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Mass Spectrometry-based Detection of Newly Emerging Psychoactive Drugs

Kenichi (Ken) Tamama, M.D., Ph.D. Associate Professor of Pathology University of Pittsburgh School of Medical Director, Clinical Toxicology Laboratory University of Pittsburgh Medical Center Learning objectives

• Describe the current drug-of-abuse situation of emerging psychoactive drugs • Summarize laboratory detection of psychoactive drugs • Explain the challenges in detecting the emerging psychoactive drugs DEA: “Drugs of abuse as a national threat in the US” http://www.post-gazette.com/ • deaths have become the leading cause of injury death in the US, surpassing the number of deaths by motor vehicles and by firearms every year since 2008. • In 2015, approximately 140 people died every day from drug poisoning. (DEA NDTA 2017) • Diacetyl (→ 6-MAM → Morphine) • Detectable by opiate EIA • Schedule I (high potential for abuse, no medical use) • Heroin overdose deaths are increasing in many cities and counties across the • Heroin availability is increasing in areas throughout the nation. • Increased demand for, and use of, heroin is being driven by both increasing availability of heroin in the US market and by some CPD abusers using heroin.

(DEA NDTA 2017) • Pittsburgh is in the western PA, facing OH and WV (Tri-State area) • PA: 5.6 death rate per 100,000 • OH: 13.3 death rate per 100,000 (Worst in the nation) • WV: 11.8 death rate per 100,000 (2nd worst in the nation)

(DEA NDTA 2017) • Synthetic • 80 to 100 times stronger than morphine • 25 to 40 times more potent than heroin • Not detected by opiate EIA (fentanyl EIA is needed) • Schedule II (high potential for abuse, acceptable medical use) • Clandestinely-produced fentanyl is sometimes added to heroin to increase its effects, or mixed with adulterants and diluents and sold as heroin • In March 2015, DEA issued a nationwide alert about the dangers of fentanyl and fentanyl analogues/ compounds, stating “Fentanyl is commonly laced in heroin, causing significant problems across the country, particularly as heroin use has increased.” • 79%↑ in synthetic opioid deaths between 2014 and 2015 (CDC)

Fentanyl analogs (Fentalogs) identified at the UPMC lab

(Li, Am J Clin Pathol. 2017) • Carfentanyl • The most potent opioid (10,000 times more potent than morphine) • Used as a tranquilizing agent for elephants • Between July 5 and July 26, 2016, paramedics in Akron, OH registered at least 236 drug overdoses with at least 14 being fatal, linked to suspected carfentanyl. (DEA NDTA 2017) Other (Mitragynine, AH-7921 and U-47700) • Mitragynine • Major psychoactive alkaloid of the plant kratom indigenous to South East Asia. • Emerging in the US as a legal psychoactive product available on-line. • In the U.S., mitragynine has been used recreationally as well as a means to treat symptoms of outside of established medical programs . • 7-hydroxymitragynine, a minor constituent of kratom, has 46 times higher than mitragynine . • AH-7921 and U-47700 • Synthetic opioids developed in the mid-70s • AH-7921 is as potent as morphine • U-47700 is 7.5 times more potent than morphine • In 2016, DEA first encountered U-47700 responsible for at least 80 deaths in the United States for the year.

(Li, Am J Clin Pathol. 2017; DEA NDTA 2017) Synthetic Designer Drugs

• Synthetic designer drugs refer to man-made substances created to mimic the effects of controlled substances, and are oftentimes unscheduled and unregulated. • The two most commonly used types of synthetic designer drugs in the United States are • Synthetic • -like including • Synthetic designer drugs might be attractive to drug users subjected to drug screening (e.g., inmates, parolees, and probationers), as drug screens often do not have the ability to identify all of these compounds.

(DEA NDTA 2015) Rapid spread of designer drugs

(DEA NDTA 2013) Synthetic cannabinoids

• Synthetic cannabinoids, also commonly known as “Spice” and “K2,” are chemicals synthesized in laboratories and simulate the biological effects of THC. • Synthetic cannabinoids are the 4th most popular drug used among 8th graders, the 3rd most popular drug used among 10th graders and the 4th most popular drug used among 12th graders.

(DEA NDTA 2015) Synthetic cannabinoids are rapidly evolving

2010 2012 (Designer_Drug_Trends_February_2014, from www.nmslab.com)

(DEA NDTA 2017) Synthetic cannabinoids F

• 4 major structural groups

AM2201 • Alkylindoles

XLR-11 • Naphthoylindoles (JWH-018, JWH-073, AM2201)

• Phenylacetylindoles (JWH-250) AM-694

• Benzolindoles (AM-694)

• Cyclopropylindoles (XLR-11)

APICA AKB-48 • Adamantyllindoles (APICA)

• Cyclohexylphenols (CP47,497)

AB-PINACA • Indazole carboxamides (AKB-48 (APINACA), AB-PINACA, AB-CHMINACA)

• Classical cannabinoids (HU-210) AB- CHMINACA Amphetamine-type stimulants • is an active compound in Catha edulis (), a flowering plant indigenous to the Horn of Africa and the Arabian Peninsula • Cathinones are also commonly known as “bath salts,” can produce pharmacological effects similar to amphetamine and MDMA. • Other types of stimulants (“2Cs or dimethoxyphenethylamines”, “Dimethoxyamphetamines”) are also emerging as well. • Synthetic cathinones are “rebranded” and sold as MDMA, or “Molly” • has been seized in all forms of supposed MDMA. Amphetamine-type stimulants • β-keto

Methylone • Pyrrolidinophenones

MDPV Amphetamine

Pyrovalerone αPVP • (dimethoxyphenethylamines)

Due to their moderate structural similarity to amphetamine, β-keto amphetamines and 2C-B 2C-I dimethoxyamphetamine appear to cross- • Dimethoxyamphetamines react weakly with some of amphetamine immunoassays. DOET DOM Arylcyclohexylamines • and hallucinogenic agents • , (PCP), and their analogs [ (MXE), 2- oxo-PCE, etc.] • Recreationally abuse • Super K (Ketamine), Wet (PCP + THC), Mexxy, m-ket, k-max(MXE) • 2-oxo-PCE cases in Hong Kong • The analogs as novel psychoactive substances (NPS) • No statistics provided in the DEA reports • 3% of the seized drug in European Drug Report 2016 • Limited scientific literature • Several cases at the UPMC

(EMCDDA, European Drug Report 2018) GABA-mimetics • GHB (γ-hydroxybutyrate​) • Originally synthesized as an active analog of GABA in 1960​ • Ingested for , relaxation, during parties • Drug rape substance as it is "color- and odorless“ • Schedule I in CSA • The total number of GHB abuse seems to be comparatively low • Pathophysiology • GHB itself is present in the and serves as both a precursor and a degradation product of GABA • γ-butyrolactone (GBL) and 1,4-butanediol (BD) are precursors of GHB • Therapeutic usage for nacrolepsy ( or Xyrem®) • Phenybut (β-phenyl-γ-aminobutyric acid) • Developed in in 60s and has been used for anxiolysis, preoperational sedation and withdrawal • Structurally similar to • Recreational abuse cases have been reported recently • Available through the Internet

(O'Connell, Am J Med. 2014; Schep Clin Toxicol. 2012; Drasbek, Acta Neurol Scand. 2006 ) GABA-mimetics

(Modified from Drasbek, Acta Neurol Scand 2006, Lapin, CNS Drug Rev. 2001) Laboratory detection of emerging drugs of abuse

• Immunoassays • Mostly not FDA-approved • Cross-reactivity of these compounds with the existing kits is often unknown • 2D molecular similarity calculation to predict cross-reactivity of these compounds • Mass Spectometry • GC-MS • Traditionally regarded as a gold standard platform • LC-MSMS or LC-high resolution MS GC-MS • GC and MS combined • Originally developed in 1950’s • Older than LC-MS • Samples have to be delivered as a gas • Analytes have to be volatile • Output from GC • Sample derivatization often needed for GC • Hard ionization J Am Soc Mass Spectrom 1993, 4, 367-371 • EI (Electron ionization) The expanding role of mass spectrometry in biotechnology

Henry’s Clinical Diagnosis and Laboratory Management. 2011 Comprehensive drug screening at UPMC

• Immune-based screening of the major drugs of abuse • Amphetamine-EIA, -EIA, -EIA, -EIA, -EIA, -EIA, Opiate-EIA, /oxymorphone-EIA, Phencyclidine-EIA, THC metabolite-EIA

• GC-MS-based screening • Untargeted comprehensive screening • For screening of the drugs not covered by immunoassays • For confirmation of immunoassays • Full scan mode and selected ion for major drugs • Laborious procedures, but offered 24/7 • Preliminary report for stat orders • Final report after the pathologist’s review in the next business day Sample preparation Comprehensive drug screening by GC-MS Opiate confirmation by GC-MS

X1

• Sample preparation is laborious for both comprehensive drug screening and drug confirmation. • CDS: >60 min for sample preparation • Opiate confirmation: >60 min for acid hydrolysis, >20 min for SPE, >20 min for derivatization • GC-MS run time: 45 min • The time for the reagent preparation is NOT included! A case of comprehensive drug screening by GC-MS

Drugs of abuse (parental and ) and impurities • Cocaine and metabolites (ecgonine methyl ester, trimethoxycocaine, cocaethylene, ethylecgonine) • 6MAM (heroin metabolite), acetylcodeine, • Fentanyl, parafluoroisobutyryl fentanyl, butanoyl-4-fluorofentanyl

Cutting agent • Levamisole • Quinine or • (?)

Others • metabolite or chlorcyclizine • • Total ion chromatogram ()

Cocaine

Ethyl ecgonine

Methyl ecgonine benzoylecgonine

Nicotine Nicotine met Fentanyl Cocaine spectrogram by GC-MS

Mass spectrum of the unknown peak

Reference mass spectrum of cocaine

• Ionization mode: EI (Electron ionization) • Hard ionization • Numerous fragment ions produced in a reproducible way • Positive identification of analytes are made by library matching of the spectrogram • Able to detect any compounds as long as it is within the library and extractable. McPherson: Henry's Clinical Diagnosis and Management by Laboratory Methods, 22nd ed. • 33 year-old male with past medical history of polysubstance abuse became unresponsive and was brought into the emergency room. He regained consciousness after receiving . He admitted to take 10 bags of “Heroin” before losing consciousness. • The initial drug screening immunoassay panel was all negative.

Which compound(s) would be detected Screening Test Case 1 by GC-MS in case 1? Amphetamine (1000 ng/mL) Negative A. Fentanyl and/or its analog(s) Barbiturate (200 ng/mL) Negative Benzodiazepine (200 ng/mL) Negative B. Heroin and its metabolite(s) Buprenorphine (5 ng/mL) Negative C. MDPV Cocaine Metabolite (300 ng/mL) Negative Opiate (300 ng/mL) Negative D. AB-CHMINACA Oxycodone (100 ng/mL) Negative E. All of the above Phencyclidine (25 ng/mL) Negative THC Metabolite (100 ng/mL) Negative Case 1 (Li, Am J Clin Pathol. 2017) • The GC-MS-based untargeted urine comprehensive drug screening detected fentanyl, methylnorfentanyl (a metabolite of 3-methylfentanyl) and quinine/quinidine . • This is a typical example of illicit drug “rebranding” in the underground market (Fentanyl and its analogs are sold as “Heroin”. ) (Li, Am J Clin Pathol. 2017) 3-methylfentanyl • 3-methylfentanyl was identified and the drug was involved in an epidemic of overdoses in California in 1984–1985. • It is 1000 – 7000 times as potent as morphine. • Allegedly it was used as the anaesthetic gas (chemical weapon) Kolokol-1 in the Moscow theater hostage crisis in 2002 (From Wikipedia)

• These were new 3-methylfentanyl cases per Allegheny County Medical Examiner at that time.

Int J Legal Med, 2008; 122:395–400 • 36 year-old male with past medical history of IVDA was found unresponsive on the street, receiving CPR by a bystander. He was defibrillated for his stable VT, and intubated by paramedic at the scene before brought into the emergency room. • The initial urine drug screening immunoassay panel was all negative.

Screening Test Case 2 Amphetamine (1000 ng/mL) Negative Which compound(s) would be detected Barbiturate (200 ng/mL) Negative Benzodiazepine (200 ng/mL) Negative by GC-MS in case 2? Buprenorphine (5 ng/mL) Negative • Please text your guess!! Cocaine Metabolite (300 ng/mL) Negative Opiate (300 ng/mL) Negative Oxycodone (100 ng/mL) Negative Phencyclidine (25 ng/mL) Negative THC Metabolite (100 ng/mL) Negative Case 2 (Li, Am J Clin Pathol. 2017) U-47700, fentanyl, and were detected

Tramadol (reference MS) U-47700 (reference MS)

RT: 23.0 min

RT: 17.7 min RT: 25.9 min

Fentanyl (reference MS) • 59 year-old male with past medical history of CAD was found unresponsive in his house. He also had seizure activity and received prehospital . He received multiple doses of naloxone with no response. He was intubated by paramedics for his respiratory failure. • The initial urine drug screening immunoassay panel was for benzodiazepine and opiate.

Which compound(s) would be detected Screening Test Case 4 by GC-MS in case 3? Amphetamine (1000 ng/mL) Negative Barbiturate (200 ng/mL) Negative A. and its metabolite(s) Benzodiazepine (200 ng/mL) Unconfirmed B. Phenibut positive Buprenorphine (5 ng/mL) Negative C. Cocaine Metabolite (300 Negative D. All of the above (A, B, and C) ng/mL) Opiate (300 ng/mL) Unconfirmed E. Only (A) and (C) positive Oxycodone (100 ng/mL) Negative Phencyclidine (25 ng/mL) Negative Case 3 THC Metabolite (100 ng/mL) Negative Phenibut (reference MS)

• The clinical team later learned that the patient purchased Phenibut online 3 days ago. • Phenibut, midazolam, hydrocodone are detected by GC-MS • 25 year-old male with past medical history of polysubstance abuse was found unresponsive in his house. He regained consciousness after receiving naloxone and was brought into the emergency room. He admitted his ingestion of “Percocet” and “Molly”. • The initial urine drug screening immunoassay panel was only positive for opiate and oxycodone.

Which compound(s) would be detected by GC-MS in case 4? Screening Test Case 4 Amphetamine (1000 ng/mL) Negative • Please text your guess!! Barbiturate (200 ng/mL) Negative Benzodiazepine (200 ng/mL) Negative Buprenorphine (5 ng/mL) Negative Cocaine Metabolite (300 ng/mL) Negative Opiate (300 ng/mL) Unconfirmed positive Oxycodone (100 ng/mL) Unconfirmed positive Phencyclidine (25 ng/mL) Negative THC Metabolite (100 ng/mL) Negative Case 4 (Li, Am J Clin Pathol. 2017) Ethylone Hydrocodone Oxycodone

• No 3,4-methylenedioxy-N- methylamphetamine (MDMA or “Molly”) was detected by GC-MS.

• This is another example of illicit drug “rebranding”. (Ethylone was sold as Molly”).

(Li, Am J Clin Pathol. 2017) • A 37-year old man with history of polysubstance abuse, presented to the hospital with altered mental status. He was last seen normal 3 days prior to admission. The patient endorsed using ketamine analogs “2-oxo-PCE” and “3-MeO- PCE,” from the dark web. • Upon presentation he was noted to be tachycardic with bilateral nystagmus, dry mucous membranes and dissociation. • The initial lab test was significant for elevated BUN (34 mg/dL), Creatinine (2.8 ng/mL), and CPK (5840 U/L) • The initial urine drug screening immunoassay panel was only positive for PCP. Screening Test Case 5 Which compound(s) would be detected by Amphetamine (1000 ng/mL) Negative GC-MS in case 5? Barbiturate (200 ng/mL) Negative Benzodiazepine (200 ng/mL) Negative A. 2-oxo-PCE and/or 3-MeO-PCE Buprenorphine (5 ng/mL) Negative B. Phencyclidine (PCP) Cocaine Metabolite (300 Negative C. ng/mL) Opiate (300 ng/mL) Negative D. All of the above (A, B, and C) Oxycodone (100 ng/mL) Negative E. Only (A) and (B) Phencyclidine (25 ng/mL) Unconfirmed positive Case 5 THC Metabolite (100 ng/mL) Negative 2-oxo-PCE Ketamine

A: Mass spectrum of the B: Reference mass unknown peak at 15.5 min spectrum of 2-oxo-PCE

2-oxo-PCE and ketamine were detected by GC-MS; however, 3-MeO-PCE was not detected.

• Ketamine does not cause a false positive PCP immunoassay result at 1 mg/mL (unpublished data). • 2-oxo-PCE causes a false positive PCP result at 1 mg/mL and above (unpublished data). • A 32-year-old male with a known history of opioid and abuse was found on the floor unresponsive at home and brought to the hospital. Prior to arrival, he received multiple doses of naloxone with minimal response. Given his persistent comatose status, he was finally intubated for airway protection and admitted to the ICU. • A CT scan of the head was performed revealing no evidence of intracranial hemorrhage, mass effect, or ischemic events. An electroencephalogram was unremarkable. • The initial urine drug screening immunoassay panel was unremarkable. • Within the first 24 hours of admission, the patient spontaneously regained consciousness without significant sequelae and was extubated. Screening Test Case 6 Amphetamine (1000 ng/mL) Negative Which compound(s) would be detected Barbiturate (200 ng/mL) Negative Benzodiazepine (200 ng/mL) Negative by GC-MS in case 6? Buprenorphine (5 ng/mL) Negative • Please text your guess!! Cocaine Metabolite (300 ng/mL) Negative Opiate (300 ng/mL) Negative Oxycodone (100 ng/mL) Negative Phencyclidine (25 ng/mL) Negative THC Metabolite (100 ng/mL) Negative Case 6 (Lopez Nunez, J Appl Lab Med. 2018) 1,4-butanediol

• A GC-MS-based comprehensive urine drug screen (CDS) test revealed the presence of 1,4-butanediol (1,4-BD). • A confirmatory test subsequently revealed the presence of 1,4-BD and gamma-hydroxybutyrate (GHB). The patient was discharged 2 days later.

(Lopez Nunez, J Appl Lab Med. 2018) 1,4-BD peak at 5.93 min (m/z: 147, 116, 177) Confirmatory test of GHB and 1,4-BD by GC-MS GHB peak at 6.46 min (m/z: 233, 204)

• Liquid-liquid extraction with acetonitrile followed by evaporation • Derivatization with BSTFA + 1% TMCS for trimethylsilation of alcohol groups • Monitoring GHB (m/z: 233, 204) and 1,4-BD (m/z: 147, 116, 177) with the SIM (selected ion monitoring) mode • Detection limit: 100 ng/mL

GHB peak-d6 at 6.40 min, buried within the peaks (m/z: 239, 204) Untargeted comprehensive drug screening by GC-MS

Pros • Powerful for drug detection, even without clinical history • Especially useful for drugs not covered by immunoassays • Fentanyl and its analogs, U47700, Bath salt (e.g., ethylone), Phenibut, GHB, etc. Cons • Laborious sample preparation and longer TAT • Analytes must be small and nonpolar to be extractable, thermostable, and volatile • Any non-volatile and/or heat-unstable compounds cannot be analyzed without chemical modification (derivatization) • Examples: synthetic cannabinoids (based on our experience) LC-MS/MS or LC-high resolution (HR)-MS

• Larger and non-volatile molecules can be analyzed • Simplified sample preparation (e.g., dilute and shoot) • Rapid TAT • LC-HR-MS: Capable of identifying unknown compounds by deducing the molecular formula from accurate mass databases

(Li, Am J Clin Pathol. 2017) Challenges for Clinical Toxicology Lab • Limited information about the chemical structure and of these newly emerging drugs • Availability of mass spectrum of the compound is a pre- requisite for the detection by MS-based screening assays • Limited utility of the immunoassays for the existing classes of drugs (e.g., Spices, U47700) • Rebranding of the illicit drugs • making the clinical histories unreliable and the targeted drug screening less useful. • Untargeted drug screening is required in these situations, but is limited to the existence of a library match for the emerging drugs.

(Li, Am J Clin Pathol. 2017) Summary • Describe the current drug-of-abuse situation of emerging psychoactive drugs • Opioid crisis (Fentalogs, U47700, etc.) • Designer drugs (Bath salts, Spice, etc.) • Others (PCP/Ketamine analogs, GABA-mimetics) • Summarize laboratory detection of psychoactive drugs • Immunoassays • Mass spectrometry • GC-MS • LC-MSMS and LC-HR-MS • Explain the challenges in detecting the emerging psychoactive drugs • Limited information of the emerging drugs of abuse • Limited utility of immunoassays for newly emerging drugs of abuse for clinical toxicology labs • “Rebranding” of illicit drugs Acknowledgments • UPMC • Special Chemistry Laboratory, UPMC CLB • Jacqueline Rymer • Spiros Giannoutsos • University of Pittsburgh SOM/UPMC • Department of Pathology • Li Liu, MD, PhD • John Skaugen, MD • Oscar Lopez Nunez, MD • Division of Medical Toxicology, Dept of Emergency Medicine • Anthony Pizon, MD • Michael Lynch, MD • Anthony Scoccimarro, MD • Kenichi (Ken) Tamama has no financial conflict of interest or other relationship with any proprietary entity (commercial, for-profit) producing health care goods or services.