Assay Technology LSD is extensively metabolized by N-demethylation, N-deethylation, and hydroxylation to inactive metabolites, and only trace amounts of unchanged drug are excreted in the urine. This, together with the minute dosage, makes detection in urine difficult, and for a long time this was feasible only by the most sensitive method, radioimmuno- assay. Over the last 3 years non-isotopic methods have emerged and are gradually gaining favor. In a number of comparative trials, including some where the LSD samples were urines collected from primates dosed with LSD, there have been significant discrepancies between isotopic and non-isotopic immunoassays, and GC–MS. Usually, the immunoassays have given a higher positive rate than TABLE 23 Concentrations of Compounds with a Negative Response to the Syva EMIT d.a.u. Cocaine Metabolite Assay Compound Concentration Tested (µg/mL) Acetaminophen 1000 Acetylsalicylic acid 1000 Amitriptyline 100 Amphetamine 500 Chlorpromazine 12* Cocaine 25 Codeine 500 Dextromethorphan 175 Ecgonine 50 Methadone 500 Methaqualone 100 Monoethylglycinexylidide 1000 Morphine 200 Oxazepam 250 p-Aminobenzoic acid 1000 PCP 750 Procainamide 1000 Propoxyphene 500 Secobarbitone 1000 *Solubility limit for chlorpromazine under assay conditions. TABLE 24 Cross-Reactivity of Cocaine and Metabolites in the CEDIA Cocaine Assay Compound Concentration (ng/mL) Cross-Reactivity (%) Benzoylecgonine 300 100 Cocaethylene 312 57 Cocaine 312 54 Ecgonine 10,000 1.1 Ecgonine methyl ester 10,000 <0.1 TABLE 25 Cross-Reactivity of Cocaine and Metabolites in the Abuscreen OnLine Assay Compound Concentration * Cross- Reactivity (%) Ecgonine 25,000 1.2 Cocaine 30,928 0.97 Ecgonine methyl ester 96,774 0.31 *Represents the approximate concentration of each compound equivalent in assay reactivity to a 300ng/mL benzoylecgonine assay cutoff. TABLE 26 Cross-Reactivity Data of Cocaine and Metabolites in the Concateno Microplate Cocaine Metabolite Assay Compound Concentration Added (ng/mL) Cross-Reactivity (%) Cocaine 10 110 100 101 1000 >30 Cocaethylene 25 64 50 56 100 41 500 24 FIGURE 10 Lysergic acid diethylamide.
  14. 14. 976 The Immunoassay Handbook GC–MS and this could well be due to cross-reactivity with LSD metabolites, which persist in urine for much longer than unchanged LSD. This may prove advantageous in future if the antibodies can be used to isolate these metab- olites and facilitate their identification. Enzyme multiplied immunoassay technique This assay is based on monoclonal antibodies raised against LSD and has a recommended cutoff level of 0.5ng/mL. Cal- ibrators containing 0, 0.5, 1.5, and 2.5ng/mL are supplied, and low and high controls are available. Cross-reactivities for some other abused drugs are given in Table 27. These are very high urine concentrations and are unlikely to be found in real samples. The manufacturers warn that samples from patients tak- ing chlorpromazine may yield false-positive results with this assay. Some workers have reported a false-positive rate of more than 10% in samples from psychiatric patients receiving antipsychotic, antidepressant, and anxiolytic drugs and a much lower and acceptable rate in the general hospital population of about 0.5%. This illustrates the dif- ficulties of evaluating the specificity of an assay which may be applied to samples containing a huge range of other pharmaceuticals and their metabolites, especially when the concentration of analyte sought is so low. Cloned enzyme donor immunoassay This assay has a range of 0–4ng/mL at a cutoff of 0.5ng/ mL in urine and is said to have an intra-assay precision of around 15% and a specificity of 99.9%. Cross-reactivity data for the assay toward other ergotamine type com- pounds are given in Table 28. There is a report of false- positive results in samples from patients on the mucolytic drug ambroxol, even though there is no similarity between this and LSD in chemical structure. Like several other immunoassays for LSD, cross-reac- tivity toward metabolites is very likely, but this has not yet been fully categorized. Abuscreen online This assay performs slightly better than some of the other non-isotopic LSD immunoassays and has good precision around the cutoff concentration of 0.5 ng/mL. The anti- body is raised against LSD itself, but there is a clear evi- dence of binding to LSD metabolites such as nor-LSD, which might explain why it can perform better than GC– MS in controlled trials (Table 29). Enzyme-linked immunosorbent assay The Concateno microplate assay is designed to detect LSD in urine samples. Precision testing at the 0.5ng/mL cutoff gave a coefficient of variation of 6%. The assay stands up well against RIA methods and may have slightly enhanced sensitivity. There is significant cross-reaction with nor- LSD (16–28% depending on the concentration), which is advantageous in a screening assay (Table 30). The assay has also been adapted for use in serum or whole blood samples. No significant interference was found for a whole range of substances similar in structure to LSD (Table 31). When these were tested at concentrations of 10,000ng/ mL the apparent LSD concentration was in all cases less than 0.5ng/mL (% cross-reactivity <0.05). METHADONE Structure See Fig. 11. TABLE 27 Concentrations of Substances Which Gave a Response Equivalent to 0.5ng/mL in the EMIT LSD Assay Compound Concentration (µg/mL) D-amphetamine 500 Ergonovine 1 Methadone 400 D-methamphetamine 100 Methylsergide 3 PCP 30 Propoxyphene 1000 TABLE 28 Cross-Reactivity of Compounds Structurally Related to LSD in the CEDIA LSD Assay Compound Concentration (ng/mL) Cross-Reactivity (%) Dihydroergotamine 125,000 <0.001 Alpha ergotamine 500,000 <0.001 Lysergic acid 100,000 <0.001 Lysergol 50,000 <0.0001 Ecgonine 100,000 <0.001 Serotonin 1,000,000 <0.001 Ecgonine methyl ester 100,000 <0.001 Psilcybin 10,000 <0.001 Psylocyn 10,000 <0.001 TABLE 29 Cross-Reactivity of LSD Metabolites and Compounds Structurally Related to LSD in the Abuscreen OnLine Assay Compound Concentration (ng/ mL) Equivalent to 0.5ng/mL LSD Cross- Reactivity (%) 2-Bromo- α -ergocryptin >20,000 <0.0025 iso-LSD 21 2.2 Lysergic acid N-(methylpropyl) amide 3.5 24 Lysergic acid N-(hydroxyethyl) amide 1560 0.034 D-Lysergic acid >20,000 0.0025 N-Demethyl-LSD (nor-LSD) 1.4 44 Methylsergide maleate 5500 <0.024 α-Ergocryptine >20.000 <0.0025 Ergotamine tartrate 13,300 0.0039 Ergonovine maleate 13,000 0.0063
  15. 15. 977CHAPTER 9.23 Drugs of Abuse Dose and Route of Administration Methadone is a favored heroin substitute in opiate with- drawal programs, and tolerant patients are given the drug orally as a linctus in doses of 40–100mg daily. It is also available as a racemic mixture of the hydrochloride salt, as tablets of 5–10mg, and as a 10mg/L solution for paren- teral injection. Pharmacological Effects Methadone has analgesic properties similar to morphine but has marked sedative effects due to drug accumulation. Although given as D,L-methadone, only the L-isomer is active. Toxic Effects Methadone overdose causes stupor, respiratory depres- sion, hypotension, coma, and circulatory collapse. Doses of 50mg can prove fatal in non-tolerant adults. Assay Technology Methadone is metabolized by mono- and di-N-demethyl- ation to unstable metabolites that cyclize spontaneously to give 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP) and 2-ethyl-5-methyl-3,3-diphenylpyrroline (EMDP). These, together with methadone, are the main urinary excretion products. The commercial immunoas- says detect methadone and cross-react poorly or not at all with EDDP or EMDP. This can be a disadvantage when testing urine samples taken a long time after the last dose, since the metabolites may still be present when methadone itself is below the detection limit. Since methadone is pre- scribed on a huge scale, there is a danger of supplying the street market if drug abusers obtain doses under false pre- tences by spiking their urine samples with the drug. Most immunoassays fail to distinguish these as adulterated sam- ples. This has resulted in the development of assays that are aimed at picking up the metabolite EDDP and have very low cross-reactivity toward methadone itself (see Table 32). Enzyme multiplied immunoassay technique The EMIT d.a.u. methadone assay is set to detect metha- done in urine at a threshold or “cutoff” concentration of 300ng/mL. High concentrations of the antihistamine drugs, doxylamine, and diphenhydramine, are also detected, but no interference from other compounds has been reported (see Table 33). Cloned enzyme donor immunoassay The CEDIA methadone assay recommends a cutoff of 150ng/mL. Again there is poor cross-reactivity with the methadone metabolites (Table 34). Abuscreen online This assay uses a cutoff value for methadone in urine of 300ng/mL, and the polyclonal antibodies are raised against the parent drug. Table 35 gives the cross-reactivity data for related compounds and other common drugs. Like most methadone immunoassays, there is the disad- vantage of very little reaction with the methadone metabo- lites EDDP and EMDP. Detection of the methadone alternative, L-α-acetylmethadol (LAAM) is possible at high doses. Enzyme-linked immunosorbent assay The Concateno microplate assay for diluted serum or whole blood has methadone calibrators of 5, 25, and 100ng/mL. Methadone toxicity can develop at a blood concentration of about 0.2mg/L (200ng/mL) which leaves plenty of scope for dilution of forensic samples into the analytical range. There is significant cross-reactivity with the methadone substitute LAAM, but in a forensic TABLE 30 Cross-Reactivity of nor-LSD in the Concateno Microplate LSD (Urine) Assay Compound Concentration (ng/mL) Cross-Reactivity (%) Nor-LSD 1 25 2.5 28 5 20 10 16 25 >20 TABLE 31 Compounds Similar in Structure to LSD Which Have Insignificant Cross-Reactivity in the Concateno Microplate LSD (Urine) Assay Dihydroergocristine Ergosine (base) Dihydroergotamine Ergosinine (base) Ergocornine Ergotamine tartrate Ergocryptine Lysergic acid Ergocristine Serotonin Ergometrinine (base) L-trytophan FIGURE 11 Methadone. TABLE 32 Cross-Reactivity of Parent Compounds and Metabolites, When Tested with the CEDIA EDDP Assay, 100ng/mL Protocol Compound Concentration Tested (ng/mL) % Cross- Reactivity EDDP EMDP α -Levo-acetylmethadol α -Levo-noracetylmethadol α -Levo-dinoracetylmethadol 100 200,000 600,000 1,000,000 1,000,000 100 0.004 0.016 0.000 0.000
  16. 16. 978 The Immunoassay Handbook case this would be discovered by additional and more dis- criminating tests. Cross-reactivity data are presented in Table 36. OPIATES Structure See Figs 12–14. Dose and Modes of Administration The opium poppy contains two narcotic analgesics, namely morphine and codeine. Synthetic derivatives such as diace- tyl morphine (heroin), hydrocodone, and dihydrocodeine TABLE 33 Concentrations of Compounds Showing a Negative Response in the EMIT d.a.u. Methadone Assay Compound Concentration Tested (ng/mL) Acetaminophen 1000 Acetylsalicylic acid 1000 Amitriptyline 50 Amphetamine 500 Benzodiazepine 400 Chlorpromazine 12* Codeine 500 Dextromethorphan 300 Diphenhydramine 100 Meperidine 200 Methaqualone 100 Morphine 200 Naloxone 500 Oxazepam 250 PCP 500 Promethazine 75 Propoxyphene 300 Secobarbitone 100 *Solubility limit for chlorpromazine under assay conditions. TABLE 34 Cross-Reactivity of Methadone-Related Substances in the CEDIA Methadone Assay Compound Concentration (ng/mL) Cross- Reactivity (%) Methadone 300 100 α-Methadol 33,333 2.65 EDDP 500,000 0.02 EMDP 100,000 0.03 LAAM 20,000 1.48 Methadol 25,000 1.50 Morphine-3-glucuronide 100,000 0.01 Norpropoxyphene 500,000 0.03 Propoxyphene 500,000 0.03 TABLE 35 Cross-Reactivity of Various Substances in the Abuscreen OnLine Methadone Assay Compound Concentration* (ng/mL) Cross-Reactivity (%) Methadol 250 120 Hydroxymethadone 577 52 LAAM 1000 30 Promethazine 12,000 2.5 Diphenhydramine 60,000 0.50 Amitriptyline 91,000 0.33 Chlorpheniramine 91,000 0.33 Doxylamine 100,000 0.30 Imipramine 100,000 0.30 D-Propoxyphene 107,000 0.28 Benzphetamine 130,000 0.23 Meperidine (pethidine) 136,000 0.22 Dextromethorphan 150,000 0.20 Chlorpromazine 250,000 0.12 EDDP 273,000 0.11 EMDP 333,000 0.09 *Represents the concentration needed to give a reaction equivalent to the methadone 300ng/mL cutoff. TABLE 36 Cross-Reactivity of Substances Related to Methadone in the Concateno Microplate Methadone Assay Compound Concentra- tion Added (ng/mL) Concentra- tion Found (ng/mL) Cross- Reactivity (%) LAAM 5 3.3 67 10 10.1 101 25 31.7 127 1000 191.6 19.1 EDDP 100 0.7 0.69 10,000 2.6 0.03 100,000 9.2 0.01 EMDP 100 0.6 0.55 10,000 2.0 0.02 100,000 13.0 0.01 FIGURE 12 Morphine.
  17. 17. 979CHAPTER 9.23 Drugs of Abuse are more properly classified as opioids, but their detection is also considered in this section. Heroin is the most commonly abused derivative and tol- erant addicts take up to 200mg daily either by intravenous injection or nasal insufflation (snorting). Inhalation of vaporized heroin, colloquially known as “chasing the dragon,” is also practiced. Pharmacological Effects These drugs are potent depressants of the CNS, causing analgesia, euphoria, and narcosis. Regular use leads to tol- erance and physical dependence. Toxic Effects Opiate poisoning is characterized by pinpoint pupils, respiratory depression, and deep coma. Following over- dosage by intravenous injection, death can occur within a few minutes. Assay Technology Heroin is rapidly metabolized to morphine, which is excreted in the urine mainly as glucuronide conjugates. The immunoassays are therefore directed toward mor- phine, which is also a metabolite of codeine. Other phen- anthrene narcotics such as codeine itself, dihydrocodeine, hydromorphine, and levorphanol and their metabolites also cross-react to varying degrees. Pholcodine (β-morpholinylethylmorphine) is widely used in cough remedies in Europe and gives a strong posi- tive response in some immunoassays. Foods containing poppy seeds, which are a source of morphine and codeine, can also yield positive urine samples by immunoassay. It is important, therefore, to reanalyze urine samples that are positive by specific chromatographic methods to discern which opioid congeners are present. Fully synthetic opioids such as meperidine (pethidine), propoxyphene, and methadone show little or no cross- reactivity in these immunoassays. Enzyme multiplied immunoassay technique The EMIT d.a.u. kit detects morphine, morphine-3-gluc- uronide, and codeine in urine. Related synthetic opiates such as dihydrocodeine, levorphanol, and pholcodine are also detected. High concentrations of meperidine (pethi- dine) and the narcotic analgesic, nalorphine, can give a positive response, but no significant interference from other compounds structurally unrelated to morphine has been reported. The kit is supplied with low and medium calibrators containing 300ng/mL and 1000ng/mL of mor- phine, respectively. A positive result is based on a response equal to or greater than that of the low calibrator (see Table 37). The compounds listed in Table 38 gave negative results. Cloned enzyme donor immunoassay The CEDIA d.a.u. Opiate kit has more sensitivity than some of the other non-isotopic assays for the target drugs morphine and codeine due to its greater rate separation (milliabsorbance units change per minute) between the negative and the 300ng/mL cutoff calibrator. It is also more selective toward morphine and codeine, but like most other immunoassays for this class of drugs, there is considerable cross-reactivity with other opiate drugs and their metabolites, the exceptions being oxycodone and its metabolite, oxymorphone (Table 39). Abuscreen online The OnLine opiates assay compares well with RIA and has somewhat better precision around the cutoff point (300ng/ mL) than, for example, EMIT II. It also has a more stable calibration curve which can reduce reagent and time costs in laboratories carrying out large numbers of assays rou- tinely. Some cross-reactivity data for opiates are given in Table 40. FIGURE 13 Codeine. FIGURE 14 Heroin. TABLE 37 Concentration of Compounds Showing a Positive Response in the Syva EMIT d.a.u. Opiate Assay Compound Concentration (ng/mL) Codeine 1000 Dihydrocodeine 260 Hydrocodone 1000 Hydromorphine 3000 Levallorphan 1000 Levorphanol 300 Monoacetylmorphine 460 Morphine 300 Morphine-3-glucuronide 3000 Oxycodone 50,000 Norlevorphanol 23,000 Oxymorphone 82,000
  18. 18. 980 The Immunoassay Handbook Enzyme-linked immunosorbent assay Concateno Bioscience produces two microplate opiate assays, both of which are for use in diluted serum or whole blood. One is for general opiate detection and picks up codeine and several other opiate drugs and the other is designed for the specific quantification of morphine and has calibrators of 0, 5, 10, 25, 50, and 100ng of morphine (Tables 41 and 42). HEROIN METABOLITE Structure See Fig. 15. Dose and Modes of Administration Heroin (diacetylmorphine, diamorphine) is a highly addic- tive Schedule 1 substance that is rarely used outside pallia- tive care. It is the most abused opiate drug and is generally administered via nasal insufflation or by intravenous or subcutaneous injection. Pharmacological Effects Esterases present in the blood rapidly metabolize heroin to 6-monoacetylmorphine and this process has a half-life of TABLE 39 Cross-Reactivity of Opiate Drugs in the CEDIA Opiate Assay Compound Concentration (ng/mL) Cross- Reactivity (%) Morphine 300 100 Codeine 300 125 Diacetylmorphine 300 53 Dihydrocodeine 300 50 Hydrocodone 300 48 Morphine-3-glucuronide 300 81 Morphine-6-glucuronide 300 47 6-Monoacetyl-morphine 300 81 Oxymorphone 20,000 1.9 Oxycodone 10,000 3.1 TABLE 40 Cross-Reactivity of Opiates in the Abuscreen OnLine Opiates Assay Compound Concentration (ng/mL)* Cross- Reactivity (%) Codeine 225 134 Ethyl morphine 265 113 6-Monoacetylmorphine 311 97 Dihydrocodeine 317 95 Thebaine 351 85 Dihydromorphine 371 81 Hydrocodone 479 63 Morphine-3-glucuronide 480 62 Hydromorphone 620 48 Norcodeine 11,744 3 Oxycodone 23,166 1 *Represents the approximate concentration equivalent in assay reactivity to the 300ng/ mL morphine cutoff calibrator. TABLE 41 Cross-Reactivity of Opiates in the Concateno Microplate Opiates Assay Compound Concentration (ng/mL) Cross- Reactivity (%) Codeine 10 577 Morphine-3-glucuro- nide 10 17.6 100 6.0 1000 3.0 6-Monoacetylmorphine 10 62.4 100 28.1 1000 34.7 Normorphine 10 23 100 2.9 1000 0.7 Nalorphine 10 12.5 100 3.0 1000 0.8 Diacetylmorphine 10 45.4 100 30.0 1000 22.7 Hydromorphone 10 45.8 100 20.1 1000 18.7 Hydrocodone 10 262 100 102 TABLE 38 Compounds Showing a Negative Response in the Syva EMIT d.a.u. Opiate Assay Compound Concentration (µg/mL) Alphaprodine 74 Amphetamine 1000 Benzoylecgonine 1000 Buprenorphine 1000 Butorphanol 1000 Chlorpromazine 12 Dextromethorphan 175 Doxylamine 1000 Meperidine 20 Meptazinol 100 Methadone 500 Nalbuphine 1000 Nalorphine 20 Naltrexone 5000 Naloxone 150 Norpropoxyphene 1000 Oxazepam 250 Papaverine 1000 Pentazocine 1000 PCP 1000 Propoxyphene 1000 Secobarbitone 1000
  19. 19. 981CHAPTER 9.23 Drugs of Abuse approximately 9min. Further metabolism of 6-monoace- tylmorphine to morphine by the liver occurs with a half- life of 40min. This relatively short half-life reduces the detection time window for heroin but the measurement of 6-monoacetylmorphine remains the only conclusive test for heroin abuse. Toxic Effects Symptoms of heroin overdosage include severe depression of the respiratory system and CNS. Assay Technology A positive urine 6-monoacetylmorphine screen is regarded as a specific marker for heroin abuse since it cannot be formed by acetylation of morphine in the body. The detec- tion time for the metabolite, although dependent upon heroin dose, is unlikely to exceed 24h after use. Cloned enzyme donor immunoassay The parent compounds, metabolites, and structurally related compounds shown in Table 43 yielded negative results against a cutoff calibrator of 10ng/mL. OXYCODONE Structure See Fig. 16. Dose and Modes of Administration Oxycodone is a semisynthetic narcotic analgesic that has been in use since 1939. The drug is available as injectable solution (10mg/mL), capsules of up to 20mg, mixtures, and various sustained-release preparations for the relief of moderate to severe pain. Pharmacological Effects The analgesic effects of oxycodone are similar to those of morphine and consequently the drug has abuse potential. Oxycodone is metabolized by N- and O-demethylation, 6-keto reduction and conjugation. One of its metabo- lites, oxymorphone, is also a potent narcotic analgesic. Toxic Effects Toxic effects are similar to those seen with morphine, i.e., depression of the respiratory system and CNS. Assay Technology Enzyme-linked immunosorbent assay Designed for use in urine, the Immunalysis microplate EIA kit has high specificity for oxycodone, but as Table 44 demonstrates there is significant cross-reactivity for the metabolite oxymorphone. PHENCYCLIDINE (PCP) Structure See Fig. 17. TABLE 42 Cross-Reactivity Data for the Concateno Specific Microplate Morphine Assay Compound Concentration (ng/mL) Cross- Reactivity (%) Codeine 1000 <1 Morphine-3-glucuronide 1000 <1 10,000 0.62 Diacetylmorphine 1000 <1 10,000 0.2 Normorphine 10 40 100 30 Nalorphine 10 490 6-Monacetylmorphine 100 <10 1000 <1 Hydromorphone 100 <10 1000 <1 FIGURE 15 6-monoacetylmorphine. TABLE 43 Compounds, Metabolites and Structurally Related Compounds Yielding Negative Results Against a Cutoff Calibrator of 10ng/mL (CEDIA) Compound Concentration Tested (ng/mL) Codeine Dextromethorphan Dihydrocodeine Heroin HCl Hydrocodone Hydromorphone Imipramine Levorphanol Meperidine Morphine Morphine-3-Glucuronide Morphine-6-Glucuronide Nalorphine Naloxone Naltrexone Norcodeine Normorphine Oxycodone Oxymorphone 500,000 100,000 500,000 80 300,000 10,000 200,000 10,000 800,000 9000 600,000 600,000 7000 300,000 300,000 600,000 30,000 400,000 80,000 FIGURE 16 Oxycodone.
  20. 20. 982 The Immunoassay Handbook Dose and Modes of Administration PCP is used in doses ranging from 2 to 6mg by smoking with tobacco, nasal insufflation (snorting), intravenous injection, and oral ingestion. Its abuse is virtually unknown outside the USA. Pharmacological Effects PCP has a legitimate use as a veterinary tranquilizer and was developed for human use as an intravenous anesthetic agent, being similar in structure to ketamine. It no longer has any therapeutic uses in humans because of its pro- nounced hallucinogenic properties. Toxic Effects Abuse of PCP causes lethargy, hallucinations, and loss of coordination. Signs of intoxication include hypertension, seizures, violent behavior, coma, and respiratory depres- sion. Doses of 100–120mg can cause death from respira- tory failure. Chronic abuse can lead to memory loss, inarticulation, depression, and a PCP psychosis that recurs on exposure to the drug. Assay Technology PCP undergoes oxidative metabolism to at least two inac- tive metabolites, which appear in the urine as glucuronide conjugates. The immunoassays are relatively specific for PCP and its metabolites. Enzyme multiplied immunoassay technique The EMIT d.a.u. PCP assay has a cutoff of 25ng/mL. The compounds in Table 45 gave negative results against the low calibrator when tested at the specified levels. Cloned enzyme donor immunoassay The CEDIA PCP assay has an average limit of detection in urine of 0.6ng/mL. Apart from one report of a false- positive finding in a sample containing diphenhydramine, the test is highly specific, and cross-reactivity data for various metabolites and related substances are listed in Table 46. Abuscreen online The Abuscreen OnLine assay has analytical sensitivity of around 5ng/mL. A limited amount of cross-reactivity data is available for this assay, but no interference from other substances has yet been reported (Table 47). PROPOXYPHENE Structure See Fig. 18. FIGURE 17 Phencyclidine. TABLE 45 Compounds Found to Give Negative Results with the Syva EMIT d.a.u. PCP Assay Drug Concentration (µg/mL) Acetaminophen 1000 Albuterol 1000 Benzoic acid 32 Buspirone 914 Cimetidine 1000 Dextrorphan 100 Diclofenac 1000 Diphenhydramine 1000 Fentanyl 100 Haloperidol 1000 Hydroxyzine 50mg dose Ketoprofen 1000 Levallorphan 10 Mesoridazine 10 Methenamine 162 Norlevorphanol 1000 Normeperidine 1000 Norpropoxyphene 1000 Orphenadrine 1000 Oxycodone 1000 Pentazocine 1000 Phenytoin 30 Salicylamide 65 Sodium salicylate 97 Terfenadine 1000 Thioridazine 1000 Tripelennamine 1000 TABLE 44 Cross-Reactivity Data for the Immunalysis Microplate EIA for Oxycodone Compound Concentration (ng/mL) Oxycodone Equivalents Cross- Reactivity % Morphine Codeine Hydromor- phone Hydroco- done Oxymor- phone Norcodeine Normor- phine Morphine-3- glucuronide Meperidine Naloxone Tramadol 10,000 10,000 10,000 10,000 200 10,000 10,000 10,000 10,000 10,000 10,000 5 7 30 50 100 ND ND 5 ND ND ND 0.05 0.07 0.3 0.5 50 ND ND 0.05 ND ND 0
  21. 21. 983CHAPTER 9.23 Drugs of Abuse Dose and Mode of Administration Dextropropoxyphene is marketed as an oral preparation either as the hydrochloride (32 or 65mg) or as the napsyl- ate (50 or 100mg) and used in daily doses of around 400mg. It is often formulated in combination with aspirin or paracetamol and prescribed on a large scale for the relief of chronic pain. There are many reports of its use success- fully in heroin maintenance or withdrawal treatment pro- grams in doses of 800–1400mg/day. Pharmacological Effects Dextropropoxyphene is a mild narcotic analgesic, less potent than codeine, and closely related to methadone in chemical structure. The L-isomer has virtually no narcotic effects and is used as an antitussive (cough suppressant). Toxic Effects These are similar to methadone in overdose with symp- toms of respiratory depression, stupor, hypotension, coma, and circulatory collapse. It has a long half-life and therefore the toxic effects are prolonged. There are many reports of fatal overdose and the minimum lethal dose in adults is estimated to be between 500 and 800 mg. Assay Technology Most commercial immunoassay systems have propoxy- phene in the portfolio and are targeted at the parent com- pound. There is usually a good cross-reactivity toward the demethylated metabolite, norpropoxyphene, but little response toward methadone despite the similarity in chemical structure. TRAMADOL Structure See Fig. 19. Dose and Modes of Administration Tramadol is a centrally-acting synthetic opioid that has been used as a narcotic analgesic since 1977. It is available as oral 50mg dispersible tablets, oral drops (100mg/mL), injection (50 and 25mg/mL), 50mg sachets, and sustained- release preparations of between 50 and 400mg. Pharmacological Effects Tramadol acts centrally as a weak opioid receptor agonist but it also inhibits the reuptake of serotonin and norepi- nephrine neurotransmitters. Tramadol has similar potency to that exhibited by codeine but causes less respiratory depression. Although considered to have lower abuse potential than opiate drugs, tramadol misuse is becoming more common. Toxic Effects Tramadol overdose may produce significant neurological toxicity, and long-term abuse may make individuals sus- ceptible to seizures. Extensive CYP2D6 metabolizers or those with renal impairment are at a greater risk of toxicity. Assay Technology Enzyme-linked immunosorbent assay Designed for use in urine, the Immunalysis microplate EIA kit has high specificity for tramadol and only limited sensitivity to the drug’s metabolites, as the Table 48 demonstrates. TABLE 46 Cross-Reactivity Data for the CEDIA PCP Assay Compound Concentration (ng/mL) Cross- Reactivity (%) 1-Phenylcyclohexyl-4- hydroxypiperidine 32 106 4-Phenyl-4-piperidine- cyclohexanol 1000 2.5 Phenylcyclohexylamine 25 68 5-(1-Phenylcyclohexyl- amine) 1000 0.6 1-[1-(2-Thienyl) cyclohexyl]-piperidine 100 31 Phenylcyclohexylamine 100 37 TABLE 47 Cross-Reactivity Data for the Abuscreen OnLine PCP Assay Compound Concentration* Cross- Reactivity (%) Dextromethorphan 272,000 0.01 Thienylcyclohexyl- piperidine 31 80 *Approximate concentration equivalent to the 25ng/mL PCP cutoff. FIGURE 18 Propoxyphene. FIGURE 19 Tramadol.
  22. 22. 984 The Immunoassay Handbook Legal Addictive Substances ALCOHOL (ETHYL ALCOHOL) AND THE USE OF CARBOHYDRATE-DEFICIENT TRANSFERRIN Toxic Effects of Alcohol The occasional bout of heavy social drinking usually causes nothing more serious than the morning-after hangover effects of headache, dehydration, and gastric upset, and most people recover as the day moves on. Deaths from overindulgence do occur, however, for example when an individual loses consciousness and aspirates vomit into the lungs. Continual heavy intake (alcoholism) leads to chronic poisoning and some of the features are liver disease (cir- rhosis), cardiac abnormalities, nerve degeneration, and loss of mental function. Those who abuse alcohol are notoriously reluctant to admit how much they imbibe, even when the information is asked for by medical person- nel trying to evaluate their health. Laboratories have been able to measure blood alcohol levels for many years and, for a long time, simple methods of measuring alcohol in saliva or breath samples have been around. The drawback of this approach is that if the abuser is given notice of the date and time of the test he/she may well refrain from drinking for a day or so and the alcohol result will be nega- tive. Even a positive alcohol measurement is only a snap- shot of the situation at the time and is no indicator of how substantial and regular the abuse is, or how long it has been going on. This has led to a search for biological markers of alcohol abuse and one of these, carbohydrate- deficient transferrin (CDT), is measured in serum by immunoassay techniques. This is now accepted as the most sensitive and specific test for diagnosing heavy alcohol consumption, but there are some flaws in that it is more specific in men than women and less sensitive when applied to non-hospital populations such as university students. Like many other diagnostic biological tests it is best com- bined with measurement of other parameters such as γ-glutamyltransferase and erythrocyte aldehyde dehydro- genase (ADH). Carbohydrate-Deficient Transferrin The glycoprotein, transferrin, takes part in the delivery of iron to the body tissues and is synthesized in liver cells. Chronic exposure to alcohol disrupts this process, possibly by acetaldehyde-mediated inhibition of glyco- syl transfer, and a proportion of the resulting transferrin is missing some carbohydrate terminal chains (e.g., sialic acid, galactose, N-acetylglucosamine). Hence the term CDT. At least 1 week of heavy drinking is needed to give raised serum CDT levels and these normalize slowly with a half-life of about 15 days after drinking stops. Assay Technology The first methods concentrated on a combination of iso- electric focusing with immunofixation techniques and were soon superceded by isocratic anion-exchange chro- matography. In this procedure iron-saturated serum is passed through a microcolumn and the separated isotrans- ferrins quantified by radioimmunoassay. The first com- mercial kit used a similar principle of separating the isoforms on a microcolumn and quantifying the eluted deficient transferrins by a double antibody immunoassay. In a second kit, serum transferrin is radiolabeled using antibody fragments and the carbohydrate-deficient form separated on an ion-exchange chromatography minicol- umn. The relative amount of CDT eluted is measured by counting the radioactivity, and any elevation above 2.5% is considered diagnostic of alcohol abuse. In comparative tri- als, the first kit was found to be more sensitive, but altera- tions in serum transferrin concentration markedly reduced the specificity of the assay. More recently, commercial competitive-binding enzyme immunoassays have been introduced. Again the transferrin isoforms are separated on an ion-exchange microcolumn and the eluted CDT then quantified by ELISA technology. An alternative approach has been to go back to isoelec- tric focusing, use direct immunofixation by a specific anti- body,andquantifybycomputerizedscanningdensitometry. This is quite an easy method to carry out, is inexpensive, and has a reputed specificity of 100% (defined as the ability to give normal results when there is no heavy alcohol use) and a sensitivity of about 95% (defined as the ability to detect heavy alcohol consumption when it really exists). The method has been described in detail by Dumon et al. (1996). The technology has now been adapted to evaluate CDT derived from dried blood spots. NICOTINE AND THE USE OF COTININE Structure See Fig. 20. Dose and Mode of Administration Tobacco is the most abundant source of nicotine and the leaves contain 1–6% by weight. Smoking is by far the most popular means of absorbing nicotine, although chewing tobacco or snorting powdered tobacco (snuff) are still practiced on a small scale. The average cigarette contains TABLE 48 Cross-Reactivity Data for Immunalysis EIA for Tramadol Compound Concentra- tion (ng/ mL) Tramadol Equivalents Cross- Reactivity % O-Desmeth- yltramadol 1000 5000 20,000 0 <50 <100 ND <1 <0.5 N-Desmeth- yltramadol 500 1000 150 225 30 22.5 Venlafaxine 50,000 100,000 0 <50 ND <0.05 O-Desmeth- ylvenlafaxine 100,000 0 ND N-Desmeth- ylvenlafaxine 100,000 0 ND
  23. 23. 985CHAPTER 9.23 Drugs of Abuse 13–19mg of nicotine and cigars have 15–40mg. During smoking a significant amount of nicotine is lost by com- bustion and in the side-stream smoke. Depending on the depth of inhalation, cigarette smokers take in 0.2–2.5mg through the lungs. Cigar and pipe smokers absorb nicotine predominantly by the mouth and take in 10–50% via this route. In recent years, the dangers of non-smokers being exposed to nicotine by inhaling smoke (passive inhalation) have been emphasized. Nicotine can also be absorbed through the skin and this property has been exploited by designing skin patches impregnated with nicotine that continually deliver small amounts to the blood stream to help stave off the craving. Nicotine patches contain up to 50mg of the drug and deliver doses of 5–21mg. An alter- native formulation is nicotine-impregnated chewing gum and each tablet contains from 2 to 4mg. Pharmacological Effects Nicotine causes stimulation of the autonomic ganglia and the CNS. Toxic Effects Nicotine is an extremely toxic substance and it has been estimated that as little as 40mg can be lethal in an adult. A lethal dose causes paralysis of the CNS, including the respiratory center, hypotension, tachycardia, muscle paral- ysis, convulsions, and death within a few minutes to 1h after ingestion. Fortunately, acute nicotine poisoning is rare and is usually associated with accidental exposure to concentrated nicotine insecticide solutions. There is far more concern about the pronounced carcinogenic effects of other components of tobacco smoke and also of the con- tribution of the smoking habit to heart disease. Assay Technology Nicotine is extensively metabolized by the liver to form largely inactive products. One of the main urinary excre- tion products is the oxidized form (cotinine) and this has been the favored antigen in developing tests for smoking patterns. A substantial amount of quantitative data has been accumulated that can be used to judge whether an individual is an active or passive smoker and how much tobacco is being consumed. From an analytical view point, cotinine measurements have the advantage being able to discount problems of equipment and biological samples being contaminated by environmental nicotine. To obtain reliable data for nicotine itself calls for the analyses to be carried out under almost aseptic conditions. Non-isotopic immunoassay is the most commonly used method for measuring cotinine in either serum, oral fluid, or urine samples, but the results of comparative trials against GC methods such as that described by Biber et al. (1987) show large discrepancies in the quantitative data. This is most likely due to problems of varying non-specific binding, although it should be pointed out that in some of these trials there have also been substantial inter-laboratory discrepancies among the GC groups. However, the differ- ence in serum cotinine concentrations between samples from non-smokers exposed to environmental tobacco smoke and people who smoke actively is substantial, and both types of assay have been shown capable of making a clear distinction. From a practical standpoint, this is often the only information required. Steroid Abuse in Sport ANABOLIC ANDROGENIC STEROIDS Anabolic androgenic steroids (AAS) are based on the naturally-occurring testosterone and numerous forms are available for legitimate use in medicine to treat a variety of conditions ranging from osteoporosis, dis- seminated breast cancer, protein deficiency states through to anemia, and post-traumatic catabolism. Abuse by athletes, in particular weight lifters, throwers (discus, shot), and sprinters, wishing to increase muscle bulk has been a cause of great concern in sports organi- zations for many years and detection of this practice presents a major challenge to analysts working in the sports testing field. This includes analysts concerned with the integrity of animal sports such as horseracing. AAS are extensively metabolized and the parent drugs are detectable only for short periods after administra- tion. As a consequence, analytical strategies are directed toward identifying the metabolites to extend the detec- tion time period. Schanzer (1996) has given a very com- prehensive account of the structures and metabolism of these compounds. A major problem is that endogenous steroids such as testosterone and dihydrotestosterone, which is an active metabolite of testosterone, are the most often used and finding an unusually high urine concentration of these is not sufficient evidence to con- firm abuse. Some headway has been made in recent years by measuring the ratios in urine samples of testosterone and dihydrotestosterone to other endogenous steroids such as epitestosterone, and Southan et al. (1992) have published a review of these and other possible markers of administration. The exogenous anabolic steroids tend to present less of a problem analytically and nandralone decanoate, which is the one most widely abused, serves as an example of the group. Structures of Testosterone and Nandralone See Figs 21 and 22. Dose and Mode of Administration Nandralone decanoate is administered for medical purposes by intramuscular injection in doses of 50 mg. Athletes may take much larger and more frequent doses. FIGURE 20 Nicotine.
  24. 24. 986 The Immunoassay Handbook Pharmacological Effects Nandralone is primarily an anabolic substance and has very little androgenic effect, although long-term use can produce acne, hirsutism, and deepening of the voice in women. There is conflicting evidence for a direct effect on muscle development in athletes by anabolic steroids, but it is accepted that, coupled with hard and continuous exercise and protein supplements, they can increase mus- cle strength. Many now take the view that there is an indi- rect effect due to the action of the steroids on the CNS, which causes increased aggression and competitiveness. Since the aggression may not always be channeled into sporting activities, this is another reason for concern. Toxic Effects In athletes anabolic steroid abuse has been linked to car- diovascular disorders such as arteriosclerosis with conse- quent coronary heart disease, fatal hypotension, carcinomas of the liver and kidney, and, in males, drastic reductions in sperm counts. Assay Technology Radioimmunoassays offered the first opportunity to screen for anabolic androgens during the Summer Olympic Games of 1976, but because of an unacceptable number of false negatives and the need for specific measurement of testosterone and epitestosterone, the technique was virtu- ally abandoned in 1980. Those laboratories which were accredited for testing by the International Olympic Com- mittee scheme moved on to chromatographic/mass spec- trometric screening methods for this group of compounds, although they still fall back on immunoassays for those such as trebolone which are difficult to detect by these techniques. It is fair to say that there is now far more inter- est in using immunoaffinity chromatography to isolate the anabolic steroids from the complex biological matrix prior to analysis by MS or to apply the immunoassay to HPLC fractions. The use and development of immunoassays for anabolic steroids are still very much in vogue in the animal sports testing laboratories, and ELISA technology has largely replaced RIA in this area. The more advanced laboratories often develop and manufacture “in-house” ELISA kits for anabolic steroids, either when these are not available com- mercially, to ensure continuity of supply, or as a cost- efficiency exercise. There will most likely be an increasing development of specific ELISA kits over the next few years, applying phage display antibody technology, which has been described in detail by Dorsam et al. (1997). It can also be predicted that the human sports testing laborato- ries will follow this lead given the increasing pressure for more intensive inter-competition monitoring. Finally, there is a less exciting, though important, prob- lem of monitoring the illegal use of anabolic steroids, such as 19-nortestosterone, in meat-producing animals. In the veterinary testing laboratories, immunoassays are an inte- gral part of the routine operation. Here again, ELISA is likely to become the favored technique. Rapid Immunoassay Tests for Drugs of Abuse Doctors have long been attracted to the idea of being able to evaluate a patient’s drug status on the spot rather than waiting until the results come back from a laboratory. Even if the laboratory is local, there are inevitable delays in sam- ple transport and processing which mean that treatment decisions are delayed, often for a day or more. The growth of drug testing in the workplace, prisons, schools, and in road traffic offences has stimulated the development of simple tests which need very little skill, give an almost immediate answer and allow action to be taken straight away. Not surprisingly, alcohol tests for use at the roadside were the first to be introduced and were based on the colo- rimetric reaction of alcohol in the breath with potassium dichromate. Since then these rather crude “blow tubes” have been replaced by hand-held devices which rely on fuel cell technology. These give a reasonable estimate of the breath alcohol level and select out those who are obliged either to undergo a more accurate evidential breath test in the police station or to opt for laboratory analysis of a blood or urine sample. Attempts to apply wet chemistry methods to detect abused drugs in urine samples were doomed to failure. Most of the tests that did reach the marketing stage were very unreliable and false-positive results were com- monplace. The emergence of dry reagent immunology sys- tems for various biochemical markers of disease has opened up this field significantly and several products are now available. The most popular versions use gold-labeled immunotechnology and are either in the form of a small hand-held disposable device that detects several drugs in the same urine sample, or as a paper test strip similar in appearance to biochemical dip sticks with separate test strips for each drug. The Alere™ Triage® is a multi-test device for 10 drug classes (11 unique tests). See Table 49. FIGURE 21 Testosterone. FIGURE 22 Nandralone decanoate.
  25. 25. 987CHAPTER 9.23 Drugs of Abuse The reaction well at the top of the device contains three reagent beads. The antibodies bead contains monoclonal antibodies for the 10 drug classes. The conjugate bead contains a representative drug from each class, bound to colloidal gold particles. The third bead contains a buffer. When a urine sample is added to the reagent well, the beads are reconstituted and the mixture is left to incubate for 10min. If the urine sample contains one or more of the target drugs at or above the threshold concentrations, the antibodies bind both free and conjugated drug to leave some unbound conjugates. The reaction mixture is trans- ferred to the detection area which has monoclonal anti- bodies immobilized on a nylon membrane in 11 discrete areas. The detection area also has two other discrete regions, one impregnated with negative control material and the other with a positive control. After the mixture has soaked into the membrane, a wash solution is added to remove any free drug-colloidal gold conjugates. Free gold conjugate binds to the immobilized antibodies producing a red bar opposite the name of whichever drug is present. The device is read in the Alere Triage® Meter. For a test to be valid, the positive control must give a red bar and the negative should be blank. The thresholds (or cutoffs) are similar to those recommended by SAMSHA for workplace testing. The kit has performed well in trials against labora- tory immunoassay techniques and chromatographic meth- ods. The test has a CLIA status of “Moderately Complex” in the USA. There can be false-positive amphetamine results due to reactions with other amphetamine-like sub- stances (e.g., phenylalkylamines in post-mortem urine), but this is not uncommon with laboratory drugs of abuse immunoassay systems. At low drug concentrations, the bands formed can sometimes be faint and may be due to the presence of artifacts rather than drugs. Concateno Bioscience, which is one of the Alere® group of companies, specializes in drug testing for oral fluid samples, including police custody suites, at the roadside and in drug treatment centers. Many of the tests are processed at their central laboratory in the UK on samples collected locally using oral fluid collection kits. The Cozart® DDSV (from Concateno) is used in police custody suites in the UK, and by the police for roadside testing in Australia, Italy and Spain. It is a multi-analyte immunochromatography/lateral flow device with a visual end point. The latest product from Concateno is the Alere™ DDS®2 Mobile Test System, which has a hand-held signal reader and data processor with a color screen. These types of rapid tests are also being used in drug dependency clinics where they are used in the context of improving patient care. However, in other areas where punitive measures may follow a positive result, the tests should be regarded as presumptive. For example, while it may be considered acceptable for an employee who tests positive to be suspended from work temporarily, any fur- ther action should be withheld until a laboratory has car- ried out confirmatory analyses by GC–MS. FUTURE TRENDS The nature of mainstream drug consumption has changed substantially in the last decade. In place of large generalized demographic groups remaining loyal to a limited variety of specific substances and suppliers, a newer generation of consumers is emerging who use the internet to research and acquire an increasing number and variety of the newer psychoactive compounds. The effects these “designer drugs” and so-called “legal highs” have on human physiol- ogy have yet to be described, and in many cases, the precise structure of these novel compounds remains unclear. This ever-changing landscape of drug misuse poses enormous problems to the regulators and those attempting to moni- tor these new substances as they emerge; immunoassay techniques for drug detection will never provide a complete solution for laboratories attempting to monitor these newer compounds, but they will undoubtedly play a key role. References and Further Reading Baselt, R.C. Urine drug screening by immunoassay: interpretation of results. In: Advances in Analytical Toxicology vol.1 (ed Baselt, R.C.). 81–123 (Biomedical Publications, Foster City, California, 1984). Biber, A., Scherer, G. and Hoepfner, I. et al. Determination of nicotine and coti- nine in human serum and urine: an interlaboratory study. Toxicol. Lett. 35, 45–52 (1987). DHHS/SAMSHA Mandatory guidelines for federal workplace drug testing pro- grams; notice. Fed. Regist. 59, 29908–29931 (1994). Dorsam, H., Rohrbach, P. and Kurschner, T. et al. Antibodies to steroids from a small human naive IgM library. FEBS Lett. 414, 7–13 (1997). Dumon, M.F., Nau, A. and Hervouet, J. et al. Isoelectric focusing (IEF) and immu- nofixation for determination of disialotransferrin. Clin. Biochem. 29, 549–554 (1996). Fraser, A.D. and Meatherall, R. Comparative evaluation of five immunoassays for the analysis of alprazolam and triazolam metabolites in urine: effects of lowering the screening and GC-MS cutoff values. J. Anal. Toxicol. 20, 217–223 (1996). Killander, J., De La Torre, R. and Segura, J. et al. Recommendations for the reliable detection of illicit drugs in urine, with special attention to drugs in the work- place, in the European Union. Scand. J. Clin. Lab. Invest. 57, 97–104 (1997). Segura, J. and de la Torre, R. Current Issues of Drug Abuse Testing. First International Symposium. (CRC Press, Boca Raton, FL, Ann Arbor, London, 1992). Schanzer, W. Metabolism of anabolic androgenic steroids. Clin. Chem. 42, 1001– 1020 (1996). Southan, G.J., Brooks, R.V., Cowan, D.A., Kicman, A.T., Unnadkat, N. and Walker, C.J. Possible indices for the detection of the administration of dihy- drotestosterone to athletes. J. Steroid Biochem. Mol. Biol. 42, 87–94 (1992). Tan, K. & Marks, V. Use of immunoassays to detect drugs in body fluids. In: The Analysis of Drugs of Abuse (ed Gough, T.A.). 311–335 (John Wiley, New York, 1991). Warner, A. Interference of common household chemicals in immunoassay methods for drugs of abuse. Clin. Chem. 35, 648–651 (1989). TABLE 49 Drugs Tested Using Triage® TOX Drug Screen Urine Test from Alere Drug Name Cutoff Level Acetaminophen 5µg/mL Amphetamines 1000ng/mL Methamphetamines 1000ng/mL Barbiturates 300ng/mL Benzodiazepines 300ng/mL Cocaine 300ng/mL Methadone 300ng/mL Opiates 300ng/mL PCP 25ng/mL Marijuana 50ng/mL Tricyclic antidepressants 1000ng/mL

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