CLINICAL & FORENSIC Toxicology News

March 2014 An AACC/CAP Educational Newsletter for Toxicology Laboratories

tency can lead to accidental deaths and overdoses Acetyl when used recreationally or unknowingly as an adul- “New ” Leads to Opiate Fatalities terant in other street drugs. The 2013 opiate deaths reflect a growing na- By Michael A. Wagner, PhD, Jeffery H. Moran, PhD, tionwide trend over the past 15 years. Opiate-related Amy L. Patton, MS, and Laurie Ogilvie, MS poisoning deaths have increased more than 650%, he past few years have seen an explosion of including from both prescription drugs and drugs of new, clandestinely produced synthetic drugs. abuse (3,4). The cycling between prescription drugs These drugs have generally fallen into two gen- and drugs of abuse has been driven by cost and eral categories—synthetic, hallucinogenic availability. In the early 2000s, as prescription drugs stimulants (commonly known as bath salts) and syn- became more available, their abuse increased. When thetic cannabinoids (known as K2 or Spice). Recently, the increase in abuse of prescription drugs led to in- a new drug from the family made a macabre vestigations of questionable prescribing practices, entrance onto the stage. street drugs such as heroin became relatively more In 2013, acetyl fentanyl was responsible for a affordable. With this increase in street drug use number of fatalities and overdoses. The Rhode Island came the added risk of dubious drug quality and le- Real-time Outbreak and Disease Surveillance system thal adulterants (5) . reported a spike in overdose deaths from March Fentanyl Analogues through May. Fourteen individuals ranging in age The recent morbidity and mortality reports in- from 19 to 57 years died from acetyl fentanyl over- volving acetyl fentanyl are not the first involving doses, with the potential that more cases went unde- fentanyl or its analogues. In 2006, a fentanyl over- tected from incomplete emergency department report- dose epidemic killed 260 individuals in Philadelphia ing. These overdoses were clustered in close prox- alone. imity to one another, indicating a possible common In 1979, the first fentanyl analogue, known as source (1). China white, appeared on the streets of California. In the first few months of 2013, the state of Penn- Since then 15 analogues have been synthesized; sylvania recorded 50 deaths of addicts using fentanyl seven of them are shown in Figure 1 (4). Through and its analogue, acetyl fentanyl. The problem quickly the years, these analogues have had various street surfaced in 13 more states. The Centers for Disease names, including Apache, China girl, China white, Control and Prevention (CDC) issued an emergency dance fever, friend, goodfella, jackpot, murder 8, warning about the appearance of this new analogue TNT, 24K, theraflu, bud ice, and recently Tango and that included a treatment recommendation that large Cash (6,7,8,9). doses of may be needed as an antidote be- Variations in the parent fentanyl structure can cause of the high potency of acetyl fentanyl (2). Continued on page 2 Fentanyl History Fentanyl is a powerful synthetic opiate with a rapid onset and short duration of action. First Inside... synthesized in the 1960s and used as a general anes- thetic, more recent applications include use to relieve Chronic Pain Management ...... 7 severe acute and chronic pain. Fentanyl’s analgesic Continuing Education Opportunities ...... 9 potency is some hundred times greater than mor- ACCENT Credit ...... 10 phine’s, and thus it is effective in low doses. This po- CLINICAL & FORENSIC TOXICOLOGY NEWS March 2014

Island, circumstances surrounding the deaths of sus- Acetyl Fentanyl pected addicts implicated opiates, but initial toxicol- Continued from page 1 ogy testing failed to confirm their presence. Immuno- assay screening of blood samples tested strongly for lead to analogues with profound pharmacological fentanyl, but gas chromatography-mass spectrometry differences. Parent fentanyl is 80 to 100 times more (GC-MS) confirmation failed to find either fentanyl potent than , and some of its analogues are or norfentanyl. A distinct chromatographic peak in up to 10,000 times more potent. Acetyl fentanyl, all the samples was consistent with acetyl fentanyl. however, is less potent than fentanyl. Many fentanyl The identity of this unknown substance was con- analogues are lipophilic, have onset of action within firmed through a qualitative reference standard re- minutes, and have short half-lives (10,11). ceived from the Drug Enforcement Administration These drugs can be highly toxic. Acetyl fen- (DEA). In addition, physical evidence from the tanyl has obvious similarities to its schedule II par- scenes confirmed positive for acetyl fentanyl. Acetyl ent, but it is not common on the illicit scene. No hu- fentanyl differs from fentanyl by a methylene group man data exists on its potency and lethality. In mice, (Figure 1). See Table 1 and Figure 2 for the analyti- the potency was 15.7 times higher than morphine but cal reference data and mass spectrum reported by the threefold lower than fentanyl. In acute toxicity stud- Rhode Island laboratory (13). ies, acetyl fentanyl is 6.7 times more lethal (LD 50 ) The Scientific Working Group for the Analysis than fentanyl, with heavy intestinal bleeding a note- of Seized Drugs (SWGDRUG) provides reference worthy symptom. In addition to analgesia, other mass spectra that differ in ion ratios for the acetyl pharmacological effects of fentanyl-like substances fentanyl-HCl powdered standard from the DEA. Ta- include euphoria, cough suppression, miosis, and ble 2 and Figure 3 show the ion ratios under the in- respiratory depression (12). strument conditions (14). Note that the ion ratios In the previously mentioned deaths in Rhode change in different pH preparations.

Fentanyl α-Methylfentanyl Parafluorofentanyl CSID 3228; 1960 CSID 459388; 2013 CSID 56081; 1979 CSID 56096; 1980s

α-Methylacetylfentanyl 3-Methylfentanyl Ohmefentanyl β-Hydroxythiofentanyl CSID 56102; 1984 CSID 55844; 1984 CSID 8649506; 1985 CSID 21106268; 1985

Figure 1 Fentanyl and some Analogues (6) CSID = ChemSpider ID; Year = Year introduced

Reproduced with permission from www.ChemSpider.com March 2014 CLINICAL & FORENSIC TOXICOLOGY NEWS tion. Eluents were dried and then re- Table 1. Rhode Island State Health Laboratories Data on Acetyl Fentanyl (13) constituted in a mobile-phase mix-

General information ture. Chemical name: N-Phenyl-N-[1-(2-phenylethyl)-4-piperidinyl] acetamide The method used two analytical col- N-(1-Phenethyl)-4-piperidyl) acetanilide umns. The first was a phenyl-hexyl Synonyms: Acetanilide (2.6 µm, 100 Å) analytical column Chemical formula: C 21 H26 N2O (50 mm x 4.6 mm) heated to 30 ºC. Molecular weight: 322.205 g/mol CAS number: 003258-84-2 The optimal flow rate for this system was set for 600 µl/min. The slow Toxicology Extraction: Recovered by routine n-butyl chloride liquid:liquid basic drug flow rate assisted in separating iso- extraction, including an acid back extraction. Sensitivity of method baric interferences. The second ana- not yet established. lytical column, which provided a sec- Detection: GC/MS electron ionization scan ondary confirmation technique, was a Ions 231, 146, 188 m/z and earlier eluter metabolite/breakdown biphenyl (2.7 µm) column (50 mm x ANPP 146, 189 m/z Elution order: Citalopram, ANPP, paroxetine, acetyl fentanyl, fentanyl, zolpidem 3.0 mm) and used the same mobile phase at a higher flow rate (800 µl/min). Electrospray Detection The electrospray mass spectrometer was set for positive ionization mode. Source (GS1/GS2), curtain, and colli- sion gases used nitrogen with settings of 50 cm/s, 50 cm/s, and 20 cm/s, re- spectively. Table 3 shows the pa- rameters for the specific reaction monitoring acquisitions. Individual enhanced product ion (EPI) settings were: 4000 Da/S; 800–600 Da; de- clustering potential (DP) 60 V; colli- sion energy spread (CES) 5 V; and collision energy (CE) 35 V. Under these conditions, spiked human urine Figure 2. Acetyl Fentanyl Basic Extraction Mass Spectrum (13) controls at the high (75 ng/mL) and low (3.75 ng/mL) concentrations pro- duced six sets of results for both acetyl fentanyl and New Analytical Method acetyl norfentanyl. For the high-concentration con- Patton et al. developed a method that couples trol, inter-day and intra-day coefficients of variation solid-phase extraction (SPE) with liquid chromatog- were below 10% and 8%, respectively. For the low- raphy tandem mass spectrometry (LC-MS/MS) to concentration control, the inter-day and intra-day val- identify acetyl fentanyl and acetyl norfentanyl in ues were below 10% and 15%, respectively. The urine (15). The researchers collaborated with Cay- lower limit of quantification was 1.06 ng/mL for ace- man Chemical (Ann Arbor, Mich.) to develop certi- tyl fentanyl and 1.62 ng/mL for acetyl norfentanyl, fied calibration and quality control standards for ace- and the limit of detection was less than 0.5 ng/mL for tyl fentanyl, acetyl norfentanyl, and their deuterated acetyl fentanyl and 1 ng/mL for acetyl norfentanyl. analogues. Prior to the LC-MS/MS analysis, they The researchers also performed in vitro experi- matrix-matched appropriate standards and quality ments using pooled human liver microsomes that control samples in human urine. They also hydro- demonstrated that acetyl fentanyl is metabolized by lyzed urine samples with ß-glucuronidase to ensure cytochrome P450s to acetyl norfentanyl. Urine sam- that the method would work in hydrolyzed solutions. ples from rats treated with a toxic dose of acetyl fen- Following these preparations, the researchers tanyl contained high concentrations of acetyl fentanyl used SPE on a polymeric strong-cationic exchange and acetyl norfentanyl. column to clean up the samples prior to LC-MS This study describes a sensitive and specific analysis. Following washes with 0.1% formic acid LC-MS/MS method that detects quantitatively acetyl and a 70:30 water/methanol solution, drugs were re- fentanyl and the predicted human metabolite, acetyl moved with a basic 50:50 methanol/acetonitrile solu- norfentanyl, in urine. In conjunction with existing CLINICAL & FORENSIC TOXICOLOGY NEWS March 2014

Table 2. Gas Chromatography-Mass Spectrometry Data on Acetyl Fentanyl (14) “1. A complete autopsy is necessary for optimal interpretation of toxicol- Sample preparation: Dilute ~1 mg/mL in chloroform ogy results, which must also be Instrument: Agilent gas chromatograph operated in split mode with MS detector Column: DB-1 MS (or equivalent): 30 m x 0.25 mm x 0.25 µm considered in the context of the cir- Carrier gas: Helium at 1 mL/min cumstances surrounding death, Temperatures: Injector: 280º C medical history, and scene findings. MSD transfer line: 280º C “2. A complete scene investigation MS source: 230º C extends to reconciliation of pre- MS quad: 150º C Oven program: scription information and pill 1) 100º C initial temperature for 1.0 min counts. 2) Ramp to 300º C “3. Blood, urine, and vitreous hu- 3) Hold final temperature for 9.0 min mor, when available, should be re- Injection parameters: Split ratio = 20:1, 1µl injected tained in all cases. Blood from the MS parameters: Mass scan range 30–550 amu femoral vein is preferable to blood Threshold: 100 Tune file: stune.u from other sites. Acquisition mode: scan “4. A toxicological panel should be Retention time: 16.843 min comprehensive and include opioid and benzodiazepine analytes, as well as other potent depressant, stimulant, and antidepressant medi- cations. “5. Interpretation of postmortem opioid concentrations requires cor- relation with medical history, scene investigation, and autopsy findings. “6. If death is attributed to any drug or combination of drugs (whether as cause or contributing factor), the certifier should list all the responsi- ble substances by generic name in the autopsy report and on the death certificate. “7. The best classification for man- ner of death in death due to the mis- use or abuse of without any Figure 3. Acetyl Fentanyl HCl Mass Spectrum (14) apparent intent of self-harm is ‘accident.’ Reserve ‘undetermined’ GC-MS techniques, the new method gives toxicol- as the manner for the rare cases in which evidence ex- ogy laboratories the capability to detect the drugs re- ists to support more than one possible determination.” sponsible for these kinds of opiate-related deaths. The panel also recommends that additional toxi- cological testing be performed in cases involving a Death Scene Investigation Guidelines known history of prescription or illicit opioid use; As the cases in Rhode Island illustrate, the certi- scene evidence of drug abuse; autopsy findings sug- fication of opiate deaths can be complicated and re- gesting drug abuse, including needle marks, liver quires a close relationship between the medical ex- damage, or other histological findings; gross patho- aminer’s or coroner’s office and the toxicology labo- logical findings such as fluid-filled lungs or foamy ratory. Although the death scenes implicated opiates, airways; smuggling or suspicion of body packing; no the initial toxicology results did not identify the obvious identified autopsy result; drug interactive drugs. Persistent toxicological testing uncovered this contributions to a natural cause; and trauma. new analogue and resulted in case interpretation and SAMHSA Recommendations a CDC warning. An expert panel from the National Association A panel of experts convened by the Substance of Medical Examiners and the American College of Abuse and Mental Health Services Administration Medical Toxicology published a guideline for these also produced guidelines for evaluations of opioid- investigations (16). The guideline recommends: related deaths (17). March 2014 CLINICAL & FORENSIC TOXICOLOGY NEWS

Table 3. Specific Reaction Monitoring Parameters for “6. The determination of free and total drug con- Acetyl Fentanyl Method (15) centrations in blood specimens, at a minimum, and ideally free and individual glucuronide metabolites. Q1 Q3 DP EP CE CXP “7. Analysis of free and total opiate/opioid concen- Analyte (m/z ) (m/z ) (V) (V) (V) (V) trations in other tissues as an adjunct to blood con- Acetyl 323 188 101 10 35 8 centrations, where appropriate, or where the blood fentanyl 323 105 101 10 57 10 concentrations may be compromised by postmortem Acetyl 328 188 56 10 33 4 artifact. fentanyl-d5 328 105 56 10 61 4 “8. Similar standards for cutoff concentrations for Acetyl 219 84 71 10 25 12 confirmatory gas chromatography-mass spectrometry norfentanyl 219 136 71 10 27 10 and liquid chromatography-mass spectrometry analy- Acetyl 224 84 66 10 25 4 sis. norfentanyl-d5 224 141 66 10 29 10 “9. Use of analytical methods that have been appro- priately validated and controlled to provide reliable DP = declustering potential; EP = entrance potential; data.” CE = collision energy; CXP = collision cell exit potential. Conclusion Reprinted with permission from Patton AL, Seely KA, Pulla S, et al. In recent years, U.S. toxicology laboratories Quantitative measurement of acetyl fentanyl and acetyl norfentanyl in human urine by LC-MS/MS. Anal Chem 2014;86:1760–6. Copy- have seen a large increase in the use of synthetic right 2014 American Chemical Society. drugs. The introduction of fentanyl analogues to a vulnerable population creates a sense of urgency be- cause these new drugs have demonstrated their lethal Its recommendations include: laboratories potential. The moving target of designer drugs illus- should develop a comprehensive screening test plat- trates how important and yet difficult it is for toxicol- form that uses technology such as immunoassays in ogy laboratory testing capabilities to keep pace with conjunction with chromatographic techniques; the what seems to be a never-ending variety of synthetic immunoassay cutoff should be set at a concentration drugs. that minimizes false-negative results; tests should be Although fentanyl analogues may go undetected capable of distinguishing between acute and chronic in initial toxicology testing, the pathological presenta- use by analyzing free drug and conjugated drug me- tion at the scene and in the autopsy suite may reveal tabolites; and blood samples should be clearly clues as to what type of toxicant a victim ingested. marked as to the site of collection, with iliac or femo- Thus, the guidelines outlined above are strongly rec- ral being preferred sources. The panel noted that the ommended as steps toward strengthening the ability variation in collection sites dramatically complicates to identify opiate-related deaths from either illicit or interpretation, and too often the term “blood” is used prescription drug use. Ultimately, proper field investi- without specifying whether it represents heart gation combined with comprehensive and technically (cardiac puncture) blood, subclavian blood, body proficient pathology and toxicology testing should cavity fluid, or spleen squeeze blood—and is some- increase communication between facilities and lead to times contaminated with embalming fluid. enhanced confidence in the final opinions. The guideline notes that standard practice for forensic toxicology includes: References “1. Identification of the site of specimen collection. If possible, samples should be collected from periph- 1. Centers for Disease Control and Prevention. eral blood vessels. Notes from the field: acetyl fentanyl overdose fa- “2. Collection and testing of admission (as op- talities—Rhode Island, March–May 2013. posed to autopsy) blood and urine specimens when MMWR Morb Mortal Wkly Rep 2013;62:703–4. applicable and available. http://www.cdc.gov/mmwr/preview/mmwrhtml/ “3. Comprehensive testing for prescription, illicit, mm6234a5.htm (Accessed February 2014). and over-the-counter drugs and alcohol. 2. Centers for Disease Control and Prevention. Rec- “4. Testing of appropriate specimens with an em- ommendations for laboratory testing for acetyl phasis on urine as a means to effectively detect drugs fentanyl and patient evaluation and treatment for and drug metabolites. overdose with synthetic opioid. http://emergency. “5. The use of an immunoassay screen with a de- cdc.gov/han/han00350.asp (Accessed March fined level of sensitivity and supplemental immuno- 2014). assays for drugs with poor cross-reactivity. 3. Warner M, Chen LH, Makuc DM. Increase in fa- CLINICAL & FORENSIC TOXICOLOGY NEWS March 2014

tal poisonings involving opioid in the deaths related to opioid drugs. Acad Forensic United States, 1999–2006. National Center for Pathol 2013;3:77–83 Health Statistics Data Brief; Centers for Disease 17. Goldberger BA, Maxwell JC, Campbell A, et al. Control and Prevention. http://www.cdc.gov/ Uniform standards and case definitions for classi- nchs/data/databriefs/db22.htm (Accessed Febru- fying opioid-related deaths: recommendations by ary 2014). a SAMHSA consensus panel. J Addict Dis 4. Jones CM, Mack KA, Paulozzi LJ. Pharmaceuti- 2013;32:231–43. cal overdose deaths, United States, 2010. JAMA 2013;309:657–9. Michael A. Wagner, PhD, is an associate profes- 5. Markon J, Crites A. Prescription-drug battle fu- sor in the department of pharmacology and toxicol- eled a rise in heroin use. Washington Post, March ogy and the department of pathology and laboratory 7, 2014;1,8. http://www.washingtonpost.com/ medicine in the Indiana University School of Medi- politics/experts-officials-missed-signs-of- cine in Indianapolis. Email: [email protected]. prescription-drug-crackdowns-effect-on-heroin- Jefferey H. Moran, PhD, is an assistant professor at use/2014/03/06/2216414a-9fc1-11e3-b8d8- the University of Arkansas for Medical Sciences and 94577ff66b28_story.html (Accessed March branch chief in environmental chemistry at the Ar- 2014). kansas Public Health Laboratory in Little Rock. 6. Royal Society of Chemistry, ChemSpider. http:// Email: [email protected]. Amy L. Patton, www.chemspider.com (Accessed February MS, is a chemist at the Arkansas Public Health Labo- 2014). ratory. Email: [email protected]. Laurie 7. National Pain Foundation. Health officials warn Ogilvie, MS, is supervisor of forensic toxicology at of fatal fentanyl overdoses. http:// the Rhode Island State Health Laboratories in Provi- americannewsreport.com/nationalpainreport/ dence. Email:[email protected]. health-officials-warn-of-fatal-fentanyl-overdoses- The authors have nothing to disclose. 8820632.html (Accessed February 2014). 8. Henderson GL. Designer drugs: past history and future prospects. J Forensic Sci 1988;33:569–75. 9. Centers for Disease Control and Prevention. Non- Poisoning Evaluation Update pharmaceutical fentanyl-related deaths—multiple AACC Press Issues New Edition states, April 2005—March 2007. MMWR Morb Mortal Wkly Rep 2008;57:793–6. The Clinical Toxicology Labora- 10. Michael Gordon. http://www.pharmacology2000. tory: Contemporary Poisoning com/General/Pharmacokinetics/kinobj6.htm Evaluation, 2nd Edition (Accessed February 2014). 11. S.I.N. Foundation. Fentanyl. http://www.drugs- Edited by Tai Kwong, Barbarajean forum.com/forum/showwiki.php?title=Fentanyl Magnani, Tom Rosano, and Les (Accessed February 2014). Shaw 12. Drug Enforcement Administration. Acetylfen- 2013, 525 pages, softcover tanyl. http://www.deadiversion.usdoj.gov/ $124 (AACC members, $99) drug_chem_info/acetylfentanyl.pdf (Accessed February 2014). This updated edition of a unique textbook on 13. Ogilvie L. New or re-emerging drug: acetyl fen- laboratory evaluation of the poisoned patient is writ- tanyl. TOXTALK, Society of Forensic Toxicolo- ten by experts in the fields of laboratory medicine, gists 2013;37(2):14–5. emergency medicine, and toxicology. The contribu- 14. Drug Enforcement Administration, Special Test- tors to the book work at poison control centers as ing and Research Laboratory. Acetyl fentanyl. well as academic, governmental, and private labora- http://www.swgdrug.org/Monographs/acetyl% tories. The tome covers a wide range of issues, in- 20fentanyl.pdf (Accessed February 2014). cluding the dramatic advances in the care of the poi- 15. Patton AL, Seely KA, Pulla S, et al. Quantitative soned patient, the advent of regional poisoning cen- measurement of acetyl fentanyl and acetyl nor- ters, and the efforts of clinical laboratories to adapt fentanyl in human urine by LC-MS/MS. Anal new technologies to support changing diagnostic and Chem 2014;86:1760–6. treatment modalities. 16. Davis GG. National Association of Medical Ex- For more information on this and other titles— aminers position paper: recommendations for the or to order—go to www.aacc.org and click on the investigation, diagnosis, and certification of AACC Store link. March 2014 CLINICAL & FORENSIC TOXICOLOGY NEWS

these drugs can go undetected. A positive opiates Chronic Pain Management class assay does not identify which opiate or opioid Urine Drug Tests Provide Important the patient is taking. A patient taking morphine, co- Tool in Monitoring Patient Compliance deine, or produces a positive result, but the clinician cannot know whether the patient is By Barbarajean Magnani, PhD, MD taking the prescribed opiate. For example, a patient taking a prescribed dose of the time-release morphine A fast-growing epidemic, prescription drug drug, MS Contin (morphine sulfate), produces the abuse has become the leading cause of unintentional same positive opiates result as someone taking co- drug overdose deaths in the U.S. (1). Drugs to treat deine, hydromorphone, or even heroin. pain have become more commonplace now that pain One reason for variability in immunoassay re- is considered the fifth vital sign. Most clinicians treat sults is that cross-reactivity varies by vendor. To cir- pain with various analgesics, including opioids for cumvent the cross-reactivity problem, some manufac- chronic noncancer pain. turers have created assays aimed at individual drugs Noncancer Pain Guidelines and Drug Testing such as , , and , but even these assays can lack specificity. For exam- Guidelines for clinicians who manage pain with ple, despite the buprenorphine assay’s low reactivity opioids recommend using urine drug screens to aid in for morphine (0.010%), the high concentrations from establishing compliance with the prescription regi- a typical morphine dose can produce a false-positive men (2). Urine drug tests supplement self-reporting buprenorphine result, particularly if the test’s thresh- and behavioral monitoring, and can identify prob- old is set low (such as 5 ng/mL). lems that would otherwise go undetected. The tests Oxycodone is another drug that can confuse a provide an objective measure that can document ab- clinician who does not understand the tests’ finer errant drug behavior. Testing can identify the non- points. A clinician who orders a standard opiates use of prescribed drugs, the use of non-prescribed screen for a patient on oxycodone may be worried at drugs, and the use of illicit drugs and alcohol. the lack of a positive result, concerned that either the Tests can help determine whether the patient is opiates screen is not working or a patient is diverting adhering to the contract that sets the terms and condi- the drug, even though the opiates screen is function- tions for chronic opioid therapy. A study by Couto ing as designed. Conversely, a clinician who orders and colleagues that examined more than 900,000 both a specific oxycodone immunoassay and an opi- urine samples from patients at a chronic pain clinic ates screen may not be aware that high concentrations underscores the need for testing (3). Using an immu- of oxycodone may produce not only a positive oxy- noassay screen with confirmation by tandem mass codone result but also a positive opiates result. spectrometry, the researchers found that 11% of the These examples illustrate the need for laboratori- samples were positive for illicit drugs, 29% indicated ans to provide guides to help clinicians understand use of a nonprescribed drug or drugs, and 38% re- which drugs are detected and which are not detected vealed no prescribed drugs. by in-house assays. Table 1 lists the drugs most com- Immunoassay Limitations monly used for pain control. Most initial drugs screens are immunoassays, Confirmation and Quantitation and despite their usefulness, immunoassays have limitations that many clinicians and even some labo- Because class immunoassays lack specificity, ratorians do not appreciate (4). Most clinicians and laboratories must use more sophisticated technologies laboratorians are aware of the need for confirmation to confirm positive results and to identify specific testing because immunoassays can be confounded by drugs. Gas chromatography-mass spectrometry and false-positive or false-negative results. A point less commonly known is that although many immunoassays are specific for a drug (such as Table 1. Drug-Test Targets in Patients Taking cocaine metabolites and cannabinoid metabolites), Prescribed Opiates many identify only a drug class (such as ampheta- Typical opioids for noncancer chronic pain mines, benzodiazepines, and opiates). Morphine, oxycodone, , hydromorphone, The standard opiates assay targets morphine. Al- , methadone, fentanyl, buprenorphine, though high concentrations of synthetic and semi- , synthetic drugs such as hydromorphone, hydro- Typical illicit drugs codone, dihydrocodeine, and oxycodone can also trigger a positive result, lower concentrations of Amphetamines, benzodiazepines, cannabinoids, cocaine CLINICAL & FORENSIC TOXICOLOGY NEWS March 2014 liquid chromatography-tandem mass spectrometry managing very challenging patients. can typically identify compounds at concentrations A laboratory ally can be especially helpful to a of 100 ng/mL or lower. These methodologies can clinician reviewing unexpected findings. On occa- identify not only parent compounds but also major, sion, a clinician may be uncertain whether a patient’s and sometimes even minor, metabolites. explanation for an unexpected positive or negative A minor metabolite that is also a prescription result is scientifically sound. For example, if a pa- opiate can complicate an interpretation. For example, tient tries to explain away a positive cocaine metabo- hydromorphone is a minor metabolite of morphine, lite result with the claim of receiving “novocaine” at so care must be taken to distinguish whether its pres- the dentist, a laboratorian who understands which ence is due to morphine metabolism or abuse of non- drugs do and do not cross-react with an assay can prescribed hydromorphone. Determining the relative provide the guidance to not believe this excuse. concentrations of hydromorphone and morphine can Close collaboration between the laboratory and help (Table 2). the clinic is a key to successful management of pa- Some analytical techniques can also identify tients on chronic opioid therapy. By adopting this pharmaceutical impurities produced in the manufac- important consultative role, laboratory medicine phy- ture of some drugs. These impurities can be confus- sicians and scientists demonstrate their importance as ing in the interpretation of the results because one members of the healthcare team who make valuable might assume that the patient is taking additional contributions beyond simply providing numbers. nonprescribed drugs. However, they are usually pre- sent in small concentrations compared with the pre- References scribed drug—the quantitation these methods provide 1. Centers for Disease Control and Prevention can resolve any ambiguity (5). (CDC). CDC grand rounds: prescription drug Consultation overdoses—a U.S. epidemic. MMWR Morb Mortal Wkly Rep Jan. 13, 2012;61(1)10–13. Laboratorians can provide important consulta- 2. Chou R, Fanciullo GJ, Fine PG, et al. Clinical tions to physicians caring for patients on chronic guidelines for the use of chronic opioid therapy opioid medications (6). By explaining the limitations in chronic noncancer pain. J Pain 2009;10 of the assays and suggesting the appropriate confir- (2):113–130. mation testing or other additional quantitative testing 3. Couto JE, Romney MC, Leider HL, et al. High needed, laboratorians can help determine whether the rates of inappropriate drug use in the chronic patient is compliant with the prescribed medication pain population. Popul Health Manag regimen. 2009;12:185–190. However, there are limitations with interpreta- 4. Kwong T, Magnani BJ. Urine drug testing in tion as well. Although tests can reveal whether pa- opioid therapy for chronic pain management. In: tients’ urine contains their prescribed medications or Kwong T, Magnani B, Rosano T, Shaw L, eds. metabolites with no nonprescribed or illicit sub- The clinical toxicology laboratory, contemporary stances, the tests cannot distinguish whether the practice in poisoning evaluation. 2nd Ed. Wash- medications are being taken as prescribed. A positive ington, D.C.: AACC Press 2013:447–458. urine test cannot indicate the amount of drug being 5. Magnani BJ, Kwong T. Urine drug testing for taken or the frequency it is being taken. Just the pain management. Clin Lab Med 2012;32 same, the results can reassure a clinician who may be (3):379–90. 6. Hammett-Stabler C, Magnani BJ. Supporting the Table 2. Interpretations of Opiate Concentrations in pain service. In: Magnani BJ, Bissell M, Kwong Patients on MS Contin T, Wu AHB, eds. Clinical toxicology testing: a guide for professionals. CAP Press, Northfield, Opiate result Possible morphine source Ill.:2012:15–26. Morphine, 500 ng/mL Poppy seeds Barbarajean Magnani, PhD, MD, FCAP, is Morphine, 50,000 ng/mL MS Contin chair and pathologist-in-chief at Tufts Medical Cen- Morphine, 50,000 ng/mL and MS Contin ter, and chair and professor at Tufts University Hydromorphone, 500 ng/mL School of Medicine. She is a member of the Clinical Morphine, 50,000 ng/mL and Ms Contin, Dilaudid & Forensic Toxicology News editorial advisory Hydromorphone, 5,000 ng/mL board. Email: [email protected].

Table courtesy of Tai C. Kwong, PhD Disclosure: The author has nothing to disclose. March 2014 CLINICAL & FORENSIC TOXICOLOGY NEWS

More information on these and other programs Continuing Education can be found at http://www.aacc.org/development/ Professional Organizations Offer certificates/massspeccertprog/pages/default.aspx. Convenient Online Opportunities Face-to-Face Education By Michael A. Wagner, PhD, and Donald L. Of course, the AACC also offers more traditional Frederick, PhD methods of continuing education. The meeting, “Mass Spectrometry in the Clinical Lab: Best Prac- Professional organizations in clinical and foren- tices and Current Applications,” will feature a presen- sic toxicology have begun offering on-line programs tation or two on toxicology applications. The confer- that make continuing education much more accessi- ence will be held Oct. 9–10 in St. Louis. Julianne Bo- ble. Many of these programs offer ACCENT ® credit, telho, PhD, of the Centers for Disease Control and which allows participants to document their continu- Prevention and Deborah French, PhD, of the Univer- ing education efforts to meet requirements for licen- sity of California, San Francisco are helping coordi- sure and certification. Participants can receive certifi- nate this event. More information is available at cates to confirm that they have completed the appro- http://www.aacc.org/events/meetings/Pages/Mass- priate modules. Spec-2014.aspx# or from the AACC’s David Sainato: The American Association for Clinical Chemis- [email protected]. try (AACC) offers two toxicology certificate pro- Several AACC webinars this year will address grams. “Clinical Toxicology” is a basic/intermediate therapeutic drug monitoring and toxicology issues. level program for the laboratory scientist. It includes On April 29, “Mass Spectrometry in Designer Drugs- four courses that can be completed online in about of-Abuse Testing” will feature Marilyn Huestis, PhD, two hours each: “Introduction to Clinical Toxicol- chief of the chemistry and drug metabolism section of ogy,” “Immunoassays to Screen for Drugs of the National Institute on Drug Abuse, discussing ad- Abuse,” “Specimens for Toxicology Testing,” and vances in LC-MS/MS and GC-MS that can help labo- “Chromatography Basics for Toxicology Testing.” ratories keep up with evolving trends in drug abuse. Each course contains a lecture, slides, transcripts, On May 13, “Mass Spectrometry 101” with Thomas and a quiz. Some include additional readings. A dis- Annesley, PhD, professor emeritus of pathology at cussion board offers the opportunity to interact with the University of Michigan, will cover basic princi- faculty and other participants. Successful participants ples of this analytical technique. A webinar later in can earn eight ACCENT ® credits for completing all the year on the investigation of poisoning will feature four modules. Barbarajean Magnani, MD, PhD, of Tufts Medical The “Using Tandem Mass Spectrometry in the Center. For more information on these and other up- Clinical Laboratory” certificate program is composed coming webinars, visit http://www.aacc.org/events/ of nine courses: “Basics of Mass Spectrometry The- webinars/pages/default.aspx#. ory,” “Basics of Liquid Chromatography Theory,” Other Organizations “Setting up a Clinical Mass Spectrometry Labora- tory,” “Maintenance and Troubleshooting of Liquid Other organizations that provide continuing edu- Chromatography-Tandem Mass Spectrometry (LC- cation for forensic toxicologists include the American MS/MS),” “Making Accurate Calibrators and High Association of Forensic Sciences (www.aafs.org), the Purity Reagents for LC-MS/MS Methods,” Society of Forensic Toxicologists (www.soft-tox. “Principles and Best Practices for Quantitation Using org), the Center for Studies of Law in Action LC-MS/MS,” “Validation of LC-MS/MS Methods,” (Borkenstein courses, www.borkensteincourse.org), “Tips for Achieving and Maintaining Consistent The Center for Forensic Science Research and Educa- High Quality Production in a Clinical Mass Spec- tion (www.forensicscienceeducation.org), and RTI trometry Laboratory,” and “Gas Chromatography- International (www.rti.org). RTI provides a number Mass Spectrometry (GC-MS) in Clinical Laborato- of web-based programs that provide certificates and ries.” ACCENT credit. Each course takes about one to three hours RTI’s programs are supported by the National online and contains a lecture, slides, transcripts, and Institute of Justice and cover a number of forensic a quiz. Some include additional reading and practice topics, including best practices, current advances, exercises. Discussion boards offer the opportunity to classic principles, interpretation, and medical legal interact with faculty and other participants. The pro- testimony. RTI has posted more than 30 courses re- gram offers 12 ACCENT credits for successful com- lated to forensic toxicology, with more added con- pletion. tinuously. The free-of-charge training modules are CLINICAL & FORENSIC TOXICOLOGY NEWS March 2014 generally about an hour and feature both live pres- • American Society of Microbiology entations and archived courses that can be accessed • American Society for Clinical Laboratory Science on-demand in a self-paced format. More informa- • American Society for Clinical Pathology tion is available at http://www.rti.org/page.cfm/ • American Medical Technologists Forensic_Science_Education • Association of Clinical Scientists Michael A. Wagner, PhD, is an associate pro- • International Federation of Clinical Chemistry fessor in the department of pharmacology and toxi- • National Registry in Clinical Chemistry cology and the department of pathology and labora- tory medicine in the Indiana University School of Learning Objectives Medicine in Indianapolis. Email: micawagn@iupui. After reading Clinical & Forensic Toxicology edu. Donald L. Frederick, PhD, DABFT, is with the News , the reader will be able to: Peoria Tazewell Pathology Group in Peoria, Illi- nois. Email: [email protected]. • Describe emerging and changing trends in drug abuse, including new designer drugs, usage patterns, Disclosure: The authors have nothing to disclose. and contaminants/adulterants.

• Identify potential analytes (drugs, metabolites, bio- markers) of clinical and/or forensic significance.

CFTN Readers Are Eligible • Evaluate methodologies for their utility and limita- To Receive ACCENT Credit tions relative to the needs of toxicology labs. • Explain the analytical and regulatory issues unique Readers of Clinical & Forensic Toxicology to specific applications, including postmortem toxi- News are eligible to receive 4.0 ACCENT® credit cology, workplace drug testing, and drug screening. hours per year (one credit per quarterly issue) of con- tinuing education. ACCENT credit allows you to How to Get Credit document your continuing education to meet require- After reading this issue’s articles, simply access ments for licensure or certification. the online evaluation form and print your continuing ACCENT credit is recognized by a wide variety education certificate: http://direct.aacc.org/ of organizations, including: customerservice/login.aspx?returnlink=http://apps. • American Association of Bioanalysts aacc.org/applications/apps2/CE/intro.aspx? • American Board of Clinical Chemistry actNum=2238465

Clinical & Forensic Toxicology News provides practical Editorial Advisory Board and timely information on the clinical, forensic, technical, and Chair regulatory issues faced by toxicology laboratories. Each Christine L. Snozek, Ph.D., Mayo Clinic, Scottsdale, issue includes articles authored by experts. Ariz., [email protected] Clinical & Forensic Toxicology News is an educational Members service of the Forensic Urine Drug Testing (FUDT) Accredi- Jennifer Collins, PhD, MedTox Laboratory, St. Paul, tation Program. Cosponsored by the American Association Minn., [email protected] for Clinical Chemistry and the College of American Uttam Garg, PhD, Children’s Mercy Hospital, Pathologists, the program includes three components: FUDT Kansas City, Mo., [email protected] accreditation, the FUDT proficiency testing survey, and this Kamisha L. Johnson-Davis, PhD, University of Utah and newsletter. The accreditation program is the responsibility of ARUP Laboratories, Salt Lake City, Utah, CAP. The surveys are sponsored jointly by AACC and CAP. [email protected] The digital newsletter is published quarterly by AACC, 1850 David J. Kuntz, PhD, Clinical Reference Laboratory, K St., N.W., Suite 625, Washington, DC 20006, (800) 892- Lenexa, Kan., [email protected] 1400 or (202) 857-0717. Email: [email protected]. Barbarajean Magnani, PhD, MD, Tufts Medical Center, Clinical & Forensic Toxicology News does not accept Boston, Mass., [email protected] advertising and is supported solely by its readers. The an- James Carl Ritchie, PhD, Emory University Hospital, nual subscription price is $65, $45 for AACC members. Atlanta, Ga., [email protected] Opinions expressed are those of the authors and do not represent the position of AACC or CAP. Readers are invited to submit questions and suggestions for articles to the editorial advisory board.

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