Author's personal copy Provided for non-commercial research and educational use only. Not for reproduction, distribution or commercial use. This chapter was originally published in the book Studies in Natural Products Chemistry, Volume 49. The copy attached is provided by Elsevier for the author's benefit and for the benefit of the author's institution, for non-commercial research, and educational use. This includes without limitation use in instruction at your institution, distribution to specific colleagues, and providing a copy to your institution's administrator. All other uses, reproduction and distribution, including without limitation commercial reprints, selling or licensing copies or access, or posting on open internet sites, your personal or institution’s website or repository, are prohibited. For exceptions, permission may be sought for such use through Elsevier’s permissions site at: http://www.elsevier.com/locate/permissionusematerial From R. Costa, The chemistry of mushrooms: A survey of novel extraction techniques targeted to chromatographic and spectroscopic screening, in: Atta-ur-Rahman (Ed.), Studies in Natural Products Chemistry. 2016, pp. 279–306. ISBN: 9780444636010 Copyright © 2016 Elsevier B.V. All rights reserved. Elsevier Author's personal copy Chapter 9 The Chemistry of Mushrooms: A Survey of Novel Extraction Techniques Targeted to Chromatographic and Spectroscopic Screening Rosaria Costa University of Messina, Messina, Italy E-mail: costar@unime.it Chapter Outline Sample Preparation: Overview Accelerated Solvent Extraction Supercritical Fluid Extraction Microwave-Assisted Extraction Pulsed Electric Field Quick, Easy, Cheap, Effective, Rugged, Safe (QuEChERS) Liquid-Phase Microextraction 282 282 283 286 287 Solid-Phase Microextraction Stir Bar Sorptive Extraction Solid-Phase Extraction Conclusion Abbreviations References 291 291 299 302 302 304 288 290 INTRODUCTION Although there is not a general consensus about the number of life kingdoms, it is sure that fungi have so peculiar characteristics to be grouped in an independent kingdom. Originally, fungi were considered as part of kingdom Plantae, fact that could not justify their morphological and biochemical properties. Classification of fungi has always been quite problematic, due to the difficulties arising from matching morphological features with biochemical and genetic findings. Indeed, fungi are complex organisms and display an impressive variety of actions. The number of fungi species is estimated to be about 1.5 million, of which 140,000 species are macrofungi [1]. Studies in Natural Products Chemistry, Vol. 49. http://dx.doi.org/10.1016/B978-0-444-63601-0.00009-0 Copyright © 2016 Elsevier B.V. All rights reserved. Studies in Natural Products Chemistry, First Edition, 2016, 279-306 279 Author's personal copy 280 Studies in Natural Products Chemistry Cavalier-Smith proposed to include in the kingdom of fungi only the macroscopic individuals belonging to Ascomycota, Basidiomycota, Zygomycota, and Deuteromycota [2]. Further, he proposed to move microscopic mushrooms (unicellular or hyphal individuals) into the Protista and Chromista kingdoms. In the following years, Cavalier-Smith proposed more complex and detailed classifications; however, a common point remained the phylogenetic position assigned to fungi, which was considered closer to animals rather than to vegetables. A recent study based on genetic analysis clarified the evolutionary position of the lower fungi Zygomycota and Chytridiomycota [3]. Based on sequential analysis, it was concluded that the four recognized phyla do not overlap with each other, and that Microsporidia would be better placed in a sister group to the fungi. Also, recent molecular studies suggested that Opisthokonta, the eukaryotic supergroup including animals and fungi, should be expanded to include some primitive Protozoa [4]. Among these, the nucleariid appears as the closest sister taxon to fungi taxa. Fungi are eukaryotic and filamentous organisms, their predominant constituent being chitin, derived from polymerization of glucose units. Fungi play a fundamental role in the daily life: they are useful for food production, for instance yeasts in beer and molds in blue cheese; they are good sources of therapeutically active molecules, such as antibiotics, cyclosporins, and so on. Helpful in agriculture, part of them promote plants nutrition through the system of mychorrhizae. Fungi result to be essential in the ecosystem for debris removal and destruction, thanks to their chemoheterotrophic character. This means that fungi are organisms that obtain energy by ingestion of organic molecules, like glucose. A peculiar trait of fungi is their ability to merge hyphae in mycelia, strictly intertwined, so to share both the nutrients and genetic heritage. Due to the consistent production of enzymes, fungi can degrade complex polymers such as cellulose and lignin. The kingdom of fungi includes also yeasts, the most famous being Saccharomyces cerevisiae or brewer’s yeast. This unicellular fungus is considered highly valuable by the various industries, which produce wine, beer, cider, and bread. Each industry holds its own selected strain as a secret recipe. By definition, “a mushroom is a macrofungus with a distinctive fruiting body that can be either epigeous or hypogeous and large enough to be seen with the naked eye and to be picked up by hand” [1]. Beyond being valuable foods, mushrooms contain substances with interesting biological and medicinal activities, such as lectins, beta-glucans (ie, lentinan and pleuran), ergosterol, antioxidants, lovastatin, GABA, and ergothioneine [5–9]. Established that the intrinsic role of mushrooms is to produce reproductive spores and to provide for their dissemination, they also play other functions in the ecosystem. Some mushrooms live in symbiosis with plants roots, indeed establishing with them a mutually beneficial rapport: mushrooms wrap around the roots under the shape of a thick web, which acts as an auxiliary absorption apparatus for the plant. The consequence is a twoway transfer of nutrients for both the mushrooms (which get sugars from the plant) and the host plant (which gets more nutrients from the soil). Studies in Natural Products Chemistry, First Edition, 2016, 279-306 Author's personal copy The Chemistry of Mushrooms Chapter | 9 281 Among wood-decaying mushrooms, Lentinus edodes (Berk.) Pegler, Omphalotaceae (Shiitake), and Ganoderma lucidum (Curtis) P. Karst., Ganodermataceae, are the most famous drugs utilized in oriental traditional medicine, although their use in food supplements is quite diffused even in Western countries. In fact, many are the medicinal properties ascribed to mushrooms (about 650 are the species with beneficial effects) [10]. The previously mentioned G. lucidum is employed in several fields of application: immunomodulation, cardiovascular disease, cancer therapy, hepatitis B treatment, enhancement of oxygen intake, free radical scavenging, and diabetes treatment are some examples. Among the bioactive substances isolated from G. lucidum are the triterpenoids (ganoderic acids) and high molecular weight polysaccharides. Antitumor properties have been demonstrated also for other mushroom species, such as Agaricus brasiliensis, which presents among its bioactive constituents a glycoprotein and three ergosterol derivatives. Other species with antitumor effects were reported to be Grifola frondosa, containing grifolan, a β-glucan; L. edodes, containing the polysaccharide lentinan; Flammulina velutipes, containing the glycoprotein proflamin; Calvatia gigantea, containing the mucoprotein calvacin. Lentinus edodes and Pleurotus ostreatus are effective in cardiovascular disorders by lowering blood pressure and the level of cholesterol. Antimicrobial activities, attributed to polysaccharides, were assessed in the mushroom species Trametes versicolor, L. edodes, Hericium erinaceus, Agaricus bisporus, and Coprinus comatus. Beyond the medicinal properties, mushrooms are today recognized as fundamental regulators of the ecosystems and biomarkers of the environmental health status. The chemistry of mushrooms can give information about particular characteristics of the soils (eg, presence of heavy metals), deterioration of the environment, biodiversity, soil fertility, plant–soil interaction. In general, the analysis of mushrooms has always been focused on cultivated and medicinal species, both for the assessment of composition and nutritional value. However, this situation has changed quite consistently in the last 15 years, due to the involvement of wild species too. As a consequence, the number of reports on mushroom analysis has grown noticeably. Papers published in this field focus on the determination of (1) dry matter, energy value, bioavailability of nutrients; (2) proteins and aminoacids; (3) lipids; (4) carbohydrates and fiber; (5) minerals and vitamins; (6) flavor and taste; (7) phenolics and pigments; (8) radioisotopes [11]. This scenario highlights great variability among different species and even within the same one. Most of the reports published through the years about composition and structural elucidation of all the different kinds of molecules detected in mushrooms are based on the exploitation of chromatographic and spectroscopic techniques. Traditional extraction with solvents and distillation as sample preparation methodologies have been somehow set apart, for a matter of both increased sensitivity requirements and environmental safety. A selection of papers published in the past 20 years on the chemical analysis of mushrooms by means of innovative and environmentally friendly sample preparation techniques will be discussed in this review. Studies in Natural Products Chemistry, First Edition, 2016, 279-306 Author's personal copy 282 Studies in Natural Products Chemistry SAMPLE PREPARATION: OVERVIEW Sampling and sample preparation are two distinct phases of an analytical procedure [12]. Sampling is the phase of collection of a representative sample from the original raw matrix, while sample preparation is the number of procedures applied to the sample in order to make it suitable to the analytical methodology chosen. Both have a fundamental role in the achievement of a successful analysis in terms of accuracy and precision. Some examples of errors deriving from sampling and sample preparation are the collection of a nonhomogeneous sample or insufficient amount; the choice of a wrong derivatization procedure or the use of none at all; the use of nonselective solvents; the loss of analytes in multistep preparation methods. Once assessed the cruciality of sampling and sample preparation, it seems worth emphasizing that, in the last decades, an increasing demand for highly sensitive and selective extraction methodologies has arisen from the scientific community. The analytical requirements have become more challenging if considering the international regulations issued for food, drug, and environmental safety. A sensitive preconcentration technique is mandatory in the case of analytes present at very low level in the sample matrix (ppb, ppt); while selectivity is necessary when targeted analyses have to be carried out. Additionally, the recent public concern about environmental and human safety dictated by the increase of pollution is a further requirement in the field of sample preparation. Techniques which are miniaturized or solventless are today welcome in the analytical facilities, due to their reduced consumption of chemicals and solvents. A new branch of chemistry, defined as “green,” includes extraction techniques, which provide solventless (or virtually solventless) extraction, make use of less harmful/toxic solvents (ie, superheated water), and exploit physical aids (ie, microwaves). On the other hand, the term “miniaturized” describes those procedures and means which perform “microextractions,” by means of small sorbent/liquid surfaces. The advantages of such techniques lie not only in human and environmental preservation but also in the costs of equipment (cheaper, unless automated systems are chosen) and easiness of use. ACCELERATED SOLVENT EXTRACTION Accelerated solvent extraction (ASE), also known as PFE (pressurized fluid extraction) or PLE (pressurized liquid extraction) or PWE (pressurized water extraction), is quite similar to supercritical fluid extraction (SFE), where the solvents used are brought near their supercritical state. Solvents are typically heated above their boiling point; however, due to pressurization, no evaporation takes place. At these extreme conditions, pressurized liquids show an efficient extraction performance, as well as supercritical fluids (SCFs). Compared to SFE, ASE is not matrix dependent and does not require any additional cosolvent (organic modifier). The high temperature applied “accelerates” the extraction process by increasing mass transfer kinetics, indeed improving solubilization of analytes into the extracting phases. The greenness of ASE lays on the low amount of Studies in Natural Products Chemistry, First Edition, 2016, 279-306 Author's personal copy The Chemistry of Mushrooms Chapter | 9 283 liquids required (15–40 mL on average) the nature of the solvent (even water can be used). Furthermore, due to the “acceleration,” a reduced analysis time can be observed with a consequent decrease of the analysis cost. However, PLE presents some disadvantages as well, such as low selectivity toward organic compounds from complex samples; sample cleanup is still required after extraction; the investment cost for the acquisition of the instrumental apparatus. Various applications of ASE to mushrooms investigation have been reported in the last years. Recently, ASE has been employed for the extraction of sterols from cultivated A. bisporus (J.E. Lange) Imbach, Agaricaceae (champignon), mushrooms [13]. At the same time, an SFE method was developed and applied to the same samples for the isolation of the sterol fraction. Mushroom sterols (ie, ergosterol) have been demonstrated to reduce the serum levels of cholesterol, as well as plant phytosterols. The two compressed liquid extraction methodologies were compared, showing the extraction yields to be higher for PLE rather than for SFE. However, the SFE fractions contained considerably higher percentages of sterols, compared to PLE. In general, PLE was suggested as the technique of choice for an exhaustive isolation of sterols from mushrooms. The same research team investigated several wild and cultivated mushroom species by using ASE as extraction technique [14]. The HMGCR activity was determined by measuring spectrophotometrically the decrease of absorbance at 340 nm. The water-soluble extracts showed an excellent (in vitro) inhibitory activity on the 3-hydroxy3-methyl-glutaryl CoA reductase, a key enzyme in the synthesis of cholesterol. The hypocholesterolemic activity was more pronounced for some mushroom species, such as L. edodes and P. ostreatus (Jacq.) P. Kumm, Pleurotaceae. A more specific analysis was conducted on samples of cultivated mushrooms (A. bisporus, P. ostreatus, and L. edodes) by applying a PWE method for the isolation of β-glucans, capable of binding bile acids [15]. Once again, the PWE extracts exerted hypocholesterolemic activity. Samples of Helvella lacunosa (Afzel.) and Helvella crispa (Scop.: Fr.) Fries, Helvellaceae, were lyophilized and extracted by ASE, using ethanol as solvent, and by SFE with carbon dioxide. Fatty acids with 16 and 18 carbon atoms and their esters were found in the SFE extracts, while the ASE extracts were characterized by the presence of longer chain fatty acids and consistent amount of crinosterol and mannitol [16]. An analytical method based on ASE extraction, followed by SPE (solid-phase extraction) cleanup, was applied to the determination of 25 organochlorine pesticides in about 1400 samples of mushrooms, retrieved from different markets in China and belonging to the genera Agaricus, Auricularia, Tremella, and Flammulina [17]. The ASE method was followed by GC-MS/MS determination of pesticides. SUPERCRITICAL FLUID EXTRACTION This extraction technique exploits the solvent properties of SCFs, which are any substances brought at a temperature and pressure above those defined as critical. Beyond the critical temperature, a gas cannot be liquefied whatever Studies in Natural Products Chemistry, First Edition, 2016, 279-306 Author's personal copy 284 Studies in Natural Products Chemistry pressure is applied. Moreover, the vapor pressure of a gas at the critical temperature is defined “critical” as well. Although named “fluid,” an SCF is neither a liquid nor a gas, but it possesses intermediate properties between the two states. Its density makes it closer to a liquid, while its diffusivity closer to a gas. Being less viscous than conventional solvents, SCFs penetrate sample matrices more consistently. In case of nonpolar and thermosensitive analytes, SCFs result to have extraction capabilities higher than conventional liquids: the extraction rate is faster and more effective than liquid–liquid approaches. In SFE procedures, an increase of the solubilization features can be obtained by adding a cosolvent or entrainer (ie, ethanol or methanol). The addition of entrainers must be monitored carefully, since in the worst cases they can modify so drastically the SCF till causing a failure of the extraction itself. In general, single fluid extractions are appropriate for nonpolar analytes, whereas fluids added with entrainers are indicated in extractions under subcritical conditions (no change in the solvation properties can be observed around the critical point). The most common SCF utilized is carbon dioxide, basically because of its critical temperature and pressure, which can be easily reached: 31°C and 73.8 bar. A fundamental advantage of SFE, although observed only at industrial scale, is that the solvent used, after supercritical extraction, can be converted into the original physical state and recycled for successive extractions. Such conversion can be obtained by tuning temperature and pressure values. These features make SFE an environmentally friendly procedure. Also, due to the low temperatures applied, SFE is very suitable for thermolabile compounds. The extract quality obtained by this technique is reportedly the same as that obtained by traditional Soxhlet extraction; however, the tunable properties of the SFE process make it unique, sensitive, and selective, compared with conventional extraction methods. In an SFE process temperature, solvent flow rate and pressure can be better regulated. Along with microwave-assisted extraction (MAE), SFE may be successfully applied to the extraction of polysaccharides. The disadvantages of this technique, which somehow set it aside, are the complexity of the instrumentation, the analyst safety (high pressures and temperatures can cause explosions) and the cost of the equipment (explosion proof equipment). Some relevant applications of SFE to mushroom analysis include the following four reports. Cultivated A. bisporus and wild Agaricus silvicola (Vitt.) Peck, Agaricaceae, were extracted by means of supercritical CO2 and analyzed by gas chromatography–mass spectrometry (GC-MS) and gas chromatography–Fourier transform infrared (GC-FTIR) spectroscopy [18]. SFE was compared to classical solvent extraction, showing to be less laborious and more selective toward some specific fatty acids. Saturated fatty acids ranging from 5 to 26 carbon atoms were determined in both species. Among these, palmitic and stearic were predominant. Benzoic acid and hexanoic acid were typically found at higher proportions in the wild species. Dried shiitake mushrooms were extracted by SFE and by conventional organic solvent extraction (COSE) [19]. Studies in Natural Products Chemistry, First Edition, 2016, 279-306 Author's personal copy The Chemistry of Mushrooms Chapter | 9 285 Different conditions and solvents were tested for SFE and COSE, as can be seen in Fig. 9.1. Extraction yields were higher for SFE. Furthermore, all the extracts were assayed for antimicrobial and antioxidant activities. Similar antioxidant activity was observed for both SFE and COSE extracts, whereas only SFE extracts exerted antimicrobial activity, against Micrococcus luteus and Bacillus cereus. Linoleic acid was the most abundant compound, the remaining fraction being constituted by palmitic acid, ergosterol, and p-menthane-1,8-diol. About thirty compounds were determined in A. brasiliensis (Peck), Agaricaceae, samples extracted by means of SFE and low-pressure methodologies, namely Soxhlet and maceration [20]. The extracts were successively analyzed by GC-MS and were basically composed of fatty acids (palmitic and linoleic), ergosterol, and derivatives. The extracts were also assayed for antioxidant and antimicrobial activity, the latter being more pronounced, particularly against Gram-positive bacteria. A low to moderate antioxidant activity was observed, fact addressed to the types of antioxidant activity tests carried out. Soxhlet gave the highest yields, although the number of compounds extracted was much lower compared to SFE. In another study, P. ostreatus mushrooms were evaluated for their content of polyphenols and ergothioneine. A statistical model was developed (response surface methodology) and applied to the SFE method optimization [21]. Analytes were quantified by using high-pressure liquid chromatography (HPLC) followed by tandem mass spectrometry (MS/MS). The 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging capacity of the SCF extracts of P. ostreatus was also tested. FIGURE 9.1 Extraction yield (w/w) results for shiitake extraction using different techniques: classical organic solvent extraction with n-hexane (Hx), dichloromethane (DCM) and ethyl acetate (EtAc); supercritical fluid extraction (SFE) (40°C/20 MPa) with ethanol (EtOH) as cosolvent at concentrations of 5%, 10%, and 15%; SFE (40°C/20 MPa) with DCM as cosolvent at concentrations of 10%, 15%, and 20%; SFE with pure CO2 at 40°C and 20 MPa; SFE (40°C/20 MPa) with EtAc as cosolvent at 15% concentration. Reproduced from C.S. Good Kitzberger, A. Smânia Jr., R. Curi Pedrosa, S.R.S. Ferreira, Antioxidant and antimicrobial activities of shiitake (Lentinula edodes) extracts obtained by organic solvents and supercritical fluids, J. Food Eng 80 (2007) 631–638. Studies in Natural Products Chemistry, First Edition, 2016, 279-306 Author's personal copy 286 Studies in Natural Products Chemistry MICROWAVE-ASSISTED EXTRACTION MAE exploits solvents heated by microwaves for the isolation of analytes from a sample matrix. The success of this technique is linked to the high temperature and to the solvent, or solvent mixture, utilized. Microwaves penetrate the molecules proportionally to their dielectric constant (ε): the higher the constant, the stronger the absorption. The extraction mechanisms of MAE place it in an intermediate state between ASE and SFE. Two options are available when extracting samples by MAE: open vessel and closed vessel. The choice is mainly dictated by the ε value of the solvent used: if it is high, then a closed vessel has to be preferred, where the solvent is heated far above its boiling point. If the solvent has a low ε value, the open-vessel mode will lead to the extraction of sample components characterized by higher ε, which will move to the surrounding cold solvent. In general, MAE exploits a small amount of solvents, therefore it is considered a “green” technique. Moreover, heating occurs in a selective manner with much less energy loss in the environment. Another green feature of MAE is the possibility of using no solvents at all (dry MAE), as in the case of essential oils from plant material, where the solvent is the water itself contained in the samples. The MAE technique is a good method for extraction of polysaccharides and fats from different sources. Agaricus bisporus samples and fungal spores and hyphae from other species were extracted by means of MAE, SFE, and classical solvent extraction [22]. HPLC was used for the determination of fatty acids. MAE provided the highest extraction yields with a correspondent reduction of analysis time and amount of solvents. Polysaccharides were extracted by MAE from himematsutake (Agaricus blazei Murril, Agaricaceae) mushrooms [23]. An experimental design-assisted method optimization involving extraction temperature and time, microwave power, and sample amount was developed. MAE extracts were then assayed for their antioxidant activity, which was found to be higher than in conventionally obtained extracts. MAE polysaccharides were suggested as ingredients of therapeutic formulations. In another study, polysaccharides from G. lucidum were extracted by MAE with the support of ultrasonication (UMAE) [24]. The extraction yields of MAE polysaccharides were higher than those of the classical hot water and single ultrasonic extraction procedures. Ultrasound extraction works on a different principle compared to microwave extraction. The solvent is generally not heated but the microbubbles formed in the solvent aggressively disrupt the sample structure and promote better mixing and contact with the solvent. The advantages support the importance and great potential of ultrasonic/ microwave technology on the polysaccharides extraction from different tissues of plant materials. Some immunological assays were also carried out, showing that UMAE extracts had no noticeable effects on phagocytosis of monocyte at the tested dosage range. A more relevant immunoresponse was observed at high dose in immunocompromised mice. Studies in Natural Products Chemistry, First Edition, 2016, 279-306 Author's personal copy The Chemistry of Mushrooms Chapter | 9 287 Again a statistical model (response surface methodology) was applied to the optimization of UMAE conditions for the extraction of polysaccharides from Inonotus obliquus (Ach. Ex Pers.) Pilát, Hymenochaetaceae [25]. The results confirmed UMAE to be more effective than traditional hot water extraction toward the isolation of polysaccharides. Furthermore, the extracts exhibited antitumor activity. PULSED ELECTRIC FIELD Recently, this technique has been successfully applied to food and agricultural processing. It exploits short (micro-to milliseconds) pulses of electricity, which impact on a product placed between two electrodes, in the PEF chamber [26]. The basic effect caused by the application of PEF on biological material is the permeabilization of cell membranes, phenomenon also known as “electroporation” or “electropermeabilization” [27]. This inevitably causes the death, more properly inactivation, of microorganisms. It is for this reason that PEF techniques have found wide diffusion as alternative pasteurization methodologies to the most common thermal treatments. Compared to thermal treatments, PEF can be applied to contrast food spoilage without undesirable effects, such as degradation and reduction of the nutritional value. On the other hand, disruption of cell membranes can be an advantageous tool for extraction purposes, since it is accompanied by acceleration of the mass transfer. In food plants, the valuable constituents, such as proteins, polysaccharides, and enzymes, are indeed enclosed in the cell compartments. The process of electroporation can be reversible (membrane discharge) or irreversible (membrane lysis), depending on the nature of the sample to be processed. A PEF system is composed of a high-voltage pulse generator; a treatment chamber, where the sample is located; a pump for introduction of fluid samples; and a cooling coil, for lowering the temperature. Numerous studies report the extraction by PEF of chemical constituents from potatoes, apple, coconuts, carrots, red bell pepper, paprika, fennel, chicory, alfalfa, red cabbage, and mango [26,27]. Since no solvents are used in this process, PEF falls within the group of green techniques. Fig. 9.2 shows an experimental setup and procedure applied to the extraction of mushrooms from the species A. bisporus by means of PEF technology [28]. The work mainly focused on the application of pressure to PEF extraction, and consequent comparison of the results with those derived from water extraction and ethanol extraction. The parameters evaluated were basically the extract stability and the extraction yields. As clearly shown in Fig. 9.2, fresh mushrooms were sliced and placed in a pressing and PEF treatment chamber. Upon pressure, a juice was expressed from the mushroom cake and collected at the bottom. It was concluded that PEF-assisted extraction from mushrooms produced extracts with higher colloid stability, high amounts of fresh-like proteins and polysaccharides, and increased nucleic acid/proteins ratio. Studies in Natural Products Chemistry, First Edition, 2016, 279-306 Author's personal copy 288 Studies in Natural Products Chemistry FIGURE 9.2 Experimental setup and pulsed electric field (PEF) protocol used for the extraction of Agaricus bisporus mushrooms. With kind permission from O. Parniakov, N.I. Lebovka, E. Van Hecke, E. Vorobiev, Pulsed electric field assisted pressure extraction and solvent extraction from mushroom (Agaricus bisporus), Food Bioprocess Technol 7 (2013) 176, Figure 1, © 2013, Springer Science and Business Media. QUICK, EASY, CHEAP, EFFECTIVE, RUGGED, SAFE (QuEChERS) This extraction protocol falls within the so-called dispersive solid-phase extraction (DSPE) techniques, originally introduced by Anastassiades et al. [29]. Samples under investigation were fruits and vegetables and target analytes were pesticides. The DSPE methodology involved a preliminary step of extraction with a small volume of acetonitrile, successively dehydrated and mixed with sorbent material, namely PSA (primary secondary amine) and magnesium sulfate. This procedure was successively validated within an international framework of research centers, in total 15 different laboratories in 7 different countries. The result of the interlaboratory study was divulgated under the acronym QuEChERS (see title) and showed excellent efficacy in terms of analyte (particularly pesticides) recovery. A QuEChERS scheme is here summarized: (1) homogenize samples; (2) extract with acetonitrile; (3) add a salt mixture to the test tube, shake, and centrifuge; (4) add the supernatant with sorbent, shake, and centrifuge; and (5) recover the upper layer containing the analytes. QuEChERS is considered an environmentally friendly sample preparation procedure, mainly because it exploits nonhalogenated solvents and in lesser Studies in Natural Products Chemistry, First Edition, 2016, 279-306 Author's personal copy The Chemistry of Mushrooms Chapter | 9 289 amount. The production of waste is almost half-reduced compared to a conventional liquid–liquid extraction process. The overall cost, considering apparatus, standards, reagents, and sample throughput, is definitely lower compared to the laborious procedures that would be needed in a conventional extraction methodology. Some limitations can be attributed also to QuEChERS protocol: the procedure results to be problematic when samples are cereals, spices, tea, and oils; some instrumental requirements, such as the need for a programmed temperature vaporizing injector or of a new generation mass spectrometer; finally, matrix effects can be observed for complex matrices. Numerous works have exploited the QuEChERS methodology for the analysis of mushrooms. Dietary supplements labeled as containing Cordyceps and Reishi were analyzed for the presence of 174 different pesticides to be determined by means of LC-MS/MS and GC-MS/MS [30]. The cultivated mushrooms A. bisporus, L. edodes, and P. ostreatus were extracted by QuEChERS to assess trace levels of pyriproxyfen, avermectin, and diflubenzuron [31]. A total of 30 samples were analyzed, including mushrooms and substrates from China. Pyriproxyfen was found in shiitake mushrooms and its substrates in a concentration range of 0.01–0.15 mg/ kg. No avermectin and diflubenzuron were detected in any samples. However, the DSPE method demonstrated to be a very valid tool for the determination by UPLC-MS/MS of ultratrace level pesticides (ppb). In combination with dispersive liquid–liquid microextraction (DLLME), QuEChERS was applied to simultaneous extraction, concentration, and derivatization of both bisphenol A and bisphenol B from canned, whole, and sliced mushrooms [32]. Tetrachloroethylene was used as extractive solvent while the acetonitrile extract, obtained from QuEChERS, was used as dispersive solvent. At the same time, acetic anhydride was added as derivatizing agent for successive GC-MS analysis. The method was considered accurate (>69% recovery), reproducible (<20% RSD), and sensitive for the target analytes (detection limits were 300 and 600 ppb, for BPA and BPB, respectively). A procedure based on the QuEChERS methodology and liquid chromatography tandem mass spectrometry (LC/MS/MS) was applied to the determination of the insecticide nicotine in fresh A. bisporus and in dried Boletus edulis Bull. (Boletaceae) samples [33]. Limit of quantification (LOQ) was 0.01 mg/ kg for both fresh and dried mushrooms. The nicotine levels for all analyzed samples were assessed as being below the limit prescribed by the EU regulations, therefore no evidence of risk for consumers could be observed. Similarly, the QuEChERS protocol was exploited for the extraction of nicotine from dried mushrooms (B. edulis) and its performance compared to liquid extraction (ethyl acetate) [34]. QuEChERS extracts were analyzed by liquid chromatography– time-of-flight mass spectrometry, whereas solvent extracts by gas chromatography–triple quadrupole-mass spectrometry. An LC-MS/MS method based on QuEChERS was developed and applied to the simultaneous determination of 33 mycotoxins in L. edodes for the first time. It was demonstrated that many samples of the mushroom, widely consumed in Studies in Natural Products Chemistry, First Edition, 2016, 279-306 Author's personal copy 290 Studies in Natural Products Chemistry dietary supplements, were contaminated by the microfungus of the Fusarium species, with highly relevant risks to the human health [35]. LIQUID-PHASE MICROEXTRACTION This extraction technique exploits a very limited amount (microliters) of solvent as extracting phase, fact that makes it environmental friendly. In single drop microextraction (SDME), a microdrop of solvent (analyte-selective, ie, able to dissolve target analytes) suspended at the tip of a GC syringe, acts as extractant. The drop is immersed in the sample solution during the extraction process; once completed this phase, the microdrop is withdrawn into the needle and successively inserted in the hot GC injection port, as a conventional sample injection procedure. Common solvents utilized in LPME are dichloromethane, chlorobenzene, n-hexane, n-octanol, xylene, and tetrachloroethane. This guarantees a wide range of selectivity for this extraction technique. LPME can be operated in various modes, from where it takes different acronyms, such as DI-SDME (direct immersion-single drop microextraction), CFME (continuous flow microextraction), DDME (drop-to-drop microextraction), DSDME (directly suspended droplet microextraction), HS-SDME (headspace single drop microextraction), LLLME (liquid–liquid–liquid microextraction), HF-LPME (hollow fiber–liquid phase microextraction), DLLME. Some of the disadvantages of LPME lie on the microdrop’s instability: it is for overcoming the undesired phenomenon of “drop-off” that in HF-LPME a hollow fiber is used, so to protect the extractant phase. Also, the hollow fiber can locate a higher amount of extracting solvent, therefore increasing both the contact area and the extraction efficiency. Furthermore, in order to increase the contact surface between the extracting solvent and the sample to be extracted, a disperser can be added to the LPME system. In DLLME, a water-immiscible extractant is dispersed in a water-soluble solvent, namely the disperser, and injected in an aqueous sample. Typical dispersers are acetone, acetonitrile, and methanol. In general, LPME appears to be an advantageous technique, basically for its simplicity, low cost, and “ready to inject” fashion. The technique is also considered quite sensitive and suitable for analytes concentrated at very low level. Among all the LPME techniques, DLLME results to be the most successful in terms of analytical performance. Nonetheless, a survey of literature evidence only a relevant work based on the use of LPME for the extraction of mushrooms of the species L. edodes [36]. As extractant, 10 μL of an ionic liquid was used, associated with a simultaneous derivatization of the target analyte (formaldehyde). In this case, the microdrop was injected into an HPLC system with very good results in terms of sensitivity (LOD: 5 μg/L). The objective of the work was to assess the amount of formaldehyde in cultivated shiitake mushrooms, knowing the carcinogenic properties of this compound and, at the same time, the wide consumption of the mushrooms in the traditional Chinese medicine. Studies in Natural Products Chemistry, First Edition, 2016, 279-306 Author's personal copy The Chemistry of Mushrooms Chapter | 9 291 SOLID-PHASE MICROEXTRACTION Introduced more than 20 years ago, this microextraction technique has become so popular to embrace different fields of applications, from forensics to analytical, from pharmaceuticals to food, and from environmental to biological [37]. SPME owes its success to a series of factors: first of all, it is a solventless technique, working at low temperature with no impact on the environment; it is simple, cheap, and can be automated. It works in a ready-to-inject fashion. It is sensitive, selective, and versatile (compatible with both GC or LC separation techniques). The “miniaturized extraction” takes place by means of a 1 cm fiber, coated with a sorbent placed inside a syringe needle. The fiber can be either exposed to a HS or immersed in a liquid sample. Fiber-coating materials of varying polarity are available, the choice depending upon the analytes to be extracted. In SPME, the extraction process is considered complete when equilibria are established between the concentration of analytes in the sample matrix and that of analytes onto the fiber sites. In the case of HS analysis, a third phase must be taken in consideration, which is the HS itself. It can be derived that SPME extraction is not exhaustive, since the amount of analytes extracted will be that allowed by the active sites of the fiber. For this reason, SPME requires method development, by tuning all the parameters affecting the entire extraction procedure: temperature, salt addition, agitation, sample volume, type of fiber coating, time of fiber exposure, and so on. Once optimized the SPME method, calibration procedures are needed for accurate quantification of extracted analytes. As already mentioned, SPME is a widely employed sample preparation technique, involving various areas of research. Even in the case of mushrooms analysis, several publications have reported the use of SPME for sample preparation. In this review, only the most relevant papers of the last decade have been summarized in Table 9.1 [38–70]. STIR BAR SORPTIVE EXTRACTION This sampling methodology is again based on the use of a small sorbent surface, as in SPME, which is coated, in this case, onto a magnetic stirrer encapsulated in glass. Stir bar sorptive extraction (SBSE) finds a good compromise between the extraction process and the need for agitation of the sample solution. Indeed, the stir bar is immersed into the sample vial and, while twisting, it adsorbs target analytes. The same parameters affecting an SPME process are valid for SBSE. The stir bar can be utilized either by immersion or by HS. Basically, SBSE differs from SPME for the higher analyte’s capacity linked to the surface of extraction: typical lengths of stir bars are in the range 10–40 mm versus 10 mm of an SPME fiber. Approximately, 55–220 μL of sorbent material are coated in a stir bar, compared to only 0.5 μL in an SPME fiber. Another difference consists of the injection mode, since the stir bar cannot be introduced directly in the injection port as for the SPME holder. A thermal desorber unit is required for analyte transfer into the GC system. One of the disadvantages of SBSE could be Studies in Natural Products Chemistry, First Edition, 2016, 279-306 Author's personal copy Studies in Natural Products Chemistry, First Edition, 2016, 279-306 Sample Type of Fiber Coating(s) Used Analytes Positive and/or Negative Outcomes Analytical Technique References Clitocybe odora (Bull.) P. Kumm, Tricholomataceae; Clitocybe fragrans (With.) P. Kumm, Tricholomataceae; Hebeloma crustuliniforme (Bulliard) Quélet, Cortinariaceae; Lepista nuda (Bull.), Tricholomataceae; Tricholoma fracticum (Batsch) Kreisel Maire, Tricholomataceae; Tricholoma terreum (Schaeff) Quél. PDMS/DVB 65 μm Volatile organic compounds 11 compounds biomarkers for taxonomic and authentication purposes. Volatile profile as discriminating tool GC-IT-MS [38] 22 different wild species, including edible and poisonous mushrooms PDMS/DVB 65 μm Amino acids, fatty acids, sterols, and volatiles 7 species/genus specific biomarkers. Partial least square discriminant analysis (PLS-DA) not suitable for discrimination of edible from toxic species GC-IT-MS [39] Lactarius fragilis (Burl.) Hesler & A.H. Sm., Russulaceae PDMS 100 μm Volatiles 3-Amino-4,5-dimethyl-2(5H) furanone (quabalactone III) is responsible for the maple syrup odor GC-MS [40] 11 edible wild species DVB/Car/PDMS 50/30 μm; Car/ PDMS 75 μm; CW/ DVB 65 μm Volatiles and semivolatiles 3 clusters: C8 derivatives; terpenic; methional GC-MS and sensorial analysis [41] 292 Studies in Natural Products Chemistry TABLE 9.1 Applications Based on the Use of Solid-Phase Microextraction (SPME) as Sample Preparation Technique for Isolation of Analytes From Mushrooms Author's personal copy Flavor Typical mushroom components were determined, along with each olfactive character. Car-PDMS and HP-5 columns extracted and separated less components GC-MS GC-O [42] Canned mushrooms Car/PDMS 75 μm Furan Amount of furan in processed mushrooms (29 ng/g). Furan variability due to dispersion in the air and to cook processing GC-MS [43] Hygrophorus spp. DVB/Car/PDMS 50/30 μm Volatiles Typical C8 compounds; sesquiterpenes only in one species GC-MS [44] Canned mushrooms PDMS/DVB 65 μm; PDMS 100 μm; DVB/Car/PDMS 50/30 μm; CW/DVB 65 μm; PA 85 μm; PEG 60 μm 2,2′-Bisphenol, bisphenol A, bisphenol S Development of a method for the determination of bisphenols from the liquid and the mushroom texture. Fibers with DVB and CW gave the lowest durability because of swelling with derivatizing agents GC-MS [45] Volvariella volvacea (Bull.) Singer, Amanitaceae; and Pleurotus ostreatus PDMS/DVB 65 μm Volatiles P. ostreatus has a higher antioxidant activity than V. volvacea. C8 compounds determined GC-MS [46] Mushroom broth In-tube SPME. CP-Sil5CB; CP-Sil19CB; CP-Wax52CB; Carboxen 1010PLOT; SupelQ PLOT Abietic and dehydroabietic acids No amounts determined in mushroom broths LC-ESI-MS [47] 293 DVB/Car/PDMS 50/30 μm The Chemistry of Mushrooms Chapter | 9 Studies in Natural Products Chemistry, First Edition, 2016, 279-306 Soup of Agaricus bisporus Continued Author's personal copy Studies in Natural Products Chemistry, First Edition, 2016, 279-306 Sample Type of Fiber Coating(s) Used Analytes Lentinus edodes PDMS 100 μm P. ostreatus PA 85 μm Agaricus bisporus Positive and/or Negative Outcomes Analytical Technique References Volatiles Very low amount of volatiles determined, which decreased after drying GC-MS [48] Polycyclic aromatic hydrocarbons (PAHs) PAHs added to the culture substrate reach the fruiting bodies GC-MS [49] Volatiles A heme-dioxygenase is involved in eight-carbon volatile production GC-MS [50] Tuber magnatum Pico DVB/Car/PDMS 50/30 μm Volatiles The aroma of truffle varies upon storage conditions (type of packaging) GC × GC; e-nose [51] T. magnatum Pico DVB/Car/PDMS 50/30 μm Flavor components Compositional variations during shelf life were in accord with sensorial changes GC-MS; e-nose [52] A. bisporus PDMS/DVB 65 μm; DVB/Car/PDMS 50/30 μm; Volatiles Volatiles were quantified in absolute physical units by means of multiple headspace extraction. Addition of water promotes enzymatic breakdown. GC-MS [53] 294 Studies in Natural Products Chemistry TABLE 9.1 Applications Based on the Use of Solid-Phase Microextraction (SPME) as Sample Preparation Technique for Isolation of Analytes From Mushrooms—cont’d Author's personal copy Volatiles Three compounds determined for the first time GC-MS [54] Tuber melanosporum, T. indicum DVB/Car/PDMS 50/30 μm Flavor The family of C8 compounds was more abundant in the less valuable T. indicum species GC-MS; GC-O [55] T. melanosporum DVB/Car/PDMS 50/30 μm Volatiles The aroma of truffles is irremediably compromised by freezing GC-MS [56] T. melanosporum; Tuber aestivum Vittad., Tuberaceae DVB/Car/PDMS 50/30 μm Volatiles The olfactive impact compounds of T. melanosporum were affected by electron-beam treatment, while T. aestivum by gamma-irradiation GC-MS [57] T. aestivum PDMS 100 μm; CW/ DVB 65 μm Volatiles Temperature and SPME fiber coating were critical parameters GC-MS [58] T. magnatum Pico DVB/Car/PDMS 50/30 μm (2 cm long); PDMS 100 μm; PDMS/DVB 65 μm Volatiles Volatile composition was affected by geographical origin GC-MS [59] T. magnatum Pico DVB/Car/PDMS 50/30 μm (2 cm long) Volatiles Rapid characterization of truffle volatile composition PTR-MS; GC [60] Volatiles Increase of alcohols and acids during storage GC-MS; GC-O [61] T. magnatum Pico 295 PDMS/DVB 65 μm; PDMS 100 μm; DVB/Car/PDMS 50/30 μm; PA 85 μm The Chemistry of Mushrooms Chapter | 9 Studies in Natural Products Chemistry, First Edition, 2016, 279-306 Tuber indicum Continued Author's personal copy Studies in Natural Products Chemistry, First Edition, 2016, 279-306 Positive and/or Negative Outcomes Analytical Technique References Volatiles Influence of contaminant (Staphylococcus pasteuri) on the volatile composition GC-MS [62] T. melanosporum Volatiles Minor compounds with high olfactive power were determined GC-MS; GC-O [63] T. melanosporum Volatiles 131 compounds determined in French truffles GC-MS [64] Sample Tuber borchii Type of Fiber Coating(s) Used DVB/Car/PDMS 50/30 μm; PDMS/ DVB 65 μm; PDMS 100 μm Analytes T. magnatum Pico DVB/Car/PDMS 50/30 μm (2 cm long) Flavor Changes of the aromatic fraction during storage at +4°C GC-MS; e-nose [65] Tuber spp. PDMS 100 μm Volatiles Determination of 75 compounds GC-MS [66] Tuber spp. DVB/Car/PDMS 50/30 μm Volatiles Determination of 89 compounds GC-MS [67] T. melanosporum PDMS 100 μm; CW/ DVB 65 μm; Car/ PDMS 85 μm 2-Methylbutan1-ol Determination of the enantiomeric excess of 2-methylbutan-1-ol EnantioGC-MS [68] 296 Studies in Natural Products Chemistry TABLE 9.1 Applications Based on the Use of Solid-Phase Microextraction (SPME) as Sample Preparation Technique for Isolation of Analytes From Mushrooms—cont’d Author's personal copy PDMS/DVB 65 μm Volatiles Quantification with standard addition method; evaluation of flavor modification during storage GC-MS [69] A. bisporus PDMS/DVB 65 μm Volatiles Assessment of biomarkers among volatiles released by diseased individuals GC-MS [70] CW, carbowax; DVB, divinylbenezene; GC-MS, gas chromatography–mass spectrometry; GC-O, gas chromatography–olfactometry; PDMS, polydimethylsiloxane; PTR-MS, proton transfer reaction–mass spectrometry; SPME, solid-phase microextraction. The Chemistry of Mushrooms Chapter | 9 Studies in Natural Products Chemistry, First Edition, 2016, 279-306 Ten wild species 297 Author's personal copy 298 Studies in Natural Products Chemistry addressed to the lower selectivity, due to the fact that only polydimethylsiloxane (PDMS) and ethylene glycol/PDMS are available as coating sorbents. In general, the lack of solvents (or use of negligible amounts) and the miniaturization make the technique as much green as SPME. SBSE was applied in HS mode and coupled with GC-MS, for the differentiation of three species of truffles, namely Tuber borchii Vittad., Tuber melanosporum Vittad., and Tuber indicum Cooke & Massee [71]. As shown in Fig. 9.3, more than one stir bar was suspended over the samples, which were the fruiting bodies or the mycelia. It was found that the volatile organic compounds (VOCs) profile showed a high intra- and interspecific variability, with alcohols and sulfur compounds dominating the HS of T. borchii and, alcohols, aldehydes, and aromatic compounds the headspace of T. melanosporum and T. indicum. Furthermore, eight VOCs markers could be identified allowing the discrimination of the three species. Interestingly, sulfur compounds, detected in the fruiting bodies, were not determined in the mycelia as well, leading to questions about their metabolic origin. SBSE was also applied to the extraction of volatile constituents (by immersion) of shiitake mushrooms [72]. The experiments were run in parallel with steam distillation and dynamic headspace extractions. It was observed that SBSE extracted the lowest number of compounds, although the extraction steps were definitely easier to be carried out, with less artifact formation. Diethyl phthalate, considered an endocrine disrupter, was determined by means of SBSE-GC-MS in canned mushrooms [73]. Samples were previously extracted by ultrasonic radiation in aqueous solution; the aqueous extracts were preconcentrated by in-sample SBSE. FIGURE 9.3 Extraction of volatiles released by truffles by means of stir bar sorptive extraction (SBSE); two or three stir bars were suspended to an iron pin over the fruiting body or mycelium for 63 h. SPME, solid-phase microextraction. Reprinted from, R. Splivallo, S. Bossi, M. Maffei, P. Bonfante, Discrimination of truffle fruiting body versus mycelial aromas by stir bar sorptive extraction, Phytochemistry 68 (2007) 2584–2598, Copyright (2007), with permission from Elsevier. Studies in Natural Products Chemistry, First Edition, 2016, 279-306 Author's personal copy The Chemistry of Mushrooms Chapter | 9 299 SOLID-PHASE EXTRACTION More than an extraction technology, SPE is rather considered a sample cleanup (from interfering substances) procedure, valid for a wide range of complex matrices. Liquid samples (ie, solvent extracts) are passed through a sorbent material and analytes are separated according to their affinity for the solid sorbent. SPE equipment consists of cartridges or disks packed with the sorbent material (stationary phase). After the sample suspension is passed through the cartridge, a selective solvent is let to flush so to retain (or elute) the target analytes (or the interfering compounds). Only the fraction containing the analytes of interest will be collected and injected into a separation system. Compared to conventional liquid–liquid extraction, SPE reduces the consumption of organic solvents, therefore dramatically reducing the wastes production. As well as in HPLC, SPE can be operated as normal phase (NP), reversed phase (RP), and ion exchange (IE) based on the chemical nature of the sorbent material. Typical sorbents are silica and alumina for NP, ion exchangers, and aminosilica for IE, octyl- and octadecylsilica for RP. Novel SPE sorbents are represented by restricted access media, immunosorbents, and molecularly imprinted polymers (MIPs). Restricted access materials, initially developed for biological applications, work in a way that is similar to size exclusion chromatography: macromolecules (ie, proteins) are prevented to enter the sorbent’s retention sites. Restricted access materials are usually porous silica gel or polymer beads. The isolation of analytes can occur by either physical access into the sorbent pores or chemical diffusion through a barrier. Restricted access media are commonly interfaced with automated injection in liquid chromatography. Immunosorbents are based on the use of biological molecules, ie, antibodies, covalently immobilized on a solid support. Immunosorbents can be packed either in a cartridge for SPE extraction or in a chromatographic column. The antigen–antibody interaction in immunosorbents is characterized by high specificity. However, they have reduced sample capacity and must be pressure proof when used as precolumns in LC. The difficulties encountered in the production process reduce the range of selectivity of such products. MIPs are considered “smart” polymers capable of a memory effect. MIPs technology consists of creating cavities in polymeric matrices that can lodge template molecules: the polymer acts as the lock and the template molecule as the key. Once the polymer has been imprinted, the template molecule is removed. The imprinted polymer saves memory and has high affinity for the template molecule over which it has been created: this makes it capable of “recognizing” and rebinding the same molecule when put in contact again with it. Some of the most relevant applications in the field of mushrooms analysis which exploit SPE techniques are summarized in Table 9.2 [74–81]. Studies in Natural Products Chemistry, First Edition, 2016, 279-306 Author's personal copy Studies in Natural Products Chemistry, First Edition, 2016, 279-306 Analytical Technique References Determination of analytes in patients who have ingested toxic Amanita spp. LC-MS-MS [74] Pesticides High sensitivity and selectivity of the SPE method for 22 pesticides LC-MS-MS [75] Primary secondary amine Carbamates High accuracy and sensitivity of the method developed UPLC-MS-MS [76] Edible mushrooms Carbon and primary secondary amine Organohalogen and pyrethroid pesticides Development of an accurate and simple method for determination of pesticides in mushrooms GC-ECD; GC-FPD [77] Omphalotus guepiniformis (Berk.) Neda, Omphalotaceae; L. edodes, P. ostreatus, Pleurotus serotinus (Pers.) Kühner, Mycenaceae; mushrooms’ soup Polymer with pyrrolidinone and phenyl groups Illudin S Development of a method suitable for the detection of illudin S in mushrooms and food meals that caused poisoning LC-MS-MS [78] Sample SPE Material Analytes Outcomes Human serum DVB copolymer with quaternary amine Muscimol, ibotenic acid Pleurotus ostreatus; Pleurotus eryngii (DC.) Quél. Pleurotaceae; Agaricus bisporus; Lentinus edodes Aminopropyl polymer A. bisporus 300 Studies in Natural Products Chemistry TABLE 9.2 Relevant Applications of Solid-phase Extraction (SPE) in Mushroom Analysis Author's personal copy Carbon and amine Pesticides Development of a method for the determination of 187 pesticides in edible mushrooms LC-MS-MS [79] P. ostreatus, L. edodes, Volvariella volvacea Polymer with aminogroups Imidacloprid, acetamiprid, thiabendazole, and carbendazim Development of a sensitive, accurate, and precise method for detection of pesticides HPLC [80] 25 species of mushrooms MIPs (caffeic acid phenethyl ester as template) Caffeic acid phenethyl ester and caffeic acid Isolation of analytes by means of MIPs and evaluation of antioxidant activity HPLC-UV [81] DVB, divinylbenzene; GC-ECD, gas chromatography–electron capture detector; GC-FPD, gas chromatography–flame photometric detector; HPLC, high pressure liquid chromatography; LC-MS-MS, liquid chromatography tandem mass spectrometry; MIP, molecularly imprinted polymer; SPE, solid-phase extraction; UPLC-MS-MS, ultraperformance liquid chromatography tandem mass spectrometry. The Chemistry of Mushrooms Chapter | 9 Studies in Natural Products Chemistry, First Edition, 2016, 279-306 Auricularia polytricha (Mont.) Sacc., Auriculariaceae; Pholiota nameko (T. Itô) S. Ito & S. Imai, Strophariaceae; Flammulina velutipes (Curtis) Singer, Marasmiaceae; L. edodes 301 Author's personal copy 302 Studies in Natural Products Chemistry CONCLUSION Mushrooms play a fundamental role in the ecosystems, besides being important sources of food and drugs. In this review, a selection of applications of the most recent and green sample preparation techniques to mushroom analysis has been presented. The newly introduced extraction techniques are characterized by low consumption of solvents, possibility of automation, simplicity, sensitivity, and selectivity. They are categorized in solvent-based (ie, ASE, MAE, LPME) and sorbent-based (ie, SPME, SBSE, SPE) techniques, with QuEChERS placed in between of them; and techniques based on physical means, such as SFE and PEF. As concerns mushrooms, part of the publications of the last 20 years focus on the development of methods suitable for the isolation and determination of pesticide residues. Another group of papers deals with the investigation of the volatile fraction with multiple purposes: differentiation of more species belonging to the same genus; determination of markers in poisonous and edible species; evaluation of parameters affecting the shelf life; and olfactometric characterization. In some cases, the analyses were targeted to the determination of specific analytes, ie, phytosterols, fatty acids, polysaccharides, and formaldehyde. Among all the techniques reported in this review, SPME resulted to be the most exploited, although this success is not exclusive of the mushroom analysis field. Overall, the utilization of miniaturized and green preconcentration techniques for mushrooms investigation appears to be attractive to the researcher for various reasons. Traditional methods are timeconsuming and multistep-based procedures. Mushrooms are biological and complex matrices, which pose analytical challenges, such as determination of target analytes at trace level. To this aim, conventional multistep approaches are not the most proper, because they can be source of irremediable errors (eg, analyte loss during sample preparation phases). Modern extraction techniques have been designed not only to support the analyst by providing simultaneously extraction and cleanup but also to reduce the impact on the environment and analysis costs. All the sample preparation techniques presented in this review are enough robust, sensitive, and reproducible to be employed either for routine or for targeted analyses. Apart from sorbent-based miniaturized techniques (ie, SPE, SPME, and SBSE), and SFE, all the extraction methods outlined in this review work at temperatures as high as to make them suitable for the isolation of thermostable components, ie, fatty acids, sterols, sugars, and so on. Their exploitation would result useless for most secondary metabolites that are strongly sensitive to high temperatures. ABBREVIATIONS ASE CFME COSE accelerated solvent extraction continuous flow microextraction conventional organic solvent extraction Studies in Natural Products Chemistry, First Edition, 2016, 279-306 Author's personal copy The Chemistry of Mushrooms Chapter | 9 DCM DDME DI-SDME DLLME DPPH DSDME DSPE e-Nose EtAc EtOH GC-ECD GC-FPD GC-FTIR GC-IT-MS GC-MS GC-O HF-LPME HS HS-SDME Hx IE LC-ESI-MS LC-MS/MS LLLME LOQ LPME MAE MIP NP PDMS PEF PFE PLE ppb ppt PSA PTR-MS PWE QuEChERS RP SBSE SCF SFE SPE SPME UMAE UPLC-MS/MS VOC 303 dichloromethane drop-to-drop microextraction direct immersion-single drop microextraction dispersive liquid–liquid microextraction 2,2-diphenyl-1-picrylhydrazyl directly suspended droplet microextraction dispersive solid-phase extraction electronic nose ethyl acetate ethanol gas chromatography–electron capture detector gas chromatography–flame photometric detector gas chromatography–Fourier transform infrared gas chromatography–ion trap–mass spectrometry gas chromatography–mass spectrometry gas chromatography–olfactometry hollow fiber–liquid phase microextraction headspace headspace single drop microextraction n-hexane ion exchange liquid chromatography–electrospray ionization–mass spectrometry liquid chromatography–tandem mass spectrometry liquid–liquid–liquid microextraction limit of quantification liquid-phase microextraction microwave-assisted extraction molecularly imprinted polymer normal phase polydimethylsiloxane pulsed electric field pressurized fluid extraction pressurized liquid extraction parts per billion parts per trillion primary secondary amine proton transfer reaction–mass spectrometry pressurized water extraction quick easy cheap effective rugged safe reversed phase stir bar sorptive extraction supercritical fluid supercritical fluid extraction solid-phase extraction solid-phase microextraction ultrasonication microwave-assisted extraction ultraperformance liquid chromatography–tandem mass spectrometry volatile organic compound Studies in Natural Products Chemistry, First Edition, 2016, 279-306 Author's personal copy 304 Studies in Natural Products Chemistry REFERENCES [1] S.-T. 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