Detection of major psychoactive compounds (safrole, myristicin, and elemicin) of nutmeg in human serum via GC-MS/MS using MonoSpin® extraction: Application in a nutmeg poisoning case.

Nutmeg is an inexpensive, readily available spice used in a variety of recipes. However, the use of nutmeg powder as a recreational drug for its hallucinogenic effects is resulting in an increase in overdose rates. We encountered a male patient being hospitalized after ingesting 75 g of commercially available nutmeg powder with the intent of committing suicide. There are no available reports documenting the toxic or comatose-fatal blood concentrations or time-course of drug action in cases of nutmeg poisoning. Therefore, to improve patient management, we endeavored to determine the blood serum levels and time-course of the major psychoactive compounds (safrole, myristicin, and elemicin) present in nutmeg. We designed a simple and reliable method using the MonoSpin® extraction kit and gas chromatography-tandem mass spectrometry to detect the presence of these psychoactive compounds in human serum. The method had detection and quantitation limits of 0.14-0.16 and 0.5 ng/mL (lowest calibration points), respectively. The calibration curves displayed excellent linearity (0.996-0.997) for all three compounds at 0.5-300 ng/mL blood concentrations. The intra- and inter-day precision values for quality assurance were in the ranges of 2.4-11 % and 2.5-11 %, respectively; bias ranged from - 2.6 % to 2.1 %. Blood serum levels of safrole, myristicin, and elemicin were measured at admission (approximately 8 h post-ingestion) and approximately 94 h after a post-admission fluid therapy to evaluate their biological half-lives. We developed this method to obtain information on the psychoactive constituents of nutmeg and, thereby, determine the toxicokinetic parameters of nutmeg in a case of nutmeg poisoning.

Rational Design of Allosteric Fluorogenic RNA Sensors for Cellular Imaging.

Fluorescence-based tools are invaluable in studying cellular functions. Traditional small molecule or protein-based fluorescent sensors have been widely used for the cellular imaging, but the choice of targets is still limited. Recently, fluorogenic RNA-based sensors gained lots of attention. This novel sensor system can function as a general platform for various cellular targets. Here, we describe the steps to rationally design, optimize, and apply fluorogenic RNA-based sensors, using the intracellular imaging of tetracycline in living E. coli cells as an example.

A new strategy based on gas chromatography-high resolution mass spectrometry (GC-HRMS-Q-Orbitrap) for the determination of alkenylbenzenes in pepper and its varieties.

Alkenylbenzenes are natural toxins with genotoxic and carcinogenic effects in rodents, which are highly present in condiments frequently consumed. The aim of this study was the development of the first multi-analyte method for the determination of eight alkenylbenzenes (eugenol, methyl eugenol, acetyl eugenol, trans-isoeugenol, safrole, estragole, myristicin and trans-anethole) in different pepper varieties by gas chromatography coupled to high-resolution mass spectrometry (GC-HRMS-Q-Orbitrap) in combination with a simple ultrasound-assisted extraction method (UAE). The method was successfully validated, and it was applied for studying the presence of these analytes in peppers as well as to elucidate the effects of the berries' maturity and the geographical origin on alkenylbenzene contents. The analysis of the pepper samples showed that eugenol (10.5-120 mg/kg), trans-anethole (10.7-42.7 mg/kg) and estragole (2.2-45.7 mg/kg) tended to be the most detected alkenylbenzenes at high levels, whereas trans-isoeugenol (0.69-3.6 mg/kg) and safrole (0.20-3.0 mg/kg) were minor components. Estragole (PubChem CID: 8815); trans-anethole (PubChem CID: 637563); Myristicin (PubChem CID: 4276); Safrole (PubChem CID: 5144); Eugenol (PubChem CID: 3314); Methyl eugenol (PubChem CID: 7127); Acetyl eugenol (PubChem CID: 7136); trans-Isoeugenol (PubChem CID: 853433); Caffeine (PubChem CID: 2519); Dicyclohexylmethanol (PubChem CID: 78197).

Widespread receptor-driven modulation in peripheral olfactory coding.

Olfactory responses to single odors have been well characterized but in reality we are continually presented with complex mixtures of odors. We performed high-throughput analysis of single-cell responses to odor blends using Swept Confocally Aligned Planar Excitation (SCAPE) microscopy of intact mouse olfactory epithelium, imaging ~10,000 olfactory sensory neurons in parallel. In large numbers of responding cells, mixtures of odors did not elicit a simple sum of the responses to individual components of the blend. Instead, many neurons exhibited either antagonism or enhancement of their response in the presence of another odor. All eight odors tested acted as both agonists and antagonists at different receptors. We propose that this peripheral modulation of responses increases the capacity of the olfactory system to distinguish complex odor mixtures.

Simultaneous separation and concentration of neutral analytes by cyclodextrin assisted sweeping-micellar electrokinetic chromatography.

An on-line cyclodextrin assisted sweeping-micellar electrokinetic chromatography (CD assisted sweeping-MEKC) was developed for the simultaneous separation and concentration of four neutral analytes (erianin, dendrophenol, naringenin and scoparone) in Dendrobium officinale Kimura et Migo (D. officinale). The D. officinale was directly determined by this on-line stacking method after simple extraction and dilution. The optimized background solution (BGS) was 50 mM phosphoric acid (PA) containing 100 mM SDS and 30% (v/v) methanol. The best separation and concentration performance of analytes dissolved in 90 mM CD and 100 mM PA was achieved in a short analysis time when injected at 50 mbar for 100 s. Compared with conventional sweeping-MEKC and MEKC method, significant improvement in enrichment efficiency was achieved by using this proposed method. A series of validation studies of the present method was performed under the optimal conditions. Good linearities were obtained with the correlation coefficients in the range of 0.994-0.999, the detection limits were ranged from 13 to 40 ng/mL. Sensitivity enhancement factors (SEFs) were in the range of 28.5-46.8 compared with traditional injection (injection time 3 s). Therefore, the proposed method was successfully applied for the separation and concentration of neutral analytes in real samples.

Detection of Nutmeg Abuse by Gas Chromatography-Mass Spectrometric Screening of Urine.

High doses of nutmeg (seeds from Myristica fragrans Houtt.) can be abused as a psychoactive drug due to phenylpropene ingredients. During controlled abstinence, e.g., in forensic psychiatric clinics, nutmeg abuse has to be distinguished from an ingestion of other spices having phenylpropene ingredients (e.g., black pepper or garden lovage) or unintentional low-dose nutmeg intake. The aim of this study was to develop an evaluation model for the estimation of time point and amount of nutmeg abuse and differentiation from ingestion of other spices or low doses of nutmeg based on the gas chromatographic-mass spectrometric (GC-MS) analysis of urine samples. A total of 3 volunteers ingested 1.5 g of freshly ground nutmeg. No symptoms were reported. Urine samples were collected for up to 3 days. In addition, 18 blank samples from volunteers with regular diet and 2 authentic samples from forensic psychiatry patients with supposed nutmeg abuse were analyzed. All samples were analyzed by GC-MS in full scan mode. Metabolites of the nutmeg ingredients safrole, myristicin and elemicin were identified via a library search. For semi-quantitative estimations, the area ratios of the analytes to the internal standard (MDMA-d5) were normalized to the creatinine concentration. Up to 8 different metabolites were detected for at least 18 hours after intake of 1.5 g of nutmeg. In the two authentic samples, the normalized area ratios of those metabolites were 0.5-14 times the maximum reached in the intake study. Two additional metabolites could be detected in authentic samples. Probably due to ingestion of other spices, 5 of the 8 metabolites after intake of 1.5 g of nutmeg were detected in blank urine samples as well. The intake of high doses of nutmeg can be differentiated from the ingestion of other spices or low doses of nutmeg via standard GC-MS analysis of urine and application of the proposed evaluation model.

Simultaneous determination of cytokinins by high performance liquid chromatography with resonance Rayleigh scattering and mechanism discussion.

A reliable, highly sensitive and highly selective method of high performance liquid chromatography associated with resonance Rayleigh scattering (HPLC-RRS) was developed to detect three cytokinins, namely, 6-benzylaminopurine (BA), kinetin (KT) and zeatin (ZT). In this work, Pd(ii) is added into the system to form ternary ion association complexes for the first time, which results in a lower limit of detection and extends the application of HPLC-RRS. The experimental conditions were optimized. In order to investigate the reaction mechanism, the ternary ion association complexes were characterized by ultraviolet-visible spectrophotometry, dynamic light scattering, scanning electron microscopy and density functional theory calculations. In a HAc-NaAc buffer solution (pH = 4.1), a ternary complex of cytokinin : Pd(ii) : EryB (1 : 1 : 2) was formed. The detection limits (S/N = 3) of BA, KT, and ZT were 0.9, 1.5 and 2.3 ng mL-1, respectively. In addition, this method was applied for the simultaneous detection of cytokinins in real samples with satisfactory results.

Inhalation exposure to volatile organic compounds in the printing industry.

This study reports on the occupational inhalation exposure to VOCs of workers in the Kuwaiti printing industry. Using the evacuated canister methodology, we targeted 72 VOCs in three printeries and compared the concentrations to previous reports and relevant occupational exposure levels (OELs). We found that recent efforts in the printing industry to reduce VOC usage had been successful, as concentrations of key hazardous VOCs were substantially lower than anticipated. On the other hand, nearly all target VOCs were found. Non-production areas were sampled along with the offset printing areas, another strength of this study, and revealed exposures to hazardous VOCs among administers and digital printer and CTP operators. Exposure to ototoxic VOCs amounted to 1-3% of the OEL, consisting mostly of ethylbenzene, which was likely in use in two of the study printeries. Exposure to carcinogenic or probably carcinogenic VOCs was 15-20% of the OEL at four locations across the three printeries, consisting mostly of vinyl chloride and benzyl chloride. Vinyl chloride VOC was partially sourced from outdoors, but was also likely used inside the study printeries. Interestingly, concentrations of vinyl chloride were similar in most sampling locations to that of CFC-114, a CFC banned by the Montreal Protocol and not commonly used as a refrigerant. This unexpected finding suggests further study is warranted to identify the use of these VOCs in printeries. Exposure to hazardous VOCs up to nearly 50% of the OEL, consisting largely of bromoform and vinyl chloride. Bromoform was found in all the study printeries, sourced partially from outdoor air. The higher concentrations found inside the study printeries likely resulted from the use of the desalinated water for washing. This finding raises of emissions from sources other than blanket washes, and inks, etc. adding to the total VOC load in printery indoor air. Implications: Results from this study indicate that efforts to reduce worker exposure to VOCs particularly dangerous to human health in recent years have been successful, but there is still much to be done to protect workers. Exposures to ototoxic and carcinogenic VOCs were identified, among both production and non-production workers. Unexpected findings included the apparent use in printing activities of the carcinogen vinyl chloride and CFC-114, banned under the Montreal Protocol. Observed lapses in safety procedures included failure to utilize ventilation systems and closing doors between work areas, indicating management and worker education should remain a priority.

Cauliflower-like resin microspheres with tuneable surface roughness as solid-phase extraction adsorbent for efficient extraction and determination of plant growth regulators in cucumbers.

New cauliflower-like phloroglucinol-glyoxylic acid resin microspheres (PGRMs) with controllable diameters and tuneable surface roughness were prepared using a one-step environmentally-friendly method without a catalyst. The PGRMs obtained exhibited a rough surface, narrow size distribution, and excellent adsorption capacity for polar compounds. The PGRMs were employed as an adsorbent for solid phase extraction (SPE) of kinetin (KT) and 6-benzyladenine (6-BA) in cucumbers and demonstrated better extraction recoveries and purification efficiency than phloroglucin-formaldehyde resin and common commercial adsorbents. Our PGRMs-SPE-HPLC method showed good linearity (r ≥ 0.9997) ranging from 0.04 to 4.00 µg/g for KT and 6-BA, and recoveries at three spiked concentration ranged from 77.8% to 104.4% with RSDs ≤ 6.8%. This PGRMs-SPE-HPLC method was applied successfully to determine of KT and 6-BA in cucumbers.

Polyhedral oligomeric silsesquioxane grafted silica-based core-shell microspheres for reversed-phase high-performance liquid chromatography.

Polyhedral oligomeric silsesquioxane (POSS) was used to modify spherical silica to fabricate core-shell POSS@SiO2 microspheres. The material was characterized by Fourier transform infrared experiments, scanning electron microscopy, thermogravimetric analysis and elemental analysis. The material was also used as a stationary phase for HPLC separation. The POSS@SiO2 column exhibits a reverse-phase liquid chromatography (RPLC) retention mechanism. The column efficiency of alkylbenzenes reaches 67,200 plates·m-1. The POSS@SiO2 column was also utilized for separation of basic anilines and polycyclic aromatic hydrocarbons. Compared with the commercial C8 column, the POSS@SiO2 column exhibits enhanced separation selectivity. The column was also used for the separation of synthetic cytokinins 6-benzylaminopurine and 6-furfurylaminopurine in bean sprout after extraction. In addition, the methacrylate groups on the surface of the POSS@SiO2 microsphere were further functionalized so as to facilitate the fabrication of versatile stationary phases with various separation mechanisms. Graphical abstract Schematic presentation of the two-step fabrication of polyhedral oligomeric silsesquioxane grafted silica-based (POSS@SiO2) core-shell microspheres for use in HPLC.

HS-SPME analysis of the volatiles profile of water celery (Apium nodiflorum), a wild vegetable with increasing culinary interest.

Water celery (Apium nodiflorum) is a wild plant traditionally harvested in some Mediterranean areas for being consumed raw. Despite its appreciated organoleptic properties, the aromatic profile of the fresh vegetable remains to be studied. In the present study, volatile compounds from five wild populations were extracted by the headspace-solid phase microextraction technique, analysed by gas cromatography-mass spectrometry, and compared to related crops. The wild species had a high number of aromatic compounds. It was rich in monoterpenes (49.2%), sesquiterpenes (39.4%) and phenylpropanoids (9.6%), with quantitative differences among populations, in absolute terms and relative abundance. On average, germacrene D was the main compound (16.6%), followed by allo-ocimene (11.9%) and limonene (11.1%). Only in one population, the levels of limonene were greater than those of germacrene D. Among phenylpropanoids, dillapiol displayed the highest levels, and co-occurred with myristicin in all populations except one. These differences may have a genetic component, which would indicate the possibility of establishing selection programmes for the development of water celery as a crop adapted to different market preferences. On the other hand, comparison with related crops revealed some similarities among individual volatiles present in the different crops, which would be responsible of the common aroma notes. However, water celery displayed a unique profile, which was in addition quantitatively richer than others. Thus, this differentiation may promote the use of water celery as a new crop.

Essential oil composition of aerial parts from Algerian Anacyclus monanthos subsp. cyrtolepidioides (Pomel) Humphries.

The chemical composition of the essential oil from the aerial parts of Anacyclus monanthos subsp. cyrtolepidioides (Pomel) Humphries (Asteraceae) growing in a semi-arid region of Algeria was investigated for the first time. The essential oil was obtained by hydrodistillation and fully characterized by gas chromatography-mass spectrometry (GC-MS). A total of 97 compounds were identified. The essential oil was found to be rich in trans-chrysanthenyl acetate (9.8 ± 2.0%), (E)-ß-farnesene (7.4 ± 1.5%), germacrene D (6.9 ± 1.3%) and myristicin (4.8 ± 0.8%).

De novo design of a fluorescence-activating ß-barrel.

The regular arrangements of ß-strands around a central axis in ß-barrels and of α-helices in coiled coils contrast with the irregular tertiary structures of most globular proteins, and have fascinated structural biologists since they were first discovered. Simple parametric models have been used to design a wide range of α-helical coiled-coil structures, but to date there has been no success with ß-barrels. Here we show that accurate de novo design of ß-barrels requires considerable symmetry-breaking to achieve continuous hydrogen-bond connectivity and eliminate backbone strain. We then build ensembles of ß-barrel backbone models with cavity shapes that match the fluorogenic compound DFHBI, and use a hierarchical grid-based search method to simultaneously optimize the rigid-body placement of DFHBI in these cavities and the identities of the surrounding amino acids to achieve high shape and chemical complementarity. The designs have high structural accuracy and bind and fluorescently activate DFHBI in vitro and in Escherichia coli, yeast and mammalian cells. This de novo design of small-molecule binding activity, using backbones custom-built to bind the ligand, should enable the design of increasingly sophisticated ligand-binding proteins, sensors and catalysts that are not limited by the backbone geometries available in known protein structures.

Surface-enhanced Raman spectroscopic analysis of N6-benzylaminopurine residue quantity in sprouts with gold nanoparticles.

A rapid and quantitative method for the determination of N6-Benzylademine (N6-BA) was established through the application of surface-enhanced Raman spectroscopy (SERS). The Raman peak intensities of N6-BA at 1002 cm-1 positively correlated to N6-BA concentrations in sprout extracts. The R2 reached 0.99, and RSDs calculated below 10% at the concentration range of 0.1 ∼5µg mL-1. The average recoveries were 80.0% ∼ 98.2% for blank samples intentionally contaminated at differing levels of 0.04, 0.4, and 1 µg g-1. The whole procedure, including sample preparation and SERS detection, did not exceed 30 min for a set of 6 samples. This study indicates that SERS is a promising technique for rapid tracing analysis and on-site testing of N6-BA.

A new composite of graphene and molecularly imprinted polymer based on ionic liquids as functional monomer and cross-linker for electrochemical sensing 6-benzylaminopurine.

Molecularly imprinted polymers prepared using traditional functional monomers and cross-linkers exhibit slow binding kinetics, low electrocatalytic activity and adsorption capacity. Herein, we report a new composite of ionic liquid-based graphene and molecularly imprinted polymer (IL-GR-MIP) with high electrocatalytic activity and adsorption capacity to construct an effective electrochemical sensor for 6-benzylaminopurine (6-BAP). Our objective was to enhance the efficiency of the sensor by incorporating more IL in the MIP framework. We synthesized IL-GR-MIP using ionic liquid 1-vinyl-3-butylimidazolium tetrafluoroborate (IL1) as functional monomer, ionic liquid 1,4-butanediyl-3,3'-bis-l-vinylimidazolium dibromide (IL2) as cross-linker, 6-BAP as template, and GR as supporter. IL-GR-MIP was characterized by Fourier transform infrared spectroscopy, thermal gravimetric analysis, Raman spectroscopy, X-ray photoelectron spectroscopy, and scanning electron microscope. Compared with GR-MIP composites based on methacrylic acid or IL1 as functional monomer, N, N'-methylenebisacrylamide and ethylene glycol dimethacrylate as cross-linker, the IL-GR-MIP (prepared with ionic liquids as functional monomer and cross-linker) sensor exhibited highest peak current for 6-BAP. The results indicate the ability of IL2 as cross-linker to enhance electrocatalytic activity and adsorption capacity for 6-BAP of IL-GR-MIP. Under the optimized conditions, the peak current of IL-GR-MIP sensor was linear to 6-BAP concentration in the range of 0.5-50 µM with a detection limit of 0.2 µM (S/N = 3). The IL-GR-MIP sensor exhibited good selectivity with the anti-interference ability of 1000-fold ascorbic acid in 6-BAP determination. Furthermore, we demonstrated practical applicability of IL-GR-MIP sensor in detecting 6-BAP in real samples with satisfactory results.

Foragers of sympatric Asian honey bee species intercept competitor signals by avoiding benzyl acetate from Apis cerana alarm pheromone.

While foraging, animals can form inter- and intraspecific social signalling networks to avoid similar predators. We report here that foragers of different native Asian honey bee species can detect and use a specialized alarm pheromone component, benzyl acetate (BA), to avoid danger. We analysed the volatile alarm pheromone produced by attacked workers of the most abundant native Asian honey bee, Apis cerana and tested the responses of other bee species to these alarm signals. As compared to nest guards, A. cerana foragers produced 3.38 fold higher levels of BA. In foragers, BA and (E)-dec-2-en-1-yl acetate (DA) generated the strongest antennal electrophysiological responses. BA was also the only compound that alerted flying foragers and inhibited A. cerana foraging. BA thereby decreased A. cerana foraging for risky sites. Interestingly, although BA occurs only in trace amounts and is nearly absent in sympatric honeybee species (respectively only 0.07% and 0.44% as much in A. dorsata and A. florea), these floral generalists detected and avoided BA as strongly as they did to their own alarm pheromone on natural inflorescences. These results demonstrate that competing pollinators can take advantage of alarm signal information provided by other species.

Ionic Liquid-Hybrid Molecularly Imprinted Material-Filter Solid-Phase Extraction Coupled with HPLC for Determination of 6-Benzyladenine and 4-Chlorophenoxyacetic Acid in Bean Sprouts.

A new method involving ionic liquid-hybrid molecularly imprinted material-filter solid-phase extraction coupled to high-performance liquid chromatography (IL-HIM-FSPE-HPLC) was developed for the simultaneous isolation and determination of 6-benzyladenine (6-BA) and 4-chlorophenoxyacetic acid (4-CPA) in bean sprouts. Sample preconcentration was performed using a modified filter, with the new IL-HIM as the adsorbent, which shows double adsorption. The first adsorption involves special recognition of molecular imprinting, and the second involves ion exchange and electrostatic attraction caused by the ionic liquid. This method combines the advantages of ionic liquids, hybrid materials, and molecularly imprinted polymers and was successfully applied to determine 6-BA and 4-CPA in bean sprouts. The adsorption of 6-BA to IL-HIM is based on selective imprinted recognition, whereas the adsorption of 4-CPA is mainly dependent on ion-exchange interactions.

Essential oil constituents and antimicrobial activity of Pycnocycla bashagardiana Mozaff. from Iran.

Pycnocycla bashagardiana is a rare endemic and endangered species that has been used in folkloric medicine in Southern Iran. This study aimed to evaluate the essential oil constituents and antimicrobial activity of wild and cultivated p. bashagardiana. The aerial parts of wild and cultivated plants were collected from two provinces of Iran. The essential oil was isolated by hydrodistillation and analyzed by a combination of capillary GC and GC-MS. The main components in wild plants were myristicin (39.12%), (E)-ß-ocimene (21.97%), sabinene (15.0%) and cis-iso-miristicin (2.67%) and in cultivated plants, (E)-ß-ocimene (55.40%), myristicin (18.27%), (Z)-ß-ocimene (12.47%) and cis-iso-miristicin (2.94%) were the main constituents in essential oil. The in vitro antimicrobial activity of the essential oil of P. bashagardiana were studied against Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli and Candida albicans for the first time. The results showed that the oil exhibited strong antimicrobial activity against all the tested pathogens.

[Determination of 4 kinds of plant growth regulator in bean sprout by solid phase extraction column coupled with ultra-high performance liquid chromatography].

OBJECTIVE: A method for the determination of 6-benzylaminopurine( 6-BAP), isopentennyladenine( z-IP), 4-fluorophenoxyacetic acid( 4-FPA), 4-chlorophenoxyacetic acid( 4-CPA) in bean sprout was developed using solid phase extraction column with ultra-high performance liquid chromatography. METHODS: The sample was extracted by acetonitrile,dehydrated by salt,then centrifugation,and purified by PXC/PWA solid phase extraction column. The chromatographic analysis was carried out on C18 chromatographic column( 100 mm ×2. 1 mm,1. 8 µm),acetonitrile and sodium dihydrogen phosphate for gradient elution,diode array detector for detection,and quantified with external standard method. RESULTS: The calibration curves showed good linearity in the range of 0. 25-25 µg/mL( 6-BAP and z-IP) and 0. 50-50 µg/mL( 4-FPA and 4-CPA) with correlation coefficients greater than 0. 999. Three levels spiked recoveries were carried out using blank bean sprout extraction as substrate,the recoveries ranged from70. 0% to 96. 4%,and the relative standard deviations( RSDs) ranged from 2. 84% to12. 10%( n = 6). The qualitative limits of detections were 0. 0082-0. 075 mg/kg and the quantitative limits were 0. 027-0. 25 mg/kg for the 4 PGRs. CONCLUSION: The method is simple and easy to operate using solid phase extraction column coupled,simultaneous determination of 4 PGRs by ultra-high performance liquid chromatography,can ensure the corresponding accuracy,sensitivity and precision.