The AAPS Journal

, Volume 18, Issue 6, pp 1489–1499 | Cite as

Metabolism of Carfentanil, an Ultra-Potent Opioid, in Human Liver Microsomes and Human Hepatocytes by High-Resolution Mass Spectrometry

  • Michael G. Feasel
  • Ariane Wohlfarth
  • John M. Nilles
  • Shaokun Pang
  • Robert L. Kristovich
  • Marilyn A. Huestis
Research Article


Carfentanil is an ultra-potent synthetic opioid. No human carfentanil metabolism data are available. Reportedly, Russian police forces used carfentanil and remifentanil to resolve a hostage situation in Moscow in 2002. This alleged use prompted interest in the pharmacology and toxicology of carfentanil in humans. Our study was conducted to identify human carfentanil metabolites and to assess carfentanil’s metabolic clearance, which could contribute to its acute toxicity in humans. We used Simulations Plus’s ADMET Predictor™ and Molecular Discovery’s MetaSite™ to predict possible metabolite formation. Both programs gave similar results that were generally good but did not capture all metabolites seen in vitro. We incubated carfentanil with human hepatocytes for up to 1 h and analyzed samples on a Sciex 3200 QTRAP mass spectrometer to measure parent compound depletion and extrapolated that to represent intrinsic clearance. Pooled primary human hepatocytes were then incubated with carfentanil up to 6 h and analyzed for metabolite identification on a Sciex 5600+ TripleTOF (QTOF) high-resolution mass spectrometer. MS and MS/MS analyses elucidated the structures of the most abundant metabolites. Twelve metabolites were identified in total. N-Dealkylation and monohydroxylation of the piperidine ring were the dominant metabolic pathways. Two N-oxide metabolites and one glucuronide metabolite were observed. Surprisingly, ester hydrolysis was not a major metabolic pathway for carfentanil. While the human liver microsomal system demonstrated rapid clearance by CYP enzymes, the hepatocyte incubations showed much slower clearance, possibly providing some insight into the long duration of carfentanil’s effects.


carfentanil metabolism norcarfentanil opioid 


Carfentanil is an ultra-potent synthetic opioid analgesic belonging to the same class of drugs as its prototype fentanyl. Little is known about human pharmacology and toxicology, or analysis of carfentanil in biological samples. While not used in human clinical medicine, carfentanil has limited use in veterinary medicine for large animal sedation, take-down, and anesthesia (1). Carfentanil, seen in Fig. 1, is 10,000 times more potent than morphine for analgesia, as measured by the rat tail withdrawal assay (2). Carfentanil has the lowest EC50 of the fentanyl analogues (3). The reasons for its potency are largely unknown but can be attributed to its high lipophilicity, ease of crossing the blood-brain barrier (BBB), high receptor efficacy, and high selectivity/specificity for the μ-opioid receptor (MOR) over other opioid receptor types, such as ƙ or ƍ (3). The μ-opioid receptor is the receptor sub-type responsible for not only the desirable opioids effects such as analgesia and euphoria but also the adverse and potentially toxic effects such as muscular rigidity, respiratory depression, and apnea (4,5).
Fig. 1

Major metabolic pathways for fentanyl (a), sufentanil, and alfentanil (b), known metabolic pathways for remifentanil and proposed pathway for carfentanil (c). Chemical structure of lofentanil (d)

Carfentanil was administered to humans to map brain μ-opioid receptors, to study addiction, and to measure pleasure responses at the receptor level (6, 7, 8). Until recently, these studies and veterinary use were the only applications for carfentanil. However, in 2002, carfentanil was reportedly administered to humans when Chechen rebels took more than 800 theatre-goers hostage at the Dubrovka Theatre in Moscow, Russia (9). After days of failed negotiations, an unknown aerosol was released into the theatre’s air conditioning system. Thirty minutes after release, police gained access to the theatre, neutralized the hostage takers, and removed hostages from the theatre. One hundred twenty-nine hostages died (10). It was later reported that an opioid derivative was used (10). In 2012, British researchers analyzed urine and clothing samples from British citizens who survived the incident, concluding that carfentanil and remifentanil were present in the incapacitating cocktail (11). Additionally, methyl-4-((propionyl)phenylamino)piperidine-4-carboxylate, the N-dealkylated product of carfentanil and remifentanil, was found in the urine sample. This report conceded that no study to date has definitively identified human carfentanil metabolites but hypothesized that norcarfentanil is a likely candidate based on known metabolites of fentanyl and other widely used congeners.

Fentanyl, sufentanil and alfentanil are primarily metabolized via CYP3A4 in the liver, generating metabolites N-dealkylated at the piperidine ring (Fig. 1a) (12,13). Whereas fentanyl is metabolized via N-dealkylation into the unique metabolite norfentanyl, alfentanil and sufentanil are metabolized to the same N-dealkylated product, making forensic distinction impossible when only this metabolite is identified (Fig. 1b). Morphine has active metabolites contributing to the therapeutic effect (14); fentanyl, sufentanil, and alfentanil’s metabolites are inactive in the opioid system (15). Remifentanil is the only family member of this class found to be ∼95% metabolized in the blood and tissues by non-CYP enzymes (16). This was attributed to an easily accessible ester group in remifentanil allowing rapid hydrolysis by circulating blood esterases, yielding remifentanil acid with 1/4000th of the parent’s analgesic potency (17). Methyl-4-((propionyl)phenylamino)piperidine-4-carboxylate is a minor metabolite from remifentanil N-dealkylation in the liver and is identical to N-dealkylated carfentanil (18). Therefore, there is no way to distinguish intake of remifentanil and carfentanil by targeting the N-dealkylated metabolite only (Fig. 1c).

To date, there are no controlled carfentanil administration studies to know how carfentanil is distributed and metabolized in the human body. We performed a comprehensive in vitro metabolism study for this fentanyl derivative with two major goals. First, as carfentanil produces its effects over hours, we assessed metabolic stability in human liver microsomes (HLM) to determine if carfentanil is toxic because of slow metabolism. Second, we aimed to identify, for the first time, carfentanil metabolites after in vitro human hepatocytes incubation. The results of our study might provide a basis for interpretation of analytical results in future opioid exposures and might confirm Riches et al.’s findings. In addition, it will enable future research on the pharmacological activity of carfentanil metabolites, which may contribute to its longer duration of action.


Chemicals and Reagents


Carfentanil is hazardous and should be handled carefully: Narcan (Naloxone HCl) intranasal injectors or alternative injectors should be readily available before working with this compound. It is highly recommended that multiple injectors be available per person.

Carfentanil citrate (95.8% pure determined by 1H and 13C NMR) was synthesized at Edgewood Chemical Biological Center (ECBC), as it was not commercially available. A 1 mg/mL solution, adjusted for the citrate salt formulation and purity, was prepared gravimetrically in LC-MS grade methanol purchased from Sigma-Aldrich (St. Louis, Missouri, USA). LC-MS grade water and formic acid were acquired from Fisher Scientific (Fair Lawn, New Jersey, USA). LC-MS grade acetonitrile was obtained from Sigma-Aldrich (St. Louis, Missouri, USA). Trypan Blue (0.4%) was used for measuring cell viability. NADPH regenerating system solutions A (part #451220) and B (part #451200) were purchased from Corning, Inc. (Corning, New York, USA).


The majority of experiments were performed according to our previous studies (19).

In silico Metabolite Predictions

MetaSite software (v.5.0.3; Molecular Discovery, Pinner, UK) predicted in silico carfentanil metabolites. Predictions were generated using the CYP P450 Liver Model with the following parameters: reactivity correction, 39 common biotransformations, and a minimum mass threshold of 100 Da for predicted metabolites. The algorithm considers both thermodynamic and kinetic factors. Predicted metabolites are assigned a probability score with 100% being the maximum likelihood of generation.

For comparison, ADMET Predictor (v.7.2.0001; Simulations Plus, Inc., California, USA) also predicted in silico carfentanil metabolites. This program uses a decision-learning-based algorithm, which differs from MetaSite’s quantitative structure-activity relationship (QSAR) algorithm. ADMET Predictor generated predictions using the modeled CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A4 enzymes. Physicochemical properties, e.g., logD, also were predicted using this software simulating conditions at pH = 4.0. This software does not calculate probability scores.


For the human liver microsome (HLM) incubations, carfentanil (5 μmol/L) was incubated for 1 h under constant shaking at 37°C with 1 mL of a solution containing HLM pooled from 50 different donors (1 mg protein/mL; Bioreclamation IVT, Columbia, MD, USA). HLM suspensions were prepared in duplicate by adding 100 μL 100 mmol/L potassium phosphate buffer, pH 7.4, 100 μL of 100 μg/mL carfentanil solution, 50 μL NADPH regenerating solution A, 10 μL NADPH regenerating solution B, and 50 μL HLM (20 mg/mL) to 690 μL purified water. One hundred-microliter samples were collected at 0, 3, 8, 13, 20, 30, 45, and 60 min and immediately mixed with an equal volume of ice-cold acetonitrile. Samples were stored at −80°C prior to analysis.

For the hepatocyte incubations, pooled cryopreserved human hepatocytes from ten different donors (Bioreclamation IVT, Columbia, MD, USA) were thawed at 37°C, transferred into pre-warmed InVitro Gro HT medium, centrifuged for 5 min at 50g, and the supernatant aspirated. As an additional washing step, the pellet was resuspended in InVitro Gro KHB buffer, 50 mL, centrifuged at 50g for 5 min again, and the supernatant aspirated. The pellet was finally resuspended in 2 mL InVitro Gro KHB buffer. For cell viability measurements, 10 μL suspended cells were dyed with Trypan blue. Viability was >77%. The final drug/cell incubation suspensions contained carfentanil drug solution (10 μmol/L) and cell suspension (500,000 cells/well). Incubation temperature was 37°C. Samples were collected at 0-, 0.5-, 1-, 2-, 4-, and 6-h incubation and were immediately mixed with 0.5 mL ice-cold acetonitrile. Positive diclofenac control samples were incubated, and formation of 4′-hydroxydiclofenac and diclofenac acyl glucuronide was evaluated to verify metabolic activity. Samples were stored at −80°C prior to analysis.

Sample Preparations

HLM samples were centrifuged at 4°C for 5 min at 15,000g to remove debris, and supernatants diluted 1:5 with mobile phase A, 0.1% formic acid in water. Ten microliters was injected into the MS system. Mobile phases A and B, 0.1% formic acid in acetonitrile (90:10, v/v), and 10 μg/L carfentanil in mobile phase (90:10, v/v) were also analyzed.

Human hepatocyte samples were thawed, vortexed, and centrifuged at 4°C for 10 min at 15,000g to remove cell debris, and supernatants diluted 1:5 with mobile phase A (0.1% formic acid in water) before injecting 10 μL into the MS system. Mobile phase (50:50, v/v) mixture and 50 μg/L carfentanil in mobile phase solution (90:10, v/v) were analyzed with hepatocyte samples.


HLM samples were analyzed on a 3200 QTRAP mass spectrometer (Sciex, Redwood City, CA, USA) and data acquired with Analyst v1.6. Chromatographic separation was performed by a Shimadzu Prominence HPLC system consisting of two LC-20AD XR pumps, a DGU-20A5R degasser, SIL-20AC XR auto-sampler, and CTO-20AC column oven (Shimadzu Corp., Columbia, MD, USA).

Hepatocyte samples were analyzed by liquid chromatography with a Shimadzu Prominence HPLC system consisting of two LC-20AD XR pumps, a DGU-20A5R degasser, SIL-20AC XR auto-sampler, and CTO-20AC column oven (Shimadzu Corp., Columbia, MD, USA) and a 5600+ TripleTOF mass spectrometer (Sciex, Redwood City, CA, USA). Data were acquired with Analyst v1.6.

LC-MS Analysis

For the HLM samples, chromatographic separation was performed on a Kinetex™ C18 column (100 × 2.1 mm, 2.6 μm, Phenomenex, Torrance, CA, USA). The HPLC mobile phases consisted of 0.1% formic acid in water (A) and acetonitrile (B), respectively. Gradient elution was as follows: 10% B from 0 to 0.5 min, ramping up to 95% B from 0.5 to 10 min, holding 95% B from 10 to 12.5 min, followed by reequilibration of 10% B at 12.5 min until completion at 15 min. LC flow was 0.5 mL/min. MS parameters were as follows: interface, positive electrospray ionization (ESI) mode; gas 1 and 2, nitrogen, 50 psi; curtain gas, nitrogen, 40 psi; source temperature 500°C; and ion spray voltage 5500 V. The transitions monitored were m/z 395.2/335.3 (collision energy CE 25 eV), m/z 395.2 /113.1 (CE 39 eV), and m/z 395.2/246.2 (CE 27 eV), all of which agree with previously published transitions for carfentanil (20). Declustering potential (DP), entrance potential (EP), and collision cell exit potential (CXP) were optimized to the following values: DP 51 V, EP 4.5 V, and CPX 4 V.

For the hepatocyte samples, chromatographic separation was performed similarly to the HLM protocol, using the same column and mobile phases. Gradient elution followed the following regimen: 10% B from 0 to 2 min, to 50% B from 2 to 20 min, then increasing to and holding at 95% B from 20 to 22 min, followed by reequilibration to 10% B at 22 min, until completion at 25 min. MS and MS/MS data were acquired via information-dependent acquisition (IDA) with dynamic background subtraction in positive ESI. For IDA, signals in the TOF-MS survey scan (range, m/z 100950; accumulation time, 0.1 s) exceeding 500 cps were selected for dependent product ion scans (range, m/z 60950; accumulation time, 0.075 s) excluding isotopes within 3 Da. Mass tolerance was 50 mDa. Ten candidates per cycle were allowed. MS source parameters were the following: gas 1 and 2, nitrogen, 50 psi; curtain gas, nitrogen, 45 psi; source temperature 500°C; ion spray voltage 5500 V; and DP 80 V. Collision energy for the product ion scans was 35 ± 15 eV. An automated calibration was performed after every third injection to maintain mass calibration.

Human Hepatocyte Data Processing

Metabolite evaluation was performed using MetabolitePilot v1.5 (Sciex, Redwood City, CA, USA). A list of theoretically possible biotransformations was created based on structures and analogous metabolism of other fentanyl-family compounds. Four different peak finding strategies were utilized: (1) search for predicted metabolites, (2) mass defect filtering with a mass defect of ±50 mDa of the parent mass and several predicted metabolites, (3) search for characteristic product ions and neutral losses (≥2), and (4) generic peak finding, which searches for intense signals not identified by the other three algorithms and suggests a number of “unexpected metabolites,” which we limited to 10. MS threshold intensity was 400 cps, MS/MS threshold intensity was 60 cps, and mass tolerance was set to 50 ppm. Minimum peak width was 2.5 s with a minimum chromatographic intensity of 1500 cps. Potential metabolites were evaluated based on three factors: mass measurement error <5 ppm; intense product ions had to either match those in parent compound fragmentation or have a mass shift corresponding to the metabolite biotransformation(s) with a mass error <10 ppm; and retention time had to be consistent with expected hydrophilic/lipophilic properties in the context of the entire metabolic profile. The in silico predicted logD was instrumental in helping explain retention times. Three controls were used in the hepatocyte experiment: (1) a mobile phase sample accounting for impurities in the solvents and the instrument, (2) a sample of carfentanil in mobile phase checking for impurities in the standard, and (3) a hepatocyte sample that was fortified with carfentanil, with hepatocytes precipitated before adding the standard accounting for matrix compounds and consumables.


Metabolic Stability

HLM half-life 7.8 ± 0.4 min was calculated from log-linear transformation of carfentanil depletion over 1 h (Fig. 2), which was translated to microsomal intrinsic clearance (CLint = 89.35 μL/min/mg protein) (21) and finally into predicted human hepatic clearance (CL = 16.2 mL/min/kg) (21).
Fig. 2

Parent compound depletion of carfentanil in human liver microsome (HLM) incubation. Performed in duplicate

However, in hepatocytes, carfentanil peak intensity did not decrease significantly over 6 h. The only slight changes in peak area probably need to be attributed to detector saturation. In contrast to HLM, hepatocytes contain intact cell membranes that must be passed prior to metabolism, which could be a possible reason for the observed differences in depletion.

Factors that may potentially contribute to the longer half-life in vivo could be carfentanil’s lipophilicity or volume of distribution. If this drug is either highly lipophilic and distributes into cellular membranes easily with slow liberation from this medium, or both, this could render less carfentanil available for hepatic extraction and metabolism. Regarding these factors, evidence exists to suggest that carfentanil is potentially highly bound by plasma proteins and has a larger volume of distribution than its less potent analogues. This is inferred from experimental data collected on more widely used fentanyl drugs: fentanyl, sufentanil, and lofentanil (Fig. 1d) that are 84.4, 92.5, and 93.6% bound, respectively (22). Based on structural similarity, we hypothesize that carfentanil should also be highly bound. With regard to volume of distribution, carfentanil caused renarcotization (23), where an agonist has a longer duration than its reversal agent. It can be inferred from carfentanil’s reputation for possessing a high risk of renarcotization that it distributes more widely than other opioids and is possibly largely sequestered by plasma proteins. These factors potentially contribute to longer exposure and duration of effects in vivo.

Carfentanil MS/MS Spectrum

Fragmentation of the parent compound is largely analogous to the collision-induced dissociation (CID)-MS of remifentanil (24), as we verified in Fig. 3. Fragment m/z 363.2075 is generated by methanol loss from the methyl ester functional group, the base peak at m/z 335.2126 by loss of the methyl ester group. Fragment m/z 279.1862 is generated by loss of both the carbomethoxy group and propenone. Fragment m/z 246.1494 results from cleavage at the central bond between carboxamide nitrogen and piperidine leading to a loss of N-phenyl-propionamide, a fragmentation common for fentanyl and derivatives (24).
Fig. 3

MS/MS of carfentanil and identified fragment structures. Protons are not indicated on metabolite fragment formulae

A 2.016 Da loss, characteristic of a carbomethoxy group loss, can be followed by a double-bond formation on the piperidine, likely through tertiary carbo-cation formation and a hydrogen shift mechanism. For instance, m/z 202.1226 generated from an inhomogeneous piperidine ring cleavage results in a protonated N-phenyl-butadienal-propionamide fragment. This fragment can undergo further propenone loss yielding m/z 146.0966. Instead of tertiary carbo-cation formation and hydrogen shifting, both C-N bonds are broken during the inhomogeneous piperidine ring cleavage and an additional double bond is formed yielding m/z 158.0964. Additional evidence for this characteristic 2.016 Da loss is from the m/z 186.1277 fragment, where the central N-piperidyl bond is cleaved and the charge resides on the phenyl-ethyl-piperidine side. Conversely to the fragment at m/z 202.1226, the charge remaining on the phenethyl side results in the methyl phenethylamine fragment at m/z 134.0969. Likewise, the m/z 113.0606 fragment has two double bonds initiated by the C‐N bond breakages, and m/z 105.0708 corresponds to the phenyl ethyl fragment indicating double bond formation, but at this specific location. Later, analysis using d5-carfentanil labeled at the N-phenyl ring helped confirm our fragment structures, as shifts of 5 Da were observed in all of the parent fragments containing the N-phenyl group: m/z 146.0966, m/z 158.0964, m/z 202.1226, m/z 279.1862, and m/z 335.2126. This helped correct for discrepancies in predicted versus elucidated fragments within MetabolitePilot.

Metabolite Profiling in Hepatocytes

Metabolite identification includes two steps: First, potential metabolites are detected by data mining the HRMS raw data with software help applying several search algorithms like predicted metabolites, mass defect filtering, and search for common product ions and neutral losses. Second, metabolite structures are elucidated based on accurate masses for the protonated molecule and product ions and mass shifts between parent and metabolite. Chemical structures of the identified metabolites and a potential pathway are shown in Fig. 4. Retention time, diagnostic fragment ions, precursor ion m/z, and MS areas for all time points for identified metabolites are shown in Table I. Metabolites were labeled M1 to M12 based on increasing elution time.
Fig. 4

Proposed metabolic pathway for carfentanil in human hepatocytes. Metabolite structures derived from MS/MS and chromatographic analysis

Table I

Detailed Information on All Identified Metabolic Products and Parent Compound

Peak ID


Elemental composition

Precursor ion (m/z)

Diagnostic fragment ions

Mass error (ppm)

RT (min)

Relative MS area, 0.5 h

Relative MS area, 1 h

Relative MS area, 2 h

Relative MS area, 4 h

Relative MS area, 6 h


N-Dealkylation + ester hydrolysis



110, 113, 175, 217, 235, 245









N-Dealklylation of piperidine ring



142, 150, 175, 231, 235, 259









N-Dealkylation of piperidine ring + hydroxylation of propanoic group



126, 173, 247, 251, 275









Ester hydrolysis + hydroxylation of piperidine ring



132, 162, 188, 232, 262, 337, 365, 379









Hydroxylation of propanoic group



186, 218, 351









Hydroxylation of phenethyl group + glucuronidation



121, 326, 351, 393, 411, 438









Hydroxylation of phenylethyl structure



121, 262, 295, 351, 379









Hydroxylation of piperidine ring



162, 232, 262, 295, 351, 393









Ketone formation on phenethyl linker



120, 148, 260, 293, 353, 349, 377









N-oxidation of piperidine N + hydroxylation of phenethyl group



166, 172, 274, 278, 303












105, 113, 134, 146, 158, 186, 202, 214, 246, 279, 335, 363









N-oxidation of 4′ nitrogen



105, 154, 247, 262, 274, 290, 303









N-oxidation of piperidine N



105, 148, 230, 262, 274, 275, 303, 379








Areas are reported as relative to maximum area for each metabolite

N-Dealkylated Metabolites

N-Dealkylated metabolites (Fig. 5) were expected based on fentanyl and other analogue metabolism studies. Indeed, carfentanil demonstrated similar metabolic products—in total, three metabolites (M1, M2, and M3) were generated by N-dealkylation.
Fig. 5

MS/MS for identified metabolites M1, M2, and M3. Fragment structures used for metabolite identification are indicated in the figure. Protons are not indicated on metabolite fragment formulae

M2 (m/z 291.1711, retention time RT 6.89 min) is the N-dealkylated metabolite, or norcarfentanil, which has completely lost the phenethyl substructure indicated by the absence of m/z 105.0704, m/z 134.0964, and m/z 186.1277. The remaining parts of the molecule are unchanged as fragments corresponding to portions of the piperidine ring and 4′ bound structures (m/z 146.0969, m/z 158.0966, and m/z 202.1226) remained in the MS/MS spectrum. M2 was the second most abundant metabolite overall at each time point. However, it should be noted that MS peak areas can be affected by ionization efficiencies and matrix effects. Based on the assumption that phase I metabolites show similar peak areas if present in the same concentrations, we estimated and compared their relative abundances. Carrying an additional acidic functional group, phase II metabolites like glucuronides and sulfates usually show lower ionization efficiencies in positive ESI mode.

M2 underwent further biotransformations yielding M1 (m/z 277.1547, RT 4.05 min) and M3 (m/z 307.1655, RT 8.20 min). Ester hydrolysis generated M1 as indicated by m/z 175.1226 corresponding to an unmodified piperidine ring and 4′ phenyl ring and m/z 217.1328 verifying that the N-propanoic group was unchanged. The fragment at m/z 113.0603 represents the hydrolyzed ester group and piperidine ring less its nitrogen. Interestingly, ester hydrolysis often is the major metabolic pathway, as esters are “built in” to make “soft” drugs more labile and easily metabolized. Remifentanil, for example, contains an additional ester moiety that when hydrolyzed results in rapid hepatic-independent clearance and subsequent offset of effects (t 1/2 = 3–10 min). However, carfentanil’s ester moiety might be more sterically hindered than remifentanil’s as it is protected by the N-phenyl ring.

M3 (m/z 307.1655, RT 8.20) was generated from M2 by monohydroxylation at the N-propanoic group. Much like M1, m/z 146.0966, m/z 158.0813, and m/z 173.1075 indicate unmodified piperidine and phenyl rings, whereas fragment m/z 275.1395 indicates an intact carbomethoxy group, leaving the propanoic group the only available site for hydroxylation. This hypothesis is further supported by m/z 247.1442, which corresponds to M3 less its carbomethoxy group.

Monohydroxylated metabolites

In total, three metabolites (M5, M7, and M8) (Supplementary Fig. 1) were generated by monohydroxylation. Hydroxylation occurred at three carfentanil sites, most dominantly at the piperidine ring, and to a lesser extent at the N-propanoic group and the phenethyl substructure.

M8 (m/z 411.2292, RT 11.20 min) is monohydroxylated at the piperidine ring, indicated by m/z 105.0706, which corresponds to an unmodified phenethyl group, and m/z 162.0920, corresponding to a hydroxylated parental fragment at m/z 134.0969 with an additional carbon atom from the piperidine ring. Finally, m/z 113.0607 indicates an unmodified carbomethoxy group, while m/z 202.1229 rules out hydroxylation of the N-propanoic group. Overall, M8 was the most abundant metabolite detected at each time point.

M5 (m/z 411.2285, RT 9.50 min) is hydroxylated at the N-propanoic group. This position was indicated by preservation of the phenethyl group (m/z 105.0704), piperidine ring (m/z 186.1275), N-phenyl ring (m/z 146.0963 and 158.0963), and carbomethoxy (m/z 113.0602) group. Absence of m/z 202.1229 and presence of m/z 218.1171, a mass shift consistent with hydroxylation, indicated hydroxylation at the N-propanoic group.

M7 (m/z 411.2279, RT 10.42) is hydroxylated at the phenethyl group and a potential precursor to M6. The hydroxylation site is concluded based on fragment m/z 121.0650, which represents the phenethyl precursor fragment m/z 105.0708 plus the mass of an oxygen atom.

Metabolites Generated by N-Oxidation

In total, three metabolites were generated through N-oxidation (M11, M12) or combination of N-oxidation and hydroxylation (M10) (Supplementary Fig. 2). These metabolites all eluted late, either slightly before or even after the parent, as is often seen for N-oxides (25,26). M10 (m/z 427.2229, RT 12.23) results from N-oxidation of M7, the phenethyl hydroxylated metabolite. The fragment at m/z 274.1434 indicates that the piperidine carbons, the carbomethoxy, and the N-propanoic groups remained unchanged. Fragment m/z 166.0854 indicates di-hydroxylation of the phenethyl region including the piperidine nitrogen. Di-hydroxylation of the aliphatic carbons would yield m/z 121.1559 and m/z 137.1553, indicating single and double hydroxylation of m/z 105.0708, respectively; however, these fragments were not found. N-oxidation at the piperidine nitrogen explains the m/z 166.0854 fragment and is further supported by M10’s late elution time.

M11 (m/z 411.2287, RT 13.48 min) is generated by N-oxidation of the 4′-position nitrogen. As indicated by parent fragments m/z 105.0703, m/z 113.0598, and m/z 132.0805, the phenethyl group, the carbomethoxy group, and the piperidine ring were unchanged. The fragment at m/z 154.0858 indicated N-oxidation of the 4′-position, further supported by m/z 274.1433 and m/z 290.1392; m/z 274.1433 represents the carbomethoxy, N-phenyl, and N-propanoic groups along with all of the piperidine carbons. Further, N-oxidation of this group led to m/z 290.1392.

M12 (m/z 411.2287, RT 13.68 min) is the N-oxide metabolic product of the piperidine nitrogen, indicated by fragments at m/z 230.1174 and m/z 262.1436. These are both N-oxide products of parent fragments m/z 214.1225 and 246.1494, both containing only the piperidine nitrogen. The phenethyl ring is determined to be unmodified (m/z 105.0703). Additionally, signature fragments at m/z 146.0966 and m/z 158.0962 are still prominent, indicating that oxidation did not occur at the piperidine carbons or any of the 4′-bound carbon positions.

Other Metabolites

Three metabolites were generated by carbonylation, or ketone formation, ester hydrolysis, and glucuronidation (Supplementary Fig. 3). M9 (m/z 409.2135, RT 11.80) was the only ketone product identified. The fragment at m/z 120.0812 indicated a double-bound oxygen on the phenethyl group, and m/z 148.0759, a double-bound oxygen added to the parent fragment (m/z 134.0969) that contained phenethyl group, piperidine nitrogen, and one carbon atom of the piperidine ring. Parent fragments m/z 113.0604, m/z 146.0967, and m/z 158.0968 suggest an intact, unmodified carbomethoxy group, N-phenyl, and piperidine ring. The most likely place for the keto group would be the ethyl-linker region between the piperidine nitrogen and phenyl ring.

M4 (m/z 397.2126, RT 9.50 min) is produced by ester hydrolysis of M8, based on the mass shift of 14.0166, corresponding to methyl group loss. The fragment at m/z 162.0909 is identical to the M8 signature fragment indicating piperidine ring monohydroxylation. The fragment at m/z 379.2004 indicates ester hydrolysis and includes the piperidine ring hydroxyl group. M6 (m/z 587.2607, RT 9.74 min) is generated by hydroxylation of the phenethyl group followed by glucuronidation and is the only phase II metabolite identified. Hydroxylation position and subsequent glucuronidation on the phenethyl group were suggested by parental fragments m/z 113.0604, m/z 146.0960, m/z 158.0966, and m/z 202.1221 indicating an intact carbomethoxy group, N-phenyl ring, and N-propanoic group. Most importantly, m/z 105.0708 was absent and m/z 121.0651 present, indicating phenethyl group hydroxylation. Glucuronic acid is bound to the phenethyl structure (m/z 326.1237).


Metabolite Intensities

Signal intensities of the 12 metabolites were tracked at 1, 4, and 6 h. The most intense peaks were M8 (piperidine ring hydroxylation) and M2 (N-dealkylation forming norcarfentanil), followed by M9 (ketone formation at phenethyl linker), M5 (N-propanoic hydroxylation), M7 (phenethyl ring hydroxylation), M12 (piperidine N-oxidation), and M11 (4-position nitrogen N-oxidation). The only phase II metabolite identified was a glucuronide conjugate of hydroxylated carfentanil (M6) with low intensity. As there was a constant high amount of parent compound available over time, signal intensities of second-generation metabolites increased, but without decreasing first-generation metabolite signal intensities. Overall, the metabolic profiles at all-time points look similar.

Support for Riches et al.?

M2, or norcarfentanil, showed the second most abundant signal in our human hepatocytes profile, ranking second after M8. These results confirm Riches et al.’s hypothesis that norcarfentanil is a carfentanil metabolite; however, norcarfentanil is a metabolite of both carfentanil and remifentanil. Riches et al. suggested use of both compounds based on positive results of clothing samples and did not attribute the presence of norcarfentanil to either. Future forensic analyses in which opioid exposure is suspected require identification of more specific metabolites. Our data suggest the piperidine ring-hydroxylated carfentanil metabolite (M8), containing the entire parent compound including the ester group and showing the most intense peak in our samples. However, verification in post exposure urine or blood is needed.

Implications for Carfentanil Toxicity

HLM half-life was 7.8 min, which indicates that toxicity would not be the result of inadequate enzymatic capacity to metabolize carfentanil. However, substrate depletion in the human hepatocyte samples was slow. According to common classifications (27), carfentanil is categorized as a “high clearance” compound. This is an interesting result as all observations about carfentanil effects point towards a long in vivo half-life. The HLM experiment definitely excludes metabolic clearance as the time-limiting factor.

Based on its calculated logP value of 3.7 (28,29) to 4.01 (30,31), indicating high lipophilicity, and the well-studied behavior of other fentanyl compounds (32), we assume that carfentanil is rapidly distributed to and stored in fatty tissues. This could prolong low-level reabsorption into the systemic circulation and prolong toxicity when combined with carfentanil’s high receptor affinity. Opioids have a central nervous system depressant effect that manifests as decreased respiration, which can lead to respiratory failure, apnea, and death (33). Except for the N-dealkylated metabolites that lost the entire phenethyl structure, many metabolites were only slight modifications of carfentanil. Metabolites with a single hydroxylation could maintain activity at the μ-opioid receptors, potentially prolonging the effect. For example, β-hydroxyfentanyl, though not a fentanyl metabolite, is a potent fentanyl analogue distributed on the black market in the 1980s (34). M7, hydroxylated at the phenethyl group (that includes the mentioned β-position), could be an active carfentanil metabolite. On the other hand, M2 and M3 are similar to fentanyl metabolites that showed little to no activity at μ-opioid receptors in vitro. Norfentanyl, analogous to carfentanil M2, and hydroxyl-norfentanyl, analogous to M3, are expected to be inactive (35), but additional research is needed for definitive conclusions.


HLM metabolic stability indicated that carfentanil is readily metabolized by CYP enzymes. We propose that slower hepatocyte metabolism was likely due to poor accessibility of the compound to these enzymes for reasons that need to be addressed in future studies. Despite slow parent depletion in hepatocytes, we identified 12 carfentanil metabolites. Phase I biotransformations dominated metabolite formation and included monohydroxylation, N-dealkylation, N-oxidation, ester hydrolysis, and carbonylation. The two most abundant metabolites were M8 and M2. M2 is the N-dealkylated carfentanil, more commonly known as norcarfentanil. Only one phase II metabolite (M6) was identified, and the glucuronide of M7. M7 was monohydroxylated at an undetermined position on the phenethyl group.

M2, or norcarfentanil, is a good indicator of exposure to carfentanil being one of the two most intense peaks during hepatocyte incubation. However, M2 is not exclusive to carfentanil, and carfentanil administration must be confirmed by looking for other more unique metabolites such as M8. This metabolite had even higher abundance than M2 and contains the entire carfentanil structure, lending itself to forensic identification if found. This study has opened the door to new questions about carfentanil and how it exhibits its toxicity. First and foremost, in vivo studies need to be conducted to confirm the metabolites identified in this study. Plasma protein binding assays can assess carfentanil binding, and in silico models might predict plasma drug concentrations, exposure duration after administration, and toxicity by physiologically based pharmacokinetic studies (PBPK). This might explain the slower than anticipated carfentanil depletion observed in hepatocytes. Finally, performing a similar study in liver spheroids, more representative of a whole liver, could compare performance of this newer technology in assessing carfentanil clearance and metabolite formation.



The authors would like to acknowledge Drs. Alison Director-Myska, Eric Moore, Neil Jensen, and Joseph Corriveau for their unwavering and continued support of this research effort. We would also like to acknowledge Simulations Plus and Molecular Discovery for the use of their software for the in silico metabolite predictions. Finally, we would like to acknowledge Tim Moeller of BioreclamationIVT for his help with the primary hepatocyte culturing procedures and technical support.

This work was funded by the Defense Threat Reduction Agency (DTRA) under project number CB3281.

Supplementary material

12248_2016_9963_MOESM1_ESM.pdf (663 kb)
ESM 1 (PDF 662 kb)


  1. 1.
    Cole A, Mutlow A, Isaza R, et al. Pharmacokinetics and pharmacodynamics of carfentanil and naltrexone in female common eland (Taurotragus oryx). J Zoo Wildl Med. 2006;37(3):318–26.CrossRefPubMedGoogle Scholar
  2. 2.
    Van Bever WF, Niemegeers CJ, Schellekens KH, Janssen PA. N-4-Substituted 1-(2-arylethyl)-4-piperidinyl-N-phenylpropanamides, a novel series of extremely potent analgesics with unusually high safety margin. Arzneimittelforschung. 1976;26(8):1548–51.PubMedGoogle Scholar
  3. 3.
    Subramanian G, Paterlini MG, Portoghese PS, Ferguson DM. Molecular docking reveals a novel binding site model for fentanyl at the mu-opioid receptor. J Med Chem. 2000;43(3):381–91.CrossRefPubMedGoogle Scholar
  4. 4.
    Ling GS, Spiegel K, Nishimura SL, Pasternak GW. Dissociation of morphine’s analgesic and respiratory depressant actions. Eur J Pharmacol. 1983;86(3–4):487–8.CrossRefPubMedGoogle Scholar
  5. 5.
    Shook JE, Watkins WD, Camporesi EM. Differential roles of opioid receptors in respiration, respiratory disease, and opiate-induced respiratory depression. Am Rev Respir Dis. 1990;142(4):895–909.CrossRefPubMedGoogle Scholar
  6. 6.
    Newman A, Channing M, Finn R, et al. Ligands for imaging opioid receptors in conscious humans by positron emission tomography (PET). NIDA Res Monogr. 1988;90:117–21.PubMedGoogle Scholar
  7. 7.
    Endres CJ, Bencherif B, Hilton J, Madar I, Frost JJ. Quantification of brain mu-opioid receptors with [11C]carfentanil: reference-tissue methods. Nucl Med Biol. 2003;30(2):177–86.CrossRefPubMedGoogle Scholar
  8. 8.
    Villemagne PS, Dannals RF, Ravert HT, Frost JJ. PET imaging of human cardiac opioid receptors. Eur J Nucl Med Mol Imaging. 2002;29(10):1385–8.CrossRefPubMedGoogle Scholar
  9. 9.
    Krechetnkiov A. Moscow theatre siege: questions remain unanswered. British Broadcasting Corporation. 24 October 2012. Web. 13 May 2015.Google Scholar
  10. 10.
    Glasser SB, Baker P. Russia confirms suspicions about gas used in raid; Potent Anesthetic Pumped Into Theater; 2 More Hostages Die From Drug’s Effects. The Washington Post 31 October 2002: A15. Print.Google Scholar
  11. 11.
    Riches JR, Read RW, Black RM, Cooper NJ, Timperley CM. Analysis of clothing and urine from Moscow theatre siege casualties reveals carfentanil and remifentanil use. J Anal Toxicol. 2012;36(9):647–56.CrossRefPubMedGoogle Scholar
  12. 12.
    Feierman DE, Lasker JM. Metabolism of fentanyl, a synthetic opioid analgesic, by human liver microsomes. Role of CYP3A4. Drug Metab Dispos. 1996;24(9):932–9.PubMedGoogle Scholar
  13. 13.
    Guitton J, Buronfosse T, Désage M, Lepape A, Brazier JL, Beaune P. Possible involvement of multiple cytochrome P450S in fentanyl and sufentanil metabolism as opposed to alfentanil. Biochem Pharmacol. 1997;53(11):1613–9.CrossRefPubMedGoogle Scholar
  14. 14.
    Hanks GW, Hoskin PJ, Aherne GW, Turner P, Poulain P. Explanation for potency of repeated oral doses of morphine? Lancet. 1987;2(8561):723–5.CrossRefPubMedGoogle Scholar
  15. 15.
    Schneider E, Brune K. Opioid activity and distribution of fentanyl metabolites. Naunyn-Schmiedeberg’s Arch Pharmacol. 1986;334(3):267–74.CrossRefGoogle Scholar
  16. 16.
    ULTIVA® (remifentanil hydrochloride) [package insert]. Rockford, IL, USA; Mylan Institutional LLC; Published March 2015. Accessed 1 June 2015.
  17. 17.
    Bürkle H, Dunbar S, Van Aken H. Remifentanil: a novel, short-acting, mu-opioid. Anesth Analg. 1996;83(3):646–51.CrossRefPubMedGoogle Scholar
  18. 18.
    Van Nimmen NF, Poels KL, Veulemans HA. Highly sensitive gas chromatographic-mass spectrometric screening method for the determination of picogram levels of fentanyl, sufentanil and alfentanil and their major metabolites in urine of opioid exposed workers. J Chromatogr B Analyt Technol Biomed Life Sci. 2004;804(2):375–87.CrossRefPubMedGoogle Scholar
  19. 19.
    Wohlfarth A, Castaneto MS, Zhu M, et al. Pentylindole/Pentylindazole synthetic cannabinoids and their 5-Fluoro analogs produce different primary metabolites: metabolite profiling for AB-PINACA and 5F-AB-PINACA. AAPS J. 2015;17(3):660–77.CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    Wang L, Bernert JT. Analysis of 13 fentanils, including sufentanil and carfentanil, in human urine by liquid chromatography-atmospheric-pressure ionization-tandem mass spectrometry. J Anal Toxicol. 2006;30(5):335–41.CrossRefPubMedGoogle Scholar
  21. 21.
    Lavé T, Dupin S, Schmitt C, et al. The use of human hepatocytes to select compounds based on their expected hepatic extraction ratios in humans. Pharm Res. 1997;14(2):152–5.CrossRefPubMedGoogle Scholar
  22. 22.
    Meuldermans WE, Hurkmans RM, Heykants JJ. Plasma protein binding and distribution of fentanyl, sufentanil, alfentanil and lofentanil in blood. Arch Int Pharmacodyn Ther. 1982;257(1):4–19.PubMedGoogle Scholar
  23. 23.
    Allen JL. Renarcotization following Carfentanil immobilization of nondomestic ungulates. J Zoo Wildl Med. 1989;20(4):423–6.Google Scholar
  24. 24.
    Thevis M, Geyer H, Bahr D, Schänzer W. Identification of fentanyl, alfentanil, sufentanil, remifentanil and their major metabolites in human urine by liquid chromatography/tandem mass spectrometry for doping control purposes. Eur J Mass Spectrom (Chichester, Eng). 2005;11(4):419–27.CrossRefGoogle Scholar
  25. 25.
    Cashman JR, Park SB, Yang ZC, Wrighton SA, Jacob P, Benowitz NL. Metabolism of nicotine by human liver microsomes: stereoselective formation of trans-nicotine N’-oxide. Chem Res Toxicol. 1992;5(5):639–46.CrossRefPubMedGoogle Scholar
  26. 26.
    Pirmohamed M, Williams D, Madden S, Templeton E, Park BK. Metabolism and bioactivation of clozapine by human liver in vitro. J Pharmacol Exp Ther. 1995;272(3):984–90.PubMedGoogle Scholar
  27. 27.
    Mcnaney CA, Drexler DM, Hnatyshyn SY, et al. An automated liquid chromatography-mass spectrometry process to determine metabolic stability half-life and intrinsic clearance of drug candidates by substrate depletion. Assay Drug Dev Technol. 2008;6(1):121–9.CrossRefPubMedGoogle Scholar
  28. 28.
    Tetko IV, Gasteiger J, Todeschini R, Mauri A, Livingstone D, Ertl P, et al. Virtual computational chemistry laboratory - design and description. J Comput Aided Mol Des. 2005;19:453–63. article.CrossRefPubMedGoogle Scholar
  29. 29.
    VCCLAB, Virtual Computational Chemistry Laboratory,; 2005.
  30. 30.
    Hansch C, Leo A. Exploring QSAR: hydrophobic, electronic, and steric constants. Vol. 2. ACS Publications. 1995.Google Scholar
  31. 31.
    ADMET Predictor v7.2.0001. Simulations Plus, Inc. 2015.Google Scholar
  32. 32.
    Hug CC, Murphy MR. Tissue redistribution of fentanyl and termination of its effects in rats. Anesthesiology. 1981;55(4):369–75.CrossRefPubMedGoogle Scholar
  33. 33.
    Dart RC. Medical toxicology 3rd Edition. Phildelphia: Lippincott Williams and Wilkins; 2004. p. 762.Google Scholar
  34. 34.
    Henderson GL. Designer drugs: past history and future prospects. J Forensic Sci. 1988;33(2):569–75.CrossRefPubMedGoogle Scholar
  35. 35.
    Mahlke NS, Ziesenitz V, Mikus G, Skopp G. Quantitative low-volume assay for simultaneous determination of fentanyl, norfentanyl, and minor metabolites in human plasma and urine by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Int J Legal Med. 2014;128(5):771–8.CrossRefPubMedGoogle Scholar

Copyright information

© American Association of Pharmaceutical Scientists 2016

Authors and Affiliations

  • Michael G. Feasel
    • 1
  • Ariane Wohlfarth
    • 2
    • 3
    • 4
  • John M. Nilles
    • 5
  • Shaokun Pang
    • 6
  • Robert L. Kristovich
    • 1
  • Marilyn A. Huestis
    • 2
  1. 1.Edgewood Chemical Biological Center, Research Development and Engineering Command, U.S. ArmyGunpowderUSA
  2. 2.Chemistry and Drug Metabolism, Intramural Research Program, National Institute on Drug Abuse, National Institutes of HealthBaltimoreUSA
  3. 3.Department of Forensic Genetics and Forensic ToxicologyNational Board of Forensic MedicineLinköpingSweden
  4. 4.Division of Drug Research, Department of Medical Health SciencesLinköping UniversityLinköpingSweden
  5. 5.Excet, Inc.SpringfieldUSA
  6. 6.SciexFoster CityUSA

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