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Journal of Addictive Diseases , 29:436–448, 2010

Copyright c Taylor & Francis Group, LLC

ISSN: 1055-0887 print / 1545-0848 online

DOI: 10.1080/10550887.2010.509277

print / 1545-0848 online DOI: 10.1080/10550887.2010.509277 Advanced Urine Toxicology Testing Peter L. Tenore, MD, FASAM

Advanced Urine Toxicology Testing

Peter L. Tenore, MD, FASAM

ABSTRACT. Urine toxicology screening testing is an important standard of care in the addiction and pain treatment setting, offering a reproducible, unbiased, and accurate laboratory test to monitor patients and provide objective support for clinical observations. It has been shown that physicians do not have proficiency in the ordering or interpretation of these tests. This article is an attempt to respond to that need. Current antibody-based enzymatic immunoassays (EIAs) used for urine toxicology screening are useful to detect classes of drugs (ex., opiate) but cannot determine which specific drug (ex., morphine) is present. Gas chromatography and mass spectroscopy can determine exactly which drugs are present, allowing prescribed (or illicit) opiates and benzodiazepines to be identified. This article will discuss principles and details of opiate and benzodiazepine EIA and gas chromatography and mass spectroscopy urine toxicology testing. The approach to detecting patients attributing positive opiate EIAs to prescription opiates who are using heroin or other opioids will be reviewed. Cases of controlled prescription drugs that do not produce the expected positive urine tests (ex., oxycodone producing negative opiate screening tests) will be discussed. How to differentiate codeine from heroin and the role of poppy seeds in toxicology will be examined. The case of an anti-depressant drug that produces false-positive benzodiazepine results and antibiotics that cause positive opiate urine toxicology results will be reviewed. Common benzodiazepines (ex., clonazepam and lorazepam) that do not reliably produce positive benzodiazepine EIAs will be discussed. The approach to detection and management of all these types of toxicology cases will be reviewed, and it is hoped that the analyses presented will impart an adequate information base to medical providers and staff members of drug treatment and pain centers, enabling them to order and interpret these tests in the clinic more effectively as an integrated part of whole patient care.

KEYWORDS. Gas chromatography, mass spectroscopy, urine toxicology, opiate, benzodiazepine, morphine, codeine, EIA, ELISA, EMIT, fluoroquinolone, sertraline, drug screening, lorazepam, clonazepam

INTRODUCTION

Rational for Drug Monitoring

Three basic reasons are used to consider urine drug monitoring in patients who are in drug treat- ment or who are prescribed opioids or benzodi- azepines.The first reason is to reassure medical providers that the patients who are prescribed opioids and benzodiazepines are taking medi-

cation as directed, evidenced by positive results in urine drug testing. 1 3 The second reason is that urine drug testing is used to detect pos- sible diversion of medication (i.e., stockpiling or selling of controlled prescribed substances to unauthorized others), evidenced by negative urine drug tests. The overdose mortality rate in the United States due to diverted prescribed opioid analgesics as reported on death certifi- cates increased dramatically (by 91.2%) from

Peter L. Tenore is affiliated with Albert Einstein College of Medicine, Bronx, NY. Address correspondence to: Peter L. Tenore, MD, FASAM, Wellness Center at Waters Place, 1510 Waters Place, Bronx, NY 10461 (E-mail: ptenore@dosa.aecom.yu.edu).

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1999 to 2002, indicating that “a national epi- demic of drug poisoning deaths began in the 1990’s and prescriptions for opioid analgesics contributed to the increases in drug poison- ing deaths.” 4 The most common opioids im- plicated in overdose deaths were, in order of frequency of occurrence, methadone, fentanyl, oxycodone, hydromorphone, hydrocodone, and morphine and were obtained by overdose vic- tims illegally from patients with legitimate pre- scriptions; therefore, it is critical for providers to ensure that patients prescribed these medi- cations are taking and not diverting them. 4 In addition to patient history and clinical presen- tation, random urine drug testing is an essential and objective part of this monitoring process. 1,2,5 The third reason is to detect the presence of illicit non-prescribed drugs, such as heroin, cocaine, methamphetamine, phencyclidine, non- prescribed opioids, and non-prescribed benzodi- azepines, according to community prevalences, the presence of which suggests that patients may not be entirely responsible to manage prescribed opioids and benzodiazepines and need further therapeutic intervention. 1,3,6 The discussion in this article will be limited to discussing two common classes of drugs that are widely abused and are widely prescribed by physicians and other medical providers: benzo- diazepines and opioids. How urine drug testing can be used to differentiate prescribed versus il- licit drugs of these classes and to contribute to the medical and psychosocial care of these pa- tients will be reviewed.

Enzyme-Immuno-Assay Testing

The most common drug screening tech- niques are antibody-based, enzyme-mediated immunoassays. These are inexpensive, auto- mated, rapid, and accurate. 1 Various tests such as Enzymatic Immunoassay (EIA), Enzyme Me- diated Immunoassay Technique (EMIT), En- zyme Linked Immunosorbent Assay (ELISA), and Cloned Enzyme Donor Immuno-Assay (CE- DIA) refer to the same basic process: a spe- cific anti-drug antibody is added to the patient’s urine and, if the specific drug is present, the antibody binds to the drug, enabling a measur- able indicator reaction to occur that is reported

as “positive.” 7 For simplicity, in this article, the term enzymatic immunoassay (EIA) will be used to represent all these antibody-mediated urine toxicology tests. The opiate EIA uses an anti-morphine anti- body to detect opiates; and the Benzodiazepam EIA uses an anti-diazepam antibody to detect benzodiazepines. 8 Because these antibodies are specific to morphine and diazepam, there will be both false-negative and false-positive re- sults with non-morphine opioids (ex., fentanyl) and non-diazepam benzodiazepines (ex., clon- azepam), which may confuse the clinical pic- ture and suggest diversion. These issues will be detailed in sections below. Although EIAs are reproducible, inexpensive, and easy to perform,

a disadvantage of EIA testing is that only the

class of drug is identified (ex., opiate or benzo- diazepine) and not the specific drug (ex., codeine or diazepam); 2 further testing, explained in the next section, is required to make a specific iden- tification.

Gas Chromatography and Mass Spectroscopy Testing

To determine specific drugs and concentra- tions, gas chromatography followed by mass spectroscopy is used. These tests are typically used in sequence. Simplistically, the urinet spec- imen is vaporized and injected into a port on the gas chromatograph where the sample is dis-

persed within a carrier gas that travels upward on

a chemically treated column. Lighter molecules

in the specimen travel up faster and heavier ones

more slowly; hence, separation occurs. As the molecules reach top, an analyzer records con- centrations as separate peaks on a graph. 2,9 Next,

each peak is sent through the mass spectroscopy, which bombards the component with electrons, creating a predictable and reproducible microex- plosion resulting in an equally reproducible pat- tern of molecular strikes on a recording plate creating energy peaks viewed as a mass spec- trograph. These spectrograph patterns are com- pared by computer to a database of thousands of mass spectrographs and the specific component

is identified (ex., morphine or diazepam). 2,9 In short, gas chromatography separates and

quantifies drug components and then mass

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spectroscopy specifically identifies them. Gas chromatography followed by mass spectroscopy is considered the “gold standard” of urine tox- icology testing because it is 99% specific and 99% sensitive. 6 The use of EIA and gas chro- matography and mass spectroscopy urine testing to monitor various commonly prescribed opioids and benzodiazepines is the subject of this article and is presented below. 10 12 Pain clinic chronic opioid patients have been shown to submit significant numbers of abnor- mal urine toxicology tests, defined as the pres- ence of illicit substances, excess levels of the prescribed substance, or the absence of the pre- scribed substance. 1 In one study of 470 pain center patients, 44% abused substances, 20% used illicit non-opioid substances, 14% used ad- ditional opioids not prescribed, and 10% did not use the opiate that was prescribed. 13 In another pain center, testing of 196 patients found 32% to be mismanaging drugs; 60% of these were using non-prescribed opiates or diverting opi- ates and 40% of the 196 patients were primarily abusing cocaine, with 11% of patients not taking the prescribed opiate medication. 14 In a review of several pain clinics, 25% to 44% of patients were found to be abusing various substances or diverting prescribed opioids, and this pattern of drug use was closely correlated with specific be- haviors, including repeated loss of prescriptions, excess pill consumption, visits without appoint- ments, frequent telephone calls to the clinic, and multiple drug intolerances or “allergies,” behav- iors of which prescribing physicians should be fully aware of and responsive to. 15 In the primary care setting, a study of 801 daily opioid patients managed by family care physicians showed 24% to be using illicit substances. 16 Accurate self-report of drug abuse and mis- use in these populations can be unreliable. 12 In a study of 110 patients in a methadone clinic, 21% reported illicit substance use in the month un- der study, yet toxicology demonstrated twice as many (39%) to be using illicit drugs. 17 A study of more than 800 chronic opiate patients found 24% to be using illicit substances, yet 46% of these patients adamantly denied substance abuse, even when guaranteed anonymity. 16 Therefore, urine drug testing has become indispensable as an ob- jective, accurate, and reproducible aid in pa-

tient monitoring patients prescribed opioids or benzodiazepines. 3,13 18

Frequency of Urine Drug Testing

In pain management settings, it is recom- mended that urine drug screening be considered at every clinic visit but actually collected on a random basis. 12 Urine collection should be random to eliminate patients’ feelings of being singled out or victimized and to assure collection of a reasonably representative set of toxicology specimens over time, yielding a toxicology profile to assist in evaluating the individual patient. 19 Urine drug tests are not intended to be the sole parameter by which patient man- agement decisions are made; rather, testing is only a small part of the greater whole of patient medical and psychosocial care and should be viewed in that context. For methadone treatment programs, federal government regulations mandate weekly urine drug testing for the first 90 days in treatment; after this period, health care providers may use clinical judgment to de- termine whether weekly testing should continue or if less frequent testing is warranted as long as 8 urine toxicology specimens are submitted annually; times of heightened patient stress or drug relapse may call for increased frequency of urine testing back to weekly. 10 Ultimately, it is the medical providers’ judgment in conjunction with the patients’ clinical status that should determine the urine testing schedule. 10,12,19

Physician Awareness

To be useful, urine toxicology test results must be interpreted properly; however, studies show that primary care physicians are not proficient at this; none of 80 physicians in one study answered all questions correctly on a brief yes-or-no rudi- mentary toxicology questionnaire, and only 20% answered half or more questions correctly. 20 In a second study, none of 114 physicians answered all questions correctly and only 30% answered half correctly. The 77 physicians who used urine drug testing in their practices performed no bet- ter than the 37 physicians who did not, and the authors called for increased physician ed- ucation regarding urine drug testing. 21 The drug

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summaries presented later in this article hope to address this deficit.

DRUGS OF INTEREST Oxycodone and Opiate EIA Testing

In a study of 52 gas chromatography and mass spectroscopy-confirmed oxycodone positive urine specimens, only 30% were determined to be positive by opiate EIA (i.e., the standard opiate EIA does not detect oxycodone in the majority of oxycodone specimens). 22 In an review of 8 commercially available opiate EIAs, the rates of detection of codeine, morphine, and semi-synthetic opiates were as follows:

morphine (100%), codeine (100%), oxymor- phone (15%), meperidine ( 3%), hydro- morphone (60%), levorphanol ( 29%), and oxycodone ( 12%). This demonstrates that the standard opiate EIA is not de- signed to and will not detect 100% of semi- synthetic opiates, including oxycodone. 6 Thus, oxycodone produces mostly negative opiate EIAs. In a survey of 359 primary care physicians, only 12% knew that oxycodone is usually not detected by most opiate EIA screen- ing immunoassays. 23 Oxycodone is a “semi- synthetic” opioid molecule, synthesized by the chemical addition of varying side-chains to a morphine molecule. 24 The side-chains alter the morphine structure to the extent that the mor- phine antibody of the opiate EIA is unable to reliably bind to or “react” with oxycodone, re- sulting in frequent negative opiate EIAs. 6,8,22,25 Patients taking oxycodone submitting the ex- pected negative opiate EIA urine in approxi- mately 90% of specimens may, because of physi- cian unawareness, stand accused of medication diversion and undergo sanctions, denial of med- ication, or clinic discharge. In one report, the authors state:

We recently encountered a patient we sus- pected of abusing or misusing prescribed OxyContin R (oxycodone). In order to de- termine whether the patient was taking the MEDICATION as prescribed, we ordered a urine-based immunoassay drug screen

(Opiate EIA). The results were negative; the patient appeared to not have oxycodone in his system. Based on these results, we dismissed the patient from our practice. 26

If these providers were aware that oxycodone and other semi-synthetics produced a significant amount of negative standard opiate EIAs, as seen in this case, and that an EIA specific for oxy- codone was available, the patient would not have experienced sanctioning. The oxycodone EIA is an extremely reliable test: in a study of 435 urine samples containing oxycontin, the oxycodone EIA demonstrated a sensitivity of 99.1% and specificity of 99.8%. 25 Medical providers can evaluate patients taking oxycodone who submit negative urine opiate EIAs with the specific oxycodone EIA; if oxy- codone is demonstrated, false accusations and unwarranted sanctions may be averted. 26,27 However, if the oxycodone EIA does not find oxycodone in an patient taking oxycodone, there are three major considerations. First, there is the possibility that the patient is diverting or stockpiling medication, indicating a need for further urine testing and clinical interven- tion. Second, some compliant patients have very low urine oxycodone levels (under the 300 ng/mL level of detection) and may inno- cently produce false-negative oxycodone EIAs. This can be seen in rapid metabolizers or in very low dose cases; in these instances, oxycodone can be detected by gas chromatog- raphy and mass spectroscopy. 3,8,9,18 Third, oxy- codone patients submitting oxycodone negative specimens may be substituting another individ- uals’ urine to avoid detection of drug abuse or diversion. If urine substitution is suspected, a supervised specimen may be collected in a re- spectful manner or an oral fluid test that can be administered by a staff member observing the oral swab in the patients’ oral cavity can be done, ensuring that the specimen belongs to the patient. 28

Semi-Synthetic Opiates

As in the case of oxycodone, other semi- synthetic opiates, including buprenorphine, oxy- morphone, oxycodone, hydromorphone, and

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levorphanol, all produce a significant number of negative standard urine opiate EIAs. 3,6 Many physicians are unaware of this fact. 17 Because, as with oxycodone, the molecular shapes of semi- synthetic morphine-based opiates differ from morphine, with side-chain substitutions they be- come poorly reactive with standard opiate EIA morphine antibodies and frequently produce negative results. 6,8,19 Therefore, negative opiate EIAs in patients taking levorphanol, hydromor- phone, buprenorphine, or other semi-synthetics do not necessarily indicate diversion. To pre- vent unfounded allegations, practitioners can monitor semi-synthetic patients with the spe- cific EIAs available for oxycodone, buprenor- phine, or hydromorphone. 18 These EIAs will verify semi-synthetic presence in those taking medication properly and will reveal absence of semi-synthetics in those not taking medication regularly or diverting it. 27 If semi-synthetic spe- cific EIAs are unavailable, gas chromatogra- phy and mass spectroscopy testing may also be used to identify semi-synthetic opiates, includ- ing oxycodone. 3,8,9 Providers should familiarize themselves with which tests their toxicology lab- oratory uses.

Hydrocodone

Hydrocodone is a semi-synthetic and will usually not be detected by most standard opi- ate EIAs, as discussed in the previous section. 18 However, there is a caveat to this statement; in 2 of 8 commercially available opiate EIAs re- viewed, the EMIT II Plus Opiate Assay and the Archetict/Aeroset Opiate Assay-hydrocodone was readily identified as an opiate, making it the exception to the rule that semi-synthetic opiates typically present as opiate-negative with stan- dard opiate EIAs. 6,18 Thus, it is critical that prac- titioners communicate with the laboratory and learn which EIAs are in use. If the opiate EIA in use is one that readily identifies the semi- synthetic hydrocodone, expect positive opiate EIAs in hydrocodone patients; if the laboratory does not use one of the 2 hydrocodone-sensitive EIAs, most opiate EIAs in patients taking hy- drocodone will be negative. Gas chromatogra- phy and mass spectroscopy may be used to identify this substance. 6,18 Given that diverted

hydrocodone has been implicated in more opi- oid analgesic overdose deaths than morphine, monitoring patients taking hydrocodone with ongoing urine drug testing and the appropri- ate hydrocodone-sensitive EIA is an important initiative. 4

Heroin

Approximately 30% of the weight of opium poppy fluid is comprised of opiate alkaloids consisting mostly of morphine with lesser amounts of codeine and other non-narcotic compounds. 24,29 By the chemical addition of 2 acetyl groups to opium morphine, heroin (di- acetyl-morphine) is synthesized. 30 Heroin is rapidly metabolized from di- acetyl-morphine to 6-mono-acetyl morphine (6- MAM), then to morphine, and then to morphine- 6-glucuronide, all with short half-lives measured in minutes such that that heroin becomes un- detectable in 40 minutes, 6-MAM in 4 to 12 hours, morphine in 8 hours, and morphine-6- glucuronide in 48 hours. Because 70% to 80% of the heroin dose is recovered in the urine and all of these metabolites are readily detected by the opiate EIA’s morphine antibody, heroin and its morphine-moiety metabolites are easily de- tected with standard opiate EIAs producing a positive opiate result at the 300 ng/mL level of detection for up to 2 days after a single intra- venous or inhaled heroin dose. 31,32

6-MAM and Heroin Detection

Heroin (3,6-diacetyl-morphine) is rapidly metabolized by liver cytochromes directly into the molecule 6-MAM, making 6-MAM an ideal candidate for specific identification of heroin use. 5 In a landmark 1988 study of heroin metabo- lites, investigators measured morphine levels and, for the first time, 6-MAM levels in 12 heroin users to quantify urine morphine levels in these cases, and to determine whether 6-MAM was useful to identify heroin use. Gas chromatog- raphy and mass spectroscopy detected 6-MAM levels of 53 to 3,390 ng/mL (10 ng/mL = “pos- itive”) in 11 of 12 cases. Only 1 case with a low morphine level (1,414 ng/mL) was 6-MAM negative. 33 In another study of heroin users, 73% of 100 specimens were found to be positive for

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6-MAM. 34 Because 6-MAM is the first product of heroin metabolism and no other compound with the exception of heroin yields 6-MAM, the presence of 6-MAM in the urine is unequiv- ocal evidence of heroin use and, thus, an ex- ceedingly important forensic urine monitoring tool. 33,34 Although evidence of 6-MAM proves heroin use, a limitation of 6-MAM testing is its short half-life, rendering it detectable for only up to 12 hours after heroin use and it can be missed if heroin use occurred prior to that. 32 Thus, re- peated testing for 6-MAM may be necessary to ultimately identify heroin use in patients with positive opiate EIAs who are suspected of but deny heroin use. 34,35 Several commercial 6-MAM EIA tests are available and, in a study of 525 6-MAM pos- itive specimens, these immunoassays proved to be 98% sensitive and 98% specific, making the 6-MAM EIA an important tool to assist patient management. 5,36

Morphine Levels in Heroin Use

After 6-MAM, heroin rapidly metabolizes to morphine and, depending when samples are col- lected in relation to heroin use, morphine lev- els vary. In one study of 12 heroin users, ran- domly collected urine morphine levels ranged from 1,400 to 87,350 ng/mL, with half of the cases having morphine levels of 8,000 ng/mL or higher. 33 In a larger study of 63 heroin users, the mean urine morphine level was 62,370 ng/mL (range: 7,100 to 476,000 ng/mL) and 80% of participants demonstrated morphine levels > 15,000 ng/mL. Thus, a morphine level greater than 15,000 ng/mL is highly suggestive of heroin use but is certainly not absolute proof because morphine use or abuse and codeine use or abuse may also produce morphine at these levels. 33,34 Because the presence of morphine in the urine specimen can indicate heroin use but can also indicate the use of morphine, codeine, or poppy seeds, to better identify heroin use, two other modalities independent of the absolute morphine level can link morphine positivity to heroin use. The first is the 6-MAM EIA test, which when present proves heroin use, but if absent does not exclude heroin use and repeat testing may

be needed. 36 The second modality is the mor- phine:codeine ratio, which can be helpful in the setting of a negative 6-MAM in a heroin user (discussed in the next section). 34

The Morphine/Codeine Ratio

Urine samples from heroin users typically contain substantial amounts of morphine with lesser amounts of codeine (reflecting poppy fluid contents) and dividing the morphine level by the codeine level yields the valuable mor- phine:codeine ratio. A morphine:codeine ratio greater than 2:1 as determined by gas chro- matography and mass spectroscopy has been shown to be a reliable indicator of heroin use. In a study of 100 heroin users, morphine:codeine ra- tios were greater than 20:1 in the majority (84%) of cases, greater than 10:1 in 90% of cases, and 2:1 or greater in 94% of cases, demonstrating that a morphine:codeine ratio greater than 2:1 is corroborative evidence of heroin use. 34 The only non-heroin drug combination that could produce a morphine:codeine ratio greater than 2:1 would be a patient using or abusing mor- phine in larger amounts and using or abusing codeine in smaller amounts simultaneously. This combination would conceivable produce a mor- phine:codeine ratio mimicking that of heroin (i.e., greater than 2:1) and a careful drug his- tory becomes critical in this case.

Codeine

Codeine is nearly identical to morphine in structure 24 so that standard urine opiate EIAs will easily detect codeine at the 300 ng/mL level of detection. 6,8 Maximum therapeutic codeine level by urine gas chromatography and mass spectroscopy should not exceed 15,000 ng/mL; levels higher than 15,000 ng/mL are indicative of codeine abuse. 37,38 Codeine is metabolized by cytochrome 2D6 to morphine; 39 thus, codeine patients will have both codeine and morphine detectable in gas chro- matography and mass spectroscopy urine sam- ples for up to 48 hours after a single dose. 24,37 Although both codeine and morphine are eas- ily identified by opiate EIAs and this informa- tion is readily available, only 29% of physicians who used urine drug testing in their practices

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were aware that codeine metabolizes to mor- phine, placing legitimate patients taking codeine at risk for accusations of morphine abuse. 21 Because codeine metabolizes to morphine, a morphine:codeine ratio can also be determined in codeine users; although heroin produces a morphine:codeine ratio of 2:1 or greater and is often much greater (ex., > 10:1), codeine pro- duces a morphine:codeine ratios of less than 2:1. If a patient taking codeine demonstrates a morphine:codeine ratio of 2:1 or greater, this is too much morphine to be explained by codeine and other morphine sources, including heroin, poppy seeds, and morphine itself, should be considered. 34,37,38

Codeine without Morphine

Rare patients lack the liver cytochrome 2D6 enzyme needed to convert codeine to morphine, meaning that they cannot metabolize codeine to morphine. These urine specimens will shown codeine only and no morphine, rendering the morphine:codeine ratio ineffective in differen- tiating drug use; supervised urine specimens or oral fluid specimens (administered directly observed by staff members) will show only codeine. 40 Another source of codeine-only specimens could be patients who are not taking their pre- scribed codeine (stockpiling or diverting) and who may spike drug-free urine samples with crushed codeine tablets to maintain the appear- ance of a positive opiate EIA. These urine sam- ples will have codeine only without morphine but examination of urine specimens will reveal a characteristic white precipitate along the bottom of the urine container. 28 Codeine normally produces positive opiate EIAs. If confirmation is desired in adherent cases, gas chromatography and mass spec- troscopy testing will reveal codeine or mor- phine levels of < 15,000 ng/mL and a mor- phine/codeine ratio of less than 2:1. The heroin metabolite 6-MAM EIA will be negative. In pa- tients taking codeine, codeine or morphine levels of > 15,000 ng/mL indicate codeine abuse 37,38 and a morphine:codeine ratio greater than 2:1 indicate heroin use. 34,41

Poppy Seed

As a product of the opium poppy, poppy seeds contain small amounts of morphine and codeine and, under proper conditions (ex., con- sumption of 1 poppy Danish streusel pastry), can readily produce positive opiate EIAs. 42 Many physicians are not aware of this poppy seed ef- fect. In a recent survey of 359 emergency room physicians, the majority (60%) were unaware that poppy seeds could produce a positive opiate EIA. 23 Goods baked with poppy seeds are widely available. Typically, poppy seed bagels, muffins, Danish, and Streusel contain morphine in amounts of 1, 2, 6, and 6 mg per piece, respec- tively, so that consumption of a single muffin, Danish, Streusel, or 2 bagels can produce a pos- itive opiate EIA at the 300 ng/mL morphine level of detection and the test can remain positive for 24 hours after ingestion. 42 In a classic 1987 study, consumption of 3 poppy seed bagels produced positive opiate EIAs with gas chromatography and mass spectroscopy morphine levels of 2,797 ng/mL at 3 hours, and 676 ng/mL at 22 hours (codeine levels were 214 and 16 ng/mL, respectively). 43 In another inves- tigation of 10 participants who consumed mea- sured amounts of whole poppy seeds reflecting amounts in baked goods, and who underwent multiple urine sampling over 24 hours, morphine was found in all cases at 24 hours. Two morphine levels were more than 2,000 ng/mL (2,635 and 2,199 ng/mL), but all other samples were less than 1,700 ng/mL, with most poppy seed mor- phine concentrations less than 1,000 ng/mL. The authors concluded that a morphine level of 3,000 ng/mL or less can be explained by poppy seed ingestion (but this can also be due to heroin or morphine use, see discussion below) and that most cases of poppy seed ingestion will have morphine levels less than 1,000 ng/mL. 44

Poppy Seed Morphine:Codeine Ratio

The morphine:codeine ratio in poppy seed ingestion, like heroin ingestion, is also 2:1 or greater (ex., 13:1, 42:1), consistent with opium poppy fluid as the opiate source for both heroin and poppy seeds. In one study, the average

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morphine:codeine ratio at 22 hours post-poppy seed ingestion was 45:1. 43 Other poppy seed studies also show morphine:codeine ratios of more than 2:1 (i.e., 19:1 to 295:1); thus, high morphine:codeine ratios with low morphine lev- els (< 3,000 ng/mL, usually < 2,000 ng/mL) are consistent with poppy seed consumption and do not automatically indicate heroin abuse. 45

Poppy Seed Ingestion versus Heroin Use

It can be difficult to determine whether pos- itive opiate EIAs are the result of poppy seed ingestion or heroin use, 38 both being derived from the same source (opium poppy). Gas chro- matography and mass spectroscopy will show that poppy seeds contain morphine and codeine indistinguishable from that of heroin and in the same ratio found in heroin (2:1 or greater), but absolute levels of morphine and codeine are typ- ically lower with poppy seeds, with morphine < 3,000 ng/mL and codeine < 300 ng/mL; most poppy seed cases produce urinary morphine lev- els less than 1,000 ng/mL. 44 High morphine levels ( > 3,000 ng/mL with a morphine:codeine ratio of 2:1 or greater) are most likely due to heroin use but any morphine level, even ones as low as 1,000 ng/mL with a morphine:codeine ratio of 2:1 or greater, can be caused by heroin use. 33 Because poppy seed in- gestion and heroin use can both produce lower morphine levels, 38 further testing for 6-MAM may be needed to identify heroin use with cer- tainty in these low level morphine cases. 33 35,46 In some cases, the only way to prove that morphine is due to poppy seed ingestion is to have patients discontinue ingesting poppy seed products and observe opiate EIAs reverting to negative levels in 48 hours. All patients are ad- vised to avoid poppy seed products to avoid un- necessary positive opiate EIAs and possible un- founded sanctions from providers. If 6-MAM is repeatedly negative and mor- phine is less than 3000 ng/mL with a mor- phine:codeine ratio of 2:1 or greater, it is impos- sible to differentiate poppy seeds from heroin. The clinical impression of the medical provider and patient history of poppy seed ingestion in conjunction with repeated gas chromatography and mass spectroscopy and 6-MAM testing is

needed to finally identify the true source of opi- ate. Nonetheless, morphine levels greater than 3,000 ng/mL, with a morphine:codeine ratio of 2:1 or greater, usually indicate heroin use and the 6-MAM EIA proves it if positive. 34,38

Fluoroquinolones

The only substances that normally produce 100% positive opiate EIAs are the morphine- based opiates such as morphine, heroin, codeine, and poppy seeds and the semi-synthetic hy- drocodone (if a hydrocodone-sensitive opiate EIA is in use). 8,19 Fluoroquinolone antibiotics, such as ofloxacin or ciprofloxacin, can also produce positive opiate EIAs, potentially leading to un- founded accusations of drug abuse and dismissal or other disciplinary action for patients enrolled in pain clinics or drug treatment programs. 47,48 Fluoroquinolone opiate EIA positivity is closely correlated to peak quinolone urine levels. In one investigation, urine collected 6 to 8 hours (peak levels) after ofloxacin or levofloxacin in- gestion produced 100% positive (false-positive) opiate EIAs in all 6 cases, with morphine lev- els registering just above the 300 ng/mL cut- off, even though there was no morphine present in the specimens. 49 Why opiate EIAs identify fluoroquinolones as morphine itself is unknown since chemical structures are completely dis- similar. 24 Individuals with positive opiate EIAs should be questioned regarding fluoroquinolone use and can be evaluated with gas chromatogra- phy and mass spectroscopy testing, which will demonstrate no opiates present in these speci- mens in the absence of opiate use or abuse. 50

Buprenorphine

Buprenorphine is a morphine-based, semi- synthetic molecule, which is a partial agonist at brain opiate mu-receptors. 24 Due to the addition of extensive side chains to the base morphine molecule to create buprenorphine, altering the appearance extremely from that of morphine; the morphine core is nearly unrecognizable in 2-dimensional depictions of buprenorphine. 24 Due to this configurational alteration, the mor- phine antibody in standard opiate EIAs will not

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react with buprenorphine, yielding negative opi- ate EIAs in these individuals. 8,51 However, fortunately highly sensitive and highly specific buprenorphine EIA tests are available and should be used to monitor patients taking buprenorphine. One buprenorphine EIA showed a specificity of 97% and a sensitivity of 99.5% 52 and another “demonstrated a sensitivity and specificity of 100%,” 54 making these tests essential to the management of the buprenor- phine patient. 53,55 However, conversely if testing in patients taking buprenorphine reveals the absence of buprenorphine, then diversion, stockpiling, or selling of medication becomes a concern. In a recent study of widespread buprenorphine di- version, medical personnel were alerted by the absence of buprenorphine in monitoring urine specimens with the buprenorphine EIA. 54 On questioning, only 43% of 71 diversion cases ad- mitted to diversion, reinforcing the importance of ongoing urine drug monitoring as a necessary objective tool to improve patient monitoring and enhance patient management. 1,3,53

Fentanyl

Fentanyl is a wholly synthetic (as opposed to semi-synthetic) opiate and, not being morphine- based, produces negative opiate EIAs. 55 Other common synthetic opiates include methadone, propoxyphene, meperedine, tramadol, and pen- tazocine. These synthetic opioids do not re- act with the opiate EIA morphine antibody and will produce negative opiate EIA results. Gas chromatography and mass spectroscopy and more specific EIA testing (discussed below) can be used to confirm the presence of these synthetics. 19,25 Because fentanyl diversion is a growing prob- lem in the United States, 56 prescribers need to be aware of fentanyl urine testing characteris- tics; fentanyl does not produce positive standard opiate EIAs. 25 The absence of positive opiate EIAs in patients taking fentanyl, although a nor- mal occurrence that is consistent with fentanyl, should not obviate the need for further con- firmatory testing to explicitly demonstrate the presence of fentanyl. A study of fentanyl patch patients showed a sensitivity and specificity

of 98% for the fentanyl urine EIA. 57 Several fentanyl-specific EIA tests available are capable of detecting minute concentrations of fentanyl in urine specimens and can be used to moni- tor patients taking fentanyl to assure this com- pound is present in patient urine specimens. 57,58 Providers should communicate with their tox- icology laboratories to determine whether a fentanyl EIA is available and apply it to pa- tients taking fentanyl in accordance with clinical needs. If patients taking fentanyl are submitting pos- itive standard opiate EIAs and these positives were attributed to fentanyl (which is not possi- ble), some morphine-based drug is being used or abused in addition to or in lieu of fentanyl, and gas chromatography and mass spectroscopy can be used for identification. 6

Methadone

Methadone is a wholly synthetic opioid molecule, which is not structurally related to morphine; therefore, methadone does not react with the standard opiate EIA morphine antibody and will yield negative opiate results in screening toxicology tests. 8 Providers who wish to mon- itor patients taking methadone can use one of the commercially available methadone EIAs that have been shown to be 98% sensitive and 98% specific. 59

Sertraline

Patients taking the anti-depressant sertraline can produce sporadic false-positive benzodi- azepine EIA urine toxicology specimens. 60 Un- founded accusations of benzodiazepine abuse in these patients may arise. In one study, 16 of 50 POSITIVE Benzodiazepine EIAs were submit- ted by patients taking sertraline who were not in- gesting benzodiazepines. In a review phase of the same study, false-positive benzodiazepine EIAs caused by sertraline were found in 26% of 2,447 patients taking sertraline urine specimens. 61 Re- cently, the benzodiazepine EIA test manufac- turer released the following statement:

patients taking therapeutic doses of Ser- traline (Zoloft R ) of 100–200 mg per day can test positive for benzodiazepines.

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An unidentified metabolite of Sertraline in urine is responsible for the cross-reactivity as unmetabolized Sertraline is not present in sufficient quantities to cause a positive test. 62

The chemical structures of sertraline and its

urinary metabolite, des-methyl-sertraline, bear a striking similarity to diazepam, 24 which likely enables the benzodiazepine EIA to react causing

a false-positive benzodiazepine result. 62 Sertra-

line is now listed as one of the compounds, along with diazepam and alprazolam, which can cause

a positive benzodiazepine EIA result in the 2008

EIA cross-reactivity list. 8 Patients taking Sertraline who submit pos- itive benzodiazepine EIAs and deny benzodi- azepine use should not automatically be accused of benzodiazepine abuse; they may undergo gas chromatography and mass spectroscopy test- ing, which can confirm the absence of benzo- diazepines, indicating a false-positive EIA due to sertraline. In cases of patients taking sertraline who are using or abusing benzodiazepines, gas chromatography and mass spectroscopy can be used to demonstrate the presence and concentra- tion of the specific benzodiazepine. 61

Clonazepam and Lorezepam

Patients taking clonazepam have been shown to produce a significant number of negative ben- zodiazepine EIAs due to lower reactivity of clon- azepam with the EIA’s diazepam antibody. 63 Similarly, lorazepam has also been show to produce mostly or all negative benzodiazepine EIAs for the same reason. In a study of 4 dif- ferent manufacturers’ benzodiazepine EIAs, 38 different benzodiazepines and their metabolites were evaluated to determine the rate of ben- zodiazepine detection for each benzodiasepine EIA. Results showed that all immuno-assays tested detected diazepam and alprazolam; how- ever, none detected clonazepam or lorazepam. 63

In an analysis of urine levels in 53 patients tak- ing benzodiazepine, 10% of whom were taking lorazepam, none of three commercially avail- able standard benzodiazepine EIAs were able to identify any lorazepam in any patient. 64 Finally,

a study of one popular commercially available

benzodiazepine EIA’s ability to react with 15 different benzodiazepines demonstrated “good” reactivity (i.e., antibody binding) to diazepam, only “moderate” reactivity to clonazepam, and “low” reactivity to lorazepam, indicating that negative benzodiazepine EIAs with clonazepam and lorazepam are expected, normal results. 65 Because these negative benzodiazepine EIAs may lead to accusations of benzodiazepine diver- sion, providers should consider gas chromatog- raphy and mass spectroscopy when needed to demonstrate clonazepam or lorazepam in these specimens. 9 However, if gas chromatography and mass spectroscopy fail to demonstrate clon- azepam or lorazepam in patients who say they are taking these medications, then diversion or missed doses should be considered. Conversely, if patients taking clonazapam or lorazepam are submitting mostly or all posi- tive benzodiazepine EIAs (not a clonazepam or lorazepam pattern) the use of other non- clonazapam or non-lorazepam benzodiazepines, such as diazepam and alprazolam, is sug- gested and gas chromatography and mass spec- troscopy can reliably determine their types and concentrations. 9

CONCLUSION

We have reviewed the use of screening urine EIAs and gas chromatography and mass spec- troscopy to verify legitimate prescriptions of specific drugs and to identify abused substances in the urine in those claiming legitimate pre- scriptions. Standard opiate EIAs identify only morphine, heroin, hydrocodone, and codeine as “positive” opiates; if the exact drug (i.e., mor- phine) and the concentration is desired, then gas chromatography and mass spectroscopy must be used to supply this information. Heroin use typically yields morphine lev- els greater than 3,000 ng/mL and poppy seed ingestion yields less than 3,000 ng/mL. Most heroin samples yield morphine levels in excess of 15,000 ng/mL, with morphine:codeine ra- tios greater than 2:1, usually 10:1 and greater. 6-MAM, a heroin metabolite, is definitive evi- dence of heroin use, although short lived under 12 hours. A morphine:codeine ratio greater than

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2:1 indicates heroin or poppy seed use; a ratio of less than 2:1 is consistent with codeine use. Synthetic opiates (non-morphine based), such as fentanyl, methadone, and meperidine, will be opiate EIA negative. Semi-synthetic opiates, such as oxycodone and hydromorphone, usu- ally yield negative opiate EIAs but, being distant morphine congeners, will also produce sporadic positive opiate EIAs (i.e., mixed positive and negative results). Specific EIAs and gas chro- matography and mass spectroscopy can identify synthetic and semi-synthetic opioids. Providers should consult with the toxicology laboratory to determine which tests are available. Fluoroquinolones such as ciprofloxacin can produce false-positive opiate EIAs. Gas chro- matography and mass spectroscopy can be used for clarification in all of these instances. Ser- traline can cause sporadic false-positive benzo- diazepine EIAs via sertraline metabolites’ re- sembling diazepam. Clonazepam and lorazepam with structures dissimilar to diazepam will fre- quently produce false-negative benzodiazepine EIAs and gas chromatography and mass spec- troscopy may be used to confirm their presence. Urine toxicology testing is an important part of comprehensive care for individuals attending drug treatment programs or receiving chronic opioid or benzodiazepine therapy and should be considered as an objective test within a greater integrated psychosocial and medical format and toxicology results should never be interpreted in a vacuum, but rather always within the broader clinical patient context.

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