Issues of Interferences With Immunoassays Used for Screening of Drugs of Abuse in Urine

Chapter 10

Issues of Interferences With Immunoassays Used for Screening of Drugs of Abuse in Urine Anu S. Maharjan1 and Kamisha L. Johnson-Davis1,2 1

Department of Pathology, University of Utah Health Sciences Center, Salt Lake City, UT, United States, 2ARUP Institute for Clinical and

Experimental Pathology, Salt Lake City, UT, United States

INTRODUCTION In recent years, immunoassays have evolved to not only detect antigens of interest, but also drugs of abuse. Drugs-ofabuse testing are important both in workplace and in medical settings. Workplace drug testing requires confirmation of a positive specimen by an alternative analytical technique preferably, liquid chromatography-tandem mass spectrometry (LC-MS/MS) or gas chromatography/mass spectrometry (GC/MS), but such confirmations may not be requested by a clinician during medical drug testing. Drug testing is important in medical settings, since most patients do not report their drug use. It has been reported that 66.6% patients who tested positive for amphetamine did not report their amphetamine use to the clinician in the emergency department (ED) [1]. A similar case was seen in 70% of patients who had an opiate positive result, but did not report their opiate abuse. Urine drug screen immunoassay applications are available on various automated analyzers as well as point-of-care devices in health care facilities or in physicians’ offices. Some immunoassays have good sensitivity and specificity; however, other assays are subjected to interferences. A study by Johnson-Davis et al. has highlighted the true-positive rate and the false-positive rate of immunoassay performance at a reference laboratory; which supports previous studies which have demonstrated that some immunoassays, such as amphetamine and opiate immunoassays, have poor specificity and a high rate of false positives [2]. As a result, positive screen results for drugs-of-abuse immunoassays with high false-positive rates should be confirmed by GC/MS or LC-MS/MS [3]. Amphetamines immunoassays are subjected to more interference that can lead to a false-positive result compared to other drugs-of-abuse assays. Dietzen et al. reviewed confirmatory results of drugs-of-abuse testing that were positive by immunoassays and reported that there are 175 instances of false-positive screening during 1-year period. Positive predictive values of various immunoassays varied from 0% (amphetamine) to 100% (tetrahydrocannabinol) [4].

INTERFERENCES IN AMPHETAMINE IMMUNOASSAY 3,4-Methylenedioxymethamphetamine (MDMA; Ecstasy) and 3,4-methylenedioxyamphetamine (MDA) are two similar synthetic designer drugs resembling amphetamine, which are widely abused. Immunoassays are the primary screening methods for the detection of amphetamines and in most of these assays, the antibodies appear to be directed toward the amino group of amphetamine, a primary amine, and methamphetamine, a secondary amine. Different commercial immunoassays use either monoclonal or polyclonal antibodies against amphetamine and methamphetamine. Both MDMA and MDA show significant cross-reactivity with commercially available amphetamine immunoassay, but there is a separate immunoassay for MDMA. In one report, the authors evaluated eight commercially available amphetamine immunoassays for their effectiveness for detecting MDMA during immunoassay screening test and observed the crossreactivity to be lower at high concentrations of these drugs (Table 10.1) [5]. In a similar study, cross-reactivity of Critical Issues in Alcohol and Drugs of Abuse Testing. DOI: https://doi.org/10.1016/B978-0-12-815607-0.00010-1 Copyright © 2019 Elsevier Inc. All rights reserved.

129

130

Critical Issues in Alcohol and Drugs of Abuse Testing

TABLE 10.1 Cross-Reactivities of MDMA and MDA With Various Commercial Amphetamine and Amphetamine/ MDMA Immunoassays Immunoassay

Manufacturer

MDMA Cross-Reactivity

MDA Cross-Reactivity

DRI-amphetamine

Diagnostics Reagents (Sunnyvale, CA)

11% 44%

51% 99%

CEDIA-amphetamine

Microgenics (Fremont, CA)

7% 46%

175% 214%

EMIT d.a.u. monoclonal

Syva (Cupertino, CA)

7% 46%

175% 214%

Synchron-CX-amphetamine

Beckman (Fullerton, CA)

49% 100%

58% 73%

COBAS Integra amphetamine

Roche Diagnostics (Indianapolis, IN)

38% 48%

24% 42%

CEDIA-amphetamine/MDMA

Microgenics (Fremont, CA)

100%

36% 63%

Online amphetamine/MDMA

Roche Diagnostics

111% 142%

75% 100%

MDMA with amphetamine immunoassay showed dependence on concentration and varied from 118% at 150 ng/mL to 18% at 10,000 ng/mL [6]. Amphetamine or methamphetamine immunoassays are created for high-sensitivity screening; therefore, there is a wide range of cross-reactivity to compounds that are structurally or physically similar to phenethylamines. Immunoassays designed for the detection of amphetamines can be classified into three general types based on antibody specificity: 1. Assays highly selective for either amphetamine and MDA or methamphetamine and MDMA. 2. Assays that are able to detect both amphetamine and methamphetamine to varying extent but that also exhibit higher levels of cross-reactivity to various over-the-counter (OTC) cold medications containing sympathomimetic amines. Medications such as bupropion [7], fluoxetine [8], labetalol [8 10], metformin [9 11], methylphenidate [8], promethazine, pseudoephedrine, ranitidine, and trazodone may cause false-positive amphetamine results. 3. Assays for amphetamine and methamphetamine with lower of cross-reactivity to OTC medications [12]. Choosing an optimal amphetamine immunoassay depends on several factors including availability of automated analyzers and intended use. Forensic or workplace laboratories prefer to use amphetamine immunoassays that are specific for amphetamine testing; whereas, hospital laboratories may need a broad spectrum immunoassay that can detect many sympathomimetic amines, including OTC drugs. According to the National Academy of Clinical Biochemistry’s (NACB) Laboratory Medicine Practice Guidelines (LMPG), the recommendations for an optimum immunoassays for amphetamine testing in ED patients are those directed toward phenethylamine as a drug class [13]. LMPG also recommends changing the name of the test from “amphetamines” to sympathomimetic amines” or “stimulant amines.” The cross-reactivity of various phenethylamine in different immunoassays has been extensively reported [14]. However, it is possible that current assays produced by the manufacturers may have different cross-reactivities from those reported due to changes in antibodies and reagent composition. The analyst should consult immunoassay package inserts or contact the manufacturer for up-to-date cross-reactivity data. It is also possible that potential interfering substances used during studies are lower than those encountered in the clinical setting and that manufacturers often do not test for crossreactivity of endogenous metabolites [15,16]. Lot-to-lot variability concerning sensitivity to targeted analytes has been reported [17].

Interferences of Sympathomimetic Amines With Amphetamine Immunoassay Interference of various sympathomimetic amines in amphetamine immunoassay is well recognized. Various OTC cold medications contain these sympathomimetic amines and use of such medications may cause false-positive test results in amphetamine immunoassay screening. In workplace drug testing, taking cold medications are not prohibited, but may cause confusion during medical drug testing, specifically if a confirmation test is not ordered or if the facility does not have the capability to confirm such drug by mass spectrometry. Unfortunately, misidentification of ephedrine/pseudoephedrine as methamphetamine has been reported during GC/MS confirmation [18]. There are several causes of misidentification, which include the following: 1. At an injector port temperature over 185 C in the gas chromatograph of the GC/MS, ephedrine and pseudoephedrine can be thermally dehydrated to methamphetamine.

Issues of Interferences With Immunoassays Used for Screening of Drugs of Abuse in Urine Chapter | 10

131

TABLE 10.2 Electron Ionization and Chemical Ionization Mass Spectral Features of Derivatized Methamphetamine and Ephedrine Derivative

Electron Impact Base Peak (m/z)

Electron Impact Other Peaks (m/z)

Chemical Ionization Base Peak (m/z)

Chemical Ionization Other Peaks (m/z)

Methamphetamine Propyl carbamate

144

102, 91, 58

236 (M 1 1)

176, 144, 119, 58

Ephedrine propyl carbamate

144

102, 77, 58

192

220, 148, 58

Methamphetamine trifluoroacetyl

154

118, 58

246 (M 1 1)

154, 119

Ephedrine trifluoroacetyl

154

118, 58

244

276, 158

Methamphetamine 4-carbethoxy

315

118, 91

400(M 1 1)

308, 119

Ephedrine 4-carboethoxy

308

118, 91

398

121

2. Electron impact mass spectrum of trifluoroacetyl, pentafluoropropyl, heptafluorobutyl, 4-carbethoxyhexafluorobutyryl, and various carbamate derivatives of methamphetamine are similar to the corresponding derivatized ephedrine/pseudoephedrine. Since ephedrine and pseudoephedrine elutes right after methamphetamine peak, ephedrine/pseudoephedrine can be misidentified as methamphetamine if the mass spectral analysis is not done carefully. Nevertheless, the misidentification can be completely avoided if the mass spectrometry is performed in the positive chemical ionization mode using methane as the reaction gas [19]. This results in ionization mass spectrum of derivatized methamphetamine that is very different from the derivatized ephedrine/pseudoephedrine. In the electron ionization mode, usually molecular ion of derivatized amphetamine is present as a very weak peak. In contrast, protonated molecular ion is the base peak (100% abundance) in the chemical ionization mass spectra of derivatized amphetamine and methamphetamine. This misidentification problem also exists with propyl carbamate derivatives of methamphetamine. In the electron ionization mode, both methamphetamine propyl carbamate and ephedrine propyl carbamate showed almost identical mass spectral fragmentation patterns. Again using chemical ionization where methamphetamine propyl carbamate showed a base peak at m/z 236 can circumvent this problem and the ephedrine propyl carbamate showed a base peak at m/z 192. The major mass spectral fragmentation pattern differences are given in Table 10.2. Recently, Ojanpera et al. described that using positive chemical ionization mass spectrometry, allowed the authors to observe protonated molecular ion peak of new amphetamine like designer drug 3,4-methylenedioxypyrovalerone in urine at m/z 276 [20]. Ephedrine or pseudoephedrine which is present in many OTC cold medications is responsible for majority of false-positive result in amphetamine immunoassay screening test. These sympathomimetic amines may be present in large amounts in urine specimens which are initially tested positive by amphetamine immunoassays. Stout et al. based on the analysis of 27,400 randomly collected urine specimens reported that 833 out of 1104 urine specimens (92% specimens) that failed to confirm amphetamine, methamphetamine, MDMA, or MDA by GC/MS contained pseudoephedrine [21]. Commonly encountered medications that interfere with amphetamines screening assays are listed in Table 10.3.

Vicks Inhaler and False Positive in Amphetamine Immunoassays Amphetamine and methamphetamine have optical isomers designated d (or 1 ) for dextrorotatory and l (or ) for levorotatory and the d isomers, the more physiologically active compounds, are the intended targets of immunoassays because d isomers are abused. Ingestion of medications containing the l isomer can cause false-positive results. For example, Vicks inhaler contains the active ingredient l-methamphetamine [22] and extensive use of this product may cause false-positive results for immunoassay screening. Specific isomer resolution procedures must be performed to

132

Critical Issues in Alcohol and Drugs of Abuse Testing

TABLE 10.3 Drugs That Interfere With Amphetamine Immunoassays Drug Class

Individual Drug

Sympathomimetic amines

Brompheniramine, benzphetamine, ephedrine, isometheptene, mephentermine, methylphenidate, pseudoephedrine, phentermine, phenylpropylamine,a propylhexedrine, phenylephrine, phenmetrazine, tyramine

Antidepressant/antipsychotic

Chlorpromazine, bupropion, desipramine, fluoxetine, perazine, thioridazine, trimipramine, trazodone

Antihistamine

Promethazine, ranitidine

Antiprotozoal

Quinacrine, chloroquine

Antiemetic

Trimethobenzamide

Beta-blocker

Labetalol

Cardioactive

N-acetyl procainamide (metabolite of procainamide) Mexiletine

Nonsteroidal antiinflammatory

Tolmetin

Norepinephrine reuptake inhibitor

Atomoxetine

Vasodilator

Isoxsuprine

Tocolytic agent

Ritodrine

a

Removed from the U.S. market in 2005.

differentiate the d and l isomers because routine confirmation by gas chromatography mass spectrometry (GCMS) does not determine isomer composition. Poklis et al. reported relatively high concentrations of l-methamphetamine in two subjects (1390 and 740 ng/mL), using chiral GC/MS and after extensively inhaling Vicks inhaler every hour for several hours. However, when such urine specimens were tested by the enzyme multiplied immunoassay technique (EMIT II amphetamine/methamphetamine assay; Dade Behring), results were negative even after such extensive use of Vicks inhaler [23]. In another study, the authors reported that nasal inhalation of Vicks inhaler following recommended dosage should not cause false-positive amphetamine/methamphetamine urine screening results using fluorescence polarization immunoassay (FPIA) for amphetamine/methamphetamine for application on TDxADX/FLx analyzer (Abbott Laboratories, Abbott Park, IL) because antibody used in this immunoassay is stereo-selective and recognized d-methamphetamine or d-amphetamine if present in the urine specimens. However, when two subjects inhaled the recommended dosage twice, positive test result was obtained by the FPIA screening assay. The concentrations of l-methamphetamine in urine specimens of these two subjects were 1560 and 1530 ng/mL, respectively [24]. However, in the case of doubt, l-methamphetamine can be distinguished from d-methamphetamine by using GC/MS and chiral derivatization technique. If greater than 80% l-methamphetamine is found in the specimen, the result is considered as consistent with use of Vicks inhaler. Abbott Laboratories discontinued FPIA assays for amphetamine/ methamphetamine assay for application on the TDX analyzer in 2010. Case Study An emergency medical team responded to the residence of a 77-year-old man who was experiencing difficulty in breathing and his oxygen saturation was 69%. A breathing treatment of albuterol and ipratropium bromide was administered and his oxygen saturation was improved to 96%. The patient was transported to the ED and was admitted to the intensive care unit. Approximately, 12 h after admission, he was unresponsive and expired. His medical history included heart failure, atrial flutter, and bronchial asthma. His urine drug testing (GC/MS) was positive for acetaminophen, nicotine, cotinine, caffeine, (Continued )

Issues of Interferences With Immunoassays Used for Screening of Drugs of Abuse in Urine Chapter | 10

133

(Continued) diltiazem, doxylamine, and methamphetamine. However, chiral analysis of methamphetamine was not initially performed raising possibility of abusing d-amphetamine by the man. To address this question further, the authors performed chiral analysis using trifluoroacetyl-l-propyl chloride as the chiral derivatizing agent and identified the methamphetamine as the l-isomer. Further investigation revealed that the decedent frequently used Vicks inhaler for his bronchial asthma [25].

Dietary Weight Loss Products and Positive Amphetamine The federal drug administration (FDA) banned ephedra alkaloids containing all products including weight loss products in April 2004. The products that were banned include ma-huang, sida, cordifolia, and Pinellia, but Chinese herbal products, herbal teas and drug containing synthetic ephedrine were not banned. Currently, there is a push for companies’ marketing herbal weight loss products to produce ephedra free diet pills. The common ingredients of these ephedra free diet pills may also contain phenylpropanolamine, tyramine, or phentermine, which like ephedrine interferes with the immunoassay screening of amphetamine/methamphetamine in urine specimens. However, other diet loss products which contain hydroxy-citric acid or bitter orange do not interfere with drugs-of-abuse testing. The FDA warned consumers in January 2006 that the Brazilian dietary supplements Emagrece Sim and Herbathin contain several active drug ingredients including fenproporex which is metabolized to amphetamine [26]. Use of other weight loss or nutritional supplements containing fenproporex has resulted in the detection of d-amphetamine in the urines of users [27]. In 2011, the US military removed all dimethylamylamine (DMAA)-containing supplements from all military exchanges worldwide. DMAA, also known as 1, 3-dimethylamylamine, is a synthetic neural stimulant used in dietary supplements that results in false-positive amphetamine [10,28]. DMAA has been linked to cases of increased blood pressure and heart rate, leading to more than two deaths in the US military [10,29]. DMAA is an aliphatic amine that resembles amphetamine and releases norepinephrine; however, it is still unknown if DMAA works similar to amphetamine. When negative drug-free urine was spiked with DMAA, both Roche Amphetamines KIMS (kinetic interaction of microparticle in solution methods) assay and Siemens Syva EMIT II Plus Amphetamines assay gave positive responses at 7500 and 3125 ng/mL [10,28]. Case Study A 25-year-old woman presented to the ED with persistent abdominal pain of 2 weeks duration and 1-day history of nausea and vomiting. Her medications included high-dose ibuprofen which she discontinued 5 days prior to her presentation and a Brazilian weight loss product, Emagrece purchased from the Internet which she also discontinued 2 weeks ago. The patient discontinued the weight loss product due to feeling mildly tremulous after taking the product and her friends also experienced similar symptoms. On admission to the hospital, results of routine blood test for electrolytes, complete blood count, amylase, lipase, and liver function tests were within normal limits. A urinary toxicology screen was positive for amphetamine. The analysis of weight loss product after extraction using GC/MS confirmed the presence of 5-cyano-amphetamine, an amphetamine derivative used outside the US for weight loss but considered illegal by the FDA. The list of ingredients only mentioned herbal components such as kava kava and ginkgo biloba but there was no mention of the amphetamine-like compound. The patient was treated with metoclopramide, pantoprazole, and acetaminophen and was discharged from the hospital 3 days later when her symptoms resolved [30].

Case Study A 35-year-old African American female with no prior history of coronary artery disease presented with sudden onset of exertional chest discomfort with radiation to the back. After arrival to the ED, she went into ventricular fibrillation-cardiac arrest (V-fib) for 6 minutes with conversion after electrical cardioversion and subsequent development of PEA arrest for a total of 4 minutes. The patient received tenecteplase and heparin prior to urgent transfer to the catheterization laboratory. A urinary toxicology screen was positive for amphetamine. The patient denied of using Adderall or any other amphetamine, and reported that she increased her physical activity to lose weight and was further supplementing with a natural weight loss dietary supplement [31].

134

Critical Issues in Alcohol and Drugs of Abuse Testing

Case Study A 22-year-old male infantry soldier arrived at the hospital with hyperthermia and dry hot skin [32]. The soldier lost consciousness while running with his battalion for 10 minutes. His laboratory values showed renal insufficiency and increased cardiac and muscle enzymes. The patient received resuscitation along with active core cooling through endovascular device, endotracheal intubation, and fluid restoration. However, the patient went into refractory asystole and passed away 4-hours later. Toxicology analyses on the autopsy showed the presence of DMAA in the antemortem blood at 0.22 mg/L and caffeine at 2.9 mg/L. He had been taking a dietary supplement for about 4 weeks which contained DMAA along with β-alanine, arginine α-ketoglutarate, and creatine. Amphetamine derivatives and creatine together may contribute to dehydration and heatstroke [33].

Other Drugs That May Cause Positive Amphetamine Screening Ranitidine is a H2-receptor blocking agent (antihistamine) which reduces acid production by the stomach and is available OTC without any prescription. Dietzen et al. reported that ranitidine if present in urine at a concentration over 43 μg/mL may produce false-positive test results with amphetamine screen using Beckman Synchron immunoassay reagents (Beckman Diagnostics, Brea, CA). This concentration of ranitidine is routinely exceeded in patients taking ranitidine [4]. Poklis also reported that ranitidine interferes with the EMIT d.a.u. amphetamine/methamphetamine assay if ranitidine concentration in urine exceeded 91 μg/mL. The concentrations of ranitidine in urine specimens collected from 23 patients receiving 150 300 mg ranitidine per day varied from 7 to 271 μg/mL [34]. Since September 2014, both Siemens Syva EMIT II Plus Amphetamine assay and Beckman Coulter Synchron AMP assays claim no positive amphetamine result if ranitidine is less than or equal to 1000 μg/mL [35]. However, there were four patient cases from the University of Pittsburgh Medical Center (UPMC) that showed positive amphetamine results due to the interference from ranitidine at a concentration as low as 160 μg/mL on Beckman Coulter platforms [35]. All four samples were negative on the Siemens platform; however, samples with 160 and 320 μg/mL of ranitidine gave positive results for amphetamine in Beckman DxC 800 and DxC600i. Similarly, trazodone interferes with amphetamine immunoassays and also causes false positives in an immunoassay specific for MDMA. A series of patients who tested positive for MDMA (using Ecstasy EMIT II assay) did not show any presence of MDMA in urine when confirmed by a specific liquid chromatography combined with tandem mass spectrometric method. However, all specimens showed the presence of trazodone and its metabolite metachlorophenylpiperazine. In addition, another hallucinogen trifluoromethyl phenylpiperazine also cross-reacts with the MDMA assay [36]. Labetalol, a beta-blocker commonly used for control of hypertension in pregnancy, caused a falsepositive amphetamine screen by immunoassay [9]. Casey et al. reported that bupropion, a monocyclic antidepressant and an aid for smoking cessation, may cause false-positive screen using amphetamine immunoassay. Out of 234 specimens screened positive by amphetamine immunoassay (EMIT II), 128 specimens did not show any presence of amphetamine during GC/MS confirmation step. Three specimens, where amphetamine could not be confirmed, contained bupropion [7]. After screening, 3571 specimens by EMI II, 389 (10.9%) samples screened positive via EMIT, but confirmed negative by LC-MS/MS, suggesting false-positive immunoassay results [8]. High-resolution mass spectrometry (HRMS) and in silico structure search identified compounds that may cause false-positive results in urine immunoassay. Compounds with same chemical formula with different molecular rearrangements may not be distinguished by immunoassay, therefore, producing false-positive results [8]. For instance, compounds with mass match for erythro-dihyrobupropion and hydroxy bupropion tested positive in immunoassay. Similarly, atomoxetine, an attention deficit hyperactivity disorder (ADHD) medication caused false-positive amphetamine immunoassay due to structure similarities between atomoxetine metabolites and amphetamine. Case Study A 27-year-old female with a past medical history of ADHD arrived at the ED following two episodes of tonic clonic movements [37]. The patient stabilized after arriving in the hospital with no visual or neurological symptoms. The patient was taking atomoxetine for ADHD, and admitted that she takes them occasionally instead of complying to her daily dosage. Prior to her arrival at the hospital, she had taken 120 mg of atomoxetine approximately 12 h before the seizure episode. The dosage was three times higher than her prescribed dose and above the maximum recommended dosage of 100 mg/day. As part of the workup, the patient’s urine was screened for illicit drugs and toxins. Her urine screen was negative for drugs such as tetrahydrocannabinol (THC), benzodiazepines, cocaine, and opiates, but screened positive for amphetamines. She denied the use of amphetamines or any other illicit drugs, and a confirmatory GC-MS test was negative for amphetamine, methamphetamine, (Continued )

Issues of Interferences With Immunoassays Used for Screening of Drugs of Abuse in Urine Chapter | 10

135

(Continued) methylene-dioxyamphetamine, 3,4-methylenedioxy-N-methylamphetamine (MDMA), 3,4-methylenedioxy-N-ethylamphetamine (MDEA). There are many prescription drugs that do result in false-positive amphetamine screen, and the authors believe atomoxetine and its metabolites to be the cause of the false-positive test results in this particular patient.

INTERFERENCES WITH OPIATE IMMUNOASSAY Certain fluoroquinolone antibiotics may cause false-positive test results with opiate immunoassay screening [38]. Baden et al. evaluated potential interference of 13 quinolones (levofloxacin, ofloxacin, pefloxacin, enoxacin, moxifloxacin, gatifloxacin, trovafloxacin, sparfloxacin, lomefloxacin, ciprofloxacin, clinafloxacin, norfloxacin, and nalidixic acid) with various opiate immunoassays and concluded that levofloxacin, ofloxacin, and pefloxacin administration would most likely cause false-positive result with opiate immunoassays [39]. Rifampicin is used in therapy for tuberculosis. In one report, the authors observed false-positive opiate immunoassay screening result in patients receiving rifampicin. Opiate immunoassay screens were conducted using KIMS assay on the Cobas Integra analyzer (Roche Diagnostics, Indianapolis, IN). The authors observed a 12% cross-reactivity of rifampicin with the opiate assay over a concentration range of 156 5000 ng/mL. A false-positive result may be observed even after 18 hours of administration of a single oral dose of 600 mg of rifampicin [40]. Naloxone, an opioid antagonist drug, is administered to reverse opioid overdose. It binds to opioid receptors and blocks the effects of other opioids. However, recent cases show naloxone cross-reacting with the opiate immunoassay, which could lead to drastic consequences for patients. Physicians order urine tests for opiate immunoassay for compliance and to ensure no illicit drug use, therefore opioid antagonist cross-reactivity may result in a false-positive result. Naloxone cross-reactivity with opiate immunoassay can also be problematic in pediatric patients that receive a dosage as high as 2 mg/dose [10,41]. Siemens’ EMIT drugs of abuse urine assays cross-reactivity listnow shows naloxone concentration greater than 11,000 ng/mL can cross-react with the opiate assay. Naloxone and naloxone glucuronide can also cause positive results for oxycodone with homogeneous enzyme immunoassay (HEIA) oxycodone (Immunalysis Corporation) and the DRI oxycodone assay (Microgenics/Thermo Fisher Scientific) [42]. Case Study A 3-year-old girl received an intravenous bolus of naloxone after presenting with decreased mental state and probable hypothermia [41]. Her initial urine toxicology report was negative for drugs of abuse; however, the next day after the administration of naloxone, the sample came out positive for opiate, the cutoff for the assay was set at 300 ng/mL. A repeated test on immunoassay screen came out positive. The urine samples were further sent out for confirmation by LC-MS/MS, which showed that naloxone was the compound that corresponded to the positive opiate results.

Case Study A 48-year-old man spent 12 days in an inpatient psychiatry facility for treatment of depression which began subsequent to his abuse of cocaine after 90 days of sobriety. He was then transferred to a residential facility. Eleven days after his transfer to the residential facility, the patient was prescribed gatifloxacin for treating urinary tract infection. His urine drug screen before initiation of gatifloxacin therapy was negative. Six days after the start of gatifloxacin therapy, his urine screen was positive for opiate. The patient denied opiate abuse or consuming poppy seed containing food. Moreover, the positive opiate screen did not show the presence of any opiate during GC/MS confirmation. Another opiate drug screen 24 days later was also positive for opiate during drug screen but GC/MS result was negative. However, when another urine sample was analyzed 13 days after cessation of gatifloxacin therapy, the urine immunoassay screen was negative for opiate indicating that the false-positive opiate screen was due to gatifloxacin [43].

INTERFERENCES WITH TETRAHYDROCANNABINOL IMMUNOASSAY Antiretroviral therapy such as efavirenz (EFV) screens positive for THC exposure despite the absence of 11-nor-tetahdryocannabinol-9-caobxylic acid (THC-COOH; the metabolite) [44]. Eight individuals who were taking 600 mg EFV/

136

Critical Issues in Alcohol and Drugs of Abuse Testing

day were randomized and analyzed for THC using six different instruments that detect THC via immunoassay. Three different immunoassays—Microgenics Corporation (Cedia Dau MultiLevel THC), BioSite Incorporated (Triage TOX Drug Screen), and Immunalysis Corporation (Cannabinoids (THC/CTHC) Direct ELISA Kit) falsely detected THC metabolites at a cutoff value greater than 50 ng/mL even though the patients were not taking THC [44]. A similar study in 2012 identified immunoassays, including Rapid Response (BTNX), CEDIA THC (Microgenics), THCA/CTHC Direct ELISA (Immunoanalysis), Triage TOX (Biosite), and Instant-view THC (Alfa Scientific) that had false-positive THC due to EFV [45]. In some of these immunoassays, cross-reactivity was linked to the position of carrier protein linkage in THC immunoassays. The low molecular weight compounds, cannabinoid haptens, require linkage to carrier proteins to produce immunogens from antibody generation. It was suggested that EFV metabolites interfered with carrier protein linkage with antibodies designed to C1-, C2-, and C5- and caused for a false-positive THC results.

INTERFERENCES WITH COCAINE IMMUNOASSAY The immunoassay for cocaine detection in drugs-of-abuse screen is designed to detect benzoylecgonine, an inactive metabolite of cocaine. The assay is unable to detect passive inhalation of cocaine; however, false-positive interferences are seen with tea products such as yerba mate or tea coming from Latin Americas. Different types of teas from Latin America are derived from coca plant, and have trace amounts of coca that may be mistaken for cocaine abuse. Five healthy volunteers (4 male and 1 female) consumed coca tealeaf steeped in 8 ounces of water for 15 minutes [46]. Participant 1 consumed 1 cup of 8 ounces tea, the second consumed 2 cups, the third consumed 3 cups, the fourth consumed 4 cups, and the fifth consumed 5 cups of coca tea. Urine from each participant was collected before the consumption and at 2, 12, 24, and 36 h after drinking the tea. Urine collected was analyzed for benzoylecgonine using Abbott AxSym System via immunoassay with a cutoff of 300 ng/mL. Each participant had a positive cocaine assay 2 hours after consuming tea. For three of the participants, the immunoassay for cocaine was positive even after 36 hours. The mean concentration of benzoylecgonine for all the participants after the consumption was 1777 ng/mL. Since yerba mate and tea from Latin American are popular in the United States, patients should abstain from drinking products with coca plants a few days before urine drug testing. Case Study A 48-year-old man was hospitalized for a pancreatic carcinoma that was confirmed via biopsy of the pancreatic mass. The patient required intensive pain control, and prior to the hospitalization, he was taking high dosage of long- and short-acting narcotics, OxyContin 40 mg orally twice daily and hydromorphone 6 mg orally every 4 hours as needed for his chronic neck and back pain. Due to his illicit drug abuse in the past (cocaine abuse), urine drug screen was performed. The patient’s urine drug test came out positive for both cocaine and oxycodone. The patient was recommended for celiac plexus block for his pain conditions, but the patient refused to take this pain management and the clinician did not think he was suitable for intrathecal pump (ITP) because of his urine drug screen and for the high risk of noncompliance. However, after analyzing the same sample with GC-MS, the results were negative for cocaine and its metabolites. The immunoassay false-positive result would have been detrimental for the patient’s pain management [47].

FALSE-POSITIVE TEST RESULTS IN OTHER DRUGS-OF-ABUSE IMMUNOASSAYS There are other drugs that may cause false-positive test results in various immunoassays used for screening of the presence of abused drugs in urine specimens. Dextromethorphan is an antitussive agent which is found in many OTC cough and cold medications. Dextromethorphan is also abused in high dosages mostly by young adults. Ingesting high amounts of dextromethorphan (over 30 mg) may result in positive false-positive test results with opiate and phencyclidine (PCP) immunoassays. In another report, the authors observed three false-positive phencyclidine tests in pediatric urine specimens using an onsite testing device (Instant-View multitest drugs-ofabuse panel; Alka Scientific, Designs, Poway, CA). The authors concluded that false-positive PCP tests were due to the cross-reactivities from ibuprofen, metamizol, dextromethorphan, and their metabolites with the PCP assay. In addition, pheniramine and methylphenidate also produced a false-positive result with the PCP immunoassay screen [48]. Studies indicate that nonsteroidal antiinflammatory drugs may produce false-positive results in immunoassays screening tests for the presence of drugs of abuse in urine specimens. Rollins et al. studied the effect of such drugs on

Issues of Interferences With Immunoassays Used for Screening of Drugs of Abuse in Urine Chapter | 10

137

TABLE 10.4 Interferences in Various Immunoassays Used for Drugs-of-Abuse Testing Immunoassay

Interfering Drugs

Benzodiazepines

Oxaprozin, sertraline

Opiates

Diphenhydramine, rifampin, dextromethorphan, verapamil, fluoroquinolones, quinine

Phencyclidine

Dextromethorphan, diphenhydramine, ibuprofen, imipramine, ketamine, meperidine, thioridazine, tramadol, venlafaxine

Tetrahydrocannabinol

Nonsteroidal antiinflammatory drugs, pantoprazole, EFV

Methadone

Diphenhydramine

drugs-of-abuse screening using 60 volunteers and 42 patients taking ibuprofen, naproxen, or fenoprofen. Out of 510 urine specimens collected from 102 individuals, only two specimens showed false-positive test for cannabinoids using enzyme-mediated immunoassay. There was one individual who ingested 1200 mg ibuprofen in three divided dosages and the other patient took naproxen regularly. Two urine specimens were false positive for barbiturates using the FPIA; one patient took ibuprofen and the other patient, naproxen. The authors concluded that there is a small likelihood of false-positive tests results in drugs-of-abuse screening assays in individuals taking nonsteroidal antiinflammatory drugs [48]. Joseph et al. studied 14 nonsteroidal antiinflammatory drugs for potential interference with EMIT and FPIA for various drugs of abuse and observed that tolmetin interferes with EMIT immunoassays at high concentrations (1800 μg/mL and higher) because of high molar absorptivity at 340 nm, the wavelength used for detection in the EMIT technology. Samples containing cannabinoid and benzoylecgonine tested negative in the presence of tolmetin but there was no effect on the FPIA assay because the detection wavelength was 525 nm [49]. Although most reports in the medical literature describe false-positive results in drugs-of-abuse testing due to the presence of OTC medications in urine specimens, Brunk et al. reported a false-negative GC/MS confirmation of marijuana metabolite as methyl derivative due to interference of ibuprofen with the methylation step. The urine specimen tested positive for marijuana metabolite using the EMIT d.a.u. assay but showed negative GC/MS confirmation result. When the specimen was analyzed by thin layer chromatography (TLC), the presence of marijuana metabolite was also observed [50]. Table 10.4 lists the drugs that interfere with various drugs-of-abuse immunoassays. Case Study A 9-year-old boy presented to pediatric ED with complaints of altered level of consciousness. He was receiving diphenhydramine for nasal congestion and sleeping difficulty in the previous week before presenting to the ED. The parents denied that he took the medicine on the evening of going to the hospital. His physical examination showed dilated pupils, dry mucous membrane, confusion and occasional hallucination. His urine drug screen showed positive for methadone and serum drug screen was positive for tricyclic antidepressant. The patient denied taking any medication other than diphenhydramine and was admitted to the hospital. His symptoms resolved within 24 h. The authors attributed positive tricyclic antidepressant serum screen due to the interference of diphenhydramine with the assay consistent with the literature reports, but only interference of diphenhydramine with EMIT opiate assay has been previously reported and the assay used (urine drug screen of the boy) was performed by a point-of-care testing device (One-Step, Multi-Drug Multi-Line Screen, ACON Laboratories, San Diego, CA). The authors then investigated the possible interference of diphenhydramine with this assay, and when the authors supplemented drug-free urine specimen with 100 ug/mL of diphenhydramine, the opiate test was positive for opiate. Therefore, the false-positive test result was due to the use of diphenhydramine [51].

CONCLUSIONS Although false-positive test results are routinely encountered in urine drug testing, such methods are widely used in clinical laboratories for initial screening for the presence of drugs, in urine or serum/plasma in patients admitted to emergency rooms and other clinical settings to support patient care. Melanson et al. commented that directors should be aware of the characteristics of their laboratories’ assays and should be able to communicate with ordering physicians

138

Critical Issues in Alcohol and Drugs of Abuse Testing

regarding proper interpretation of qualitative results. In addition, manufacturer’s claim must be interpreted with caution and if possible should also be validated using the patient population of the hospital [52]. However, false-positive results can be avoided in drugs-of-abuse testing by using more specific analytical methods such as chromatography combined with mass spectrometry. Eichhorst et al. described an improved cost-effective high-throughput method for drugs-ofabuse screening by tandem mass spectrometry replacing immunoassay screening [53] or another approach would be to create a large drug testing panel by utilizing mass spectrometry, to replace poor performing drug immunoassays, in combination with drug immunoassays that have a low false-positive rate (,5%) [54].

REFERENCES [1] Chen WJ, Fang CC, Shyu RS, Lin KC. Underreporting of illicit drug use by patients at emergency departments as revealed by two-tiered urinalysis. Addic Behav 2006;31:2304 8. [2] Johnson-Davis KL, Sadler AJ, Genzen JR. A retrospective analysis of urine drugs of abuse immunoassay true positive rates at a national reference laboratory. J Anal Toxicol 2016;40:97 107. [3] Standridge JB, Adams SM, Zotos AP. Urine drug screening: A valuable office procedure. Am Fam Physician 2010;81:635 40. [4] Dietzen DJ, Ecos K, Friedman D, Beason S. Positive predictive values of abused drug immunoassays on the Beckman synchron in a veteran population. J Anal Toxicol 2001;25:174 8. [5] Hsu J, Liu C, Liu CP, Tsay WI, Li JH, Lin DL, et al. Performance characteristics of selected immunoassays for preliminary test of 3,4-methylenedioxymethamphetamine, methamphetamine, and related drugs in urine specimens. J Anal Toxicol 2003;27:471 8. [6] Kunsman GW, Manno JE, Cockerham KR, Manno BR. Application of the syva emit and abbott tdx amphetamine immunoassays to the detection of 3,4-methylene-dioxymethamphetamine (mdma) and 3,4-methylene-dioxyethamphetamine (mdea) in urine. J Anal Toxicol 1990;14:149 53. [7] Casey ER, Scott MG, Tang S, Mullins ME. Frequency of false positive amphetamine screens due to bupropion using the syva emit II immunoassay. J Med Toxicol 2011;7:105 8. [8] Marin SJ, Doyle K, Chang A, Concheiro-Guisan M, Huestis MA, Johnson-Davis KL. One hundred false-positive amphetamine specimens characterized by liquid chromatography time-of-flight mass spectrometry. J Anal Toxicol 2016;40:37 42. [9] Yee LM, Wu D. False-positive amphetamine toxicology screen results in three pregnant women using labetalol. Obstet Gynecol 2011;117:503 6. [10] Saitman A, Park HD, Fitzgerald RL. False-positive interferences of common urine drug screen immunoassays: A review. J Anal Toxicol 2014;38:387 96. [11] Fucci N. False positive results for amphetamine in urine of a patient with diabetes mellitus. Forensic Sci Int 2012;223:e60. [12] Butler D, Guilbault GG. Analytical techniques for ecstasy. Analytical Letters 2004;37:2003 30. [13] Wu AH, McKay C, Broussard LA, Hoffman RS, Kwong TC, Moyer TP, et al. National academy of clinical biochemistry laboratory medicine practice guidelines: Recommendations for the use of laboratory tests to support poisoned patients who present to the emergency department. Clin Chem 2003;49:357 79. [14] Ka M. Amphetamines/sympathomimetic amines. In: Levine B, editor. Principles of forensic toxicology. Washington DC: AACC Press; 2003. p. 341 8. [15] Poklis A. Unavailability of drug metabolite reference material to evaluate false-positive results for monoclonal emit-d.A.U. Assay of amphetamine. Clin Chem 1992;38:2560. [16] Williams RH, Erickson T, Broussard LA. Evaluating sympathomimetic intoxication in an emergency setting. Lab Medicine 2000;31:497 508. [17] Singh J. Reagent lot-to-lot variability in sensitivity for amphetamine with the syva emit ii monoclonal amphetamine/methamphetamine assay. J Anal Toxicol 1997;21:174 5. [18] Hornbeck CL, Carrig JE, Czarny RJ. Detection of a GC/MS artifact peak as methamphetamine. J Anal Toxicol 1993;17:257 63. [19] Dasgupta A, Gardner C. Distinguishing amphetamine and methamphetamine from other interfering sympathomimetic amines after various fluoro derivatization and analysis by gas chromatography-chemical ionization mass spectrometry. J Forensic Sci 1995;40:1077 81. [20] Ojanpera IA, Heikman PK, Rasanen IJ. Urine analysis of 3,4-methylenedioxypyrovalerone in opioid-dependent patients by gas chromatography-mass spectrometry. Ther Drug Monit 2011;33:257 63. [21] Stout PR, Klette KL, Horn CK. Evaluation of ephedrine, pseudoephedrine and phenylpropanolamine concentrations in human urine samples and a comparison of the specificity of dri amphetamines and abuscreen online (kims) amphetamines screening immunoassays. J Forensic Sci 2004;49:160 4. [22] Solomon MD, Wright JA. False-positive for (1)-methamphetamine. Clin Chem 1977;23:1504. [23] Poklis A, Jortani SA, Brown CS, Crooks CR. Response of the emit ii amphetamine/methamphetamine assay to specimens collected following use of vicks inhalers. J Anal Toxicol 1993;17:284 6. [24] Poklis A, Moore KA. Stereoselectivity of the tdxadx/flx amphetamine/methamphetamine ii amphetamine/methamphetamine immunoassayresponse of urine specimens following nasal inhaler use. J Toxicol Clin Toxicol 1995;33:35 41. [25] Wyman JF, Cody JT. Determination of l-methamphetamine: A case history. J Anal Toxicol 2005;29:759 61. [26] FDA News P06-07, January 13, 2006.

Issues of Interferences With Immunoassays Used for Screening of Drugs of Abuse in Urine Chapter | 10

139

[27] Jemionek JBT, Jacobs A, Holler J, Magluli J, Dunkley C. Five cases of d-amphetamine positive urines resulting from ingestion of “Brazilian nutritional supplements” containing fenproporex have been reported. ToxTalk SOFT newsletter 2006;30:11. [28] Vorce SP, Holler JM, Cawrse BM, Magluilo Jr. J. Dimethylamylamine: A drug causing positive immunoassay results for amphetamines. J Anal Toxicol 2011;35:183 7. [29] Lattman Peter, Singer Natasha. Army studies workout supplement after deaths. New York Times 2012;February 2:B1. [30] Nguyen MH, Ormiston T, Kurani S, Woo DK. Amphetamine lacing of an internet-marketed neutraceutical. Mayo Clin Proc 2006;81:1627 9. [31] Perez-Downes J, Hritani A, Baldeo C, Antoun P. Amphetamine containing dietary supplements and acute myocardial infarction. Case Rep Cardiol 2016;2016:6404856. [32] Eliason MJ, Eichner A, Cancio A, Bestervelt L, Adams BD, Deuster PA. Case reports: Death of active duty soldiers following ingestion of dietary supplements containing 1,3-dimethylamylamine (dmaa). Mil Med 2012;177:1455 9. [33] Bailes JE, Cantu RC, Day AL. The neurosurgeon in sport: Awareness of the risks of heatstroke and dietary supplements. Neurosurgery 2002;51:283 6 discussion6-8. [34] Poklis A, Hall KV, Still J, Binder SR. Ranitidine interference with the monoclonal emit d.A.U. Amphetamine/methamphetamine immunoassay. J Anal Toxicol 1991;15:101 3. [35] Liu L, Wheeler SE, Rymer JA, Lower D, Zona J, Peck Palmer OM, et al. Ranitidine interference with standard amphetamine immunoassay. Clin Chim Acta 2015;438:307 8. [36] Logan BK, Costantino AG, Rieders EF, Sanders D. Trazodone, meta-chlorophenylpiperazine (an hallucinogenic drug and trazodone metabolite), and the hallucinogen trifluoromethylphenylpiperazine cross-react with the emit(r)ii ecstasy immunoassay in urine. J Anal Toxicol 2010;34:587 9. [37] Fenderson JL, Stratton AN, Domingo JS, Matthews GO, Tan CD. Amphetamine positive urine toxicology screen secondary to atomoxetine. Case Rep Psychiatry 2013;2013:381261. [38] Zacher JL, Givone DM. False-positive urine opiate screening associated with fluoroquinolone use. Ann Pharmacother 2004;38:1525 8. [39] Baden LR, Horowitz G, Jacoby H, Eliopoulos GM. Quinolones and false-positive urine screening for opiates by immunoassay technology. JAMA 2001;286:3115 19. [40] de Paula M, Saiz LC, Gonzalez-Revalderia J, Pascual T, Alberola C, Miravalles E. Rifampicin causes false-positive immunoassay results for urine opiates. Clin Chem Lab Med 1998;36:241 3. [41] Straseski JA, Stolbach A, Clarke W. Opiate-positive immunoassay screen in a pediatric patient. Clin Chem 2010;56:1220 3. [42] Jenkins AJ, Poirier III. JG, Juhascik MP. Cross-reactivity of naloxone with oxycodone immunoassays: Implications for individuals taking suboxone. Clin Chem 2009;55:1434 6. [43] Straley CM, Cecil EJ, Herriman MP. Gatifloxacin interference with opiate urine drug screen. Pharmacotherapy 2006;26:435 9. [44] Rossi S, Yaksh T, Bentley H, van den Brande G, Grant I, Ellis R. Characterization of interference with 6 commercial delta9tetrahydrocannabinol immunoassays by efavirenz (glucuronide) in urine. Clin Chem 2006;52:896 7. [45] Oosthuizen NM, Laurens JB. Efavirenz interference in urine screening immunoassays for tetrahydrocannabinol. Ann Clin Biochem 2012;49:194 6. [46] Mazor SS, Mycyk MB, Wills BK, Brace LD, Gussow L, Erickson T. Coca tea consumption causes positive urine cocaine assay. Eur J Emerg Med 2006;13:340 1. [47] Kim JA, Ptolemy AS, Melanson SE, Janfaza DR, Ross EL. The clinical impact of a false-positive urine cocaine screening result on a patient’s pain management. Pain medicine 2015;16:1073 6. [48] Marchei E, Pellegrini M, Pichini S, Martin I, Garcia-Algar O, Vall O. Are false-positive phencyclidine immunoassay instant-view multi-test results caused by overdose concentrations of ibuprofen, metamizol, and dextromethorphan?. Ther Drug Monit 2007;29:671 3. [49] Joseph R, Dickerson S, Willis R, Frankenfield D, Cone EJ, Smith DR. Interference by nonsteroidal anti-inflammatory drugs in emit and tdx assays for drugs of abuse. J Anal Toxicol 1995;19:13 17. [50] Brunk SD. False negative GC/MS assay for carboxy thc due to ibuprofen interference. J Anal Toxicol 1988;12:290 1. [51] Rogers SC, Pruitt CW, Crouch DJ, Caravati EM. Rapid urine drug screens: Diphenhydramine and methadone cross-reactivity. Pediatr Emerg Care 2010;26:665 6. [52] Melanson SE, Baskin L, Magnani B, Kwong TC, Dizon A, Wu AH. Interpretation and utility of drug of abuse immunoassays: Lessons from laboratory drug testing surveys. Arch Pathol Lab Med 2010;134:735 9. [53] Eichhorst JC, Etter ML, Rousseaux N, Lehotay DC. Drugs of abuse testing by tandem mass spectrometry: A rapid, simple method to replace immunoassays. Clin Biochem 2009;42:1531 42. [54] McMillin GA, Marin SJ, Johnson-Davis KL, Lawlor BG, Strathmann FG. A hybrid approach to urine drug testing using high-resolution mass spectrometry and select immunoassays. Am J Clin Pathol 2015;143:234 40.