Norcarfentanil: carfentanil misuse or remifentanil treatment?

Toxivial A^® extraction devices were purchased from Interchim (Montluçon, France). N,O-Bis(trimethylsilyl)trifluoroacetamide with trimethylchlorosilane, trimethylsilyl chloride (BSTFA 1% TMCS), β-glucuronidase from Helix pomatia and ammonium formate were purchased from Sigma-Aldrich (St Quentin-Fallavier, France). Methanol (MeOH) (LiChrosolv), acetonitrile (CH₃CN) (Pestipure), ethyl acetate (HiPerSolv) and ammonia solution 25% were purchased from Merck (Darmstadt, Germany). Formic acid (analytical grade) was provided by Prolabo (Paris, France). Sulfosalicylic acid was provided by Carlo Erba (Val de Reuil, France). Ultrapure water (resistivity ≥ 18.0 MΩ cm) was produced using a Milli-Q Plus (Millipore, Molsheim, France). Carfentanil (5 mg), norcarfentanil (1 mg), 1-hydroxyl-fentanyl (2.5 mg), acetyl fentanyl (10 mg), acrylfentanyl (5 mg), benzyl carfentanil (5 mg), desomorphine (1 g/L in acetonitrile), furanyl fentanyl (1 g/L in acetonitrile), normethylfentanyl (1 mg), ocfentanil (1 mg), ohmefentanyl (1 mg), remifentanil (100 mg/L in methanol), thienyl fentanyl (1 mg), U-47700 (1 mg), valeryl fentanyl (1 mg) and fentanyl-d5 (100 mg/L in methanol) were purchased from LGC Standards (Molsheim, France).

Blood specimens were collected into 5-mL propylene tubes containing sodium heparin as anticoagulant, and urine specimens were collected into 5-mL propylene dry tubes.

All plasma and urine samples were collected for routine toxicological analyses.

Immunoassays of the urine samples were performed using a Vista analyzer (Dimension Vista system, Siemens Healthcare Diagnostics Inc., Siemens). Syva^® EMIT^® II Plus AMPH Flex^®, Syva^® EMIT^® II Plus EXTC Flex^®, Syva^® EMIT^® II Plus COC Flex^®, Syva^® EMIT^® II Plus OPI Flex^®, Syva^® EMIT^® II Plus THC Flex^® (Flex Reagent Cartridge; Siemens Diagnostics) were used to search for amphetamines, 3,4-methylenedioxymethamphetamine (MDMA), cocaine, opiates and 11-nor-9-carboxy-Δ9-tetrahydrocannabinol (THCCOOH), respectively. Buprenorphine in urine samples was analyzed using immunochromatography (TOX/See™, BIO-RAD). Plasma and urine were tested for benzodiazepines and ethanol using the Syva^® EMIT^® tox™ kit and the ETOH kit (Flex Reagent Cartridge; Siemens Diagnostics), respectively.

Unknown drug screenings were performed using gas chromatography–mass spectrometry (GC–MS) and liquid chromatography coupled with diode array detection (LC-DAD) [24]. The drugs were compared with both in-house (constructed from reference standards) and commercial libraries [25‐27].

Synthetic opioids were identified in biological samples (plasma and urine) using liquid chromatography–tandem mass spectrometry (LC–MS/MS) methods.

The LC system consisted of a Dionex Ultimate 3000 RS quaternary pump and a Dionex Ultimate 3000 quaternary pump, equipped with a Dionex Ultimate 3000 RS autosampler and a Dionex Ultimate 3000 RS column compartment (Thermo Fisher Scientific, Germering, Germany). Online sample clean-up was performed on an Oasis HLB column (2.1 mm × 20 mm; 25 µm, Waters, Milford, MA, USA). Chromatographic separation was performed on a Waters analytical column, XSelect^® HSS T3 (100 × 2.1 mm; 2.5 μm) maintained at 60 °C. The operating procedure for the HPLC-integrated online sample clean-up consisted of two steps: the sample was first injected into the system and transferred onto the Oasis HLB column, where the analytes were adsorbed. Potentially interfering matrix compounds (mainly salts and protein residues) were washed directly into waste by mobile phase A (water + 0.2% ammonium hydroxide) delivered at a flow rate of 2.0 mL/min. After this first step, the six-port valve was switched. To obtain a good separation between the compounds, the extract was then eluted in back-flush mode and transferred to the analytical column. The elution gradient profile used was performed at 0.3 mL/min and had following conditions using water + 20 mM ammonium formate adjusted to pH 2.8 with formic acid (mobile phase C) and CH₃CN + 0.2% formic acid (mobile phase D): 0–1 min: 20% D; 1–4 min: 20–70% D; 4–4.2 min: 70–95% D; 4.2–6.4 min: 95% D; 6.4–6.5 min: 95–20% D; 6.5–7.6 min: 20% D. During this chromatographic step, the valve was switched back on its original configuration allowing a mobile phase B (MeOH + water + 0.2% formic acid) to wash the Oasis HLB purification column, before being equilibrated using mobile phase A for the next run. A run was completed within 7.6 min.

MS/MS analyses were performed on an API 4000 mass spectrometer (Sciex, Toronto, Ontario, Canada) equipped with an electrospray probe, on a Turbo V^® ion source.

The mass spectrometer was operated under the following conditions: positive electrospray ionization (ESI) voltage: 5500 V; nebulization gas flow rate: 45 psi; turbo heater gas flow rate: 60 psi; ion source temperature: 500 °C. The analyses were performed in selected reaction monitoring (SRM) mode. The monitored transitions and collision energies are presented in Table S1 (Supplementary data).

The calibration curves were built using a 200-µL drug-free plasma sample spiked with standard solutions. Calibration curves were generated with calibrators of 0.01, 0.05, 0.1, 0.5, 1, 5, 10 and 20 ng/mL. The regression equations and correlation coefficients were obtained from the peak area ratios (analyte/internal standard) plotted against the analyte concentrations. Quality control (QC) samples were prepared in the same manner at high (8 ng/mL), medium (2 ng/mL) and low concentrations (0.5 ng/mL).

Validation of the LC–MS/MS assay was performed in compliance with both the French Analytical Toxicology Society (SFTA) and international recommendations for the validation of new analytical methods [28, 29]. Six independent calibrations were conducted on different days, using different drug-free matrix samples. The selectivity was tested by the analysis of 10 blank samples of each matrix. The limit of detection (LOD) was defined as the lowest concentration with retention time within ±0.2 min from the average of all calibrator concentrations and a signal-to-noise ratio of at least three for all selected ion transitions. The lower limit of quantitation (LLOQ) was the lowest concentration that could be quantified with acceptable imprecision [coefficient of variation (CV) % ≤ 20%] and acceptable accuracy (within ±20% of the theoretical concentration). Within-day precision and accuracy were calculated from six repeated analyses of spiked plasma or urine samples (at three levels) during one working day. Between-day precision and accuracy were calculated from six analyses of spiked plasma or urine samples (at three levels), one analysis being performed a day. The precision was expressed as the CV (in  %), and the accuracy as the percentage of deviation between nominal and measured concentrations. Two dilution factors (10- and 100-fold) were tested by spiking blank plasma or urine (n = 6) with all analytes at a concentration above the highest calibrator (20 ng/mL). Potential endogenous interferences were determined by the analysis of ten different blank plasma and urine specimens. For the assessment of exogenous interferences, several opioids and metabolites were tested. Indeed, exogenous interferences could be due to several reasons (close chemical structures, same ion transitions, coelutions responsible for ion suppression and/or enhancement, etc.). However, not all drugs could be tested, and we decided to test drugs from the same chemical family (opioid drugs). The following compounds were added to blank specimens at 50 ng/mL: methadone, 2-ethylene-1,5-dimethyl-3,3-diphenylpyrrolidine, dextropropoxyphene, oxycodone, pethidine, tramadol, O-desmethyltramadol, N-desmethyltramadol, whereas buprenorphine, norbuprenorphine, buprenorphine glucuronide, norbuprenorphine glucuronide, fentanyl, norfentanyl, sufentanil, naloxone, and alfentanil were tested at 5 ng/mL.

Qualitative matrix effects were studied by analyzing ion suppression and the enhancement phenomenon. Extracted double-blank plasma (n = 6) and urine (n = 6) samples were injected into the LC system, while a methanolic solution that contained all compounds and fentanyl-d5 (each at 20 ng/mL) was continuously post-column infused in the ionization source through a tee [30].

Linear regression with 1/× weighting showed the standard curves for all compounds to be linear from their LLOQ to 20 ng/mL, with r² > 0.992. Intra- and inter-day precision and accuracy for three levels of QC are shown for plasma and urine, respectively, in Tables S2 and S3 (Supplementary data). Variation coefficients for precision and accuracy are under 20% and 15% for LLOQ and other values, respectively. LOD and LLOQ are listed in Table S4 (Supplementary data).

No additional peaks were observed due to endogenous substances that could have interfered with the detection of the compounds of interest. Opioid and metabolite tests for the assessment of exogenous interference showed no additional peaks. Any carryover was observed by analyzing a blank sample injected after analysis of either the top calibrator or an authentic positive sample.

This targeted analysis allowed the detection of 15 synthetic opioids, namely carfentanil, norcarfentanil, 1-hydroxy-fentanyl, acetyl fentanyl, acrylfentanyl, benzyl carfentanil, desomorphine, furanyl fentanyl, normethylfentanyl, ocfentanil, ohmefentanyl, remifentanil, thienyl fentanyl, U-47700 and valeryl fentanyl, with good sensitivity and a short sample run time (7.6 min); online solid-phase extraction enabled relatively quick pretreatment of samples.

Qualitative matrix effects study using post-column infusion was based upon the change in signal response. SRM signals did not always follow the same tendency of the internal standard one. However, this experimental observation did not affect significantly performances of the method as intra- and inter-day precision and accuracy remained acceptable.

Toxicological screening of urine (sampled at 1 h) using GC–MS revealed the presence of cocaine and its metabolites (benzoylecgonine, ecgonine methylester, anhydroecgonine methylester), lidocaine, levamisole and midazolam. Quantitation results for cocaine, benzoylecgonine and ecgonine methylester using LC–MS/MS were 570 ng/mL, 1230 ng/mL and 367 ng/mL, respectively. Despite patient opioid toxidrome, no opioid was highlighted using screening, so further analyses were performed on plasma and urine using two targeted LC–MS/MS techniques: one to search for opioids, the other to detect fentanyl analogs. The targeted LC–MS/MS screening for fentanyl analogs revealed the presence of carfentanil (2.88 ng/mL) and norcarfentanil (8.8 ng/mL) in urine, and below the method’s lower quantitation limits (< 0.01 ng/mL for carfentanil and < 0.05 ng/mL for norcarfentanil) in plasma (Table 1). The opioid search by LC–MS/MS allowed exclusion of the presence of tramadol, O-desmethyltramadol, N-desmethyltramadol, methadone, EDDP, propoxyphene, norpropoxyphene, pethidine, oxycodone, buprenorphine, buprenorphine-glucuronide, norbuprenorphine, norbuprenorphine-glucuronide, naloxone, fentanyl, norfentanyl, sufentanil and alfentanil.

Case 2 In May 2017, a 48-year-old woman, with a known history of depression and drug abuse, was found unconscious at home by relatives. Two empty boxes of drugs (pregabalin—Lyrica^® and buprenorphine—Subutex^®) were found near her. The emergency unit provided first aid, and the medical care included sufentanil delivery. She was admitted to the hospital ICU with a Glasgow coma scale score of 3. She presented myosis, and administration of a usual dose of intra-nasal naloxone (Nalscue^®) had no effect. During the hospitalization she was intubated, ventilated and treated by lidocaine, etomidate, midazolam and propofol. Her recovery was complete after 8 days in the ICU and 12 days on an internal medicine ward. She admitted that she bought “fentanyl” on the internet. Plasma and urine were collected on the day of her hospital admission.

Qualitative urine immunoassays were positive for THCCOOH and negative for MDMA, amphetamine, cocaine, methadone and opiates. Paracetamol, tricyclic antidepressant and ethanol immunoassays in the plasma were negative, and pregabalin was found in non-toxic concentration. LC-DAD urine screening initiated during the early stages of hospitalization revealed the presence of fentanyl and sufentanil derivatives and lidocaine. The targeted opioid LC–MS/MS technique detected fentanyl (< 0.05 ng/mL in plasma and 1.7 ng/mL in urine), sufentanil (< 0.05 ng/mL in plasma and urine), buprenorphine (0.48 ng/mL in plasma and 6.04 ng/mL in urine), norbuprenorphine (< 0.5 ng/mL in plasma and 19.4 ng/mL in urine), buprenorphine glucuronide (2.1 ng/mL in plasma and 264 ng/mL in urine), norbuprenorphine glucuronide (2.7 ng/mL in plasma and 116 ng/mL in urine) and naloxone (0.442 ng/mL in plasma and 4.59 ng/mL in urine). Targeted fentanyl analog screening by LC–MS/MS revealed large quantities of carfentanil and its metabolite, norcarfentanil, in both urine and plasma (Table 1). These two targeted screening campaigns enabled the exclusion of the other screened molecules.

Two plasma samples were taken on day 1 and 2 of hospitalization; a urine sample was taken on day 2.

Qualitative urine immunoassays were negative for amphetamine, MDMA, THCCOOH, cocaine, methadone and buprenorphine. Immunochemistry-based testing for opiates was strongly positive, justifying a targeted analysis. LC–MS/MS opiate determination indicated the presence of morphine (1,660 ng/mL) and codeine (< 5 ng/mL) in the urine sample from day 2, whereas no opiates were detected in the plasma sample from the same day.

GC–MS urine screening revealed the presence of propofol, laudanosine, lidocaine, morphine, codeine and norcarfentanil. Fentanyl analog analysis using the LC–MS/MS technique enabled the detection and quantitation of norcarfentanil and remifentanil in both the urine and the two plasma samples (Table 1).