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.

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.

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.

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.

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.

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).

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.

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.

Cytogeography of essential oil chemotypes of Eremophila longifolia F. Muell (Scrophulariaceae).

Previous studies have demonstrated that the widely distributed desert plant Eremophila longifolia has at least six geographically defined essential oil chemotypes. The focus of the present study is to extend and enhance information concerning known chemotypes and to investigate the involvement of cell nuclei ploidy in this variation. Forty field collected specimens of E. longifolia were taken from most of the mainland states of Australia then subjected to hydrodistillation to produce essential oils, which were then chemically characterised. Ploidy was determined using relative fluorescence of cell nuclei stained with propidium iodide, measured in a flow cytometer. Using principal component analysis (PCA), at least three essential oil chemotypes, in addition to the six already described, were identified in the present study. Previously described high yielding essential oil chemotypes were also characterised in terms of diploidy. For the first time diploid populations were identified in New South Wales, correlating with high yielding isomenthone/menthone and karahanaenone chemotypes. Furthermore, the separate diploid population previously described from Western Australia was demonstrated to be the safrole/methyl eugenol type, which is restricted to a small geographic range in far north-west Western Australia (Murchison District). All other chemotypes were shown to be tetraploid, including apparently randomly emerging individuals, representative of chemotypes producing low yields of isomenthone/menthone and karahanaenone similar in composition to the high yielding diploid types.

Chemical composition and major odor-active compounds of essential oil from PINELLIA TUBER (dried rhizome of Pinellia ternata) as crude drug.

The chemical composition of the essential oil from PINELLIA TUBER (Japanese name: Hange), the dried rhizome of Pinellia ternata, was investigated by capillary gas chromatography (GC) and GC-mass spectrometry (MS) analyses. The oil obtained from Pinellia tuber was revealed the presence of 114 compounds, representing 90.6% of the total oil identified. This colorless oil had a spicy and woody odor. The main components of the oil were ß-cubebene (8.8%), atractylon (7.8%), methyl eugenol (6.2%), and δ-cadinene (5.3%). Fifteen major odor-active compounds were identified in the essential oil from PINELLIA TUBER by the GC-olfactometry (GC-O) and aroma extract dilution analysis (AEDA). Among these, safrole (spicy) and ß-vatirenene (woody) showed the highest flavor dilution (FD) factor (128), followed by paeonol (FD = 64; woody, spicy), α-humulene (FD = 64; woody), and ß-phenylnaphthalene (FD = 64; spicy).

Chemical composition and bioactivity of Piper auritum and P. multiplinervium essential oils against the red flour beetle, Tribolium castaneum (Herbst) / Composición química y bioactividad de los aceites esenciales de Piper auritum y P. multiplinervium contra el escarabajo rojo de la harina, Tribolium castaneum (Herbst)

Stored grain insects have shown resistance to synthetic insecticides, fact that has promoted the use of vegetable species for integrated pest management. Piper auritum and P. multiplinervium are two plants from the Piperaceae family present in the department of Chocó, Colombia, one of the most important hot spots of biodiversity in the world. This study was conducted to determine the repellent activity and toxicity of essential oils (EOs) isolated from these plants against Tribolium castaneum, using the area preference and contact toxicity methods, respectively. P. auritum EO presented greater repellency than P. multiplinervium, the first showed 100 percent lethality at minimum tested exposure period (24 h) whereas the second reached 16 percent at 72 h. EOs were analyzed by gas chromatography-mass spectrometry. P. auritum major components were safrole (93.2 percent) and miristicine (4.3 percent), whereas for P. multiplinervium were beta-elemene (9.0 percent), trans-beta-caryophyllene (5.3 percent) and caryophyllene oxide (4.1 percent). It is speculated that the repellent effect of P. auritum may be related to its safrole content, a known repellent. These results evidenced Piper species could be used for development of repellents against T. castaneum.

Los insectos de los granos almacenados han mostrado resistencia a los insecticidas sintéticos, hecho que ha promovido el uso de especies vegetales para el manejo integrado de plagas. Piper auritum y P. multiplinervium son dos plantas de la familia Piperaceae presentes en el departamento del Chocó, Colombia, uno de los puntos de biodiversidad más importantes del mundo. En este estudio fue determinada la actividad repelente y toxicidad de los aceites esenciales (AE) aislados de estas plantas contra Tribolium castaneum, utilizando el método de área de preferencia y toxicidad por contacto, respectivamente. El AE de P. auritum presentó mayor repelencia que el de P. multiplinervium, el primero mostró 100 percent de letalidad al menor tiempo de exposición (24 h), mientras que el segundo alcanzó el 16 percent a las 72 h. Los AEs fueron analizados por cromatografía de gases-espectrometría de masas. Los componentes principales de P. auritum fueron safrol (93.2 percent) y miristicina (4.3 percent), mientras que para P. multiplinervium fueron beta-elemene (9.0 percent), trans-beta-cariofileno (5.3 percent) y óxido de cariofileno (4.1 percent). Se cree que el efecto repelente de P. auritum puede estar relacionado con su contenido de safrol, un repelente conocido. Estos resultados evidencian que las especies de Piper podrían ser utilizadas para el desarrollo de repelentes contra T. castaneum.

Quantitation of tr-cinnamaldehyde, safrole and myristicin in cola-flavoured soft drinks to improve the assessment of their dietary exposure.

Quantitation of tr-cinnamaldehyde, safrole and myristicin was carried out in 70 samples of cola-flavoured soft drinks purchased in eight European countries with the purpose of assessing the variability in the levels of these substances. Results indicated a limited variability in the content of the three substances: the ratio between the 90th and the 10th percentile concentration amounted to 21, 6 and 13 for tr-cinnamaldehyde, safrole and myristicin, respectively. The uncertainty in the assessment of dietary exposure to these substances due to the variability of their level in cola-flavoured drinks was low. Based on these analytical data and on refined food consumption data, estimates of exposure to safrole associated to cola drink consumption, along with Margin of Exposure (MOE) values, were obtained. For high consumers of cola-flavoured soft drinks in certain age groups, within some European countries, MOE values lower than 10,000 resulted, MOE values of 10,000 or higher having been stated by the EFSA as a quantitative criterion to identify low concern from a public health point of view and low priority for risk management actions. The lowest MOE values, from 1900 to 3000, were observed for children and teen agers in the United Kingdom and Ireland.

[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.

[Determination of aldicarb and its metabolites in peanuts by high performance liquid chromatography-linear ion trap triple stage mass spectrometry].

A high performance liquid chromatography-linear ion trap triple stage mass spectrometry (HPLC-IT/MS3) method was established to detect aldicarb, aldicarb sulfoxide, and aldicarb sulfone in peanuts. The samples were extracted by acetonitrile saturated with cyclohexane, followed by clean-up with gel permeation chromatography (GPC). The determination was performed using HPLC-IT/MS3 for the identification and quantification of the compounds. The separation was carried on a Capcell PAK CR column with gradient elution using 5 mmol/L acetic acid/ammonium acetate/acetonitrile as mobile phase. The ionization of molecules was performed by electrospray mode. Selective reaction monitoring (SRM) was the acquisition mode used for the monitoring of MS3 transitions for each compound using aldicarb-d3 as internal standard for three analytes. Matrix effects were evaluated by comparing the recovery of matrix-matched and solvent-based calibration curves. The calibration graphs were linear in the ranges of 10 - 500 microg/L and the detection limits ranged from 4 to 5 microg/kg. The average recoveries ranged between 81.5% and 115% at three different spiked levels (10, 20 and 40 microg/kg). Satisfactory results were obtained in the determination of real peanut samples by this method.

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.

Chemical variation of the essential oil of Prangos uloptera DC. at different stages of growth.

In the current investigation, the influence of the plant growth stages (prior to emergence of flower heads or vegetative, anthesis and fruiting) on the essential oil content and composition in the aerial parts of Prangos uloptera DC. was studied. Quantitative and qualitative differences were found among oil of aerial parts at three phenological stages. The principle compounds of the oil before emergence of flower heads were saferole (21.6%) and α-pinene (20%), while the oil at anthesis was composed of α-bisabolol (30.5%), saferole (19.11%) and (+)-spathulenol (12.9%). The oil at fruiting stage displayed γ-terpinene (35.5%) and trans-anethole (23.5%) as major constituents. These findings may confer valuable information regarding chemical composition of P. uloptera DC., which could be beneficial in the fields of herbal medicine.

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.

Reduction of safrole and methyleugenol in Asari radix et rhizoma by decoction.

INTRODUCTION: Asari radix et rhizoma (Xixin, Manchurian Wildginger, Asarum spp) is a herbal drug commonly used as an ingredient in traditional Chinese medicine (TCM). Many species of Asarum contain safrole and methyleugenol as the main components of their volatile oils. However, toxicological studies have shown that safrole and methyleugenol may be a hepatocarcinogen and/or genotoxic leading to concerns regarding the habitual consumption of this herbal drug. MATERIALS AND METHODS: An HPLC method was established to assess the levels of safrole and methyleugenol in five batches of Asari radix et rhizoma and two TCM formulae containing this herbal drug as an ingredient. Analyses showed that the content of safrole in the dried herbal drugs tested ranged from 0.14-2.78 mg/ g whilst the content of methyleugenol ranged from 1.94-16.04 mg/g. RESULTS: The present study demonstrated that following a 1-hour decoction, the amount of safrole was decreased by more than 92% resulting in the equivalent of no more than 0.20 mg/g safrole remaining in the aqueous extract. Similarly, the content of methyleugenol was decreased to the equivalent of 0.30-2.70 mg/g. Furthermore, both TCM formulae, after decoction, showed negligible amounts of safrole (maximum, the equivalent of 0.06 mg/ g), and only 1.38-2.71 mg/g of methyleugenol. CONCLUSIONS: The present study shows that a decoction procedure, similar to that traditionally used for Chinese herbal preparations, is able to effectively reduce the amount of safrole and methyleugenol effectively. Such a reduction in the content of safrole should be acceptable for therapeutic use.

Investigation of sulfur forms and transformation during the co-combustion of sewage sludge and coal using X-ray photoelectron spectroscopy.

X-ray photoelectron spectroscopy was used to investigate the characteristics and evolution of sulfur (S) in mixtures of bituminous coal and sewage sludge (SS) and their chars during isothermal combustion. Five groups of mixtures with SS content of 0%, 10%, 20%, 30% and 100%, were examined at different burn-off ratios (beta) of 0, 30%, 50%, 70% and 100%. The S in the coal mainly exist as the forms of mercaptan (S1), sulfide (S2), thiophene (S3), sulfoxide (S4), sulfone (S5) and sulfate (S6). During the coal combustion process, the content of S1 and S2 decreased, while that of S3 and S5 increased in the early stage and decreased in the late stage. The S4 content increased throughout the entire process of combustion. Small amount of S6 was detected, showing a fluctuated pattern. The trend of S1, S2, S5 and S6 in SS was alike with that in coal, whereas S4 decreased at the end of combustion. The changing process of S3 in SS was opposite to that of coal, while the composition of S in the mixtures resulted from the mixing of coal and SS. The transformation of each functional group during co-combustion were correlated with their transformation characteristics during the mono-combustion of coal and SS, and no obvious interaction was observed, which demonstrated that the coal-origin and SS-origin sulfur in mixtures kept their own characteristics in the combustion. SS may accumulate on the solid surface as alpha increase, resulting its significant influence on the evolution of each form of S. When alpha was low, most of the S-contained functional groups presented the characteristics of coal. The percentage of coal-origin functional groups declined as alpha increased. The transforming trends of most functional groups were similar with that of SS when alpha reached 30%.

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.