Enantioseparation of phenethylamines by using high-performance liquid chromatography column permanently coated with methylated ß-cyclodextrin.

Cyclodextrins and their derivatives have been used for chiral high-performance liquid chromatography selectors, while they are costly to use as mobile phase additives in high-performance liquid chromatography. Here, we report application of phenyl column coated permanently with methylated ß-cyclodextrin for chiral high-performance liquid chromatography. A 0.1% (v/v) phosphoric acid solution containing 1 M NaCl and 0.5% (w/v) methylated ß-cyclodextrin was subjected to a phenyl column at a flow rate of 0.5 mL/min at 30°C for 2 h. Using the precoating phenyl column, all the enantiomers of the four phenethylamines (norepinephrine, epinephrine, octopamine, and synephrine) were successfully separated simultaneously by high-performance liquid chromatography with a mobile phase without methylated ß-cyclodextrin at a flow rate of 0.2 mL/min at 30°C. The enantioseparation ability was retained for successive analyses during 1 week. It is suggested that inclusion complex of methylated ß-cyclodextrin with a phenyl group on the surface of the stationary phase could be formed and that the inclusion complex could form the ternary complex with the injected analytes. The longer retention time of (S)-enantiomers of analytes than corresponding (R)-enantiomers for high-performance liquid chromatography could be explained from the higher stability of the methylated ß-cyclodextrin complexes with (S)-enantiomers, which were confirmed by capillary electrophoresis and 1 H NMR spectroscopy experiments.

In-tube solid phase microextraction and determination of ractopamine in pork muscle samples using amide group modified polysaccharide-silica hybrid monolith as sorbent prior to HPLC analysis.

A facile in-tube solid phase microextraction (in-tube SPME) procedure was developed to enrich ractopamine before HPLC-UV analysis. This was achieved by employing amide groups modified polysaccharide-silica hybrid monolith as an efficient sorbent. The monolith was synthesized by a simple reaction with agarose oxide and tetramethoxylisane, followed by the modification of amide groups via subsequent ring opening, "thiol-ene" click and dehydration reactions. Under the optimized extraction conditions, the enrichment factors for ractopamine, dopamine, clenbuterol, para-methylphenol and phenol were determined to be 50.5, 32.2, 4.8, 2.1 and 1.8, respectively. The monolithic column has ideal selectivity for ractopamine. Coupled with HPLC-UV, this method demonstrated a linearity within 2.0-800 ng/g for ractopamine with spiking in pork muscles (R2 = 0.9958). The LOD was 0.64 ng/g (S/N = 3) and recoveries ranged from 85.2 to 108.1% (n = 3). This approach provides a feasible way for analysis of trace ractopamine in biological samples.

Effective Removal of Clenbuterol and Ractopamine from Water with a Stable Al(III)-Based Metal-Organic Framework.

Clenbuterol (CLE) and ractopamine (RAC) are two kinds of typical ß2-adrenergic agonists which pose a serious threat to the health of human beings. In this work, 10 kinds of metal-organic frameworks (MOFs) with high stability and various pore features are screened to assess adsorption performance for CLE and RAC. An Al(III)-MOF (BUT-19) with abundant ethyl groups exhibits exceptional performance in removing CLE and RAC from water. The maximum adsorption capacity for CLE and RAC are up to 294.1 and 366.3 mg/g under the optimum adsorption conditions, respectively. Meanwhile, the adsorption mechanism effects of pH, temperature, and coexisted ions are investigated systematically. It is found that the MOF pore size and weak hydrogen-bond interactions between CLE/RAC molecules and the MOF are the main causes leading to the extraordinary adsorption. This study provides a new idea for the purposeful design and synthesis of MOFs for removing environmental pollutants and sheds light on the depuration of contaminated water.

Analysis of bitter orange dietary supplements for natural and synthetic phenethylamines by LC-MS/MS.

Citrus aurantium, commonly known as bitter orange, is a popular dietary supplement ingredient sold worldwide. Bitter orange supplements are sold primarily as weight management and sports performance products and have gained popularity after Ephedra products were banned from the US market. Supplements containing synephrine are reported to exhibit adverse cardiovascular effects especially in the presence of caffeine. In this study, an LC-MS/MS method was established to quantify five natural amines (synephrine, octopamine, tyramine, N-methyltyramine, and hordenine) and four synthetic phenethylamines (phenylephrine, methylsynephrine, etilefrine, and isopropyloctopamine) in dietary supplements sold in the US. The method was validated and found to have acceptable performance to accurately measure analytes in complex botanical products. The average recoveries from a blank matrix were 88-125% with an RSD of 0.5-7.0%. Fifty-nine products labeled to contain bitter orange peel, extract, or its amines were purchased and their amine content was measured. Several products were found to contain higher amounts of amines than that expected from a typical bitter orange extract. Of the 23 products that made label claims for synephrine, only 5 products (22%) were within 80-120% of labeled synephrine content. The presence of synthetic amines, methylsynephrine (up to 240 mg/daily serving), and isopropyloctopamine (up to 76 mg/daily serving), whose effects in humans are not known, were detected in six products and one product, respectively. While the use of methylsynephrine and isopropyloctopamine are not permitted in dietary supplements, hordenine, N-methyltyramine, and octopamine are currently listed on the FDA's Dietary Supplement Ingredient Advisory List.

Schwarzinicines A-G, 1,4-Diarylbutanoid-Phenethylamine Conjugates from the Leaves of Ficus schwarzii.

Schwarzinicines A-G (1-7), representing the first examples of 1,4-diarylbutanoid-phenethylamine conjugates, were isolated from the leaves of Ficus schwarzii. The structures of these compounds were determined by detailed analysis of their MS, 1D and 2D NMR data. Compounds 1-4 exhibited pronounced vasorelaxant effects in the rat isolated aorta (Emax 106-120%; EC50 0.96-2.10 µM). However, compounds 1 and 2 showed no cytotoxic effects against A549, MCF-7, and HCT 116 human cancer cells (IC50 > 10 µM).

Perylene Bisimide Aggregates as Probes for Subnanomolar Discrimination of Aromatic Biogenic Amines.

Perylene bisimide derivatives show peculiar physical chemical features, such as a highly conjugated system, high extinction coefficients and elevated fluorescence quantum yields, making them suitable for the development of optical sensors of compounds of interest. In particular, they are characterized by the tendency to aggregate into π-π stacked supramolecular structures. In this contribution, the behavior of the PBI derivative N, N'-bis(2-(trimethylammonium)ethylene)perylene bisimide dichloride was investigated both in aqueous solution and on solid support. The electronic communication between PBI aggregates and biogenic amines was exploited in order to discriminate aromatic amines down to subnanomolar concentrations by observing PBI fluorescence variations in the presence of various amines and at different concentrations. The experimental findings were corroborated by density functional theory calculations. In particular, phenylethylamine and tyramine were demonstrated to be selectively detected down to 10-10 M concentration. Then, in order to develop a surface plasmon resonance (SPR) device, PBI was deposited onto a SPR support by means of the layer-by-layer method. PBI was deposited in the aggregated form and was demonstrated to preserve the capability to discriminate, selectively and with an outstanding analytical sensitivity, tyramine in the vapor phase and even if mixed with other aromatic amines.

Dopamine/2-Phenylethylamine Sensitivity of Ion-Selective Electrodes Based on Bifunctional-Symmetrical Boron Receptors.

Piperazine-based compounds bearing two phenylboronic acid or two benzoxaborole groups (PBPA and PBBB) were applied as dopamine receptors in polymeric membranes (PVC/DOS) of ion-selective electrodes. The potentiometric sensitivity and selectivity of the sensors towards dopamine were evaluated and compared with the results obtained for 2-phenylethylamine. Since the developed electrodes displayed strong interference from 2-phenylethylamine, single-molecule geometry optimizations were performed using the density functional theory (DFT) method in order to investigate the origin of dopamine/2-phenylethylamine selectivity. The results indicated that phenylboronic acid and benzoxaborole receptors bind dopamine mainly through the dative B⁻N bond (like 2-phenylethylamine) and the potentiometric selectivity is mainly governed by the higher lipophilicity of 2-phenylethylamine.

Design, synthesis and characterization of tracers and development of a fluorescence polarization immunoassay for the rapid detection of ractopamine in pork.

In this study, 10 fluorescein-labeled ractopamine (RAC) derivatives (tracers) were synthesized and characterized to develop a rapid fluorescence polarization immunoassay (FPIA) for the detection of RAC in pork, using previously produced RAC polyclonal antibodies. The effect of the tracer structure on the sensitivity of the FPIA was investigated. The specificity of the FPIA was evaluated with 70 ß-agonists and ß-blockers. The FPIA showed a limit of detection of 0.56 µg kg-1 for RAC in pork, with recoveries ranging from 74.8% to 86.6% in spiked samples. The total analysis time, including sample pretreatment, was less than 1 h. The FPIA was used to screen 150 commercial pork samples for RAC residues and the results were consistent with those of an enzyme-linked immunosorbent assay (ELISA) and ultra-high-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). Our results demonstrate that the FPIA developed here is a rapid, accurate, and sensitive screening method for RAC residues in pork.

Application of Enzyme Digestion and Deconjugation Followed by Quick, Easy, Cheap, Effective, Rugged, Safe Extraction and Liquid Chromatography-Tandem Mass Spectrometry Methodology To Determine Ractopamine Residue in Pork.

A new methodology is proposed for ractopamine residue analysis in pork. It consists of enzyme-mediated digestion and deconjugation steps; modified quick, easy, cheap, effective, rugged, and safe (QuEChERS) extraction; and liquid chromatography-tandem mass spectrometry (LC-MS/MS). In brief, the samples were digested with protease and then deconjugated with ß-glucuronidase enzyme; they were then subjected to extraction and cleanup by QuEChERS and underwent sequential analysis by LC-MS/MS. The method performance was evaluated in accordance to the validation guidelines regulated by the Brazilian Ministry of Agriculture and Food Supply. The limit of detection was 0.15 µg/kg and limit of quantification was 0.5 µg/kg. When the method was applied to real samples, ractopamine residue was found in concentrations (up to 7.86 µg/kg) below international recommendation limits up to 10 µg/kg. The method is sensitive, accurate, quick, simple, and suitable for routine analysis; therefore, it is a monitoring tool that may be adopted by laboratories to achieve compliance levels.

Determination of ractopamine and salbutamol in pig hair by liquid chromatography tandem mass spectrometry.

A liquid chromatography tandem mass spectrometric method was developed for the determination of two ß-agonists (ractopamine and salbutamol) in pig hair samples. An isotope of ractopamine-d5 or salbutamol-d6 as an internal standard was used to carry out quantitative analysis. Concentrated sodium hydroxide was used to pretreat hair samples and then purified by the solid phase extraction (SPE) procedure. The extracted solution was evaporated and reconstituted for injection in the instrument with electrospray ionization (ESI) operating in a positive multiple-reaction-monitoring (MRM) mode. Ractopamine and salbutamol separation were performed on C18 analytical column under gradient condition. The internal standard calibration curve was linear in the range of concentration from 0.5 to 100 ng mL-1 (R2 > 0.995). Recoveries of this method estimated at three spiked concentrations of 100, 250 and 500 ng mL-1 in pig hair samples, were 79-82% for ractopamine and 77-96% for salbutamol. The corresponding inter-day and intra-day precisions expressed as relative standard deviation (RSD %) were 3.8-6.4% and 3.8-8.6%, respectively. The analytical time for one sample was 8 min. The detection limit of this method was 0.6 and 8.3 ng mL-1 for ractopamine and salbutamol, respectively. This developed method can be applied for monitoring the use of the ß-agonists salbutamol and ractopamine in swine feed incurred pig hair.

A new cinnamoylphenethylamine derivative from a Mongolian Allium species, Allium carolinianum.

A new cinnamoylphenethylamine derivative, compound 1, was isolated as the main HPLC peak after partitioning the methanol extract of bulbs of a Mongolian onion species, Allium carolinianum DC. The chemical structure of this substance was determined by spectroscopic analysis including MS, and 1D and 2D NMR. Compound 1 showed weak cytotoxic activity in the murine leukemia cell line P388.

Unusual phenethylamine-containing alkaloids from Elaeocarpus tectorius.

From the leaves of Elaeocarpus tectorius (Lour.) Poir. four previously undescribed phenethylamine-containing alkaloids were isolated, namely, tectoricine, possessing an unprecedented isoquinuclidinone ring system incorporating a phenethylamine moiety, tectoraline, representing a rare alkamide incorporating two phenethylamine moieties, and tectoramidines A and B, representing the first naturally occurring trimeric and dimeric phenethylamine alkaloids incorporating an amidine function. The structures of these alkaloids were established by detailed spectroscopic analysis. The absolute configuration of tectoricine was determined by comparison of the experimental and calculated ECD spectra. Plausible biosynthetic pathways to the four alkaloids are proposed.

Discovery and Synthesis of Caracolamide A, an Ion Channel Modulating Dichlorovinylidene Containing Phenethylamide from a Panamanian Marine Cyanobacterium cf. Symploca Species.

A recent untargeted metabolomics investigation into the chemical profile of 10 organic extracts from cf. Symploca spp. revealed several interesting chemical leads for further natural product drug discovery. Subsequent target-directed isolation efforts with one of these, a Panamanian marine cyanobacterium cf. Symploca sp., yielded a phenethylamide metabolite that terminates in a relatively rare gem-dichlorovinylidene moiety, caracolamide A (1), along with a known isotactic polymethoxy-1-alkene (2). Detailed NMR and HRESIMS analyses were used to determine the structures of these molecules, and compound 1 was confirmed by a three-step synthesis. Pure compound 1 was shown to have in vitro calcium influx and calcium channel oscillation modulatory activity when tested as low as 10 pM using cultured murine cortical neurons, but was not cytotoxic to NCI-H460 human non-small-cell lung cancer cells in vitro (IC50 > 10 µM).

Comparison of three S-ß-CDs with different degrees of substitution for the chiral separation of 12 drugs in capillary electrophoresis.

Three kinds of sulfated ß-cyclodextrin (S-ß-CD), including a single isomer, heptakis-6-sulfato-ß-cyclodextrin (HS-ß-CD), degree of substitution (DS) of 7, which was synthesized in our laboratory and another two commercialized randomly substituted mixtures, a sulfated ß-cyclodextrin with DS of 7 to 11, as well as a highly sulfated-ß-cyclodextrin with DS of 12 to 15, were used for the enantioresolution of 12 drugs (the ß-blockers, phenethylamines, and anticholinergic agents) in capillary electrophoresis. The enantioseparation under varying concentrations of S-ß-CD and background electrolyte pH were systematically investigated and compared. Based on the experimental results, the effect of the nature of S-ß-CD and analyte structure on the enantioseparation is discussed.

Tyramine and ß-phenylethylamine, from fermented food products, as agonists for the human trace amine-associated receptor 1 (hTAAR1) in the stomach.

The aromatic amines tyramine and ß-phenylethylamine are abundant in fermented foods. Recently, a family of human trace amine-associated receptors (hTAARs) was discovered that responds to these compounds. This study examined the expression of hTAAR genes in five human organs. Among them, the stomach expressed hTAAR1 and hTAAR9. Interestingly, more hTAAR1 was expressed in the pylorus than in the other stomach regions. The CRE-SEAP reporter assay revealed that only hTAAR1 functioned as a Gs-coupled receptor in response to tyramine and ß-phenylethylamine stimulation. The ß-phenylethylamine-mediated hTAAR1 activity could be potentiated using 3-isobutyl-1-methylxanthine. These data suggest that tyramine and ß-phenylethylamine in fermented foods act at hTAAR1 as agonists in the pylorus of stomach.

Analysis of 25 C NBOMe in Seized Blotters by HPTLC and GC-MS.

Use of unauthorized synthetic drugs is a serious, forensic, regulatory and public health issue. In this scenario, consumption of drug-impregnated blotters is very frequent. For decades, blotters have been generally impregnated with the potent hallucinogen known as lysergic acid diethylamide (LSD); however, since 2013 blotter stamps with N-2 methoxybenzyl-substituted phenylethylamine hallucinogen designated as "NBOMes" have been seized in Chile. To address this issue with readily accessible laboratory equipment, we have developed and validated a new HPTLC method for the identification and quantitation of 25-C-NBOMe in seized blotters and its confirmation by GC-MS. The proposed method was validated according to SWGTOX recommendations and is suitable for routine analysis of seized blotters containing 25-C-NBOMe. With the validated method, we analyzed 15 real samples, in all cases finding 25-C-NBOMe in a wide dosage range (701.0-1943.5 µg per blotter). In this situation, we can assume that NBOMes are replacing LSD as the main hallucinogenic drug consumed in blotters in Chile.

A novel aptasensor for electrochemical detection of ractopamine, clenbuterol, salbutamol, phenylethanolamine and procaterol.

ß-agonists are phenylethanolamines with different substituent groups on the aromatic ring and the terminal amino group which have the effect of nutrition redistribution and can accumulate in body tissues causing acute or chronic poisoning when consumed. Therefore, it is very important to establish a fast screening method for the detection of several kinds of ß-agonists in food safety control. In this study, the aptamer-agonists (AP-Ago) has screened out by Isothermal Titration Calorimetric method. AP-Ago was a single-strand DNA with 22 base-pairs. The dissociation constant (Kd) to phenylethanolamine (PHL) was 3.34 × 10(-5)mol L(-1). The AP-Ago based electrode was constructed by self-assembling on gold electrode. A label-free electrochemical aptasensor was then developed with AP-Ago-based gold electrode, which was sensitive to phenylethanolamine(PHL), clenbuterol (CLB), ractopamine (RAC), salbutamol (SAL) and procaterol (PRO). The detection limits were 0.04 ng/mL (RAC), 0.35 pg/mL (CLB), 1.0 pg/mL (PHL), 0.53 pg/mL (SAL) and 1.73 pg/mL(PRO), respectively, The detection time was 15 min. The reproductivity of the mentioned aptasensor is good with RSD of 2.09%. Comparing with ELISA and HPLC on ß-agonists detection in actual sample, this aptasensor is advantage of fewer steps and fast screen-detection of these five ß-agonists or their mixtures. This study suggests that the aptasensor can be developed to a rapid screening means with multi-ß-agonists (may be one or more) in sample.

Enhancement of surface plasmon resonance signals using a MIP/GNPs/rGO nano-hybrid film for the rapid detection of ractopamine.

A novel surface plasmon resonance (SPR) sensor that uses molecular imprinted polymers (MIPs) coated with gold nanoparticles (GNPs) and reduced graphene oxide (rGO) as a sensing nano-hybrid film was developed for detection of ractopamine. The MIPs were synthesized by precipitation polymerization and characterized by scanning electron microscopy and Scatchard analysis. The GNPs/rGO composite was synthesized by a single-step reduction of graphene oxide and HAuCl4 solution. The MIP/GNPs/rGO nano-hybrid film was immobilized onto a bare sensor chip and exhibited remarkable sensitivity and stability by the "grafting to" method with the assistance of ionic liquid (IL) as a binder. The prepared sensor showed class-specific selectivity for ractopamine (RAC) and its analogs under optimized conditions. The novel SPR sensor had a wide linear range over an RAC concentration from 20 to 1000 ng/mL with a detection limit of 5 ng/mL (S/N=3). The results demonstrated that the MIP/GNPs/rGO nano-hybrid film was suitable as the recognition element of the SPR sensorfor rapid screening and detection of beta-agonists such as RAC.

Enhanced simultaneous detection of ractopamine and salbutamol--Via electrochemical-facial deposition of MnO2 nanoflowers onto 3D RGO/Ni foam templates.

In this paper, we report a facile method to successfully fabricate MnO2 nanoflowers loaded onto 3D RGO@nickel foam, showing enhanced biosensing activity due to the improved structural integration of different electrode materials components. When the as-prepared 3D hybrid electrodes were investigated as a binder-free biosensor, two well-defined and separate differential pulse voltammetric peaks for ractopamine (RAC) and salbutamol (SAL) were observed, indicating the simultaneous selective detection of both ß-agonists possible. The MnO2/RGO@NF sensor also demonstrated a linear relationship over a wide concentration range of 17 nM to 962 nM (R=0.9997) for RAC and 42 nM to 1463 nM (R=0.9996) for SAL, with the detection limits of 11.6 nM for RAC and 23.0 nM for SAL. In addition, the developed MnO2/RGO@NF sensor was further investigated to detect RAC and SAL in pork samples, showing satisfied comparable results in comparison with analytic results from HPLC.

Riparin A, a compound from Aniba riparia, attenuate the inflammatory response by modulation of neutrophil migration.

Inflammation is a local tissue response to attacks characterized by vascular and cellular events, including intense oxidative stress. Riparin A, a compound obtained from Aniba riparia, has been shown to have antioxidant activity and cytotoxicity in vitro. This study was aimed at evaluating the anti-inflammatory effect of riparin A against acute inflammation. The results of our evaluations in various experimental models indicated that riparin A reduced paw edema induced by carrageenan, compound 48/80, histamine, and serotonin. Furthermore, it decreased leukocyte and neutrophil counts, myeloperoxidase activity, thiobarbituric acid reactive substance (TBARS) levels, and cytokine (tumor necrosis factor-α and interleukin-1ß) levels increased by carrageenan-induced peritonitis, and reversed glutathione levels. Riparin A also reduced carrageenan-induced adhesion and rolling of leukocytes on epithelial cells and did not produce gastric-damage as compared with indomethacin. In conclusion, the data show that riparin A reduces inflammatory response by inhibiting vascular and cellular events, modulating neutrophil migration, inhibiting proinflammatory cytokine production, and reducing oxidative stress.