Method Validation and Evaluation of Safrole Persistence in Cowpea Beans Using Headspace Solid-Phase Microextraction and Gas Chromatography.

Bioinsecticides are regarded as important alternatives for controlling agricultural pests. However, few studies have determined the persistence of these compounds in stored grains. This study aimed at optimizing and validating a fast and effective method for extraction and quantification of residues of safrole (the main component of Piper hispidinervum essential oil) in cowpea beans. It also sought to assess the persistence of this substance in the grains treated by contact and fumigation. The proposed method used headspace solid-phase microextraction (HS-SPME) and gas chromatography with a flame ionization detector (GC/FID). Factors such as temperature, extraction time and type of fiber were assessed to maximize the performance of the extraction technique. The performance of the method was appraised via the parameters selectivity, linearity, limit of detection (LOD), limit of quantification (LOQ), precision, and accuracy. The LOD and LOQ of safrole were 0.0057 and 0.019 µg kg-1, respectively and the determination coefficient (R2) was >0.99. The relative recovery ranged from 99.26 to 104.85, with a coefficient of variation <15%. The validated method was applied to assess the persistence of safrole residue in grains, where concentrations ranged from 1.095 to 0.052 µg kg-1 (contact) and from 2.16 to 0.12 µg kg -1 (fumigation). The levels measured up from the fifth day represented less than 1% of the initial concentration, proving that safrole have low persistence in cowpea beans, thus being safe for bioinsecticide use. Thus, this work is relevant not only for the extraction method developed, but also for the possible use of a natural insecticide in pest management in stored grains.

Rapid Method for The Gas Chromatographic Quantitative Analysis to Determinate Safrole in Commercial Essential Oils.

Safrole is a well-known carcinogenic agent that is present in camphor trees. In this study, a gas chromatographic method was established to quantitate the levels of safrole in essential oils using n-decyl alcohol as an internal standard. The method used a nonpolar column and was able to detect concentrations of safrole as low as 5 µg/ml in the samples. Following addition of 2-10 mg of safrole into 1 g of essential oil extracted from Stout Camphor wood (Cinnamomum kanehirai Hayata) or 1-10 mg of safrole into 1 g of essential oil extracted from Small-flower Camphor wood (Cinnamomum micranthum Hayat), the recovery rates of safrole were determined. With direct injection of samples into the gas chromatograph, the results showed that the recovery was more than 96.1%, with a coefficient of variation below 5.6%. We then analyzed 23 commercially available Stout Camphor and other essential oil samples and found that 21 of them contained safrole in the range of 37.65-355.07 mg/g. In addition, in the heavier essential oil distilled from Small-flower Camphor wood, the safrole level was up to 642.98 mg/g. Our results demonstrated that most camphor essential oils on the market have a carcinogenic potential due to their high safrole levels.

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.

[Genetic diversity and volatile components of Asarum sieboldii in Qin-ba region].

OBJECTIVE: To analyze the genetic diversity and the volatile components of Asarum sieboldii from seven habitats in Qin-ba region. METHODS: The genetic diversity of the herb was analyzed by ISSR (inter simple sequence repeat) markers; The relative content volatile components of the herb were dectected by head space solid-phase microextraction gas chromatogrphy-mass spectrometry (HS-SPME-GC-MS). The contents of the 3 main components were analyzed by steam distillation gas chromatography-mass spectrometry (GC-MS). RESULTS: 57 bands were amplified from 7 populations by 6 reliable primers, 51 of which were polymorphic (89.47% of the total). The cluster analysis presented that these resources were divided into two main groups. There were differences in the chemical components and the contents of Asarum sieboldii from the 6 wild habitats. Except for some same components, many unique components were identified in them respectively. In addition, some components could be detected only in some populations which had smaller genetic distance. CONCLUSION: Cluster analysis shows no direct correlation between genetic distance and geographic distance of Asarum sieboldii in Qin-ba region. The accumulations of some volatile components of Asarum sieboldii are possibly related to genetic diversity.

Chemical compositions, antioxidant and antimicrobial activities of essential oils of Piper caninum Blume.

Chemical composition, antioxidant and antimicrobial activities of the fresh leaves and stems oils of Piper caninum were investigated. A total of forty eight constituents were identified in the leaves (77.9%) and stems (87.0%) oil which were characterized by high proportions of phenylpropanoid, safrole with 17.1% for leaves and 25.5% for stems oil. Antioxidant activities were evaluated by using ß-carotene/linoleic acid bleaching, DPPH radical scavenging and total phenolic content. Stems oil showed the highest inhibitory activity towards lipid peroxidation (114.9 ± 0.9%), compared to BHT (95.5 ± 0.5%), while leaves oil showed significant total phenolic content (27.4 ± 0.5 mg GA/g) equivalent to gallic acid. However, the essential oils showed weak activity towards DPPH free-radical scavenging. Evaluation of antimicrobial activity revealed that both oils exhibited strong activity against all bacteria strains with MIC values in the range 62.5 to 250 µg/mL, but weak activity against fungal strains. These findings suggest that the essential oils can be used as antioxidant and antimicrobial agents for therapeutic, nutraceutical industries and food manufactures.

Feasibility study on recycled vegetable oil waste and recycled polyethylene for the modification of aged asphalt.

The application of reclaimed asphalt pavement has been widely encouraged due to its significant economic and environmental benefits. However, it is necessary to add rejuvenators to ensure its performance. Currently, bio-oil-based regenerants have attracted attention owing to their advantages of renewability and cost savings. The purpose of this paper is to study the use of recycled vegetable oil waste (R-oil) and recycled polyethylene particles for the regeneration and modification of aged asphalt. Physical, rheological, and chemical tests were used to figure out their influence on the pavement performance of aged asphalt. According to the physical test indices (penetration, softening point, and ductility), the performance of the rejuvenated asphalt was better than that of virgin asphalt. The workability and low-temperature performance of the rejuvenated asphalt were basically the same as those of virgin asphalt, and its fatigue and high-temperature performance were better. Infrared spectroscopy showed that R-oil diluted the high-polarity sulfoxide base of aged asphalt. Gel permeation chromatography showed that its molecular weight dispersion was better than that of aged asphalt. Therefore, R-oil and polyethylene can improve the pavement performance and chemical properties of aged asphalt.

Abuse of nutmeg seeds: Detectable by means of liquid chromatography-mass spectrometry techniques?

Numerous case reports of intoxications with nutmeg seeds (Myristica fragrans, Houtt.) can be found in literature often following their abuse, as psychotropic effects were described after ingestions of large doses. The successful detection of the main ingredients of the nutmeg seeds essential oil elemicin, myristicin, and safrole, as well as their metabolites in human urine by gas chromatography coupled to mass spectrometry (GC-MS) was already described. The aim of this study was to investigate the detectability of the main ingredients of nutmeg seeds and their metabolites in human blood and urine samples using liquid chromatography coupled to linear ion trap mass spectrometry (LC-LIT-MSn ) and liquid chromatography coupled to high-resolution mass spectrometry (LC-HRMS/MS) after nutmeg seed abuse. Sample material of three individuals was retrospectively investigated after a systematic screening approach indicated an intoxication with nutmeg seeds as a likely cause of symptoms. Metabolic patterns in plasma and urine using GC-MS were comparable with those described in earlier publications. Investigations using hyphenated liquid chromatography techniques lead to the detection of myristicin and safrole, as well as further metabolites not described using GC-MS and revealed sulfation as an additional Phase II metabolic pathway. These results might help to detect or confirm future intoxications with nutmeg seeds by using LC-MS techniques.

Biological evaluation of Safrole oil and Safrole oil Nanoemulgel as antioxidant, antidiabetic, antibacterial, antifungal and anticancer.

BACKGROUND: Safrole is a natural compound extracted from various plants, and has shown various biological activities. The current study aimed to investigate the antioxidant, antidiabetic, antimicrobial, and anticancer activity of safrole oil and to study the influence of safrole nanoemulgel on these activities. METHODS: The antioxidant and antidiabetic in-vitro assays were conducted using standard biomedical methods. The safrole oil nanoemulgel was developed using a self-emulsifying technique. Then the antimicrobial activity of the safrole oil and safrole nanoemulgel were performed on different microbial species, and cytotoxicity was determined against Hep3B cancer cell lines using the MTS assay. RESULTS: Safrole oil showed moderate antioxidant activity compared with standard Trolox, with IC50 value 50.28 ± 0.44 and 1.55 ± 0.32 µg/ml, respectively. Moreover, it had potent α-amylase inhibitory activity (IC50 11.36 ± 0.67 µg/ml) compared with Acarbose (IC50 value 5.88 ± 0.63). The safrole nanoemulgel had pseudo-plastic behaviour, droplet sizes below 200 nm, a polydispersity index (PDI) below 0.3, and a zeta potential of less than - 30 mV. Safrole oil has potential antimicrobial and anticancer activities, and these activities were improved with safrole nanoemulgel. CONCLUSION: The safrole oil may be applied for the prevention and treatment of oxidative stress, diabetes, different microbial species and cancer, and these activities could be improved by nano-carriers.

Pressurized liquid extraction and GC-MS analysis for simultaneous determination of seven components in Cinnamomum cassia and the effect of sample preparation.

A pressurized liquid extraction and GC-MS method was developed for simultaneous quantitative determination of the seven components, including cinnamaldehyde, copaene, cinnamic acid, coumarin, 2-methoxycinnamaldehyde, 2-methoxycinnamic acid and safrole in Cinnamomum cassia. The results showed that methanol and ethanol was not available for extraction of cinnamaldehyde and 2-methoxycinnamaldehyde due to aldol reaction. The developed method was validated to be sensitive, accurate and simple, and was successfully employed for the analysis of 15 samples of C. cassia. The contents of the investigated components were significantly variant and cinnamaldehyde is the most abundant compound, but safrole was not detected in all 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.

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).

Safrole-DNA adduct in hepatocellular carcinoma associated with betel quid chewing.

Betel quid chewing, which contributes high concentration of safrole in saliva, is a popular oral habit in Taiwan. Safrole is a documented rodent hepatocarcinogen, yet its hepatocarcinogenic potential in human is not known. Here, we used LC/ESI-ITMS(n) and LC/QTOF-MS confirmed safrole-dGMP as reference standard to detect the safrole-DNA adduct in hepatic tissues from HBsAg-/HCV-seronegative hepatocellular carcinoma patients by (32)P-postlabeling. We first synthesized and confirmed safrole-dGMP by LC/MS. Two isomeric safrole-dGMPs were characterized as N(2)-(trans-isosafrol-3'-yl) deoxyguanosine and N(2)-(safrol-1'-yl) deoxyguanosine. This technique was able to detect hepatic safrole-DNA adduct in mice that were treated with safrole but not sensitive enough to detect safrole-DNA adduct in human samples. Using the nuclease P1 version of the (32)P-postlabeling technique, we detected the presence of safrole-DNA adduct in two out of 28 hepatic tissues from hepatocellular carcinoma patients, and only these two patients had a history of betel quid chewing lasting more than 10 years. From co-chromatography with the mass confirmed safrole-dGMPs, this safrole-DNA adduct was identified as N(2)-(trans-isosafrol-3'-yl) deoxyguanosine. These results suggest that betel quid-containing safrole might be involved in the pathogenesis of hepatocellular carcinoma in human beings and LC/MS has the potential to identify DNA adducts in clinical samples.

[Analysis of disulfoton sulfoxide in chingentsuai by pulsed splitless mass spectrometry using programmed temperature vaporization (PTV) inlet].

A peak considered to be due to disulfoton sulfoxide as a metabolite of disulfoton was detected in the analysis of the chingentsuai extraction solution of vegetables by GC-FPD. In the analysis by GC/MS for identification, though the retention time and the mass spectrum were the same as those of the standard, the spectrum was different from MS library data. It appeared that decomposition of disulfoton sulfoxide occurred at the inlet. Therefore, we used a programmed temperature vaporization (PTV) inlet in the pulsed splitless mode to achieve a low inlet temperature and high injection pressure. As a result, the expected mass spectrum of disulfoton sulfoxide was obtained. Under this condition, the peak obtained from chingentsuai was identified as disulfoton sulfoxide. Disulfoton sulfoxide was detected from 2 of 25 chingentsuai samples, at concentrations of 0.66 microg/g and 0.14 microg/g.

[Comparing analysis of components in volatile oils of nutmeg and prepared nutmeg by GC-MS].

OBJECTIVE: To compare components in volatile oils of nutmeg and prepared nutmeg. METHOD: Volatile oil from nutmeg and prepared nutmeg were extracted by vapor distillation. The chemical components in two kinds of volatile oils were determined and indentified by GC-MS. RESULT: The change in quantity and quality of components in volatile oils were observed after processing. 13 new components occurred and 4 components disappeared in volatile oils after processing. The contents of methyleugenol and methylisoeugenol that are active ingredients were increased. The contents of myristicin and safrol that are toxic ingredients in volatile oils were decreased. CONCLUSION: The processing method of nutmeg by soaking with water and roasting with bran is scientific.

A traditional method of Cinnamomum carolinense preparation eliminates safrole from a therapeutic Pohnpean tea.

Cinnamomum carolinense, locally known as madeu, is a tree endemic to the volcanic mountains of the Island of Pohnpei in the Eastern Carolines of the South Pacific. The bark is harvested from trees and brewed to make a medicinal tea and hot beverage that is regularly consumed. Many species of Cinnamomum contain the known hepatocarcinogen safrole, sparking concern regarding habitual consumption of this beverage. HPLC-PDA analysis confirmed the presence of the carcinogen in alcoholic extracts of Cinnamomum carolinense bark shavings (0.435%, w/w), but safrole was not detected in the tea. The limit of detection and limit of quantitation of safrole were determined to be 1.25 and 3.75 microg/mL, respectively. The traditional preparation method, which boils the bark shavings, degrades the safrole.

Determination of safrole, dihydrosafrole, and chloromethyldihydrosafrole in piperonyl butoxide by high-performance liquid chromatography.

An HPLC method for the simultaneous determination of safrole (S), dihydrosafrole (DHS), and chloromethyldihydrosafrole (CM-DHS) in piperonyl butoxide, with fluorimetric detection and elution gradient, is described. Samples with internal standard (piperonyl isobutyrate) are adsorbed on silica cartridges, then eluted with an appropriate solvent. Internal standard, S, DHS, and CMDHS are eluted together and injected into a reversed-phase chromatography column. The concentrations of the three products are calculated from calibration graphs, and linearity and reproducibility are verified. The limit of detection is 2 mg.kg-1. This analytical method allows for control of the synthesis process of piperonyl butoxide and determination of some carcinogenic substances, such as S and DHS.

Quantitation of flavor-related alkenylbenzenes in tobacco smoke particulate by selected ion monitoring gas chromatography-mass spectrometry.

Little is known about the possible health effects associated with inhaling alkenylbenzenes through cigarette smoking, even though these flavor-related compounds have known toxic effects in animals. We developed a rapid and sensitive solid-phase extraction (SPE) method to quantify seven alkenylbenzenes and piperonal in mainstream cigarette smoke particulate. The smoke particulate fraction of a single cigarette was collected on Cambridge filter pads, solvent extracted, concentrated, purified with SPE, and analyzed by selected ion monitoring gas chromatography-mass spectrometry. We positively identified and quantified five alkenylbenzenes compounds (eugenol, isoeugenol, methyleugenol myristicin, and elemicin) and piperonal in the smoke particulate from eight U.S. brands with mean levels (measured in triplicate) ranging from 6.6 to 4210 ng per cigarette. Additionally, complete blocking of nearly invisible ventilation holes in the cigarette filter increased 2- to 7-fold the percent transfer of alkenylbenzenes from tobacco to the particulate fraction of mainstream smoke.

In vitro metabolic products of RWJ-34130, an antiarrythmic agent, in rat liver preparations.

The in vitro metabolism of RWJ-34130, an antiarrhythmic agent, was conducted using rat hepatic 9000 x g supernatant (S9) and microsomes in an NADPH-generating system, and the rat liver perfusion. The 100 and 20 microg ml(-1) concentrations of RWJ-34130 aqueous solution were used for microsomal incubation and liver perfusion, respectively. Unchanged RWJ-34130 (approximately 77-78% of the sample in both S9 and microsomes) plus a major metabolite, RWJ-34130 sulfoxide (20% of the sample in both S9 and microsomes) were profiled, isolated and identified from both hepatic S9 and microsomal incubates (60 min) using HPLC and mass spectrometry (MS), and by comparison to a synthetic RWJ-34130 sulfoxide, which was synthesized by reacting RWJ-34130 with MCPBA (meta-chloroperoxy benzoic acid). No unchanged RWJ-34130 was detected in the 3 h liver perfusate, however, 1-phenyl-2-oxo-pyrrolidine was profiled, isolated and identified as a major hydrolyzed metabolite of liver perfusate. RWJ-34130 is not extensively metabolized in vitro in rat hepatic S9 and microsomes. All HPLC metabolic profiles of hepatic S9 and microsomal samples (30 min, 60 min) were qualitatively and nearly quantitatively identical.

Liquid chromatographic determination of safrole in sassafras-derived herbal products.

A liquid chromatographic (LC) method was developed for determining safrole in herbal products derived from sassafras (Sassafras albidum), as well as related compounds such as isosafrole and dihydrosafrole. The procedure involves solvent extraction and isolation of analyte by reversed-phase LC with UV detection at 235 nm. Safrole is resolved from related compounds and other sample constituents including thymol, a component of thyme. A linear concentration range of 0.003-0.200 mg/mL was obtained for safrole, isosafrole, and dihydrosafrole. Limits of detection (LOD) and quantitation (LOQ) were e0.0015 and 0.0051 micrograms/mL for safrole, 0.0018 and 0.0061 micrograms/mL for isosafrole, and 0.0038 and 0.0125 micrograms/mL for dihydrosafrole, respectively. Intraday relative standard deviations (RSDs) for safrole (n = 5) from various samples ranged from 1.30 to 5.39% at analyte levels of 0.01-1.5%. Safrole contents of 26 samples including root bark powder, leaves, oils, tea concentrate, herbal extract tinctures, and herbal powder capsules ranged from < LOD for most leaf samples to 92.4% for an oil. Recoveries of safrole from fortified samples ranged from 83.6% for an oil to 117.2% for a tincture preparation. Safrole contents of 0.09-4.66 mg/cup were found for brewed teas prepared from sassafras root bark powders and tinctures.

SFE with GC and MS determination of safrole and related allylbenzenes in sassafras teas.

Safrole (4-allyl-1,2-methylenedioxybenzene), a natural plant component of the aromatic oil of sassafras root bark, possesses carcinogenic and mutagenic activity. Legal restrictions have been placed on safrole as a food additive. However, sassafras teas continue to be accessible from health food establishments in the United States. Supercritical fluid extraction (SFE) with gas chromatographic-mass spectrometric (GC-MS) determination is utilized in the formulation of a rapid, accurate, and specific method for the determination of safrole and related allylbenzenes in unbrewed sassafras teas. Samples are extracted in a static-dynamic mode with CO2 at 690 bar and 80 degrees C with methanol as an extractor-added modifier. Levels of safrole exceeding 10,000 mg/kg (1.0%) are commonly encountered. Lesser amounts of other allylbenzenes, including eugenol and 4-allyl-1,2-dimethoxybenzene, are also reported. Recoveries of safrole and related compounds from previously extracted tea samples fortified at 100 and 1000 mg/kg ranged from 96 to 101%.