Cover story

Analytical challenges for forensic screening of drugs of ahuse

By Maria Luz Rodrguez, PhD

orensic toxicology focuses on the medico-legal and nonmedical aspects of the harmful effects of chemicals and poisons. There are three major subdisciplines of forensic toxicology; human performance, post-mortem, and forensic drug testing. Human petformance refers to mental or physical effects of drugs that may impair judgment or coordination: post-mortem refers to toxicologie analysis in circumstances of death to determine the cause of death; and forensic drug testing is used most often in the workplace, the military, and the emergency department,' The importance of drug testing, due to the increase in the use and abuse of drugs for recreational purposes and in sports, represents a challenge for testing laboratories.
Drug detection

(SAMHSA) and use cut-offs established by the federal workplace drug testing program. For the practice of forensic toxicology and for the analytical aspects of drug analysis, guidelines are in place.'
Sample Matrices

Drug detection involves initial screening of samples for drugs, and in medical applications the screening tests results are directly used for medical evaluation. For legal purposes, the screening procedure eliminates all negatives, and positive results are regarded as presumptive and require confirmation using confirmatory methods such as high performance liquid ehromatography and mass spectrometry,- Confirmatory methods offer an accurate detection of drugs, but due to cost and labor requirements their application to routine and large-scale analysis is limited. Screening methods are employed to enable rapid routine analysis of multiple samples. The samples are assigned as positive if the value is above a defined cut-off concentration. Many laboratories perform testing under the Substance Abuse and Mental Health Services Administration

Samples examined for toxicological purposes include urine, blood, oral fluid, meconium, hair, nails, tears, and sweat. Vitreous humor, gastric fluid, and solid organ samples are also used in post-mortem examinations,' Urine is the most widely used specimen for drug testing, as it is produced in large quantities, is easy to collect, and offers a wide window of detection. Technological improvements have made blood accessible as a matrix for screening: this matrix provides information about how much of the drug is present in the circulating blood, which could give an indication of whether the donor of the specimen is likely to be affected by the drug at the time of collection. The collection of oralfluidis easy and non-invasive with detection time of drugs similar to that of blood, Meconium is the first fecal matter passed by a neonate. It is formed between the twelfth and sixteenth weeks of gestation and then accumulates in the fetal bowels until birth. Its

The importance of drug testing, due to the increase in the use and abuse of drugs for recreational purposes and in sports, represents a chaiienge for testing laboratories.
sampling is easy and noninvasive, and the analysis of meconium is used for assessing the risk of gestational exposure to drugs and other substances in newborn infants. Hair and nail analysis provides information about consumption over a long period.''
Immunoassay-based drug screening

CONTINUING

EDUCATION

To earn CEUs, see test on page 16 or online at www.mlo-onUne.com under the CE Tests tab. LEARNING OBJECTIVES Upon completion of this article, the reader will be able to: 1. Define steps and criteria used in the detection of drugs of abuse. 2. Identify simultaneous determination in various sample matrices for drugs of abuse. 3. Differentiate between immunoassays for screening versus confirmation techniques for detection of drugs of abuse. 4. List and define the sample preparation approaches for drugs-of-abuse testing.

Immunoassays are highly selective antibody-based tests that provide high throughput determination of a range of drugs and their metabolites in different matrices. Several studies comparing performance of available screening methods have been reported,' " The use of biochip array technology provides a multi-matrix platform for the simultaneous screening of drugs from a single sample and allows the handling of batches of samples, which is advantageous in test settings,'- " For drug testing, competitive immunoassays in matrix-dedicated kits are employed and the cut-off levels ean be individually selected, avoiding recalibration and the use of different calibrator series. This overcomes limitations found with generic methodologies for the analysis of different matrix types, which require different sample dilutions to achieve the particular cut-offs that correspond to the matrix type analyzed. Moreover, preparation of calibrators and buffers to achieve the analytical requirements for a particular sample type can introduce errors and variations in the measurements. continued on page 10
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Cover story
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The capture antibodies are immobilized and stabilized in precise locations on the biochip surface (9mm x 9mm), defining microarrays of discrete test regions (DTRs). The drugs in the specimen and enzyme-labeled-conjugate compete for the binding sites of the capture antibody in the DTRs. The chemiluminescent signals produced in the different DTRs are simultaneously detected by digital imaging technology. The signal generated in the DTRs is inversely proportional to the analyte concentration in the sample. The immunoassays can be applied to several dedicated analyzers. Automation facilitates the integrity, reliability, and accuracy of the drug-testing process.
Simultaneous determination in different sample matrices

Tricyclic antidepressants (TCAs) are indicated for the treatment of clinical depression, neuropathic pain, nocturnal enuresis, and attention deficit hyperactivity disorder (ADHD). While therapeutic drug monitoring (TDM) of TCAs is well established in the treatment of depression, TCA overdose remains the most common cause of death from prescription drugs.'* " Buprenorphine has been widely prescribed since the 1980s for the treatment of moderate to severe pain and in anesthesiology for premedication and/or anesthetic induction."" Soon after buprenorphine became clinically available there were reports of abuse, as it was frequently used as the drug of choice when there was a shortage of heroin supplies." Studies then showed that when this drug was used for opiate dependence treatment, the withdrawal syndrome on di.scontinuing buprenorphine was milder than that of methadone, and fewer symptoms emerged during detoxification. A subhngual tablet of buprenorphine hydrochloride was then developed and registered for opiate dependence treatment. The psychostimulant drug amphetamine is used as a recreational drug and as a performance enhancer. Methamphetamine is also a psychostimulant drug and has high potential for abuse and addiction. The 3,4-Methylenedioxymethamphetaniine (MDMA), widely known as "Ecstasy," is used as a recreational drug and can induce euphoria and diminished anxiety. Barbiturates are central nervous system depressants that can be used as sedatives, hypnotics, and anticonvulsants. They are still used in general anesthesia and have physical and psychological addiction potential. The benzodiazepines, other central nervous system depressants, have replaced the barbiturates in routine medical practice (e.g., treatment of anxiety and insomnia), as they are less dangerous in overdose. However, when combined with other nervous system depressants such as alcohol and opiates, the benzodiazepines' potential for toxicity increases.'* The cannabinoids are a group of compounds that activate cannabinoid receptors, the primary psychoactive compound being A'tetrahydrocannabinol (THC), which produces effects of euphoria, sedation, and altered time sense. THC is the main active chemical in marijuana, which is a commonly abused drug. Cocaine stimulates the central nervous system. Its effects include increased alertness, euphoria, and a sense of confidence and physical strength that may encourage risk-taking behavior. Chronic abuse and overdose of cocaine can cause acute intoxication, which can lead to profound central nervous system stimulation, seizures, and cardiac arrest."-" Opiates such as morphine and codeine are narcotic analgesic drugs; heroin and hydrocodone are semi-synthetic derivatives. These drugs are potent central nervous system depressants. Heroin is the most commonly abused derivative. Phencyclidine (PCP) is a synthetic drug developed as an anesMarch 2012 MLO

thetic and analgesic, but it was removed from the market due to its hallucinogenic properties and the unpredictable behavioral reactions which occurred following anesthesia.-"^" It is a drug of abuse due to its euphoric and hallucinogenic effects. In urine, TCAs, buprenorphine, methadone and other drugs of abuse (amphetamine, methamphetamine, barbiturates, benzodiazepines, cannabinoids, cocaine metabolite, MDMA, opiates, and phencyclidine) are simultaneously detected on a biochip array with limits of detection ranging from 0.005 ng/mL for buprenorphine to 84.5 ng/mL for amphetamine. The broad specificity profile for the TCA immunoassay on biochip array is shown in Table 1. The percentage agreement of the simultaneous immunoassays with the confirmatory method gas chromotography/mass spectrometry (GC/ MS) ranges from 91% (amphetamine) to 100% (buprenorphine, MDMA). To facilitate detection of adulteration in urine by waterloading, an additional test site is present on the biochip surface, which allows the determination of creatinine. The determination of creatinine adds an advantage to the screening of urine samples as it allows detection of false negative samples because an intake of large volume offluidcan dilute the concentration of the drugs below a level determined to be positive. -' -''

te.

^^^^^^^f

% cross-reactivity
100 1335.7 386.0 375.0 217.4 170.6 131.2 71.8 71.4 49.9 48.7 39.4 28.3 20.2

Nortriptyline Imipramine N-oxide

Opipramol Trimipramine Imipramine Cyclobenzaprine Desipramine Amitriptyline Doxepin Protrlptyline Promazine Dothiepin Maprotiline Cyproheptadine
Table 1. Generic TCAs detection on the biochip array test site

A recent evaluation of a biochip-based immunoassay for the measurement of the anxiolytic agent meprobamate in whole blood or plasma reports specificity of 100%, sensitivity of 97.2%, and accuracy of 97.6%.-'' The meprobamate is the main active metabolite of carisoprodol.-' Both compounds induce depressant effects on the central nervous system and have the potential to impair driving.-^ Overdose of both drugs is often serious and sometimes lifethreatening; this immunoassay is therefore useful in test settings for the detection of intoxication. A study evaluating the screening of drugs of abuse in 128 forensic blood samples on biochip array reports an efficient detection of benzodiazepines, cannabinoids, cocaine, and opiates with specificity (% of true negative samples) of 100% for benzodiazepines, cocaine, and opiates and 98.1% for cannabinoids, and sensitivity (% true positive satnples) values of 95.2%, 93.5%, 93.2%, and 78.4% for cocaine, opiates, cannabinoids, and benzodiazepines respectively, when compared with GC/MS results.-* The applicability of the biochip array to the screening of meconium specimens has been reported, and the data indicate an accuracy >90% (amphetamines,
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Drugs-of-AbuseTesting
barbiturates, benzodiazepines, cocaine metabolite, methadone, and phencyclidine) and 88% (cannabinoids) when compared with other methodologies.-'' This technology has been also applied to the screening of amphetamine and methamphetamine in oralfluidfor an analysis by GC/MS."' Other matrices such as vitreous humor, liver, and muscle from post-mortem samples have also been investigated, and the evaluation showed favorable agreement (80% to 100% for barbiturates, benzodiazepines. opiates, and methadone) between the biochip-based immunoassays and confirmatory analysis."
Conclusion
1 8 . Fraser AD, Use and abuse of the benzodiazepines. Ther Drug Monit. 1998:20:481-489,
19. Beselt RC, Urine drug screening by immunoassay: interpretation of results. In: Besett RC, Advances in Analytical Toxicology, vcl 1, ch 5,81-123. 20. Eskridge KD, Guthrie SK. Clinical issues associated with urine testing of substances of abuse. P/iarmacotf7er-3pn1997:l7l3):497-510. 21. Schneider S, Kuffer P, Wennig R, Determination of lysergide {LSD} and phencyclidine in biosamples. J. Chromatogr. B Biomed. Sei Appl. 1998:713:189-200. 22. Wild (ed). The Immunoassay Handbook, second edition. Nature Publishing Group, London, Basingstoke, New York, 2001:808-810. 23. Lafolie P, et al. Importance of creatinine analyses of urine Vi/fien screening for abused drugs, C/mC/iem, 1991:37:1927-1931. 24. Needleman SB, Porvaznik M, Ander D, Creatinine analysis in single collection urine specimens, J. Forensic Sei. 1992:37:1125-1133, 25. Alvarez JC, et al. Evaluation of the first immunoassay for the semi-quantrtative measurement of meprobamate in human whole blood or plasma using biochip array technology. Clin. Chim. /Icta. 2012:413 273-277, 26. Olsen H, Koppang E, Alvan G, Morland J, Carisoprodol elimination in humans. 77ier: Drug Mon/t. 1994:16:337-340. 27. Logan BK, Case GA, Gordon AM, Carisoprodol, meprobamate, and driving impairment. J. Forensic Sei 2000:45:619-623, 28. Grassin DS, Mathieu B, Abe E, Alvarez JC. Evaluation of screening for drugs of abuse and benzodiazepines in forensic blood samples using Evidence Investigator analyser, Ann. roxicoi/Ana/. 2008:20:17-24. 29. Marin SJ, Merrell M, McMillin GA, Drugs of abuse detection in meconium: a comparison between ELISA and biochip microarray, J. Anal Toxico! 2011:35:40-45, 30. Choi H, Baeck S, Jang M, Lee S, Choi H, Chung H, Simultaneous analysis of psychotropic phenylalkylamines in oral fluid by GC-MS with automated SPE and its application to legal cases. Forensic Sei Int. 2011. 31. McLaughlin PA, Pounder DJ, Osselton MD, Evaluation of the Randox whole blood drugs of abuse arrays I and II far the analysis of alternative post-mortem toxicology samples, S16, SOFTAnnual Meeting 2010. Maria Luz Rodriguez, PhD, has been involved for more than fifteen years in the research and development of clinical, drug residue, and drugs-of-abuse irtimunoassays in Randox Laboratories.

Forensic toxicology aids medico-legal investigations of death and poisoning and. in a broader sense, workplace drug testing and testing for driving under the influence of alcohol and drugs. At present, testing for common drugs of abuse, whether in a clinical, forensic, sport, or workplace setting, is usually undertaken as a two-step process. The first involves a screening test followed by a confirmatory conclusive analysis. Immunoassays represent useful tools in the forensic toxicology investigation. They can be used for the rapid screening of a large number of samples for the potential presence of a drug, and the main challenge is to provide rapid and accurate results for the increasing number of target compounds in different complex biological matrices. The implementation of simultaneous immunoassays on a biochip surface allows the multiplex determination of drugs from a single sample, which increases the test result output. The application of the biochip-based immunoassays to automated analyzers facilitates the integrity, reliability, and accuracy of the drug testing process. This instrumental analytical platform can be applied to the multiplex screening of drugs in different sample matrices, which facilitates the testing process and represents a flexible technology to accommodate the constant increase in new substances of abuse and alternative matrices. D
References 1. Kolecki PR "Forensic toxicology" in f^anual of forensic emergency medicine: a guide for clinicians. Riviello RJ. ed. Jones & Bartlett Publishers, 2010:6:36-43, 2. Hawks RL Analytical Methodology. NIDA. Research Monogr 1986:73:30-41. 3. Penders J, Verstraete A. Laboratory guidelines and standards in clinical and forensic toxicology. Accred. Qual. Assur. 2006:11:284-290. 4. Mali N, Karpe M, Kadam V, A review on biological matrices and analytical methods used for determination of drug of abuse, J. Appi. Pharm. Sei 2011:01(06):58-65. 5. BuchanBJ,WalshJM,LeavertonPE. Evaluation of the accuracy of on-sitemutti-analyte drug testing devices in the determination of the prevalence of illicit drugs in drivers, J. Forensic Sei 1998:43:395-399, 6. Taylor EH, Oertii EH, Wolfgang JW, Mueller E, Accuracy of five on-site immunoassay drugs of abuse testing devices. J. Anal. Toxicol. 1999:23:119-124, 7. Crouch DJ, Frank JF, Farretl LJ, Karsch HM, Klaunig JE, A multiple-site laboratoiv evaluation of three on-site urinalysis drug-testing devices. J Anal Toxicol. 1998:22:493-502. 8. Crouch DJ, Cheever N/IL Andrenyak DM, Kuntz DJ, Loughmiller DL A comparison of DNTRAK TESTCUP, Abuscreen ONTRAK, Abuscreen ONLINE and GC/MS urinalysis test results, J. Forensic Sei. 1998:43:35-40. 9. Peace MR, Tarnai LD, Poklis A. Performance evaluation of four on-site drug testing devices for the detection of drugs of abuse in urine. J. Anal. Tbx/'co/. 2000:24:589-594. 10. Leino A, Saarimies J, Gronholm M, Ullsunde P Comparison of eight commercial on-site screening devices for drugs of abuse testing. Scand. J. Clin. Lab. /nvest 2001:61:325-331. 11. Luzzi VI, et al. Analytic performance of immunoassays for drugs of abuse below established cutoff values. Clin. Ctem. 2004:50:717-722. 12. Fitzgerald SP, Lamont JV, fVicConnell Rl, Benchikh EO, Development of a high throughput automated analyzer using biochip array technology. Clin. C/iem. 2005:51:1165-1176, 13. Molloy RM, McConnell Rl, Lamont JV, Fitzgerald SP Automation of biochip array technology for quality results. Clin. Chem. Lab. Med 2005:43:1303-1313. 14. Haddad LM, Managing tricyclic antidepressant overdose. Am. Fam. Physician. 1992:46:153-159, 15. Pimentel t, Trommer L Cyclic antidepressant overdoses: a review. Emerg. tAed. Clin. North Am. 1994:12:533-547, 16. Cirimele V, Kintz P, Lohner S, Ludes B, Enzyme immunoassay validation for the detection of bupreorphine in urine, J. Anal. Toxicol 2003:27:103-105, 17. Debrabandere L. van Boven M, Daenens P Development of a fluoroimmunoassay for tfie detection of buprenorphine in urine. J. Forensic Sei. 1995:40:250-253.

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