Quantification and distribution of 4-fluoroisobutyryl fentanyl (4-FiBF) in postmortem biological samples using UHPLC–QqQ-MS/MS

Synthetic opioids pose a high risk of intoxication to consumers and are an example of progressive drug market adaptability. International legal regulations related to the illicit distribution of drugs have resulted in increasing structural modifications to new psychoactive substances (NPS) released onto the global drug market. Since 2009, there have been 57 new synthetic opioids introduced to Europe’s drug market. According to the latest European Monitoring Centre for Drugs and Drug Addiction (EMCDDA) report [1], in 2019, 8 new opioids were seized for the first time, including two totally novel fentanyl analogs. The opioid crisis can also be observed in North America. The present and most deadly wave of fentanyl analog use began in the United States around 2013. Similarly, in Canada, the levels of opioid-related deaths has increased since 2016. Between January and December 2019 alone, there were 77% intoxications involving fentanyl or fentanyl analogs, while seizures of fentanyl increased from about 96 kg in 2015 to more than 2.9 tons in 2018 [2]. Like in North America and Europe, East and Southeast Asia is also observing a large amount of fentanyl analogs reported annually. Japan reported the largest number of NPS, followed by China and Singapore [3]. According to an United Nations Office on Drugs and Crime (UNODC) report (October 2020), the opioid crisis is a global complex problem, whose nature seems to be a combination of factors, including lowering costs by organized crime groups through substitution of pharmaceutical opioids with fentanyl analogs and other synthetic opioids [2].

4-Fluoroisobutyryl fentanyl (4-FiBF) is one of the fentanyl analogs, which differs from the original drug by the presence of a fluorine atom and isobutyramide in place of the propenamide group. The first official EMCDDA-Europol notification of 4-FiBF was on August 26, 2016 from the Slovenian National Forensic Laboratory. White powder (5 g) was collected as test purchased and shipped from China as a part of the EU co-funded project RESPONSE [4]. As a seizure, 4-FiBF was first identified in November 2016 by German Police in Berlin. From 2016 to 2017 in total, there were 22 other seized drugs reported in Sweden (20), Belgium (1) and the United Kingdom (1) [5]. Due to the varied manufactured formulas in which 4-FiBF can be found (powders, tablets, liquids, blotters and herbal smoking mixtures), there are different ways to take this drug. It can be administered orally, rectally, intranasally via a spray, inhaled by smoking or injected intravenously and intramuscularly. For this reason, 4-FiBF distribution and its concentrations in different tissues and biological fluids can vary significantly [6]. In Europe, one of the first fatal intoxications which was associated with 4-FiBF occurred in September 2016 and was reported by the Swedish STRIDA project. Postmortem serum concentration of 4-FiBF after intravenous injection was 38 ng/mL [7], however, it is worth noting that the patient was reanimated with naloxone before his death. Twenty-six deaths in the US in which 4-FiBF was detected as one of the fentanyl analogs were also reported during a similar time period [8].

Progressively, this analog became increasingly popular on the drug market, especially due to its easy availability for purchase on the Internet. The eDarkTrends project developed by Lamy et al. [9] revealed that between 2018 and 2019, 4-FiBF was third the most commonly advertised fentanyl analog among darknet customers. Forensic testing of illicit drug seizures in Hong Kong also confirmed this trend. In the years 2016–2018, 4-FiBF along with cyclopropylfentanyl were the mostly detected fentanyl-related substances [10].

A large toxicological study performed by Pardi et al. [11] based on evaluation of 4-FiBF in blood samples from 247 authentic cases in New York City occurring between 2017 and 2018, revealed that postmortem concentration levels of these drug can reach the value even of 331 ng/mL. Compared to the fentanyl, 4-FiBF concentrations are an order of magnitude higher. In part, it can be explained by binding and functional activity data of opioid receptors in the context of fentanyl and its analogs. The half-maximal effective concentration, EC50 (nM), value represents the concentration that causes a half-maximal response of the receptor agonist. In vitro studies of pharmacodynamics shows that for fentanyl these values are around 28.8, 996 and 347 nM for µ-, δ- and ĸ- opioid receptors, respectively. In contrast, the values of EC50 for 4-FiBF in context of µ-, δ- and ĸ- opioid receptors are 115, 2490 and 1330 nM, respectively. Accordingly, the study indicates that more 4-FiBF is needed to achieve half of the maximum receptor response compared to fentanyl. Similar impressions are shared by participants in online forums, who noted that 4-FiBF is long lasting and less euphorigenic when compared to other opioids [6]. This fact might lead users to increase the doses and may be the reason for such high concentrations of this drug in postmortem samples.

Drug detection in health care centers in acute poisoning cases is often performed using commercial immunochemical assays. However, a major limitation is their cross-reactivity. Wharton et al. [12] performed an extended study of the reactivity of fentanyl analogs using immunoassay kits. The investigations showed that analogs with N-aryl substitutions (such as 4-FiBF) are relatively poorly detectable by immunochemical methods. Despite its high structural similarity to the fentanyl, p-fluoro substituent on the phenyl ring with a methyl group in the acyl fragment of the anilide is not well tolerated in commercially available kits. Furthermore, these NPS exhibited average cross-reactivities of 10–30% across all fentanyl-targeting kits and almost 50% across individual kit types. For this reason, to date, the most sensitive, selective and reliable methods for the determination of fentanyl analogs in biological samples are those based on chromatographic separation with mass spectrometry detection.

The determination of 4-FiBF in biological samples was performed using chromatographic methods: high-performance liquid chromatography coupled with triple quadrupole tandem mass spectrometry (HPLC–QqQ-MS/MS) in blood samples [11, 13, 14]; ultra-high-performance liquid chromatography coupled with ion trap and tandem mass spectrometry (UHPLC–Ion Trap-MSⁿ) in blood, urine and tissues samples [8]; UHPLC coupled with tandem mass spectrometry and triple quadrupole linear ion trap (UHPLC–QTRAP-MS/MS) in blood [15] and hair samples [16]. In turn, the determination of 4-FiBF in seized drugs was performed through the use of gas chromatographic methods: coupled with tandem cold electron ionization mass spectrometry (GC–cold EI-MS/MS) and with vacuum ultraviolet detection (GC–VUV) [17] as well as gas chromatography with infrared detector (GC–IRD) [18].

The bodies of a 33-year-old male (case 1) and a 38-year-old male (case 2) were discovered in an apartment. No detailed information regarding the circumstances of the incident was available. Internal and external examinations of the corpses showed edema of the brain tissue, pulmonary emphysema as well as acute gastritis in both the deceased.

An ambulance was called for an unconscious 38-year-old male (case 3). The physician diagnosed sudden cardiac arrest. After transport to the hospital, the man was still unconscious and unresponsive to impulses. Resuscitation was carried out unsuccessfully, and finally, the man’s death was confirmed. The autopsy did not reveal any pathological lesions.

A 22-year-old female (case 4) was found in an apartment. According to witnesses’ statements, 2 days before her death the woman had been using recreational drugs. The next day, she complained about feeling unwell. Furthermore, the woman had been taking analgesics after an accident and lower limb injury. A plastic bag containing a white powder was found in the woman's apartment. The total weight of seized powder was 57.8 mg. External and internal examination of the corpse revealed cerebral tissue edema, pulmonary emphysema, and acute mucositis.

In each case, the participation of third parties in the deaths were excluded. For further toxicological analysis, biological fluids and tissues were collected. In cases 1 and 2: blood, urine, vitreous humor, bile, brain, kidney, liver and gastric content; in case 3: blood, vitreous humor, bile, brain, kidney, liver and stomach wall; in case 4: blood, urine, vitreous humor, brain, liver and gastric content. All the blood samples were collected in tubes containing sodium fluoride as the preservative agent. Toxicological investigations found 4-FiBF in all collected postmortem materials along with other drugs and NPS. Analysis for low molecular volatile compounds did not reveal the presence of ethanol and its congeners (methanol, acetone, and isopropanol) in any of the reported cases.

This paper aims to apply an UHPLC–QqQ-MS/MS method for quantification of 4-FiBF and its distribution in authentic postmortem biological fluids (blood, urine, bile, vitreous humor, gastric content) and tissues (brain, liver, kidney, stomach wall) in four fatal intoxications which occurred in September 2018 in Poland.

The most widely used method for the determination of 4-FiBF in biological samples is LC–MS, which are characterized by high levels of selectivity, sensitivity and accuracy. These parameters are essential for reliable verification of fentanyl analog ingestions. A comparison of the methods applied for analysis of biological fluids in cases of 4-FiBF intoxication is presented in Table 5. Most of the articles described the methods with the use of triple quadrupole [11, 13, 14], however, ion trap [8] and triple quadrupole linear ion trap [15] systems have also been applied. Furthermore, the most widespread ion source was ESI, while detection of 4-FiBF was performed based on its fragmentation in the MRM mode [11, 13‐15] or auto MSⁿ [8]. All methods concerned on determination of 4-FiBF in whole blood samples [11, 13‐15], however, Shoff et al. [8] applied their method for analysis of both, whole blood and urine. All methods described to date have used fentanyl-d₅ as an IS. Due to its structural similarity to the determined 4-FiBF, it was possible to achieve very good recovery values ranging from 73.6% (in the work of Strayer et al.) [13] up to 99.7% (in the method described by Qin et al.) [15]. The lowest LOQ of the method (0.05 ng/mL) with the smallest sample volume (100 µL) was achieved by Qin et al. [15]. However, the authors were the only ones who applied UHPLC–QTRAP-MS/MS, which allows for much higher sensitivity through the availability of trapping ions before its further fragmentation. Shoff et al. [8] and Adamowicz et al. [14] determined only LOD of their methods, which were 0.5 ng/mL and 0.13 ng/mL, respectively. Nevertheless, it is difficult to compare both LOD and LOQ values reported by the authors due to the different injection volumes used. Most of the UHPLC–MS/MS procedures described to date, as well as our method, achieved a LOQ at the concentration of 0.1 ng/mL. However, the injection volumes ranged from 2.0 µL (presented method) to 5.0 µL [8, 15] to as much as 10 µL [11, 14]. Of the techniques described to date, more than half have been based on the use of solid-phase extraction (SPE) as a sample preparation procedure [8, 11, 13]. The most important highlights of this procedure is very good purification of the sample as well as obtaining a clear extract with concentrated analyte content. However, this preanalytical sample procedure is complicated and time consuming. Furthermore, the SPE techniques described by Shoff et al. [8] and Strayer et al. [13] required 1000 µL of biological material. In this paper, as well as the two previous described methods [14, 15], an application of LLE technique in alkaline medium (pH 9–11) with ethyl acetate as the organic phase was achieved. This technique of sample pre-treatment is fast and simple, while reducing of multi-stage preparation procedure allows to obtain better precision, accuracy and recovery values.

The results of toxicological analysis presented in this paper are the most detailed characterization of 4-FiBF concentrations in various biological samples. The reports on postmortem concentrations are limited to serum, blood and urine concentrations [7, 11]. Helander et al. [7] reported fatal ingestion of a 250-mg dose of 4-FiBF by a 27-year-old male. Toxicological analysis of biological material collected 3.5 h after intoxication revealed 4-FiBF concentrations of 38 ng/mL and 31 ng/mL in the serum and urine, respectively. In turn, Pardi et al. [11] presented 247 cases in which the presence of 4-FiBF in blood samples was found. Concentrations of this synthetic fentanyl analog in postmortem blood samples ranged from 0.1 up to 331 ng/mL. The 4-FiBF concentrations described in this paper (76.1–257 ng/mL) are within those previously reported. The presented four fatal intoxications were associated with rapid (within minutes) loss of consciousness and death after the drug administration. Our results suggest that the distribution of 4-FiBF is very rapid, and concentrations of the xenobiotic in the liver, bile, and kidney are many orders of magnitude higher than in blood concentrations. However, in the case of co-administered cathinone analogs (4-CMC and N-ethylpentylone), the differences were not as significant. Nevertheless, taking into account the coincidence of 4-FiBF with cathinones (which are unstable in blood samples [21, 23‐25]), and the high concentrations of 4-FiBF in the liver, it is appropriate to collect the fragment of this tissue together with blood samples for further toxicological studies. The elimination of 4-FiBF occurs rapidly, and the urinary concentrations are extremely high. It appears to be inversely proportional to the blood concentration—in our cases, as the blood concentration of 4-FiBF decreased, an increase in the urinary concentration was observed, which may suggest that this is the main pathway of elimination of this compound from the body. An interesting finding was observed in the case of quantification of 4-FiBF in the gastric contents and stomach wall, as particularly high concentrations of this substance were found there. The results are even more surprising, as all four decedents ingested 4-FiBF by inhalation before their deaths (this fact was described in detail by the witnesses). Therefore, it seems that the presence of 4-FiBF in the gastrointestinal tract was the result of saliva or smoke swallowing while smoking.

The examination of the seized white powder (from case 4) revealed the presence of a mix of two NPS belonging to the group of synthetic cathinones (α-PiHP) and fentanyls (4-FiBF). To our knowledge, such an unusual combination of these two drugs has not been reported to date. The local nature of this trend seems to be confirmed by the fact that in 2020 Adamowicz et al. [14] reported a fatal intoxication case which occurred in Poland. The determined concentrations of 4-FiBF along with α-PiHP in the postmortem blood sample were 74 ng/mL and 56 ng/mL, respectively. In our cases, the intoxication with a mixture of 4-FiBF and α-PiHP, and in addition with 4-CMC and tramadol was found. However, the blood concentrations of 4-FiBF and α-PiHP described in this paper’s UHPLC–QqQ-MS/MS analysis (119 ng/mL and 6.1 ng/mL, respectively) differed from those obtained by Adamowicz et al. [14]. These differences were most likely due to the different proportions of psychoactive substances in the drugs used.