Chemistry

Carfentanil is a synthetic opioid (Fig. 1) that is an analogue of fentanyl (a carboxylated fentanyl). Its IUPAC name is 4-[(1-oxopropyl)-phenylamino]-1-(2-phenylethyl)-4-piperidinecarboxylic acid methyl ester, but it is also marketed under the names carfentanyl, carfentanila, carfentanilum, Wildnil, UNII-LA9DTA2L8F and 59708-52-0. Other Medical Subject Headings synonyms include 4-carbomethoxyfentanyl, carfentanyl, (4-methoxycarbonyl)fentanyl, 11C-carfentanil, carfentanil citrate, carfentanil oxalate, carfentanil (+−) isomer, R31833 and R33799 [23, 93, 94]. It is sold either as its base with the molecular formula C 24 H 30 N 2 O 3 (molecular weight 394.515 g/mol) or as its citrate salt with the molecular formula C 30 H 38 N 2 O 10 (molecular weight 586.638 g/mol) [93, 95, 96]. The CAS number of carfentanil is 59708-52-0, and there are no data concerning its boiling and melting points [93, 94, 97]. Carfentanil base is a white powder and has also been identified in seized white, pink, and brown powders along with other substances [43, 46, 98,99,100,101]. Carfentanil citrate is highly water soluble with no distinguishing odor [45]. Carfentanil can be structurally confirmed by means of infrared spectroscopy, nuclear magnetic resonance, GC–MS, and isotope ratio mass spectrometry [101].

Synthesis

Carfentanil was synthesized by a team of chemists, including Paul Janssen, at Janssen Pharmaceuticals in 1974 [102]. Its synthesis was firstly described in the literature by Van Bever et al. [21] in 1976 and was patented by Janssen et al. (US patent No. 4,179,569) in 1979 [22]. Several synthesis routes for carfentanil have been described in the literature [21, 22, 103,104,105,106]. The first developed synthetic procedure starts with the reaction of 1-phenylethyl-4-piperidine with KCN and aniline in acetic acid. The resulting compound is hydrolyzed with cool H 2 SO 4 and gives the corresponding diamine which is further hydrolyzed with KOH in refluxing ethylene glycol. The acid reacts with methanol under acidic conditions and the resulting methyl ester is acylated with propionic anhydride yielding carfentanil (Fig. 3) [21, 22, 103].

Fig. 3 Synthesis of carfentanil according to Janssen and Van Daele [22] Full size image

Labeled carfentanil have been also synthesized in order to be used mainly in pharmacology studies on the µ opioid receptor (mOR) binding [104, 105]. A simple synthesis of [11C] carfentanil was described by Jewett in 2001. Initially the tetrabutylammonium salt of 4-[N-(1-oxopropyl)-N-phenylamino]-1-(2-phenylethyl)-4-piperidinecarboxylic acid reacted with [11C] methyl triflate in dimethyl sulfoxide (DMSO). The resulting [11C] carfentanil is extracted via an Empore SPE extraction disc and all radioactive contaminants are removed. The product is then eluted by a mixture of ethanol and water and passed through an anion exchange column to remove any remains of contaminants [105].

Carfentanil can be also synthesized via the Siegfried route in which 1-phenethyl-4-piperidone is used as a precursor [107], but it is slightly more complex than fentanyl’s [108], because it requires the introduction of a carbomethoxy group [107].

Prevalence and use

Carfentanil, the active ingredient of Wildnil, is a fentanyl analogue that is approved for veterinary use as a tranquilizing agent for sedation, as a hypnotic, and as anesthesia of animals such as elephants, gazelles, goats, horses, pigs, polar bears, rhinoceroses, seals, and wolves [23,24,25,26,27,28,29]. It has been extensively studied in animals since it was first synthesized [23, 28,29,30, 32, 37, 38, 109], but a few human studies have been also reported [31, 33, 34]. Carfentanil has been characterized as the most potent, dangerous, and commercially available fentanyl analogue. It has been also used as a chemical weapon, making it a very serious hazard to public safety [19, 20, 45]. It has been reported through seizures, intoxication cases, and illicit drug trafficking in Europe, Asia, the USA, and Australia [14, 40, 41, 43, 45, 46, 110,111,112,113].

Most of fentanyl analogues including carfentanil are usually manufactured in China and exported from there to all over the world [19, 20, 99, 113, 114]. In October 2016, Associated Press news reported finding 12 Chinese laboratories willing to export carfentanil to the United States, Canada, the United Kingdom, France, Germany, Belgium, and Australia for the price of US$2750/kg [20, 114]. Until March of 2017, carfentanil was not regulated in China; it was openly and legally manufactured and sold by Chinese companies [20, 94]. The drug can be also found easily online, on the darknet, through related websites in which it is often labeled as a “research chemical” and sold through direct mail shipments in prices from US$800 to 2500 per gram [19, 20, 111, 113]. More specifically in 2016, one darknet search engine gave 118 websites selling carfentanil [115]. Because carfentanil is more potent than heroin, its trafficking quantities are significantly less than those of heroin. Therefore, it is easier and cheaper to be smuggled without necessarily being cheap to manufacture [19, 116]. Carfentanil arrives from China in powdered and tablet form, but it also comes in many other forms such as blotter papers, patches, and sprays. In some cases, it has been accidentally absorbed through the skin, inhaled, or ingested [45, 117,118,119].

Carfentanil has not been reviewed up to now by the WHO ECDD [14]. It was reported for the first time in EMCDDA in February 2013, when it was identified in a seized powder by the Latvian police. In the same period, the Latvian National Local Point issued an alert on carfentanil, which noted that the drug was related to several unconfirmed deaths throughout the country [44].

On September 22, 2016, the Drug Enforcement Administration (DEA) issued a worldwide warning to the public and law enforcement agencies about the risks and hazards of carfentanil, based on the increasing number of carfentanil-related deaths throughout the United States [120]. Carfentanil has been identified across eight states in more than 400 seized materials from July to October 2016 [121]. Most of these seizures were located in Ohio [115]. The DEA recorded prevalence of the drug in a number of states including Florida, Georgia, Rhode Island, Indiana, Pennsylvania, Kentucky, West Virginia, New Jersey, and Illinois, while an intoxication outbreak linked to carfentanil was recorded in July 2016 in Cincinnati, OH [19, 43, 111]. The drug possibly arrived in Ohio through Canada and Mexico, as transshipment points [19, 113, 122]. Since the DEA’s report, carfentanil-related deaths have been also recorded in Florida, Illinois, Colorado, Wisconsin, Minnesota, Michigan, West Virginia, New Hampshire, Virginia, and Maryland, but also some unconfirmed cases have been reported in some other states [119].

In Australia, carfentanil was noted for the first time within a seizure in Sydney in December 2016, where it was identified in a package at a Sydney mail center. Australian police issued an alert in February 2017, when the drug was identified in a package at a mail center in Brisbane [114, 116, 123, 124].

Carfentanil is often used to adulterate heroin, cocaine, and fentanyl. In some other cases, it is labeled and sold as heroin [19, 42]. In Cincinnati, OH, a new drug appeared in 2016 under the street name of “gray death”. It looks like cement and often contains cocaine, heroin, fentanyl, carfentanil, furanylfentanyl, and acrylfentanyl [125, 126]. Carfentanil has also been identified in mixtures along with caffeine, antihistamines, furanylfentanyl, or acrylfentanyl, while in some other cases it has been found to be laced with ketamine [113, 126]. Unconfirmed reports of marijuana laced with carfentanil have been found in northeast Ohio and Canada [125]. Furthermore, the drug has been found in counterfeit pills. The DEA reported that carfentanil had shown up in counterfeit prescription pills sold as OxyContin and Xanax [125, 127].

In some cases of opioid use, the users developed a tolerance to the drug, and they began to chase opioids offering a more intense outcome. However, in the case of carfentanil, more than any other fentanyl analogue, users do not know that its intensified effect can kill them [42]. A user mentioned that he tried a nasal spray of carfentanil [48], while in another drug forum a user sought information about making a carfentanil solution, which was probably intended to be used intravenously [49]. However, many drug users commented: “Never use carfentanil. If someone vends it, he should be banned, because it’s poison” or “Warning: using carfentanil is stupid and deadly and no one should ever do it. Ever” [47]. These comments indicate that opioid addicts are also alerted regarding the harmful potential of this drug.

Metabolism

To the best of our knowledge, there is only one published metabolic study of carfentanil. Feasel et al. [128] described the possible metabolic pathways of carfentanil in humans for the first time in 2016. Two prognostic models, MetaSite software (Molecular Discovery, Pinner, UK) and ADMET Predictor (Simulations Plus Inc., CA, USA), were applied to predict possible in silico carfentanil metabolites. Initially carfentanil (5 μmol/L) was incubated with human liver microsomes (HLM) in order to determine carfentanil’s clearance and to assess its possible toxicity due to its slow metabolism. The HLM samples were treated properly and analyzed via an HPLC system. The HLM half-life was calculated by observation of carfentanil’s depletion over 1 h. The study concluded that carfentanil is readily metabolized by the CYP enzymes; its long in vivo half-life was attributed to other factors. They suggested that the drug’s high lipophilicity and larger volume of distribution makes it less available for hepatic metabolism than its less potent fentanyl analogues. Evidence from other fentanyl analogues suggests that carfentanil probably binds to plasma proteins strongly. They conducted further studies to identify the metabolites of carfentanil after incubation with human hepatocytes. An HPLC system coupled with a triple time-of-flight (TOF) mass spectrometer was used for identification of the metabolites and the analysis was performed under the same chromatographic conditions (column and mobile phases) as with the HLM analysis. Twelve phase I and phase II metabolites were identified through the following metabolic pathways: N-dealkylation and ester hydrolysis (M1), N-dealkylation of piperidine ring (M2), N-dealkylation of piperidine ring and hydroxylation of propanoic group (M3), ester hydrolysis and hydroxylation of piperidine group (M4), hydroxylation of propanoic group (M5), hydroxylation of phenethyl group and glucuronidation (M6), hydroxylation of phenethyl structure (M7), hydroxylation of piperidine ring (M8), ketone formation of phenethyl linker (M9), N-oxidation of piperidine and hydroxylation of phenethyl group (M10), N-oxidation of 4′-nitrogen (M11) and finally N-oxidation of piperidine (M12). The most abundant metabolites were the phase I metabolites of piperidine ring hydroxylation (M8) and N-dealkylation (M2), followed by ketone formation of phenethyl linker (M9), N-propanoic hydroxylation (M5), phenethyl ring hydroxylation (M7), piperidine N-oxidation (M12) and 4′-position nitrogen N-oxidation (M11). The only phase II metabolite identified was a glucuronide conjugate of hydroxylated carfentanil (M6). The chemical structures of the identified metabolites are presented in Fig. 4 [128].

Fig. 4 In vitro metabolic pathways for carfentanil in human hepatocytes proposed by Feasel et al. [128] Full size image

Pharmacology and toxicology

Carfentanil was pharmacologically studied for the first time in 1979 by a scientist group of Janssen Pharmaceuticals, who synthesized it. Its ED 50 value was determined via a rat hot tail withdrawal test and found to be about 10,000 and 100 times more potent than morphine and fentanyl, respectively [22]. Since then it has been studied extensively among other opioids, but it was never approved for clinical use in humans [23, 28,29,30,31,32,33,34,35,36,37,38,39]. Carfentanil is mainly used as an anesthetic agent in large animals [45].

Several studies have been conducted for the elucidation of the action of carfentanil through opioid receptors [30,31,32,33,34,35,36]. In 1993, [3H] carfentanil and [3H] [d-Ala2-MePhe4-Gly-ol5]enkephalin (DAMGO) were used as radioligands in order to image high-affinity binding sites in sections of the rat brain. For that purpose 30 brain sections were examined in which absolute and relative densities of high affinity carfentanil binding sites were measured and the autoradiographic image was obtained in order to determine the distribution pattern of the two substances. The highest levels of binding were observed in the striatum section and the lowest levels of binding were observed in the cerebellum. The autoradiographic images showed close distribution patterns for [3H] carfentanil and [3H] DAMGO, but a remarkable difference in the interaction of the substances with mOR was observed. It is not clear whether the different sensitivity, between [3H] carfentanil and [3H] DAMGO, in the proteins of the mOR can be attributed to the structural differences [30].

[11C] carfentanil and [11C] diprenorphine were also used as radioligands in a study measuring the total binding capacity to the opioid receptors, in human volunteers, before and after the administration of naloxone. Twenty-eight volunteers participated in the study. Twenty-one received [11C] carfentanil, three [11C] diprenorphine, and four received both radioligands. A simple dual detector coincidence system was used for the measurements of the two ligands in the brain [31]. [11C] carfentanil was synthesized according to a previously described method [104]. The total binding of [11C] diprenorphine was found greater than [11C] carfentanil’s, possibly due to either easier transportation of the former through the blood-brain barrier or more binding sites of it in the brain [31].

In a study conducted by Jewett et al. [32], eight derivatives of [11C] carfentanil were evaluated as potential mOR agonists within the research for substances with better pharmacodynamics than the parent one. Derivatives were prepared via substitution of aryl- or alkyl-group on a [11C]-labeled form of carfentanil and were evaluated for their mOR binding capacity and their pharmacokinetics in mouse brain. Another group of 2-chloro, 2-methoxy and 2-methyl derivatives was also evaluated for their binding within specific brain regions and for their distribution by using an equilibrium infusion rat model [32]. All the [11C] derivatives were prepared through O-[11C] methylation [105] of the corresponding free carboxylic acid [32].

Weltrowska et al. [35] studied the binding affinity of the two isomers of the “carba-analogue” of carfentanil (c-carfentanil) with the opioid receptors. They replaced the nitrogen in the piperidine ring of carfentanil with a carbon in order to assess how the electrostatic interaction of nitrogen with Asp protein of the opioid receptor affects binding and activation of opioid receptors. The trans isomer of c-carfentanil was found to result in a 4000-fold decrease in mOR binding affinity than carfentanil, but was still significant. Its δ opioid receptor (dOR) binding affinity was found to be similar to that of mOR, but no receptor selectivity was observed between mOR and dOR receptors. This isomer did not show any significant binding affinity with the κ opioid receptor (kOR). The cis isomer did not show any significant binding affinity with any one of the three receptors [35].

Labeled [11C] carfentanil has been used in some pharmacological studies as a positron emission tomography (PET) scan radiotracer [33, 34, 39]. More specifically, [11C] carfentanil was used in a study for evaluating the response of cocaine users to mOR agonists, including the respective adverse effects. The initial hypothesis was that chronic cocaine exposure might lead to increased brain mOR binding potential. As a result, chronic cocaine users might have a different response to mOR agonists. Carfentanil’s plasma half-life was calculated and found to be 51.4 (±16.2) and 41.8 (±17.5) min for cocaine users and non-drug using controls, respectively. As there were not any significant differences in its half-life among the two groups, they concluded that these differences are most likely pharmacodynamically based. Furthermore, cocaine users were found to have fewer adverse effects (nausea, dizziness, headache, vomiting, and itchiness) than the control ones [33]. [11C] carfentanil was again used as a radiotracer in a PET study for the measurement of endogenous opioid release in the brain during painful proximal gastric balloon distension. The volunteers were chosen based on specific criteria as history of drug abuse or psychiatric and gastrointestinal disorders. The study showed up that no endogenous opioid release was detected in the brain during sustained visceral pain and concluded that endogenous opioid levels are more associated with somatic pain [34]. [11C] carfentanil was also used as a radioligand in a PET study, in which the mOR availability was measured in the psychiatric disorder of pathological gambling. The results provided an evidence that dysregulation of endogenous opioids might have an important role in the pathophysiology of gambling addiction [39].

Carfentanil is used extensively for immobilizing and tranquillizing mostly large wild animals. Because of this, several animal studies have been conducted in order to clarify the effects of carfentanil on different animal species [45]. Carfentanil was initially evaluated as an anesthetic in guinea pigs, combined with etomidate. Its performance was investigated upon 20 animals of this species weighting from 600 to 800 g. A solution of both substances was prepared and was administrated intramuscularly into the hind leg. Eight of the animals underwent hemodynamic monitoring as well. All the observations showed that this combination of carfentanil and etomidate was sufficient in producing anesthesia and did not influence the circulation and respiration of the animals in dangerous levels. The increased heart rate was attributed to the excitement of the animals, and the recovery period from this drug combination was 90 min [23].

In some other studies ten female and six male captive-born dama gazelles were administrated carfentanil in order to assess the cardiovascular response to it. The drug was administered intramuscularly at doses of 18.4 ± 2.2 μg/kg, and no food was given for 24 h before recumbency. A significant decrease in heart rate, beginning from 5 min after immobilization, was observed. Fifteen minutes after anesthesia was induced, a decrease in respiratory rate became apparent, while hypertension was present over the whole time. Analysis of the arterial blood samples showed that Pa CO2 and Pa O2 were within normal limits [28].

Carfentanil is the 4-carbomethoxy derivative of fentanyl. It is of great interest that the 4-carbomethoxy derivatization gives as much as 100-fold enhancement of potency of fentanyl as mOR agonist. Vučković et al. [37] synthesized other regioisomers of carfentanil i.e., (±) cis and (±) trans 3-carbomethoxy fentanyls, and tested their potency as a function of antinociceptive action in rats. As results, the study revealed that the introduction of the 3-carbomethoxy group in the piperidine structure of fentanyl shortened the duration of its action and reduced its potency. It was clarified that the carbomethoxy derivatization at the carbon in 4-position of the piperidine ring is essential to gain such high potency of about 100 times higher than that of fentanyl. However, they suggested that carfentanil regioisomers have to be further evaluated as antinociceptive compounds within the frame of structure-activity relationship studies [37].

Eight domestic goats were used for the clarification of the pharmacokinetic profile of carfentanil and naltrexone by Mutlow et al. [29]. The animals were administrated with 40 μg/kg carfentanil intramuscularly, and after 30 min naltrexone (100 mg naltrexone/mg carfentanil) was administered for anesthesia reversal in several routes of administration (intravenous, intramuscular, and subcutaneous). Blood samples were collected before and after carfentanil administration at different times up to 5 days. Hemodynamic responses were monitored though the entire procedure and also during the collection of the blood samples. The pharmacokinetic profiling of plasma showed rapid carfentanil absorption and a rapid reversal of immobilization by naltrexone after all routes of administration. Carfentanil’s half-life (5.5 h) did not differ according to different administration routes [29].

Naloxone and naltrexone are opioid receptor antagonists and had been used in several cases for the reversal of the respiratory depression caused by carfentanil [29, 109, 129]. Naltrexone was evaluated for antagonizing carfentanil in mOR binding by Miller et al. [109] in an animal study conducted in captive Rocky Mountain elk. Considering the mean immobilization induction time, the mean recovery time and the observed adverse effects, the study concluded that a naltrexone dose of 100 mg/mg of carfentanil is quite effective in reversing carfentanil’s immobilization effects [109]. In another PET scan study conducted by Saccone et al. [129], naloxone was evaluated for its ability to displace [11C] carfentanil in the mOR after intranasal and intravenous administration. PET imaging showed that intravenous and intranasal naloxone produced similar decrease in mOR occupancy caused by [11C] carfentanil [129]. However, naloxone has a shorter duration of action than that of carfentanil, and multiple doses may be needed to reverse carfentanil’s effects [38, 42, 99, 111, 113, 117, 130]. One or two doses of naloxone are considered enough to treat a heroin overdose. In the case of carfentanil, six or maybe more doses are needed. The DEA suggested the continuous administration of naloxone until the individual’s breathing is resumed for at least 15 min or until emergency medical care arrives [19, 117]. On the other hand, the efficacy of naltrexone in reaching mOR is not as good as that of carfentanil; there is a need to find new opioid antagonists with fewer disadvantages. Yong et al. [38] investigated the efficacy of different doses of nalmefene for antagonizing carfentanil-induced loss of righting reflex and respiratory depression using naloxone as a control in rats. Respiratory parameters and parameters of arterial blood gases were monitored through the procedure. Nalmefene was found to dose-dependently decrease the duration of loss of righting reflex and reverse the respiratory depression caused by carfentanil [38].

Carfentanil is very potent and its effects in humans appear rapidly [43]. These effects include symptoms and signs very similar to those of an opioid intoxication, like cold and clammy skin, nausea, vomiting, pinpoint pupils, disorientation, dizziness, lethargy, sedation, sudden drowsiness, respiratory disorders, possible heart failure, and weak pulse. The DEA advised that anyone experiencing any of the mentioned symptoms should immediately seek medical care [42, 43, 117]. The mentioned symptoms are dose dependent. High doses can lead to severe intoxications and consequently to death [44, 46, 119, 120].

Most adverse effects that carfentanil causes originate from its mOR binding [36]. Mosberg et al. [131] suggested that one way to overcome such adverse effects is by synthesizing compounds with mixed mOR agonist and dOR antagonist properties [131]. Purington et al. [132] had suggested in 2009 that such compounds are certain peptides that had proven to display such properties. In 2015, Váradi et al. [36] conducted a study in which they synthesized ten carfentanil amide analogues and assessed if these analogues could provide analgesia with fewer adverse effects. All of these amides displayed high affinity to the mOR binding, while one displayed high affinity to dOR and low affinity to kOR. This compound was found to provide moderate analgesic efficacy in vivo with no signs of physical dependence and less respiratory depression than morphine [36].

Intoxications and fatal cases

Carfentanil is a very toxic fentanyl analogue. In veterinary medicine the drug is used as a tranquilizer agent for large animals, but has no proven medical use in humans. It has lately entered the illicit drug market and is responsible for many intoxication cases and deaths all over the world. Since it was first introduced as a chemical weapon at a theater in Moscow, it has been a part of the recently emerged fentanyl analogue crisis. Carfentanil has led to a significant number of deaths, mostly in the United States, and also in Europe [40, 44,45,46].

The first fatal cases possibly related to carfentanil were reported in Russia, in 2002, when more than 120 people died in the Moscow Dubrovka Theater, in which they were held as hostages by Chechen rebels. During a rescue attempt, the Russian military forces released a “poison” gas though the ventilation system in order to subdue the rebels. Hundreds of hostages were submitted to hospitals suffering from “sleeping gas” poisoning. In the hospital, doctors took several hours testing various antidotes, and after 4 days, the mysterious gas was identified as a fentanyl analogue. Despite the fact that the Russian Health Minister announced that the drug used cannot be characterized as fatal, collectively 127 of the 800 hostages died and 650 required hospital monitoring. The deaths were initially attributed to bad captivity conditions. Evidence suggested that the mysterious “sleeping gas” contained carfentanil and an anesthetic agent like halothane [40]. An LC–MS/MS analysis of clothing and biological fluids from three survivors of the siege revealed the presence of carfentanil along with remifentanil on a survivor's shirt, and a metabolite identified as norcarfentanil was found in a urine sample [41].

Later, in 2013, a series of carfentanil-related deaths was reported in Latvia, and a related alert was issued by the Latvian National Focal Point [44].

In the United States, the first reported carfentanil-related intoxication took place in 2010 in Chicago. It was an unintentional intoxication where a veterinarian was accidentally exposed to carfentanil. A 42-year-old man was anesthetizing elk in order to test them for tuberculosis by using shooting darts containing 1.5 mg of carfentanil citrate and 50 mg of xylazine. When he tried to dislodge a dart, the substance was accidentally splashed into his eyes, face, and mouth. Although he immediately washed his face, he began to feel drowsiness 2 min after. He was at once administered parenterally 100 mg naloxone by his coworkers and was transferred to the medical care unit. On his arrival to the medical center, he only complained of mild and transient chest pain, and his vital signs were evaluated. The size of his pupils and his heart, lung, neurological, and abdominal examinations were within normal limits. He was monitored for 24 h and when stabilized, he was discharged. That was the first reported carfentanil intoxication internationally [45].

In September 2016, the DEA issued an alert informing the police and the public about the dangers of carfentanil. According to the DEA, local law enforcement, and first responders, several overdose deaths were linked to the drug in many parts of the country [120]. Because the DEA published the report on carfentanil, several intoxication deaths have been recorded throughout the states of Florida, Illinois, Colorado, Wisconsin, Minnesota, Michigan, West Virginia, New Hampshire, Virginia, and Maryland. In other states, more unconfirmed cases have been reported [119].

In Ohio, more than 4000 deaths linked to opioids appeared in 2016. In particular, a 36% increase from the previous year was reported. This increase was attributed to heroin and carfentanil abuse. For example, in Akron’s Summit County, nearly half of its 308 overdose deaths were attributed to carfentanil intoxication [133]. In Hamilton County, Ohio, law enforcement agencies recorded 50–70 intoxication cases per week in early 2016. When carfentanil appeared, the number increased to 175–200 per week [119].

In 2017, carfentanil was confirmed as the cause of death in two intoxication cases in Florida. In the first case, a 34-year-old man was found dead in the driver’s seat of a van. A syringe, spoon, and a yellow bag containing a brown powder were found in the cup holder near the seat. He had a history of tobacco, alcohol, marijuana, and heroin abuse, and mild hypertensive heart disease and a mild hepatic steatosis were found at autopsy. Toxicological analysis revealed the presence of carfentanil in heart blood along with furanylfentanyl, fentanyl, morphine, and hydromorphone at concentrations 1.3 ng/mL for carfentanil, 0.34 ng/mL for furanylfentanyl, 6 ng/mL for fentanyl, and <20 ng/mL for both morphine and hydromorphone. Additionally, 6-acetylmorphine along with hydrocodone and hydromorphine was present in his vitreous humor, and morphine, hydromorphone, 6-acetylmorphine, hydrocodone and hydromorphine were present in his urine. The cause of death was pronounced as intoxication due to heroin, fentanyl, carfentanil, and furanylfentanyl. In the second case, a 25-year-old man was found unconscious on his mattress by his mother in a tent where he was living. The mother called emergency services, and on arrival of medical responders, he was pronounced dead. A bag with a brown powder was found next to the deceased. The toxicological analysis showed a concentration 0.12 ng/mL of carfentanil in heart blood, 460 ng/mL of benzoylecgonine in peripheral blood, 510 ng/mL of benzoylecgonine, and 40 ng/mL of cocaine in the vitreous humor. The medical examiner declared accidental carfentanil intoxication as the cause of death [46].

Several other reports concerning fatal cases involving carfentanil can be found on web sources. All these cases occurred in the United States [134,135,136,137,138,139,140,141].

Analysis of carfentanil in seized materials and biological specimens

Many analytical methods for determining carfentanil in seized materials and biological specimens have been described through the years. Some have been specifically developed for this purpose, while others within the frame of the investigation of specific carfentanil-related intoxication cases [26, 41, 46, 90, 101, 128, 142,143,144].

An 125I-radioimmunoassay (125I-RIA) detection method was published in 1989, in which the ability of sevn antibodies to fentanyl derivatives developed to react with carfentanil was investigated. The seven antibodies were evaluated in vivo for their binding ability with fentanyl, carfentanil, and four other analogues. The ability was evaluated by measuring the concentration of carfentanil that is required to reduce maximum binding to 50%. Carfentanil cross-reacted well with only one antibody, while less satisfactory cross-reactivity was observed with the six other antibodies [26].

Tobin et al. [142] developed and evaluated a one-step ELISA test for sufentanil, which also can cross-react with carfentanil and detect it in horse urine several hours after its administration [142]. Carfentanil is included in a screening test based on ELISA principles that were developed recently by Randox Laboratories Ltd. for “Designed fentanyl and opioids” along with other fentanyl analogues in an NPS panel as described before in this review [90].

Hunter et al. [143] developed and validated the first chromatographic method for determining carfentanil and naltrexone in goat plasma. Plasma samples were extracted two times with toluene after pretreatment with 1 M NaOH. The extracts were dried under N 2 in a warm water bath and injected into the LC–MS chromatographic system. The analysis was isocratically performed with acetonitrile/(10 mM ammonium acetate and 0.1 mM citrate) (30:70, v/v). A Zirchrom PBD column was used and the flow rate was set in 0.3 mL/min. The mass spectrometer was set at a single ion monitoring mode. The lower limit of quantification was 8.5 pg/mL for carfentanil and 0.21 ng/mL for naltrexone [143].

Another chromatographic method was developed and validated by Wang et al. [144] to analyze carfentanil and norcarfentanil along with 11 other fentanyl analogues in human urine. All urine samples were extracted through SPE Oasis HLB® C18 columns. Initially, 0.5-mL urine samples were spiked with an IS mixture of deuterated analytes including carfentanil-d 5 and norcarfentanil-d 5 and acetate buffer (pH 4.0). The samples were then extracted via SPE columns, concentrated and transferred in autosampler microvials. The eluents were dried, reconstituted in water and injected into the LC–MS/MS chromatographic system. The chromatographic column was a Waters Xterra MS C18 column and the system was operated in a gradient mode with a flow rate 0.5 mL/min. Mobile phase A was ammonium acetate in HPLC water and mobile phase B was ammonium acetate in acetonitrile/methanol (95:5, v/v). The method was validated and the limit of detection (LOD) for each analyte was determined. The LODs for carfentanil and norcarfentanil were found to be 0.003 and 0.027 ng/mL, respectively [144].

An LC–MS/MS method was used for the analysis of the clothing extracts of two survivors of the Moscow theater siege, and urine from a third survivor. A jumper and a leather jacket were collected from one victim, a shirt from another, and two blood samples from each. A single urine sample was obtained from the third survivor. Clothing samples and blood samples were initially screened by a GC–MS system and a GC–MS/MS system, respectively, for fentanyl, cis-3-methylfentanyl, carfentanil, sufentanyl, lofentanil, and remifentanil. Clothing samples were extracted with solvent (CH 2 Cl 2 ) or water and blood samples were liquid–liquid extracted with 1-chlorobutane prior to screening. Results after analysis of clothing and blood samples were negative for these substances. All clothing samples and the urine from the third victim were further cleaned with SPE according to a previously published method by Shou et al. [145] prior to LC–MS/MS analysis. The SPE columns were preconditioned with methanol/water/5% acetic acid solution and after that the cartridges were washed with 5% aqueous acetic acid and methanol. Subsequently, they were eluted twice with 2% aqueous NH 4 OH in chloroform/isopropanol (4:1, v/v). The eluents were dried, reconstituted with acetonitrile/water/trifluoroacetic acid (TFA) (95:5:0.05, v/v/v) and injected to the chromatographic system. The system performed in isocratic mode with 7% of 0.05% TFA in water/93% of 0.05% TFA in acetonitrile. A Betasil Silica-100 chromatographic column was used at a flow rate of 200 μL/min [41].

Feasel et al. [128] used both low-resolution and high–resolution mass spectrometry systems for the assessment of carfentanil’s metabolic clearance and for the determination of in vitro carfentanil’s metabolites, respectively. For the low-resolution HPLC–MS/MS, the samples were injected into the HPLC chromatographic system, which was set in gradient mode with 0.1% formic acid in water as phase A and 0.1% formic acid in acetonitrile as phase B. A Kinetex™ C18 chromatographic column was used and the flow rate was set at 0.5 mL/min. For the identification of possible metabolites, an HPLC–triple TOF analysis was performed under the same chromatographic conditions (column and mobile phases) as with the HLM analysis [128].

Seized powders from three exhibits were subjected to profiling analysis via infrared spectroscopy, nuclear magnetic resonance spectroscopy, GC–MS, and isotope ratio mass spectrometry. Spectral data for carfentanil citrate and carfentanil hydrochloride were provided in the study. The quantification of carfentanil was performed by GC combined with flame ionization detection. For the GC–MS analysis, CH 2 Cl 2 extract of carfentanil was injected via split mode into the system with a flow rate at 36.5 cm/s of helium. A DB-1 fused silica capillary column was used. Fentanyl, furanylfentanyl, acetylcarfentanil, heroin, 6-acetylmorphine, acetylcodeine, noscapine, and diphenhydramine were detected in the seized materials along with carfentanil [101].

A GC–MS screening method was used for the determination of carfentanil in two fatal intoxication cases involving carfentanil and furanylfentanyl. The urine and blood samples were pretreated with alkaline borate buffer and a mixture of toluene/hexane/isoamyl alcohol (78:80:2, v/v/v). Subsequently, the samples were back extracted into ethyl acetate using sulfuric acid and neutralization with NaHCO 3 /K 2 CO 3 . An Rtx-5 column was used for the chromatographic analysis. Case 1 had carfentanil and furanylfentanyl at concentrations of 1.3 and 0.34 ng/mL in blood, respectively. Case 2 had a carfentanil concentration at 0.12 ng/mL in blood [46].

Legal status

Carfentanil is one of the most dangerous fentanyl analogues to come into the drug market in recent years. As a result, there is an undeniable need for the safety of the public for it to be regulated in order to minimize the potential of an imminent health hazard as much as possible. The drug is not scheduled as of now under the 1961 United Nations Convention, but it is a controlled substance in many countries in Europe, as well as the United States, Canada, Australia, and China [121, 146,147,148,149,150,151,152,153,154,155,156,157].

In Czech Republic, carfentanil is regulated under the Government regulation concerning lists of drugs issued on December 18, 2013 [147]. Carfentanil was included in Annex 1 of Danish Executive Order No.557 of 31 May 2011, which came in force on November 24, 2016; the drug was included on the B List, and it can be used only for medical and scientific purposes [148]. In the United Kingdom carfentanil is controlled under the Misuse of Drugs Act 1971 [149].

In the United States, the DEA classifies carfentanil as a Schedule II substance according to the Federal Controlled Substances Act issued on October 28, 1988 [150,151,152]. On September 29, 2016 the Department of Health in Pennsylvania issued the placement of carfentanil in Schedule II under the Controlled Substance, Drug, Device, and Cosmetic Act [150].

In Canada, carfentanil is a Schedule I substance under Canada’s Controlled Drugs and Substances Act, which was last amended on May 18, 2017 [153, 157].

In Australia, carfentanil has been included in the Poisons Standard in June 2017, by the Australian Department of Health. It has been clarified as a schedule 8 poison, and legal restrictions apply to its manufacture, supply, distribution, possession, and any type of abuse, physiological misuse, or physical dependence [154].

China started to discuss scheduling carfentanil 4 months after the Associated Press discovered 12 Chinese laboratories willing to export carfentanil to the United States, Canada, and Europe in October 2016. The scheduling came into force in March 2017 when it was added to the list regulated by the “Administrative Measures on Narcotics and Psychotropic Substances without Medical Use” [121, 155, 156].