Determination of volatile organic compounds as biomarkers of lung cancer by SPME-GC-TOF/MS and chemometrics

A method for qualitative and quantitative the determination of concentrations volatile organic compounds (VOCs) in human breath samples using solid phase microextraction (SPME) and gas chromatography-time of flight-mass spectrometry (GC-TOF/MS) has been carried out. They are employed for the preconcentration, separation and analysis of biological samples. The technique to rapid determination compounds present in human air, at the level of parts per billion (ppb) is applied. This method was optimized and evaluated. It showed linear correlations ranging from 0.83 to 234.05 ppb, limit of detection in the range of 0.31 to 0.75 ppb and precision, expressed as the RSD, was less then 10.00%. The unique combination of statistical methods allowed reduce the number of compounds to significant ones only and indicate the potential way to find the biomarkers of the lung cancer. Presented an analytical and statistical methods for detection composition of exhaled air could be applied as a potential non-intrusive tool for screening of lung cancer.     

Biological degradation of diesel fuel in water and soil monitored with solid-phase micro-extraction and GC-MS

Solid-phase micro-extraction (SPME) was used for monitoring degradation of hydrocarbons in diesel-fuel-contaminated (1% v/v) water and soil. Natural soil bacteria with and without external addition of inoculum were used. Directly after a 10-s exposure of the sample, the polydimethylsiloxane fibre was injected into the GC-MS. This method strongly reduced the time of analysis compared to a conventional liquid/liquid extraction. A comparison of SPME and pentane extraction of diesel oil was made and found to be consistent. The degradation of diesel fuel in water was monitored for 10 weeks using SPME. After 5 weeks all hydrocarbons were degraded except for the decahydronaphthalenes. These compounds were approximately 3% of the total hydrocarbons in the diesel oil used and remained undegraded throughout the study although none of the chemical or physical parameters was limiting. In the soil study the degradation of diesel fuel in normal soil was completed after 3 weeks, when the only remaining substances were decahydronaphthalenes. All samples were compared to sterile references to make up for evaporation losses. SPME proved to be a fast and reliable method to monitor changes in concentrations of semi-volatile organic compounds. Received: 23 December 1997 / Received revision: 5 February 1998 / Accepted: 27 February 1998     

Microwave-assisted extraction of homoharringtonine from <Emphasis Type="Italic">Cephalotaxus koreana</Emphasis>

An efficient method using microwave energy was developed to extract homoharringtonine (HHT), an alkaloid component effective in the treatment of leukemia, from Cephalotaxus koreana. The effects of major process parameters on extraction efficiency were also investigated. Using a fixed biomass-to-methanol ratio of 1:8 (w/v), an extraction temperature of 30°C, an extraction time of 20 min, and a stirrer velocity of 250 rpm, a 25% higher yield of HHT was achieved using microwave-assisted extraction (MAE) than using conventional solvent extraction. It was possible to recover more than 95% of the HHT by extracting twice using MAE. In addition, the HHT yield increased as the extraction temperature increased, but the content of plant-derived tar and waxy compounds increased as well. Removal of these impurities and of the pigments from extracts was most effectively accomplished at a mixing ratio of biomass-to-sylopute of 1:1.5 (w/w). The effects of using different organic solvents (acetone, chloroform, ethanol, or methanol) for MAE were also assessed; the highest extraction efficiency was obtained using methanol. When the agitation speed was altered, most of the HHT (> 99%) was recovered at 250 rpm. A mixing ratio of biomass-to-methanol of 1:6 (w/v) at an extraction temperature of 40°C and an extraction time of 10 min proved to be the most effective for reducing processing time and organic solvent usage while enabling nearly all of the HHT (> 99%) to be recovered.     

The study on the chromatographic fingerprint of Fructus xanthii by microwave assisted extraction coupled with GC-MS

The chromatographic fingerprint of Fructus xanthii, a kind of Traditional Chinese Medicines (TCMs), was studied by microwave assisted extraction (MAE) coupled with gas chromatography-mass spectrometry (GC-MS). The optimized conditions of MAE were examined. The method of MAE was evaluated in contrast to heat reflux extraction (HRE) method and by the validation tests of precision and repeatability. The relative standard deviations (RSDs) of retention time and peak area of each component were less than 0.2% and 6%, respectively. Twenty-five different batches of samples collected from different producing areas and the toasting process of F. xanthii were studied. The characteristic differences in the producing areas and the chemical variances in the toasting process were obtained and studied by principal components analysis (PCA) and similarity analysis. The trends of main varying components were attempted to be described in order to specify the related pharmacology and toxicology in crude and toasted samples. The results suggest that the chromatographic fingerprint developed by MAE coupled with GC-MS provides useful information to reveal the quality of F. xanthii and evaluate the quality changes in the producing process.     

Combined microwave-assisted extraction and high-speed counter-current chromatography for separation and purification of xanthones from Garcinia mangostana

A microwave-assisted extraction (MAE) method is presented for the extraction of xanthones, α-mangostin and γ-mangostin from Garcinia mangostana. The MAE conditions including extraction temperature, liquid/solid ratio, extraction time and concentration of ethanol were optimized with an orthogonal test, and 5 g sample was extracted with the optimized conditions. The crude extraction of MAE was successfully isolated and purified by high-speed counter-current chromatography (HSCCC) with a two-phase solvent system composed of petroleum ether-ethyl acetate-methanol-water (0.8:0.8:1:0.6, v/v) in one-step separation. The separation yielded 75 mg of α-mangostin at 98.5% purity, and 16 mg of γ-mangostin at 98.1% purity from 360 mg crude extract of G. mangostana in less than 7h. The purity of the two xanthones was determined by HPLC. Their structures were further identified by ESI-MS, (1)H NMR and (13)C NMR.     

Use of gas chromatography-mass spectrometry/solid phase microextraction for the identification of MVOCs from moldy building materials

Gas chromatography-mass spectrometry/solid phase microextraction (GC-MS/SPME) was applied to identify microbial volatile organic compounds (MVOCs) in water-damaged, mold-infested building materials (gypsum board papers (n=2), mineral wool, and masonite) and in cultivated molds (Aspergillus penicillioides, Stachybotrys chartarum, and Chaetomium globosum). Three SPME fibers (65-microm PDMS-DVB, 75-microm Carboxen-PDMS, and 70-microm Carbowax-stableflex) designed for automated injection were used of which the latter showed best performance. A number of previously reported MVOCs were detected both in the building materials and the cultivated molds. In addition, methyl benzoate was identified both in the S. chartarum and A. penicillioides cultures and in the building materials. SPME combined with GC-MS may be a useful method for the determination of MVOCs emitted from mold-infested building materials.     

Microwave-assisted extraction of glycyrrhizic acid from licorice root

In the present study, a microwave-assisted extraction (MAE) technique has been developed for the extraction of glycyrrhizic acid (GA) from licorice root. Various experimental conditions, such as extraction time, different ethanol and ammonia concentration, liquid/solid ratios, pre-leaching time before MAE and material size for the MAE procedure were investigated to optimize the efficiency of the extraction. Under appropriate MAE conditions, such as extraction times of 4-5min, ethanol concentrations of 50-60% (v/v), ammonia concentrations of 1-2% (v/v) and liquid/solid ratios of 10:1(ml/g), the recovery of GA from licorice root with MAE was equivalent with conventional extraction methods. Those methods include extraction at room temperature (ERT), the traditional Soxhlet extraction, heat reflux extraction and ultrasonic extraction. Due to the considerable savings in time and solvent, MAE was more effective than the conventional methods. This novel method is suitable for fast extraction of GA from licorice root.     

The study of fingerprint characteristics of the emanations from human arm skin using the original sampling system by SPME-GC/MS

An efficient and noninvasive method consisting of an original sampling device, solid phase microextraction (SPME) and gas chromatography-mass spectrometry (GC-MS) was developed to analyze volatile organic emanations from the skin of human arms. The emanations were sampled by SPME connected with the active sampling device for 30 min and transferred into GC-MS immediately for the consequent analysis. The sampling projects for 15 candidates were scheduled in both winter and spring with the same optimized conditions. Thirty-five compounds were finally identified according to various degrees of certainty. Different emission behaviors specified with principal component analysis (PCA) and similar fingerprint characteristics were observed clearly by comparisons of chromatograms of different seasons. Top ten emanations contributing to characteristics in different seasons were attempted to be described using comparisons based on common model strategy. The large amounts of experimental data were all handled by the corresponding chemometrics strategies with the homemade chromatographic data processing system. The results suggest that the analysis based on fingerprint characteristics of human skin emanations could provide useful and important clues to reveal biomarkers among the mixture of human skin emanations.     

Investigation of volatile biomarkers in lung cancer blood using solid-phase microextraction and capillary gas chromatography-mass spectrometry

In the present work, solid-phase microextraction (SPME) and gas chromatography-mass spectrometry (GC-MS) was developed for investigation of lung cancer volatile biomarkers. Headspace SPME conditions (fiber coating, extraction temperature and extraction time) and desorption conditions were optimized and applied to determination of volatiles in human blood. To find the biomarkers of lung cancer, investigation of volatile compounds in lung cancer blood and control was performed by using the present method. Concentrations of hexanal and heptanal in lung cancer blood were found to be much higher than those in control blood. The two molecules of hexanal and heptanal were regarded as biomarkers of lung cancer. By comparison of volatiles in breath and in blood, it is demonstrated that hexanal and heptanal in breath were originated from blood and screening of lung cancer by breath analysis be feasible. These results show that SPME/GC-MS is a simple, rapid and sensitive method very suitable for investigation of volatile disease markers in human blood.     

鱼腥草抑菌有效成分的醇提、鉴定及抑菌效果研究

以大肠埃希菌抑菌效果为指标,采用正交实验的方法对鱼腥草抑菌有效成分的醇提条件进行优化,同时对鱼腥草醇提液进行分极分离,采用HPLC和GC-MS的方法对抑菌有效部的成分进行鉴定,并对该有效部的抑菌效果进行初步研究。实验结果表明鱼腥草抑菌有效成分醇提的最佳条件为醇浓度70%,温度80℃、料液比1?15,提取时间4 h,该条件下所获得的醇提液分极分离所获得的氯仿部抑菌效果最好,GC-MS和HPLC分析表明氯仿部中含有13种酚类物质及少量的鱼腥草素和甲基正壬酮,该有效部对大肠埃希菌(MIC:16.0 mg/mL)、金黄色葡萄球菌(MIC:8.0 mg/mL)、白色念珠菌(MIC:4.0 mg/mL)、黄曲霉(MIC:8.0 mg/mL)以及黑曲霉(MIC:8.0mg/mL)均有较好的抑菌效果。     

Microwave-assisted extraction of cyclotides from Viola ignobilis

Cyclotides are an interesting family of circular plant peptides. Their unique three-dimensional structure, comprising a head-to-tail circular backbone chain and three disulfide bonds, confers them stability against thermal, chemical, and enzymatic degradation. Their unique stability under extreme conditions creates an idea about the possibility of using harsh extraction methods such as microwave-assisted extraction (MAE) without affecting their structures. MAE has been introduced as a potent extraction method for extraction of natural compounds, but it is seldom used for peptide and protein extraction. In this work, microwave irradiation was applied to the extraction of cyclotides. The procedure was performed in various steps using a microwave instrument under different conditions. High-performance liquid chromatography (HPLC) and matrix-assisted laser desorption/ionization time-of-flight (MALDI–TOF) results show stability of cyclotide structures on microwave radiation. The influential parameters, including time, temperature, and the ratio of solvents that are affecting the MAE potency, were optimized. Optimal conditions were obtained at 20 min of irradiation time, 1200 W of system power in 60 °C, and methanol/water at the ratio of 90:10 (v/v) as solvent. The comparison of MAE results with maceration extraction shows that there are similarities between cyclotide sequences and extraction yields.     

Development of a Headspace Solid‐Phase Microextraction Gas Chromatography‐Mass Spectrometry Method to Study Volatile Organic Compounds (VOCs) Emitted by Lavender Roots

A headspace solid‐phase microextraction (HS‐SPME) method combined with gas chromatography‐mass spectrometry (GC/MS) was developed and optimized for the extraction and the analysis of volatile organic compounds (VOCs) from lavandin and fine lavender roots. Optimal parameters to extract volatile molecules from ground and intact roots were determined using a divinylbenzene‐carboxen‐polydimethylsiloxane (DVB/CAR/PDMS) coating fiber at 70 °C for 60 min. A total of 99 VOCs, including 40 monoterpenoids, 15 sesquiterpenoids, 1 diterpenoid and 2 coumarins were detected. The main compounds detected in lavandin roots were fenchol, borneol, and coumarin. Performances of the optimized SPME GC/MS method were evaluated via the comparison of VOC emissions between roots from different cultivars of fine lavender (7713 and maillette) and lavandin (abrial and grosso). Chemometric analysis, using partial least squares‐discriminant analysis (PLS‐DA), suggests fifteen significant features as potential discriminatory compounds. Among them, β‐phellandrene allows discrimination between lavender and lavandin varieties.     

Microwave assisted extraction of ursolic acid and oleanolic acid from Ocimum sanctum

Present study deals with the microwave assisted extraction (MAE) of ursolic acid (UA) and oleanolic acid (OA) from Ocimum sanctum leaves. UA and OA have been reported to possess significant medicinal properties. Various experimental parameters such as selection of solvent, solvent composition, irradiation time, microwave power, solid to solvent ratio, preleaching time and number of cycles were investigated to optimize the extraction process. Under optimum conditions of irradiation time (3 min), microwave power (272 W), solid to solvent ratio (1:30), preleaching time (10 min), maximum UA and OA has been extracted in one extraction cycle with ethanol: water (80:20) as a solvent. Maximum 86.76 and 89.64% of UA and OA was extracted under above mentioned optimized experimental conditions. MAE was also compared with the batch and ultrasound assisted extraction (UAE) method. As compared to batch and UAE, higher extraction yield of these important phytochemicals have been obtained through MAE in only 3 min.     

松毛虫性信息素的固相微萃取及质谱和触角电位分析

松毛虫性信息素成分在腺体中含量很低。本研究利用固相微萃取（SPME）技术近距离长时间采集单头云南松毛虫Dendrolimus houi处女雌蛾释放的性信息素成分, 并进一步利用气相色谱-质谱（GC-MS）和气相色谱-触角电位联用（GC-EAD）技术分析SPME采集的性信息素成分。 结果显示: 在云南松毛虫处女雌蛾求偶召唤期间SPME聚乙二醇/二乙烯基苯（CAR/DVB）萃取头吸附了大量的反5, 顺7-十二碳二烯醇（E5, Z7-12:OH）和反5, 顺7-十二碳二烯乙酸酯（E5, Z7-12:OAc）成分及微量的反5, 顺7-十二碳二烯醛（E5, Z7-12:Ald）成分, 这3种腺体成分均能激起云南松毛虫雄虫触角电位反应。与溶剂浸提昆虫性信息素成分的方法比较, SPME活体采样技术和GC-MS及GC-EAD分析方法联用研究昆虫的信息素成分具有采集的样品代表性强、样品量能满足色谱分析需要和无溶剂干扰等方面的优点。最后还着重讨论了SPME技术的应用价值、效果及实际应用中应该注意的一些问题。      

Microwave-assisted extraction of embelin from <Emphasis Type="Italic">Embelia ribes</Emphasis>

A rapid and efficient microwave-assisted extraction (MAE) process for the selective extraction of embelin from Embelia ribes was developed. Solvent selection, microwave energy input and solid loading were optimized. The rate of extraction and purity of embelin depended upon the solvent used and exposure time to microwaves. Maximum MAE was achieved in acetone with total yield of 92% (w/w) embelin with 90% (w/w) purity with 1% (w/v) raw material loading at 150 W power level in 80 s. Non-polar solvents, such as hexane and dichloromethane, were not effective for the selective extraction of embelin.     

Rapid determination of volatile compounds emitted from Chimonanthus praecox flowers by HS-SPME-GC-MS

A novel rapid, simple and solvent-free method was developed for determination of the volatile compounds from the flowers of Chimonanthus praecox Link using headspace solid-phase microextraction (HS-SPME) and gas chromatography-mass spectrometry (GC-MS). The SPME conditions were firstly optimized and applied to sampling of the volatile compounds emitted from living Chimonanthus praecox L. flowers and excised Chimonanthus praecox L. flowers. Thirty-one compounds emitted from living flowers were identified for the first time, which mainly included 4-methyl-1,3-pentadiene (2.0%), a-phellandrene (4.7%), benzyl methanol (11.1%), trans-linalool oxide (furanyl ring) (5.3%), a-linalool (36.0%), methyl salicylate (24.5%) and acetic acid benzyl ester (5.9%). Comparing the emission from living flowers and excised flowers, twenty-eight compounds were found to be detected in the two emissions, and three compounds, n-pentadecane, n-cetane and n-heptadecane, were only found in the emission from the living flowers, which shows that they might be biomarker compounds.     

Characterisation of volatile organic compounds in stemwood using solid-phase microextraction

Solid-phase microextraction (SPME), hydrodistillation and dynamic headspace combined with GC and GC-MS were applied and compared for the analysis of volatile organic compounds (VOCs) from coniferous wood. The SPME conditions (type of fibre, size of wood sample, temperature and exposure time) were optimised, and more than 100 VOCs and semi-volatile compounds extracted and identified from the sapwood and heartwood of Norway spruce (Picea abies). The total number of mono- and sesquiterpenes eluted and identified was similar for the SPME and hydrodistillation methods, but more semi-volatile compounds were released by hydrodistillation. By applying dynamic headspace at room temperature, it was possible to analyse only the most volatile compounds. The qualitative composition of VOCs was similar in spruce sapwood and heartwood, although Z-beta-ocimene occurred only in sapwood while fenchol was present only in heartwood. SPME sampling coupled with GC, applied here to the analysis of VOCs released from stemwood of firs for the first time, is a convenient, sensitive, fast, solvent-free and simple method for the determination of wood volatiles. The technique requires much smaller sample amounts compared with hydrodistillation, and the total amount of VOCs extracted and identified is higher than that obtained by hydrodistillation or dynamic headspace. The relative ratios of the main mono- and sesquiterpenes and -terpenoids were similar using the SPME-GC and hydrodistillation methods.     

Rapid determination of solanesol in tobacco by high-performance liquid chromatography with evaporative light scattering detection following microwave-assisted extraction

Solanesol is the starting material for many high-value biochemicals, including coenzyme Q(10) and Vitamin-K analogues. The aim of the current study was to develop a reliable and fast analytical procedure for the determination of solanesol in tobacco using high-performance liquid chromatography (HPLC) with evaporative light scattering detection (ELSD) coupled with microwave-assisted extraction (MAE) as an efficient sample preparation technique. The HPLC conditions were Agilent C18 column using acetonitrile-isopropanol (60:40, v/v) as mobile phase at a flow rate of 1 ml/min. ELSD conditions were optimized at nebulizer-gas flow rate of 1.5 l/min and drift tube temperature of 65 degrees C. The method was validated to achieve the satisfactory precision and recovery, and the calibration range was 0.1-1.5 mg/ml. The developed analytical procedure was successfully applied to determine solanesol content in tobacco samples from different growing regions in China.     

油菜花粉总黄酮的微波辅助提取及其抗氧化活性研究

采用微波辅助提取法提取油菜花粉总黄酮,通过单因素和正交试验,得到最佳提取条件：80％的乙醇、1：20的料液比、提取时间140s、微波功率242W、提取3次,总黄酮质量分数为（2．64±0．05）％。同时还研究提取物对羟自由基、DPPH自由基的清除作用,以及对小鼠肝组织脂质过氧化的抑制作用,自由基半清除质量浓度和脂质过氧化丰抑制盾奄浓度分剐为0．115、0．325和0．065mg／mL。     

Headspace analysis identifies indole and 1-octen-3-ol as the “coal tar” odor of <Emphasis Type="Italic">Tricholoma inamoenum</Emphasis>

The odor emanating from sporocarps of Tricholoma inamoenum has been described as resembling “coal tar”. To characterize the compounds responsible for this odor, volatile chemicals released from T. inamoenum sporocarps were collected using solid phase microextraction (SPME). Subsequent analysis by gas chromatography-mass spectrometry (GC-MS) showed only indole and 1-octen-3-ol, so these compounds must be responsible for the “coal tar” odor of T. inamoenum. Mushroom pileus size was a factor in the amount of indole produced; larger mushrooms released 25-times more indole than smaller ones. A comparison of SPME and CH₂Cl₂ solvent extraction of sporocarps showed major differences in the volatile organic compounds. Benzaldehyde and phenyl acetaldehyde were the major compounds in the solvent extracts, but were not detected in the SPME experiments. Tissue disruption of the mushroom before solvent extraction showed up to a 40-fold increase in the amount of 1-octen-3-ol present.