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Joshua Seither, Lisa Reidy, Confirmation of Carfentanil, U-47700 and Other Synthetic Opioids in a Human Performance Case by LC–MS-MS, Journal of Analytical Toxicology, Volume 41, Issue 6, July-August 2017, Pages 493–497, https://doi.org/10.1093/jat/bkx049
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Abstract
Recently, it has been documented that there has been a rise in synthetic opioid abuse. Synthetic opioids are compounds that were created to act as agonists for the opioid receptors. Like synthetic cannabinoids, most of these compounds were created by research groups or pharmaceutical companies in an attempt to find compounds that have medicinal use. Synthetic opioids have severe health implications when abused that can include hospitalization and death. Due to the high potency and the low dose required to produce the desired effects for these compounds, it was hypothesized that they may not be detectable in human performance case samples. However, this report documents a male driver who was involved in a single-vehicle incident. First responders treated the subject with naloxone as opioid drug impairment was suspected and he was transported to the local emergency room. The subject consented to a blood draw for a driving under the influence (DUI) investigation. Initial routine testing identified alprazolam at 55 ng/mL and fentanyl at less than 0.5 ng/mL. Further testing using a validated liquid chromatography–tandem mass spectrometry (LC–MS-MS) assay, confirmed the presence of carfentanil, furanyl fentanyl, para-fluoroisobutyryl fentanyl, U-47700 and its metabolite. To the author's knowledge, this is the first report of a DUI cases where carfentanil, U-47700 and other synthetic opioids were confirmed and described in a human performance blood sample. This case demonstrates the need to supplement routine toxicological analyses with a sensitive methodology that can detect synthetic opioids in human performance cases where opioid use may be implicated.
Introduction
There has been an increased emergence of novel psychoactive substances (NPS) worldwide over the last decade (1). The availability and detailed information of these compounds on the Internet have dramatically increased the spread and use of these compounds (2). NPS are intended to mimic the effects of illicit compounds and have been identified from the various pharmacological and structural drug classes including arylcyclohexylamines, benzodiazepines, cathinones, cannabinoids, indanes, opioids, phenethylamines, piperazines and tryptamines (3). While cost, availability and exploration have been cited as reasons for the use of these compounds, it was initially hypothesized that the use of these compounds was to avoid potential legal consequences of creating, supplying and using illicit compounds (2, 4).
Opioids encompass a group of compounds that either act as an agonist or antagonist on the various opioid receptors found in the body (5). Structurally, there is considerable variability in this group of compounds. Semi-synthetic opioids are synthesized using natural opioid alkaloids and are typically structurally similar to morphine, whereas synthetic opioids are synthesized using synthetic precursors and may not share the similar structure. Among the opioid compounds, there are various legal statutes under the United States Controlled Substances Act (CSA) ranging from schedule I to non-controlled substances. Medicinal use may also vary as some opioids are approved to be used in humans and animals, while others are not.
The first synthetic opioid, meperidine, was discovered in the late 1930s (6). Since then, like other novel psychoactive classes such as the synthetic cannabinoids, there has been a considerable amount of research performed in an attempt to create similar compounds that can be used medicinally (7). While this research yielded compounds that are successfully used therapeutically in medicine such as fentanyl and methadone, other analogs were not deemed medically safe or useful. Unfortunately, clandestine laboratories can obtain information regarding the synthesis of these analogs as it is documented in open access scientific literature and websites such as Erowid (8).
In an attempt to combat the current opioid epidemic, the United States passed laws that render it challenging to obtain prescription opioids from legitimate sources. However, individuals that suffer from opioid dependency have turned to illicit opioids to satisfy their addiction due to their availability and cost. The consumption of newer synthetic opioids may occur from two possible situations. Drug users may consume different synthetic opioids intentionally or without knowledge when laced with either another opioid or substituted for one (9). The increased potency of these compounds can lead to an unintended overdose which can result in hospitalization or death (10).
While there is limited human pharmacological data available in the literature for the new fentanyl analogs and synthetic opioids, it is thought that they act similar to fentanyl and are μ-opioid receptor agonists. There are estimates on potency and metabolism predictions for some analogs based on in vitro and in vivo animal studies (11, 12). Potency estimates vary greatly among these compounds, with acetyl fentanyl estimated to be five times more potent than morphine, while carfentanil was estimated to be 10,000 times more potent than morphine, and 100 times more potent than fentanyl (13).
Acute intoxications involving synthetic opioids have been documented in previous literature. Cole described a butyrfentanyl overdose in an emergency department setting (14). Helander and Backberg reported on multiple intoxications involving 4-methoxybutyrfentanyl, 4-fluorobutyrfentanyl, acetyl fentanyl, butyrfentanyl and furanyl fentanyl from the Swedish STRIDA project (15, 16). Schneir describes a patient who recreationally used U-47700 and needed medical attention (17). McIntyre reported a fatality where a male was found dead after ingesting U-47700 (18). Coopman reported a fatality that was presumed accidental due to U-47700 and fentanyl ingestion (19). Polkis reported on two fatalities involving butyryl fentanyl (20).
Due to effects induced by opioids, including analgesia, euphoria, sedation, mental clouding and depressed reflexes, the identification of these compounds is necessary for human performance forensic cases when impairment questions arise (5). Routine screens performed by many laboratories could fail to identify these drugs as most synthetic opioids are structurally different when compared to morphine, and therefore have limited cross-reactivity with immunoassays that typically target morphine (6). Fentanyl specific immunoassay have been shown, to some degree, to cross react to acetyl fentanyl, furanyl fentanyl and butyryl fentanyl but at a lesser extent than fentanyl (21). Due to the lower doses of these opioids consumed, it is hypothesized that they will be present at lower concentrations than fentanyl and therefore pose to be problematic for the Enzyme Linked Immunosorbent Assay (ELISA) test. In addition, the sensitivity of typical broad drug screens using Gas Chromatography–Mass Spectrometry (GC–MS) may not be sufficient to detect some of these very potent synthetic opioids due to their low concentrations in urine and blood samples (22). This paper addresses the detection of new synthetic opioids and presents a human performance case where multiple synthetic opioids were confirmed.
Case Report
A police officer responded to a call around 4:15 pm, where he found an unconscious 28-year-old male in the driver seat of a vehicle. While waiting for fire rescue to arrive, the police officer observed a needle on the subject's lap. A further search yielded two clear plastic baggies that contained suspected heroin. The subject was transported to the local hospital's emergency room and was eventually released. The subject was subsequently interviewed by the police officer. He stated that he was with another male who was buying heroin for him. While the other male left to purchase more drugs the subject took a “bump of heroin” and subsequently passed out behind the wheel of the vehicle. Field sobriety tests and a DRE evaluation were not performed in this case due to the driver being unconscious when the police officer arrived. The subject voluntarily provided a blood sample for the driving under the influence (DUI) investigation over 2 h post arrest. Samples submitted to the toxicology laboratory included two blood vials collected in gray stopper vacutainers which contain sodium fluoride and potassium oxalate for sample preservation.
Materials and Methods
Chemicals and reagents
β-hydroxythiofentanyl, 4-methoxy-butyryl fentanyl, butyryl fentanyl, carfentanil, despropionyl fentanyl (4-ANPP), furanyl fentanyl, isobutyryl fentanyl, N-desmethyl U-47700, para-chlorofentanyl, para-fluorobutyryl fentanyl, para-fluoroisobutyryl fentanyl (FIBF), U-47700 and valeryl fentanyl were purchased from Cayman Chemical Company (Ann Arbor, MI). Acetyl fentanyl, fentanyl, fentanyl D5, norfentanyl D5, norfentanyl and acetyl fentanyl 13C6 were purchased from Cerilliant Corporation (Round Rock, TX, USA). Ammonium hydroxide, ethyl acetate, glacial acetic acid, potassium phosphate monobasic and potassium phosphate dibasic were purchased from VWR International (Randor, PA). HPLC-grade water and HPLC-grade acetonitrile were purchased from Avantor (Center Valley, PA). Formic acid (98–100%) was purchased from EMD Millipore Corp. (Billerica, MA). Ammonium formate and methanol were purchased from Alfa Aesar (Ward Hill, MA).
Solid phase extraction
An aliquot of 0.5 mL of blood or urine sample was pretreated with 2 mL of 0.1 M Potassium Phosphate (pH = 6.0) before extraction. All samples were fortified with an internal standard mix containing Norfentanyl-D5, Fentanyl-D5 and Acetyl fentanyl-13C6 at a concentration of 5ng/mL. The samples were then sonicated for 15 min and centrifuged at 1,308 g for 10 min at −10°C. Cerex Trace B columns (35 mg) were conditioned with 1 mL of methanol and 1 mL of 0.1 M Potassium Phosphate (pH = 6.0). The supernatant of the pretreated samples was then added to the solid phase extraction cartridges. The cartridges were washed with 2 mL of DI water and 1 mL of 100 mM Acetic Acid. The cartridges were dried under max pressure for 1 min. Further wash steps included 1 mL of methanol and 1 mL of ethyl acetate. The cartridges were then dried under max pressure for 1 min for the second time. Then 600 μL of an elution solvent consisting of 93:5:2 ethyl acetate:methanol:ammonium hydroxide (v:v:v) was added to the cartridge and was collected. This elution step was repeated for a total of 1,200 μL of an elution solvent added to each cartridge. The solvent was evaporated to dryness at 35°C, 10 psi. The dried extract was reconstituted with 100 μL of the initial mobile phase and transferred to a limited volume auto-sampler vial (ALS) and analyzed by LC–MS-MS. Positive and negative controls were prepared in-house using matrix from UTAK Laboratories (Valencia, CA). Blank solvents were injected in between case to ensure that carryover did not occur.
LC–MS-MS
An Agilent 1260 liquid chromatogram system (Agilent Technologies, California, USA) equipped with a Poroshell analytical column, 120 EC-C18 μm (2.1 × 100 mm) 2.7 μm (Agilent, California, USA) was utilized for chromatographic separation of the various opioids. The analytical column was maintained at 40°C in the thermostat column compartment. The mobile phase consisted of 5 mM Ammonium Formate with 0.1% formic acid in HPLC-grade water (A) and acetonitrile with 0.1% formic acid (B). The following gradient elution was programmed: 20% B at time 0 min, this composition was held until 0.5 min. At 0.5 min the percentage of B started to increase until it reached 55% B at 8.0 min. Mobile phase B was increased again until it reached 90% B at 8.50 min. A 3-min post-run time was used to re-equilibrate the column before the subsequent injection. The injection volume was set at 5 μL.
An Agilent 6460 triple quadrupole mass spectrometer with a Jetstream electrospray source operated in positive ion mode with the following parameters: gas temp of 320°C, flow 8 L/min, nebulizer 27 psi, sheath gas heater 380°C and flow 12 L/min, capillary voltage at 3,750 V, and the nozzle voltage 500 V was used for mass spectral analysis. Three transitions were monitored for each compound and internal standard. MS1 and MS2 quadrupoles were set to unit resolution. The fragmentor voltage and collision energy used for each transition vary based on the results from the optimization study. The instrument was operated in dynamic MRM (Multiple Reaction Monitoring) mode.
MassHunter® quantitative software was used to analyze the acquired data. Identification criteria included retention time and qualifier transition ratios. This is a qualitative method only, as Certified Reference Material (CRM) was not available during the validation experiments.
Results and Discussion
This case was analyzed by the normal routine protocol for suspected DUI cases. Standard protocol includes a Headspace Gas Chromatography Flame Ionization Detector (HS-GC-FID) volatile analysis, 8 panel (amphetamine, cocaine, benzodiazepine, opiates, fentanyl, cannabis, buprenorphine and barbiturates) ELISA screen and a basic drug screen using GC–MS for that can detect over 700 compounds.
Volatile compounds, including ethanol, were not detected in the blood sample. The ELISA screen elicited a presumptive positive response for the benzodiazepines and fentanyl groups. The benzodiazepine presumptive positive result was confirmed and quantitated by Liquid Chromatography–Tandem Mass Spectrometry (LC–MS-MS), with 55 ng/mL of alprazolam. The fentanyl presumptive positive result was further analyzed using an extended opiate panel with analysis by Gas Chromatography–Tandem Mass Spectrometry (GC–MS-MS) methodology which confirmed the identification of fentanyl, but at a concentration less than 0.5 ng/mL (Lower limit of quantitation—LLOQ).
Due to the lack of correlation with the confirmed toxicology results and the case information provided by the police officer, the sample was subsequently reflexed to a more comprehensive opioid method. This method utilized a LC–MS-MS and targeted sixteen synthetic opioids, primarily fentanyl derivatives that have been reported in the literature and local drug seizure reports. The method validation, including Limit of Detection (LOD) studies (Table I), carryover, interference and ionization suppression/enhancement was carried out in accordance with the Scientific Working Group for Forensic Toxicology (SWGTOX) working document (23). The target analytes and their respective LOD's are listed in Table I. Ionization enhancement and suppression observed were within acceptable ranges. No appreciable interference was observed in endogenous and exogenous interference studies. This method successfully identified the presence of carfentanil, fentanyl, furanyl fentanyl, para-fluoroisobutyryl fentanyl, U-47700 and its metabolite in the subject's blood sample.
Compound . | LOD (pg/mL) . | |
---|---|---|
Blood . | Urine . | |
4-Methoxy-butyryl fentanyl | 100 | 100 |
Acetyl fentanyl | 500 | 500 |
Butyryl fentanyl | 100 | 100 |
Carfentanil | 10 | 10 |
Despropionyl fentanyl | 100 | 10 |
Fentanyl | 500 | 500 |
Furanyl fentanyl | 100 | 100 |
Isobutyryl fentanyl | 100 | 100 |
N-Desmethyl U-47700 | 100 | 100 |
Norfentanyl | 100 | 100 |
para-Chlorofentanyl | 100 | 100 |
para-Fluorobutyryl fentanyl | 100 | 100 |
para-Fluoroisobutyryl fentanyl | 100 | 100 |
U-47700 | 100 | 100 |
Valeryl fentanyl | 100 | 100 |
β-Hydroxythiofentanyl | 10 | 10 |
Compound . | LOD (pg/mL) . | |
---|---|---|
Blood . | Urine . | |
4-Methoxy-butyryl fentanyl | 100 | 100 |
Acetyl fentanyl | 500 | 500 |
Butyryl fentanyl | 100 | 100 |
Carfentanil | 10 | 10 |
Despropionyl fentanyl | 100 | 10 |
Fentanyl | 500 | 500 |
Furanyl fentanyl | 100 | 100 |
Isobutyryl fentanyl | 100 | 100 |
N-Desmethyl U-47700 | 100 | 100 |
Norfentanyl | 100 | 100 |
para-Chlorofentanyl | 100 | 100 |
para-Fluorobutyryl fentanyl | 100 | 100 |
para-Fluoroisobutyryl fentanyl | 100 | 100 |
U-47700 | 100 | 100 |
Valeryl fentanyl | 100 | 100 |
β-Hydroxythiofentanyl | 10 | 10 |
Compound . | LOD (pg/mL) . | |
---|---|---|
Blood . | Urine . | |
4-Methoxy-butyryl fentanyl | 100 | 100 |
Acetyl fentanyl | 500 | 500 |
Butyryl fentanyl | 100 | 100 |
Carfentanil | 10 | 10 |
Despropionyl fentanyl | 100 | 10 |
Fentanyl | 500 | 500 |
Furanyl fentanyl | 100 | 100 |
Isobutyryl fentanyl | 100 | 100 |
N-Desmethyl U-47700 | 100 | 100 |
Norfentanyl | 100 | 100 |
para-Chlorofentanyl | 100 | 100 |
para-Fluorobutyryl fentanyl | 100 | 100 |
para-Fluoroisobutyryl fentanyl | 100 | 100 |
U-47700 | 100 | 100 |
Valeryl fentanyl | 100 | 100 |
β-Hydroxythiofentanyl | 10 | 10 |
Compound . | LOD (pg/mL) . | |
---|---|---|
Blood . | Urine . | |
4-Methoxy-butyryl fentanyl | 100 | 100 |
Acetyl fentanyl | 500 | 500 |
Butyryl fentanyl | 100 | 100 |
Carfentanil | 10 | 10 |
Despropionyl fentanyl | 100 | 10 |
Fentanyl | 500 | 500 |
Furanyl fentanyl | 100 | 100 |
Isobutyryl fentanyl | 100 | 100 |
N-Desmethyl U-47700 | 100 | 100 |
Norfentanyl | 100 | 100 |
para-Chlorofentanyl | 100 | 100 |
para-Fluorobutyryl fentanyl | 100 | 100 |
para-Fluoroisobutyryl fentanyl | 100 | 100 |
U-47700 | 100 | 100 |
Valeryl fentanyl | 100 | 100 |
β-Hydroxythiofentanyl | 10 | 10 |
To the author's knowledge, this is the first human performance case report where carfentanil, fentanyl, furanyl fentanyl, para-fluoroisobutyryl fentanyl, U-47700 and its metabolite were all confirmed in an antemortem blood sample. Except para-fluoroisobutyryl fentanyl, each compound found in this case has been confirmed in post-mortem cases that have been reported in scientific literature (6, 10, 22, 24, 25). While para-fluoroisobutyryl fentanyl has not yet been documented in a case, it has been cited in several press releases suspecting its involvement in drug seizures and overdoses.
As observed in the cases reported, the concurrent detection of other psychoactive compounds present in confirmed synthetic opioid cases has been well documented in the literature (26). Typically, synthetic opioids have been identified with other opioids and opiates. Armenian et al. describe a case where both fentanyl and U-47700 was confirmed in a serum sample after a patient ingested an illicit “Norco” pill (27). The detection of compounds related to heroin, fentanyl or other synthetic opioids may lead to the hypothesis that substitution or lacing of heroin with synthetic opioids may be occurring. There are cases where synthetic opioids have been detected with other NPS. For example, a forensic laboratory in Japan detected the presence of both acetyl fentanyl and 4-methoxy PV8 in both drug material and human matrices from a post-mortem case (28). However, without analyzing drug paraphernalia or the drug material itself, it would be difficult to conclusively determine if the drug user took the confirmed drugs at the same time as these compounds may have different pharmacokinetic properties that could lead for a compound to remain in the system from a previous drug exposure.
In addition to a large number of synthetic opioids confirmed in this case, the detection of carfentanil is of interest to the human performance toxicology field. The authors initially hypothesized that carfentanil might not be detectable in human performance cases due to the compound's potency. However, after a literature review, another case report involving two human performance toxicology case identified carfentanil. These cases demonstrated support of the use of poly-opioids as in conjunction with carfentanil; one case also contained fentanyl and acetyl fentanyl, the other confirmed 6-acetylmorphine and morphine (29). The subjects in Tiscione's reported cases and this case report were all administered naloxone, and an argument could be made that without the intervention of first responders all three of these cases would have been post-mortem cases instead of human performance cases. However, as these are DUI cases, human performance toxicology laboratories should be aware of the identification of carfentanil in these cases. If the case history suggesting opioid use and toxicological findings are not consistent, it may be necessary to screen for carfentanil and other synthetic opioids using sensitive instrumentation.
Another point of interest, in this case, is that, to the author's knowledge, this is the first reported human consumption of para-fluoroisobutyryl fentanyl (4-FiBF) in both human performance and post-mortem toxicology fields. Previously, the Swedish STRIDA project identified para-fluorobutyryl fentanyl (4-FBF), which is a regioisomer of 4-FiBF, in both serum and urine clinical samples (16). Similar to other classes of NPS, there are compounds in the synthetic opioids that are structural isomers. These structural isomers can complicate the identification of the presence of a compound as retention time, and mass spectral data can be very similar if not identical. In the method presented in this case report, 4-FiBF and 4-FBF are monitored using the same transitions as their breakdown patterns are similar due to their structure similarity. Retention times were needed to distinguish these regioisomers (Figure 1) successfully.
As new synthetic opioids appear frequently, targeted methods such as this method may not be comprehensive enough. Due to this reason, it is important for toxicology laboratories to compliment the targeted analysis with sensitive methods that can detect unknown compounds such as LC–MS instrumentation operating in the scan acquisition mode. This is especially important with cases where the case history is not consistent with the toxicological results.
Conclusions
Carfentanil, fentanyl, furanyl fentanyl, para-fluoroisobutyryl fentanyl, U-47700 and its metabolite were confirmed in this DUI antemortem blood case. Without utilizing the additional targeted methodology described, the four new psychoactive opioids would not have been detected resulting in <0.5 ng/mL of fentanyl as the psychoactive compound to account for the opioid like condition. A concentration of fentanyl <0.5 ng/mL in the blood, would not correlate with the subjects observed symptoms and behavior. This case report illustrates the need to supplement routine toxicological analyses with increased sensitivity and specificity to target synthetic opioids when analyzing human performance samples when opioid use is suspected.