ABSTRACT
The recent emergence of new synthetic opioids (NSOs) compounds in the illicit market is increasingly related to fatal cases. Identification and medical care of NSO intoxication cases are challenging, particularly due to high frequency of new products and extensive metabolism. As the study of NSO metabolism is crucial for the identification of these drugs in cases of intoxication, we aimed to investigate the metabolism of the piperazine NSO AP-237 (= bucinnazine). Two complementary approaches (in silico and in vitro) were used to identify putative AP-237 metabolites which could be used as consumption markers. In silico metabolism studies were realized by combining four open access softwares (MetaTrans, SyGMa, Glory X, Biotransformer 3.0). In vitro experiments were performed by incubating AP-237 (20 µM) in differentiated HepaRG cells during 0 h, 8 h, 24 h or 48 h. Cell supernatant were extracted and analyzed by liquid chromatography coupled to high-resolution mass spectrometry and data were reprocessed using three strategies (MetGem, GNPS or Compound Discoverer®). A total of 28 phase I and six phase II metabolites was predicted in silico. Molecular networking identified seven putative phase I metabolites (m/z 203.154, m/z 247.180, m/z 271.180, two m/z 289.191 isomers, m/z 305.186, m/z 329.222), including four previously unknown metabolites. Overall, this cross-disciplinary approach with molecular networking on data acquired in vitro and in silico prediction enabled to propose relevant candidate as AP-237 consumption markers that could be added to mass spectrometry libraries to help diagnose intoxication.
Subject(s)
Opiate Alkaloids , Mass Spectrometry , Analgesics, Opioid/metabolism , PiperazinesABSTRACT
OBJECTIVES: To establish a rapid method for the analysis of bucinnazine in blood by UPLC-MS/MS and to apply the method to the practical case. METHODS: After the internal standard was added to blood, the protein was precipitated with 900 µL mixed solution (Vacetonitrileâ¶Vwater=8â¶2). After vortex and centrifugation, the protein was measured through 0.22 µm filter membrane. The separation was performed on C18 chromatography column, with acetonitrile and 5 mmol/L ammonium acetate containing 0.1% formic acid aqueous as mobile phase gradient elution at the flow rate of 0.4 mL/min. Multiple reaction monitoring scan was performed in electrospray positive ion mode, quantitative measurement was performed by internal standard method, and methodological verification was carried out. RESULTS: The linear relationship of bucinnazine in blood was good in the range of 0.5-200 µg/L, the correlation coefficient (r) was 0.999 7, the limit of detection was 0.1 µg/L, the limit of quantitation was 0.5 µg/L, and the recovery was 78.3%-83.8% at 1, 10 and 100 µg/L mass concentration levels. The matrix effect was 69.4%-73.8%, the intra-day precision was 1.9%-2.8%, and the inter-day precision was 2.8%-3.2%, the accuracy was 3.1%-3.5%. The stability test results of 1 and 100 µg/L mass concentrations at -25 â showed that the accuracy (bias) of 10 d was less than 4.5%. CONCLUSIONS: This method has the advantages of simple pre-treatment process, fast sample processing speed, high sensitivity of instrument analysis, good stability of content determination and reliable identification results, and can meet the needs of case identification.
Subject(s)
Tandem Mass Spectrometry , Tandem Mass Spectrometry/methods , Chromatography, Liquid , Chromatography, High Pressure Liquid/methods , AcetonitrilesABSTRACT
Novel synthetic opioids (NSOs) are a class of opioid agonists that include analogs of fentanyl and structurally distinct non-fentanyl compounds normally used as standalone products, heroin adulterants, or constituents of counterfeit pain pills. Most NSOs are not currently scheduled in the U.S., are predominantly illegally synthesized, and sold on the Darknet. Among them, the cinnamylpiperazine derivatives such as bucinnazine (AP-237), AP-238, and 2-methyl-AP-237 and the arylcyclohexylamine derivatives, analogs of ketamine, such as 2-fluoro-deschloroketamine (2 F-DCK) have appeared in several monitoring systems. Two white powders purchased on the internet as bucinnazine were first analyzed with polarized light microscopy followed by direct analysis in real time-mass spectrometry (DART-MS) and gas chromatography-mass spectrometry (GC-MS). Both powders were white crystals with no other significant microscopic properties. The DART-MS analysis showed the presence of 2-fluorodeschloroketamine in powder #1, and AP-238 in powder #2. Identification was confirmed by GC-MS. The purity of each substance was 78.0% for powder #1, and 88.9% for powder #2, respectively. The toxicological risk associated with the misuse of NSOs still needs further study. The absence of bucinnazine and the presence of different active compounds in internet purchased samples raises public health and safety concerns.
Subject(s)
Analgesics, Opioid , Fentanyl , Powders , Chromatography, Liquid/methods , Analgesics, Opioid/analysisABSTRACT
OBJECTIVES@#To establish a rapid method for the analysis of bucinnazine in blood by UPLC-MS/MS and to apply the method to the practical case.@*METHODS@#After the internal standard was added to blood, the protein was precipitated with 900 μL mixed solution (Vacetonitrile∶Vwater=8∶2). After vortex and centrifugation, the protein was measured through 0.22 μm filter membrane. The separation was performed on C18 chromatography column, with acetonitrile and 5 mmol/L ammonium acetate containing 0.1% formic acid aqueous as mobile phase gradient elution at the flow rate of 0.4 mL/min. Multiple reaction monitoring scan was performed in electrospray positive ion mode, quantitative measurement was performed by internal standard method, and methodological verification was carried out.@*RESULTS@#The linear relationship of bucinnazine in blood was good in the range of 0.5-200 μg/L, the correlation coefficient (r) was 0.999 7, the limit of detection was 0.1 μg/L, the limit of quantitation was 0.5 μg/L, and the recovery was 78.3%-83.8% at 1, 10 and 100 μg/L mass concentration levels. The matrix effect was 69.4%-73.8%, the intra-day precision was 1.9%-2.8%, and the inter-day precision was 2.8%-3.2%, the accuracy was 3.1%-3.5%. The stability test results of 1 and 100 μg/L mass concentrations at -25 ℃ showed that the accuracy (bias) of 10 d was less than 4.5%.@*CONCLUSIONS@#This method has the advantages of simple pre-treatment process, fast sample processing speed, high sensitivity of instrument analysis, good stability of content determination and reliable identification results, and can meet the needs of case identification.
Subject(s)
Tandem Mass Spectrometry/methods , Chromatography, Liquid , Chromatography, High Pressure Liquid/methods , AcetonitrilesABSTRACT
Bucinnazine (1-butyryl-4-cinnamylpiperazine) is a synthetic opioid recently discovered in heroin seized samples in the U.S and in Europe. It was first synthesized in the late 1960s and has been used for the treatment of cancer-associated chronic pain in China for many years. Bucinnazine is one of the most potent compounds among the series of piperazines, which also include other relevant compounds, such as MT-45, AD-1211, and 2-methyl-AP-237, a methylated derivative of bucinnazine. Bucinnazine is considered a µ-selective opioid, binding primarily to the µ-opioid receptor. However, bucinnazine also may share several characteristics with other piperazines, which act primarily on dopamine, serotonin, and norepinephrine neurotransmission. At the present, bucinnazine is not scheduled in the U.S., as it is not a therapeutic choice for the treatment of pain. Nevertheless, with the advent of the cryptocurrency and the easy access of substances on the Darknet, bucinnazine is a real threat to the public health. This review discusses the main aspects of bucinnazine's chemistry, pharmacology, and toxicology and brings attention to the risk of the presence of this opioid in seized samples. Further studies on bucinnazine are still required to better evaluate its toxicity mechanisms, potential for drug-drug interactions, and abuse liability. Such information will be of utmost importance to guide future policies concerning the legal status of bucinnazine in the U.S.
Subject(s)
Analgesics, Opioid , Neurosciences , Dopamine , Humans , Pain , Public HealthABSTRACT
In this investigation, surface-enhanced Raman spectroscopy (SERS) technology was performed to detect bucinnazine hydrochloride (BH) injection in water and urine. The theoretical Raman spectrum of BH with characteristic peaks was calculated and identified by density functional theory (DFT). Employing an improved silver sol as a SERS active substrate, the SERS spectra of a BH solution with different concentrations were acquired with a 0.5 mol/L KI solution as an aggregation agent. It was determined that the limit of detection (LOD) was low, to 0.01 µg/mL. A good linear relationship of BH between the Raman intensity and the concentrations was obtained in water at a concentration range from 0.5 to 6 µg/mL (R2 = 0.9914), which laid a favorable foundation for quantitative analysis. In addition, the recovery rate of spiked samples from 95.13 to 120.54% were calculated. Finally, the detection of BH injection in artificial urine was completed and the detection limit could reach 0.5 µg/mL, which met the requirements of a rapid on-site detection of drugs in urine. As a result, it indicates that the inspection of BH by the SERS method is with simplicity and high sensitivity, having a great potential for real-time detection.
ABSTRACT
Objective To investigate the effects of Bucinnazine Hydrochloride on the pain behavior and the expression of caveolin-1 (Cav-1) in the anterior cingulate cortex of neuropathic pain mice.Methods 64 adult male Kunming mice (20-25g) were divided randomly into 4 groups with 16 in each group:Sham+BH(Bucinnazine Hydrochloride) group,Sham+NS (Normal Saline) group,CCI+ BH group and CCI+ NS group.The corresponding drugs were administered by intraperitoneal injectionfrom the forth day after CCI once a day for three days.Paw thermal withdrawal latency was measured by Hargreaves methods.Mechanicalwithdrawal threshold was assayed by electronic dolorimeter.c-Fos protein in anterior cingulate cortex was detected by immunohistochemistry staining and the expression of t-Cav-1,p-Cav-1was detected by Western blot.Results Bucinnazine Hydrochloride administered by intraperitoneal injection(0.1 mg/10 g,mice) alleviated thermal hyperalgesia and mechanical allodynia of CCI mice.Compared with the forth day (4.92±0.41) s of CCI+BH group,paw withdrawal latency on the fifth day(5.92±0.61) s was increased(P<0.05),and on the sixth day(7.93±0.91) s and seventh day (9.12±0.69)s were increased more(P<0.01,P<0.01).The paw withdrawal mechanical threshold on the sixth and seventh day of CCI+BH group mice((2.54 ±0.41)g,(3.68±0.61)g) were increased significantly (P<0.01,P<0.01)compared with the forth day(1.55± 0.31)g.Immunohistochenistry results showed that the expression of c-Fos decreased after treated with Bucinnazine Hydrochloride in the anterior cingulate cortex of CCI mice(P<0.001).Western Blotting showed that the expression of t-Cav-1 (1.97±0.31) and p-Cav-1 (0.11 ±0.09) in the anterior cingulate cortex of CCI +BH group mice decreased compared with that of in CCI+NS group mice(t-Cav-1:2.87±0.15,p-Cav-1:0.48± 0.09) (P<0.01,P<0.01).Conclusion Bucinnazine Hydrochloride can alleviate both thermal hyperalgesia and mechanical allodynia of neuropathic pain of mice,and reduce the expression of c-Fos,t-Cav-1,p-Cav-1 in the anterior cingulate cortex of neuropathic pain mice.