Drug Screens. 6.1 Drug Testing

Transcription

1 Drug Screens 6 In this chapter, drug screens will be briefly discussed keeping in mind different requirements between clinical toxicology concerned with patient management and forensic toxicology. The sample requirements and the methods available for drug testing are presented. The cutoff values as well as the detection periods of several drugs of abuse in urine and blood are presented. A case is presented to illustrate the usefulness of the cutoff values for drug screens. The cutoff values for drugs of abuse by rapid immunoassay techniques and for confirmations generally done by gas chromatography/mass spectrometry (GC/MS) are presented. For clinical requirements, the drug screens may or may not be confirmed, while for forensic purposes confirmations are always required. 6.1 Drug Testing In order to identify individuals abusing drugs in healthcare, the workplace, and criminal settings, drug testing is performed. Federal government guidelines through SAMHSA (Substance Abuse, and Mental Health Services Administration), previously known as NIDA (National Institute of Drug Abuse), mandate that at least five classes of drugs be tested. These are: 1. Cannabinoids (marijuana) 2. Cocaine (cocaine, benzoylecognine) 3. Amphetamines (amphetamines, methamphetamines) 4. Opiates (heroin, opium, codeine, morphine) 5. Phencylidine (PCP) In addition to the above-described five classes of drugs, most laboratories have facilities in place to test the following class of drugs: 1. Barbiturates 2. Hydrocodone 3. Methaqualone 4. Benzodiazepines 5. Propoxyphene 37

2 38 Forensic Toxicology: Medico-Legal Case Studies 6. Ethanol 7. Ecstasy There are five types of samples in which drug tests are performed. These are urine, blood, hair, saliva, and sweat. Among these, urine drug testing is inexpensive and quick and therefore is the preferred sample (1). Urine drug testing is also performed as a requirement for pre-employment, the military, athletics, and legal and criminal situations such as post-accident. Drug testing is useful for rehabilitation of ex-convicts, treatment compliance, and establishing the cause of death. False-positive drug tests, as well as misinterpretation of the results, have serious consequences, such as risk of prison term, loss of employment, inappropriate exclusion of athletes from sporting events, inappropriate medical treatment in emergency situations, and loss of custody of a child by the social service agencies (1). 6.2 Immunoassays Urine drug screens are performed by immunoassay techniques and confirmed by GC-MS. Until confirmed, a positive drug result by immunoassay is designated as a presumptive positive. Generally, the hospital laboratories have several different drug screens available to the clinicians and the choice depends on their needs. Confirmations are not always necessary for the clinician for patient management. Therefore, the clinician has the choice to request drug screens with or without confirmations (1-3). Immunoassays are cheaper and, because of automation, quite rapid. They can be used to test several samples at the same time. There are several immunoassay techniques available on the market. These include cloned enzyme donor immunoassay, enzyme multiplied immunoassay techniques (EMIT), fluorescence polarization immunoassay (FIPA), immunoturbidimetric assay, and radioimmunoassay (RIA). The main disadvantage is that immunoassays sometimes give false-positive results. For this reason, they need to be confirmed by GC-MS, which can detect small quantities of drugs. This technique is accurate, sensitive, and reliable (2, 3). The EMIT assay principle, developed by Syva for urine drug screens, is shown in Figure 6.1. The EMIT procedure uses the enzyme glucose-6-phosphate dehydrogenase (G6PD) tagged with a drug that retains the G6PD activity. Antibodies are made for the drug. When the enzyme G6PD tagged with the drug combines with the antibody, it forms a complex. The complex so formed loses G6PD activity. On the other hand, the drug tagged with G6PD competes with the drug present in the urine for the antibody. The antibody then forms a complex with the drug in the urine and releases the drug tagged with

3 Drug Screens 39 Enzyme Multiplied Immunoassay Technique (EMIT) Drug G6PD + Ab Ab:Drug G6PD Inactive Enzyme Drug G6PD + Ab + Drug Ab:Drug + Drug G6PD Active Enzyme G6PD Glucose-6-Phosphate + NAD - 6-Phosphogluconate + NADH NADH generated is measured at λ 340nm and is then converted to the amount of drug present in ng/ml. Figure 6.1 EMIT assay. G6PD. This drug-tagged G6PD retains the enzyme activity and reacts with the substrate glucose-6-phosphate in the presence of NAD forming 6-phosphogluconate and NADH. The instrument measures NADH at a wavelength of 340 nm by a spectrophotometer, which is converted to a qualitative number indicating the presence of the drug in the urine (1-3). EMIT drug screens can be performed with as little as 200 µl of urine. EMIT assay is quick, inexpensive, and automated (4). Many times the clinicians in the emergency room need only rapid urine screens by immunoassay without confirmatory tests. EMIT assay is known to give false-negative results (5). This technique can also give false-positive results due to interference of lactate and LDH in the urine as in postmortem urine samples (6) and in diabetes mellitus. Oftentimes urine samples are adulterated, necessitating confirmatory or alternative tests. 6.3 Gas Chromatography-Mass Spectrometry (GC-MS) Confirmatory drug testing is done mostly by GC-MS. The method can detect a small amount of drug present in the sample. The method is accurate, sensitive, and mostly without interfering substances. However, the method is time consuming and costly, and requires expertise and the watchful eye of an experienced technologist. Essentially, the method separates several compounds present in the urine or serum by GC and the separated compounds are ionized. The ionized compounds are broken down to stable charged fragments, which can be separated further and detected by a detector. Relative abundance of a particular ion is plotted

4 40 Forensic Toxicology: Medico-Legal Case Studies as mass spectrum as a function of mass/charge (m/z). Generally, approximately 5 ml of urine are treated with a small amount of activated charcoal and acidic and basic buffers in the presence of methylene chloride. The methylene chloride layers from the acidic and basic buffers are combined and dried. The dried extract is treated with 50 µl of methanol, of which 1 µl is injected into the instrument. In the case of serum, 3 ml are treated with 5 ml of methylene chloride. The methylene chloride layer is pulled off and dried. The dried extract is treated with 50 µl of methanol, out of which 1 µl is injected into the instrument (7). The identity of the compounds can be determined by comparing the spectra of the unknown compound with the spectra of known compounds from spectral libraries (8). Spectral libraries can be developed in-house and stored. In addition, approximately 4000 to 5000 spectra of compounds can be purchased from the Wiley Library or approximately 40,000 to 50,000 spectra of compounds can be purchased from the Pflieger Library. Spectral libraries of additional compounds are purchased when available, added to the spectral libraries, and stored in the computer. Figure 6.2 illustrates the identification of an unknown compound as physostigmine by comparing it with the spectral library. Drugs of abuse, like any other drugs, are metabolized by the body and eventually are excreted and dissipated. The Table 6.1 illustrates the length of time drugs of abuse can be detected in blood and urine (8). Scan 2102 ( min) of f32068jeffr.d SUBTRACTED SCALES Abundance Abundance #875: Physostigmine P1331 SCALED CONDENSED Mass/Charge Figure 6.2 Identification of unknown compound.

5 Drug Screens 41 Table 6.1 Detection Periods for Drugs of Abuse in Blood and Urine Drug Detection Period Alcohol, ethyl 3 10 h Amphetamine Barbiturates Up to 2 6 weeks Benzodiazepines 3 5 days Cocaine 5 h Benzoylecgonine (cocaine metaboline) 2 4 days Codeine Heroine as morphine Hydromorphone (Dilaudid) Methaqualone (Quaalludo) 2 weeks Methadone (Dolophene) 2 3 days Morphine PCP (Phencyclidine) 2 8 days Propoxyphene (Darvone) 6 h Propoxyphene metabolites 6 48 h THC metaboline (marijuana) 1 joint, urine 2 days 3 times weekly, urine 2 weeks Daily, urine 3 6 weeks Blood 8 h Table 6.2 Immunoassay Cutoff Levels (ng/ml) Cocaine (as Benzoylecogonine) 150 Phencyclidine (PCP) 25 Opiate 2000 Cannabinoids (THC) 50 Amphetamines 1000 Source: National Institute of Drug Abuse (NIDA), Substance Abuse and Mental Health Services Administration (SAMHSA). In order to avoid false-positive drugs of abuse levels, SAMHSA has established workplace cutoff levels by immunoassay, as well as by GC-MS (see Table 6.2 and Table 6.3) (8).

6 42 Forensic Toxicology: Medico-Legal Case Studies Table 6.3 GC/MS Cutoff Levels (ng/ml) Cocaine (as Benzoylecogonine) 150 Phencyclidine (PCP) 25 Opiate 2000 Cannabinoids (THC) 50 Amphetamines 500 Source: National Institute of Drug Abuse (NIDA), Substance Abuse and Mental Health Services Administration (SAMHSA). 6.4 Workplace Presumptive Positive Alcohol and Drugs A Case Report* Ira M. Shroud was employed as Outsource Staffer for Ordinary Personnel Services (OPS) Corporation. This is a major national company in Pennsylvania with 500 employees. This company has an employee rights manual, but it is not unionized. The manual states that an employee injured at work must submit to mandatory drug testing as soon as possible immediately after the injury. The company has a no tolerance policy for drugs, which calls for immediate dismissal if a worker tests positive for drugs. Mr. Shroud works from 8 a.m. to 5 p.m., five days a week. He drives 25 miles each way from a nearby major city. He lives with his mother, two sisters, and their eight children. His sisters boyfriends frequently spend the night in his apartment. Over the Memorial Day weekend, Mr. Shroud comes home to find a party in progress. The partygoers and his sisters boyfriends are bringing down the house. Drugs and alcohol are in use. The apartment is filled with smoke from marijuana and crack. Mr. Shroud does not do drugs but he drinks a lot of tequila. He passes out on the sofa at 3 a.m. Tuesday morning while the party is still going on around him. He wakes up at 7 a.m. reeking from the smell of pot, crack, and alcohol. He knows that he has to drive to work, and he is short of time. He quickly changes clothes and hurriedly leaves for work. Mr. Shroud stops at Chartwell Bagel Drive Through for his usual coffee and a poppy seed bagel, which he eats while driving to work. He realizes that he is a few minutes late. He rushes and parks his car, enters the building, and sprints up the steps to get to his desk. He trips on the last step. He hits his head on the water cooler and knocks it over, spilling water with a thundering * This hypothetical case illustrates the importance of cutoff levels for drugs of abuse established by SAMHSA. This hypothetical case was used and published in the course materials for the Pennsylvania Bar Institute s Annual Workers Compensation Program sponsored by the Workers Compensation Section of the Pennsylvania Bar Association. Permission to use this case in this book is graciously given by Attorney Brian R. Steiner who was the program planner for the 2004 annual meeting in Hershey, Pennsylvania.

7 Drug Screens 43 noise. His co-workers rush to the scene to help him. They call a supervisor to report the accident. Mr. Shroud has horrible pain in his head, and he thinks that both his arms are broken. The supervisor calls for mobile assistance, and asks them to take Mr. Shroud to the company s treatment facility at the University of Marcus Hook Medical Center. He tells them to make sure that drug testing is done before Mr. Shroud gets any treatment. At the medical center s emergency room, the doctor diagnoses Mr. Shroud with severe concussion and broken arms. He also notes in the chart that Mr. Shroud smells of alcohol and marijuana. Mr. Shroud s blood and urine are drawn at 8:30 a.m. and is sent to Sum Tymes Reliable Labs for toxicology analysis. Immunoassay performed by the laboratory indicates detectable levels of THC, cocaine metabolite, and opiates in the blood and urine. Enzymatic assay results show a BAC of 0.05%. Immunoassay for drugs in urine showed cocaine, 100 ng/ml, opiates, 250 ng/ml, and THC, 40 ng/ml. Mr. Shroud is discharged from the hospital without being advised of his toxicology results and is asked to return to the medical center in two weeks to see an orthopedist. He is given pain medication and is asked to rest for four days and then return to work for modified duties on the fifth day. The doctor restricts him for light work with no repetitive use of the hands. On the fifth day, Mr. Shroud has blurred vision, and terrible pain in his head and arms. He calls work to tell the supervisor that he cannot drive to work. The supervisor tells him that is not necessary, as he has been fired for violation of the company s drug policy. Several days later, Mr. Shroud receives a Notice of Denial for his work injury from the Human Resources Department Conclusions Based on the evidence available, Mr. Shroud s drug screens were below the cutoff levels and his BAC was also below the legal limit. Therefore, his removal from employment was not justified. References 1. Erowid. Drug testing basics. info1.html. 2. Bowers, L.D., Ambruster, D.A., Caims, T., Cody, J.T., Fitzgerald, R., Goldberger, B.A., Lewis, D., and Shaw, L.M. Gas chromatography/mass spectrometry (GC/ MS) confirmation of drugs: Approved guidelines. NCCLS. 22:1 33, Moeller, K.E., Lee, K.C., and Kissack, J.C. Urine drug screening: Practical guide for clinicians. Mayo Clin. Proc. 83:66 76, Hamilton, C.R. A rapid toxicology screen for them for emergency and routine care of patients. Clin. Chem. 34: , 1988.

8 44 Forensic Toxicology: Medico-Legal Case Studies 5. Wagner, R.E., Linder, M.W., and Valdes, R. Decreased signal in Emit assays of drugs of abuse in urine after ingestion of aspirin: Potential for false-negative results. Clin. Chem. 40(4): , Sloop, G., Hall, M., Simmons, G.T., and Robinson, C.A. False positive postmortem, EMIT drugs of abuse assay due to lactate dehydrogenase and lactate in urine. J. Anal. Toxicol. 19: , Ulluci, P.A., Cardoret, R., Stasiowski, P.D., and Martin, H.F. A comprehensive GC/MS drug screening procedure. J. Anal. Toxicol. 2:33 38, Fenton, J.J. Toxicology: A Case-Oriented Approach. CRC Press, Boca Raton, FL, 2002.