Sampling volatile compounds from natural products with headspace/solid-phase micro-extraction

Recent advances in the development of analytical methods based on headspace/solid-phase micro-extraction (HS/SPME) of natural aroma compounds are reviewed, with special emphasis on increasing reproducibility. Representative examples of applications of HS/SPME to the determination of components at trace levels, strategies for fiber selection, and improvement of SPME sampling conditions are presented. Examples of applications to quality control and sample classification, based on aroma profiling were selected to highlight the use of multivariate statistical methods.     

An alternative method for irones quantification in iris rhizomes using headspace solid-phase microextraction

Introduction – The essential oil obtained from iris rhizomes is one of the most precious raw materials for the perfume industry. Its fragrance is due to irones that are gradually formed by oxidative degradation of iridals during rhizome ageing. Objective – The development of an alternative method allowing irone quantification in iris rhizomes using HS‐SPME‐GC. Methodology – The development of the method using HS‐SPME‐GC was achieved using the results obtained from a conventional method, i.e. a solid–liquid extraction (SLE) followed by irone quantification by CG. Results – Among several calibration methods tested, internal calibration gave the best results and was the least sensitive to the matrix effect. The proposed method using HS‐SPME‐GC is as accurate and reproducible as the conventional one using SLE. These two methods were used to monitor and compare irone concentrations in iris rhizomes that had been stored for 6 months to 9 years. Conclusion – Irone quantification in iris rhizome can be achieved using HS‐SPME‐GC. This method can thus be used for the quality control of the iris rhizomes. It offers the advantage of combining extraction and analysis with an automated device and thus allows a large number of rhizome batches to be analysed and compared in a limited amount of time. Copyright © 2010 John Wiley & Sons, Ltd.     

Pattern recognition and genetic algorithms for discrimination of orange juices and reduction of significant components from headspace solid‐phase microextraction

Introduction – Orange (Citrus sinensis L.) juice comprises a complex mixture of volatile components that are difficult to identify and quantify. Classification and discrimination of the varieties on the basis of the volatile composition could help to guarantee the quality of a juice and to detect possible adulteration of the product. Objective – To provide information on the amounts of volatile constituents in fresh‐squeezed juices from four orange cultivars and to establish suitable discrimination rules to differentiate orange juices using new chemometric approaches. Methodology – Fresh juices of four orange cultivars were analysed by headspace solid‐phase microextraction (HS‐SPME) coupled with GC‐MS. Principal component analysis, linear discriminant analysis and heuristic methods, such as neural networks, allowed clustering of the data from HS‐SPME analysis while genetic algorithms addressed the problem of data reduction. To check the quality of the results the chemometric techniques were also evaluated on a sample. Results – Thirty volatile compounds were identified by HS‐SPME and GC‐MS analyses and their relative amounts calculated. Differences in composition of orange juice volatile components were observed. The chosen orange cultivars could be discriminated using neural networks, genetic relocation algorithms and linear discriminant analysis. Genetic algorithms applied to the data were also able to detect the most significant compounds. Conclusions – SPME is a useful technique to investigate orange juice volatile composition and a flexible chemometric approach is able to correctly separate the juices. Copyright © 2009 John Wiley & Sons, Ltd.     

Headspace Solid Phase Microextraction Coupled to GC/MS for the Analysis of Volatiles of Honeys from Arid and Mediterranean Areas of Algeria

The volatile composition of seven honey samples collected from various regions of Algeria and feeding on different plants have been determined. The Headspace Solid‐Phase MicroExtraction (HS‐SPME) coupled with Gas Chromatography‐Mass Spectrometry (GC/MS) was used to achieve the purpose. In this work, different parameters of the HS‐SPME analytical method were investigated in order to reach maximal sensitivity, and thus to obtain maximum information about the volatile profile of Algerian honey. These parameters include saline medium, HS extraction temperature, and the nature of the fiber used. The results showed a great diversity in the chemical composition, in total 124 compounds from different chemical classes were identified, including compounds found for the first time in honey. The Ascending Hierarchical Classification (AHC) demonstrated the importance of choosing SPME extraction conditions to find volatile compounds, which could be as specific markers of the floral or geographical origin of honey, the latter was optimized in the parameter PDMS‐55 °C.     

Trends in solventless sample preparation techniques for environmental analysis

The paper presents recent trends in solventless sample preparation techniques for environmental analysis. First, a general classification of solventless methods is given. Next, three of them, treated as preferable techniques, i.e. SPME, SDME and HS, are presented in detail, with respect to their usability and effectiveness for environmental samples. Examples of all discussed techniques are given in the tables.     

Trends in solventless sample preparation techniques for environmental analysis

The paper presents recent trends in solventless sample preparation techniques for environmental analysis. First, a general classification of solventless methods is given. Next, three of them, treated as preferable techniques, i.e. SPME, SDME and HS, are presented in detail, with respect to their usability and effectiveness for environmental samples. Examples of all discussed techniques are given in the tables.     

Volatile Compounds of Viola odorata Absolutes: Identification of Odorant Active Markers to Distinguish Plants Originating from France and Egypt

Absolutes isolated from Viola odorata leaves, valuable materials for the flavor and fragrance industry, were studied. Violets are mainly cultivated in France and Egypt and extracted locally. The absolutes of the two origins showed different olfactory profiles both in top and heart notes, as evidenced by sensory analysis. The aims of this study were i) to characterize the volatile compounds, ii) to determine the odorant‐active ones, and iii) to identify some markers of the plant origin. Two complementary analytical methods were used for these purposes, i.e., headspace solid‐phase microextraction (HS‐SPME) using different fiber coatings followed by GC/MS analysis and gas chromatography – olfactometry/mass spectrometry (GC‐O/MS) applied to violet leaf extracts. From a total of 70 identified compounds, 61 have never been reported so far for this species, 17 compounds were characterized by both techniques (with seven among them known from the literature), 23 compounds were solely identified by HS‐SPME GC/MS (among them only two being already mentioned as components of violet absolutes in the literature), and, finally, 30 compounds were only identified by GC‐O/MS. According to the HS‐SPME GC/MS analyses, ethyl hexanoate and (2E,6Z)‐nona‐2,6‐dienol were specific volatile compounds of the sample with French origin, while (E,E)‐hepta‐2,4‐dienal, hexanoic acid, limonene, tridecane, and eugenol were specific of the samples with Egyptian origin. Additional compounds that were not detected by HS‐SPME GC/MS analysis were revealed by GC‐O analyses, some of them being markers of origin. Pent‐1‐en‐3‐ol, 3‐methylbut‐2‐enal, 2‐methoxy‐3‐(1‐methylethyl)pyrazine, 4‐ethylbenzaldehyde, β‐phenethyl formate, and 2‐methoxy‐3‐(2‐methylpropyl)pyrazine revealed to be odorant markers of the French sample, whereas cis‐rose oxide, trans‐rose oxide, and 3,5,5‐trimethylcyclohex‐2‐enone were odorant markers of the Egyptian samples.     

Aroma Profile of Rubus ulmifolius Flowers and Fruits During Different Ontogenetic Phases

The chemical composition of spontaneous volatile emission from Rubus ulmifolius flowers and fruits during different stages of development was evaluated by HS‐SPME‐GC/MS. In total, 155 chemical compounds were identified accounting 84.6 – 99.4% of whole aroma profile of flowers samples and 92.4 – 96.6% for fruit samples. The main constituents were α‐copaene, β‐caryophyllene, germacrene D, (E,E)‐α‐farnesene, 1,7‐octadien‐3‐one,2‐methyl‐6‐methylene, tridecane, (E)‐2‐hexenol acetate, (E)‐3‐hexenol acetate and cyperene. The results give a chemotaxonomic contribution to the characterization of the VOCs emitted from flowers and fruits during their ontogenic development.     

Pollen aroma fingerprint of two sunflower (Helianthus annuus L.) genotypes characterized by different pollen colors

Samples of fresh pollen grains, collected from capitula in full bloom from two genotypes of sunflower (Helianthus annuus L.) and characterized by a different color, i.e., white‐cream (WC) and orange (O), were analyzed by the HS‐SPME (headspace solid phase microextraction)/GC/MS technique. This study defined for the first time the fingerprint of the sunflower pollen, separated from the disc flowers, to define its contribution to the inflorescence aroma. In the GC/MS fingerprints of the WC and O genotypes, 61 and 62 volatile compounds were identified, respectively. Monoterpene hydrocarbons (34% in O vs. 28% in WC) and sesquiterpene hydrocarbons (37% in O vs. 31% in WC) were ubiquitous in all samples analyzed and represented the main chemical classes. α‐Pinene (21% in O vs. 20% in WC) and sabinene (11% in O vs. 6% in WC) were the dominant volatiles, but also a full range of aliphatic hydrocarbons and their oxygenated derivatives gave a decisive contribution to the aroma composition (10% in O vs. 12% in WC). In addition, dendrolasin (3% in O vs. 4% in WC) and some minor constituents such as (E)‐hex‐2‐en‐1‐ol (0.4% in O vs. 0.1% in WC) were pointed out not only for their contribution to the pollen scent, but also for their well‐known role in the plant ecological relationships. Having evaluated two pollen morphs with different carotenoid‐based colors, the study sought to highlight also the presence of some volatile precursors or derivatives of these pigments in the aroma. However, the pollen aroma of the two selected genotypes made a specific chemical contribution to the sunflower inflorescence scent without any influence on carotenoid derivatives.     

成熟度对印度块菌香气成分的影响

研究成熟度对印度块菌Tuber indicum香气成分的影响,并测定成熟印度块菌的关键香气成分。将3种不同成熟度的印度块菌,以固相微萃取(SPME)技术为香气富集方法,利用气相色谱-质谱联用(GC-MS)分析其香气成分。结果表明未成熟印度块菌中仅检测出4种香气成分,中度成熟阶段检测出8种,成熟阶段检测出13种,而且成熟阶段检测出的香气成分大多都是前人报道过对块菌香气有贡献的成分;通过计算成熟块菌各香气组分的香气活度值(OAV),可知二甲基硫醚、2,3-丁二酮、3-甲基正丁醛、2-甲基正丁醛、己醛、1-辛烯-3-醇这6种物质是印度块菌的关键香气成分(OAV>1)。     

贮藏保鲜中SO2伤害对红提葡萄香气组分的影响

采用固相微萃取技术及气相-质谱联用技术对不同SO2漂白伤害程度的红提果实进行香气成分的提取与鉴定,以探讨SO2漂白伤害对贮藏葡萄果实中香气组分的影响.结果表明,果实经漂白伤害后其具有芳香气味的气体所占比重明显下降,主要成分醇类化合物下降最多,而具有刺激难闻气味的酸类化合物所占比重明显上升;此外,4-萜烯醇、正己醇、月桂醇、松油醇、芳樟醇、橙花醇、乙酸萜烯酯及青叶醛等具有葡萄特征性香味的化合物在香气中所占比例明显下降或消失,而异辛醇、1-辛烯-3-醇、壬酸、辛酸、正癸酸、乙酸、己酸、2,6-二叔丁基对甲酚及2-丁基-5-(2-甲基丙基)-噻吩等具有臭味或刺激性气味的化合物出现或所占比重上升.说明,SO2伤害对红提果实香气的影响主要表现为香味化合物所占比重的减少或消失及异味化合物的产生.     

燕山山脉一种香料型乳菇的香味成分分析

利用固相微萃取技术（SPME）偶联气相色谱-质谱（GC-MS）分析燕山山脉一种香料型乳菇——香亚环乳菇Lactarius subzonarius的挥发性成分。共检出25种挥发性成分,其中氧杂环化合物3种、醛类1种、酯类13种、烯烃类3种、芳香族化合物2种和烷烃类3种。具有葫芦巴感官气味的挥发性组分3-羟基-4,5-二甲基-2(5H)-呋喃酮的相对含量35.76%,该成分是其近缘种Lactarius helvus的主要香味化合物,故推测3-羟基-4,5-二甲基-2(5H)-呋喃酮是其关键香味成分。     

Fine‐scale spatial genetic structure analysis of the black truffle Tuber aestivum and its link to aroma variability

Truffles are symbiotic fungi in high demand by food connoisseurs. Improving yield and product quality requires a better understanding of truffle genetics and aroma biosynthesis. One aim here was to investigate the diversity and fine‐scale spatial genetic structure of the Burgundy truffle Tuber aestivum. The second aim was to assess how genetic structuring along with fruiting body maturation and geographical origin influenced single constituents of truffle aroma. A total of 39 Burgundy truffles collected in two orchards were characterized in terms of aroma profile (SPME‐GC/MS) and genotype (microsatellites). A moderate genetic differentiation was observed between the populations of the two orchards. An important seasonal and spatial genetic structuring was detected. Within one orchard, individuals belonging to the same genet were generally collected during a single season and in the close vicinity from each other. Maximum genet size nevertheless ranged from 46 to 92 m. Geographical origin or maturity only had minor effects on aroma profiles but genetic structuring, specifically clonal identity, had a pronounced influence on the concentrations of C₈‐ and C₄‐VOCs. Our results highlight a high seasonal genetic turnover and indicate that the aroma of Burgundy truffle is influenced by the identity of single clones/genets.     

NMR,HS‐SPME‐GC/MS,and HPLC/MSn Analyses of Phytoconstituents and Aroma Profile of Rosmarinus eriocalyx

In this work, a comprehensive study on the chemical constituents of the aerial parts of Rosmarinus eriocalyx (Lamiaceae), an aromatic shrub traditionally consumed as a food and herbal remedy in Algeria, is presented. The aroma profile was analysed by headspace solid phase microextraction (HS‐SPME) coupled with gas chromatography‐mass spectrometry (GC/MS), whereas the crude extract constituents were analyzed by ¹H‐NMR and by high performance liquid chromatography coupled with mass spectrometry (HPLC/MSⁿ). Thirty‐nine volatile compounds, most of them being monoterpenes, have been identified, with camphor, camphene, and α‐pinene as the most abundant constituents. ¹H‐NMR analysis revealed the presence of phenolic compounds and betulinic acid while HPLC/MSⁿ allowed the identification of glycosilated and aglyconic flavonoids as well as phenylpropanoid derivatives. Some of these constituents, namely as betulinic acid, rosmanol, and cirsimaritin were reported for the first time in R. eriocalyx.     

软儿梨果酒发酵过程中挥发性风味物质变化分析

软儿梨是西北地区特有的一种“冻果”产品,酸甜可口、果香浓郁,是酿造果酒的上乘原料。为明确果酒发酵过程中果香的保留情况,并探究发酵时间的延长对软儿梨果酒品质的影响,以青海省民和县软儿梨冻果为原料,基于带皮渣发酵工艺,先采用顶空固相微萃取（solid?phase microextraction, SPME）,再结合气相色谱?质谱联用技术（gas chromatography?mass spectrometry, GC?MS）对原汁（0 d）、前发酵（7、14、28 d）、后发酵（100 d）3个不同时期样品中挥发性风味物质进行了动态跟踪分析,并结合相对气味活度值（relative odor activity value,ROAV）和主成分分析（principal component analysis,PCA）法分别探讨了软儿梨果酒原果香味保留情况和后发酵时间的延长对果酒风味的影响。结果显示：整个发酵过程共检出88种挥发性化合物,其中酯类33种、醇类29种、酸类6种、萜烯类7种以及13种其他类化合物,且风味物质的总含量随发酵的进行呈先上升后略有下降的趋势。ROAV结果表明：软儿梨原汁关键风味物质共8种,分别是丁酸乙酯、己酸乙酯、2?甲基丁酸乙酯、正辛醇、大马士酮、芳樟醇、丁香酚和癸醛;其中果香物质己酸乙酯、正辛醇、芳樟醇、丁香酚和大马士酮在果酒发酵中得到了很好的保留,是决定软儿梨果酒风味的关键物质。主成分分析表明：发酵初期主要的香气贡献物质是具有水果香的丁酸乙酯、3?羟基丁酸乙酯、2?甲基丁酸乙酯;发酵100 d时,乙酸乙酯、α?松油醇、柠檬烯和芳樟醇对香气贡献较大,这些香气化合物共同赋予软儿梨果酒幽香清雅、馥香浓郁的独特风味品质。研究获得了软儿梨果酒关键风味物质及其在不同发酵阶段特征风味物质的变化规律,可为研发高品质软儿梨果酒产品、改进软儿梨果酒发酵工艺提供理论支撑和参考。     

Application of solid-phase microextraction combined with derivatization to the determination of amphetamines by liquid chromatography

This work evaluates the utility of solid-phase microextraction (SPME) in the analysis of amphetamines by liquid chromatography (LC) after chemical derivatization of the analytes. Two approaches have been tested and compared, SPME followed by on-fiber derivatization of the extracted amphetamines, and solution derivatization followed by SPME of the derivatives formed. Both methods have been applied to measure amphetamine (AP), methamphetamine (MA), and 3,4-methylenedioxymethamphetamine (MDMA), using the fluorogenic reagent 9-fluorenylmethyl chloroformate (FMOC) and carbowax-templated resin (CW-TR)-coated fibers. Data on the application of the proposed methods for the analysis of different kind of samples are presented. When analyzing aqueous solutions of the analytes, both approaches gave similar analytical performance, but the sensitivity attainable with the solution derivatization/SPME method was better. The efficiencies observed when processing spiked urine samples by the SPME/on-fiber derivatization approach were very low. This was because the extraction of matrix components into the fiber coating prevented the extraction of the reagent. In contrast, the efficiencies obtained for spiked urine samples by the solution derivatization/SPME approach were similar to those obtained for aqueous samples. Therefore, the later method would be the method of choice for the quantification of amphetamines in urine.     

Analysis of biological samples using solid-phase microextraction

Solid-phase microextraction (SPME) has gained widespread acceptance for analyte-matrix separation and preconcentration. SPME is a simple, effective adsorption/desorption technique that eliminates the need for solvents or complicated apparatus for concentrating volatile or non-volatile compounds in liquid samples or headspace. SPME is compatible with analyte separation/detection by gas chromatography and high performance liquid chromatography and provides linear results for a wide range of concentrations of analytes. By controlling the polarity and thickness of the coating on the fiber, maintaining consistent sampling time, and adjusting several other extraction parameters, an analyst can ensure highly reliable results for low concentrations of analytes. This review provides updated information on SPME with chromatographic separation for the extraction and measurement of different analytes in biological fluids and materials. Firstly the background to the technique is given in terms of apparatus, fibers used, extraction conditions and derivatisation procedures. Then the different matrices, urine, blood, breast milk, hair and saliva are considered separately. Finally, the future potential of SPME for the analysis of biological samples in terms of the development of new devices and fiber chemistries as well as applications for in vivo studies are discussed.     

Perceptual characterization and analysis of aroma mixtures using gas chromatography recomposition-olfactometry

This paper describes the design of a new instrumental technique, Gas Chromatography Recomposition-Olfactometry (GC-R), that adapts the reconstitution technique used in flavor chemistry studies by extracting volatiles from a sample by headspace solid-phase microextraction (SPME), separating the extract on a capillary GC column, and recombining individual compounds selectively as they elute off of the column into a mixture for sensory analysis (Figure 1). Using the chromatogram of a mixture as a map, the GC-R instrument allows the operator to "cut apart" and recombine the components of the mixture at will, selecting compounds, peaks, or sections based on retention time to include or exclude in a reconstitution for sensory analysis. Selective recombination is accomplished with the installation of a Deans Switch directly in-line with the column, which directs compounds either to waste or to a cryotrap at the operator's discretion. This enables the creation of, for example, aroma reconstitutions incorporating all of the volatiles in a sample, including instrumentally undetectable compounds as well those present at concentrations below sensory thresholds, thus correcting for the "reconstitution discrepancy" sometimes noted in flavor chemistry studies. Using only flowering lavender (Lavandula angustifola 'Hidcote Blue') as a source for volatiles, we used the instrument to build mixtures of subsets of lavender volatiles in-instrument and characterized their aroma qualities with a sensory panel. We showed evidence of additive, masking, and synergistic effects in these mixtures and of "lavender' aroma character as an emergent property of specific mixtures. This was accomplished without the need for chemical standards, reductive aroma models, or calculation of Odor Activity Values, and is broadly applicable to any aroma or flavor.     

Comparison of headspace techniques for sampling volatile natural products in a dynamic system

Commonly used dynamic sorption techniques for collecting biologically active volatile compounds have been compared. Solid phase microextraction (SPME) using two types of fibers (polydimethylsiloxane, PDMS, 100 microm, and carbowax/divinylbenzene, CW/DVB, 65 microm) was compared to purge and trap methods (Porapak Q, Tenax TA and charcoal) and a technique based on absorption in methanol in a cooling bath. Sampling was done in a stream of purified air (20 ml/min) in a closed and temperature-regulated (27 degrees C) glass tube, passing over a capillary tube containing a hexane solution of tridecane, heptadecane, 1-octen-3-ol, 1-hexadecanol, ethyl tetradecanoate, alpha-pinene, linalool, terpinen-4-ol, cis-verbenol, verbenone, beta-caryophyllene, E,E-farnesol, and geranylgeraniol. With all of the methods, the sampling was performed for a period of 30 min before extraction and analysis was done on a GC-FID system. In general, SPME gave a higher response for all compounds except for alpha-pinene, which was only extracted by the CW/DVB fiber. Purge and trap methods and methanol absorption gave the same response for all substances extracted. None of the methods extracted hexadecanol and geranylgeraniol under the conditions used. However, the SPME equipped with the PDMS coating extracted heptadecane, E,E-farnesol and ethyl tetradecanoate. Our results show that SPME, when selecting the fibers to fit the polarity and volatility of the compounds, is an outstanding extraction method compared to purge and trap and methanol absorption, especially for a qualitative analysis. The best conditions for storing fibers exposed to compounds of high volatility were at low temperatures (6 degrees C) in sealed vials, while the worst way was to leave the exposed fiber unprotected at room temperature (22 degrees C). The dynamic sampling system was effectively tested on a fruiting body of a polypore fungus (Ganoderma applanatum) emitting 1-octen-3-ol, and again SPME showed to be the most sensitive technique.