VOCs Are Relevant Biomarkers of Elicitor-Induced Defences in Grapevine

Grapevine is susceptible to fungal diseases generally controlled by numerous chemical fungicides. Elicitors of plant defence are a way of reducing the use of these chemicals, but still provide inconsistent efficiency. Easy-to-analyse markers of grapevine responses to elicitors are needed to determine the best conditions for their efficiency and position them in protection strategies. We previously reported that the elicitor sulphated laminarin induced the emission of volatile organic compounds (VOCs) by grapevine leaves. The present study was conducted to characterise and compare VOC emissions in response to other elicitors. Bastid^® was first used to test the conditions of VOC collection and analysis. Using SBSE-GC-MS, we detected several VOCs, including the sesquiterpene α-farnesene, in a time-dependent manner. This was correlated with the induction of farnesene synthase gene expression, in parallel with stilbene synthesis (another defence response), and associated to resistance against downy mildew. The other elicitors (Redeli^®, Romeo^®, Bion^®, chitosan, and an oligogalacturonide) induced VOC emission, but with qualitative and quantitative differences. VOC emission thus constitutes a response of grapevine to elicitors of various chemical structures. Therefore, VOC analysis is relevant for studying the impact of environmental factors on grapevine defence responses and optimising the performance of elicitors in vineyards.     

Determination and imaging of metabolites from Vitis vinifera leaves by laser desorption/ionisation time‐of‐flight mass spectrometry

Analysis of grapevine phytoalexins at the surface of Vitis vinifera leaves has been achieved by laser desorption/ionisation time‐of‐flight mass spectrometry (LDI‐ToFMS) without matrix deposition. This simple and rapid sampling method was successfully applied to map small organic compounds at the surface of grapevine leaves. It was also demonstrated that the laser wavelength is a highly critical parameter. Both 266 and 337 nm laser wavelengths were used but the 266 nm wavelength gave increased spatial resolution and better sensitivity for the detection of the targeted metabolites (resveratrol and linked stilbene compounds). Mass spectrometry imaging of grapevine Cabernet Sauvignon leaves revealed specific locations with respect to Plasmopara viticola pathogen infection or light illumination. Copyright © 2010 John Wiley & Sons, Ltd.     

The pivotal role of high-resolution mass spectrometry in the study of grape glycosidic volatile precursors for the selection of grapevines resistant to mildews

A breeding program to produce new grape varieties tolerant to main vine fungal pathogens (Plasmopara viticola and Erysiphe necator) is carrying out by crossing Vitis vinifera cv. “Glera” with resistant genotypes such as “Solaris,” “Bronner,” and “Kunleany.” Firstly, resistance gene-based markers analyses allowed the identification of five genotypes, which have inherited the resistance loci against mildews. To select those that also inherited the phenotype as close as possible to ‘Glera’ suitable to be introduced in the Prosecco wine production protocols, the grape glycosidic derivatives were studied by UHPLC/QTOF mass spectrometry. Targeted identification of the metabolites was performed using a database expressly constructed by including the glycosidic volatile precursors previously identified in grape and wine. A total of 77 glycosidic derivatives including many aroma precursors and some variety markers, were identified. Original resistant genotypes had distinct metabolomic profiles and different to ‘Glera’, while the crossings showed varying similarity degrees to V. vinifera parent. Findings demonstrated the Glera × Bronner and Glera × Solaris crossings are more suitable to produce high-sustainable Prosecco wines. Coupling of glycosidic volatile precursors profiling to multivariate statistical analysis was effective for phenotypic characterization of grapes and to evaluate their enological potential.     

Identification and quantification of VOCs by proton transfer reaction time of flight mass spectrometry: An experimental workflow for the optimization of specificity,sensitivity, and accuracy

Proton transfer reaction time of flight mass spectrometry (PTR‐ToF‐MS) is a direct injection MS technique, allowing for the sensitive and real‐time detection, identification, and quantification of volatile organic compounds. When aiming to employ PTR‐ToF‐MS for targeted volatile organic compound analysis, some methodological questions must be addressed, such as the need to correctly identify product ions, or evaluating the quantitation accuracy. This work proposes a workflow for PTR‐ToF‐MS method development, addressing the main issues affecting the reliable identification and quantification of target compounds. We determined the fragmentation patterns of 13 selected compounds (aldehydes, fatty acids, phenols). Experiments were conducted under breath‐relevant conditions (100% humid air), and within an extended range of reduced electric field values (E/N = 48–144 Td), obtained by changing drift tube voltage. Reactivity was inspected using H₃O⁺, NO⁺, and O₂⁺ as primary ions. The results show that a relatively low (<90 Td) E/N often permits to reduce fragmentation enhancing sensitivity and identification capabilities, particularly in the case of aldehydes using NO⁺, where a 4‐fold increase in sensitivity is obtained by means of drift voltage reduction. We developed a novel calibration methodology, relying on diffusion tubes used as gravimetric standards. For each of the tested compounds, it was possible to define suitable conditions whereby experimental error, defined as difference between gravimetric measurements and calculated concentrations, was 8% or lower.     

Detecting changes in arabidopsis cell wall composition using time‐of‐flight secondary ion mass spectrometry

Time‐of‐flight secondary ion mass spectrometry (ToF‐SIMS) was previously used to characterize lignocellulosic materials, including woody biomass. ToF‐SIMS can acquire both rapid spectral and spatial information about a sample's surface composition. In the present study, ToF‐SIMS was used to characterize the cell walls of stem tissue from the plant model organism, Arabidopsis thaliana. Using principal component analyses, ToF‐SIMS spectra from A. thaliana wild‐type (Col‐0), cellulose mutant (irx3), and lignin mutant (fah1) stem tissues were distinguished using ToF‐SIMS peaks annotated for wood‐derived lignocellulose, where spectra from the irx3 and fah1 were characterized by comparatively low polysaccharide and syringyl lignin content, respectively. Spatial analyses using ToF‐SIMS imaging furthermore differentiated interfascicular fiber and xylem vessels based on differences in the lignin content of corresponding cell walls. These new data support the applicability of ToF‐SIMS peak annotations based on woody biomass for herbaceous plants, including model plant systems like arabidopsis. Copyright © 2015 John Wiley & Sons, Ltd.     

Volatile compounds profiling by using proton transfer reaction‐time of flight‐mass spectrometry (PTR‐ToF‐MS). The case study of dark chocolates organoleptic differences

Direct‐injection mass spectrometry (DIMS) techniques have evolved into powerful methods to analyse volatile organic compounds (VOCs) without the need of chromatographic separation. Combined to chemometrics, they have been used in many domains to solve sample categorization issues based on volatilome determination. In this paper, different DIMS methods that have largely outperformed conventional electronic noses (e‐noses) in classification tasks are briefly reviewed, with an emphasis on food‐related applications. A particular attention is paid to proton transfer reaction mass spectrometry (PTR‐MS), and many results obtained using the powerful PTR‐time of flight‐MS (PTR‐ToF‐MS) instrument are reviewed. Data analysis and feature selection issues are also summarized and discussed. As a case study, a challenging problem of classification of dark chocolates that has been previously assessed by sensory evaluation in four distinct categories is presented. The VOC profiles of a set of 206 chocolate samples classified in the four sensory categories were analysed by PTR‐ToF‐MS. A supervised multivariate data analysis based on partial least squares regression‐discriminant analysis allowed the construction of a classification model that showed excellent prediction capability: 97% of a test set of 62 samples were correctly predicted in the sensory categories. Tentative identification of ions aided characterisation of chocolate classes. Variable selection using dedicated methods pinpointed some volatile compounds important for the discrimination of the chocolates. Among them, the CovSel method was used for the first time on PTR‐MS data resulting in a selection of 10 features that allowed a good prediction to be achieved. Finally, challenges and future needs in the field are discussed.     

Metabolic study of grapevine leaves infected by downy mildew using negative ion electrospray – Fourier transform ion cyclotron resonance mass spectrometry

Grapevine is of worldwide economic importance due to wine production. However, this culture is often affected by pathogens causing severe harvest losses. Understanding host–pathogen relationships may be a key to solve this problem. In this paper, we evaluate the direct flow injection by electrospray – Fourier transform ion cyclotron resonance mass spectrometry (MS) of leaf extracts as a rapid method for the study of grapevine response to downy mildew (Plasmopara viticola) attack. The comparison of MS profiles obtained from control and infected leaves of different levels of resistant grapevines highlights several classes of metabolites (mainly saccharides, acyl lipids, hydroxycinnamic acids derivatives and flavonoids) which are identified using high resolution MS and tandem MS (MS/MS). Statistical analyses of 19 markers show a clear segregation between inoculated and healthy samples. This study points out relative high levels of disaccharides, acyl lipids and glycerophosphoinositol in inoculated samples. Sulfoquinovosyl diacylglycerols also emerge as possible metabolites involved in plant defense.     

Banchromene and other secondary metabolites from the endophytic fungus Fusarium sp. obtained from Piper guineense inhibit the motility of phytopathogenic Plasmopara viticola zoospores

A new chromene, (S)-banchromene (1), together with seven known compounds, ergosterol, beauvericin (2), fusaproliferin (3), radicinin (4), poly(3-hydroxybutyric acid) (PHB, 5), N-methylpyrrolidone and an inseparable mixture of isochromene derivatives 6a, 6b, were isolated from a culture of Fusarium sp. strain CAMKT24b1, an endophytic fungus from the leaves and twigs of Piper guineense (Piperaceae). The structures of these metabolites were elucidated on the basis of their spectroscopic data; the absolute configuration of 1 was determined by ab initio-calculation of the optical rotation. In tests with the zoospores of the grapevine downy mildew pathogen Plasmopara viticola, compounds 1–4 showed moderate to high levels of motility-impairing activity at concentrations as low as 2.5 μg/mL. Compound 2 was the most active, exhibiting both motility-halting and lytic activities. Furthermore, compounds 2 and 3 displayed significant cytotoxic activity against brine shrimp larvae (Artemia salina) at 10 μg/mL. This is the first report on motility inhibitory and lytic activities of metabolites from an endophytic Fusarium species against the zoospores of the downy mildew pathogen P. viticola.     

Investigation of block depth distribution in PS‐b‐PMMA block copolymer using ultra‐low‐energy cesium sputtering in ToF‐SIMS

Directed self‐assembly of block copolymers (BCPs) is a promising candidate for next generation nanolithography. In order to validate a given pattern, the lateral and in‐depth distributions of the blocks should be well characterized; for the latter, time‐of‐flight (ToF) SIMS is a particularly well‐adapted technique. Here, we use an ION‐TOF ToF‐SIMS V in negative mode to provide qualitative information on the in‐depth organization of polystyrene‐b‐polymethylmethacrylate (PS‐b‐PMMA) BCP thin films. Using low‐energy Cs⁺ sputtering and Bi₃⁺ as the analysis ions, PS and PMMA homopolymer films are first analyzed in order to identify the characteristic secondary ions for each block. PS‐b‐PMMA BCPs are then characterized showing that self‐assembled nanodomains are clearly observed after annealing. We also demonstrate that the ToF‐SIMS technique is able to distinguish between the different morphologies of BCP investigated in this work (lamellae, spheres or cylinders). ToF‐SIMS characterization on BCP is in good agreement with XPS analysis performed on the same samples. Copyright © 2013 John Wiley & Sons, Ltd.     

Temperature‐dependent release of volatile organic compounds of eucalypts by direct analysis in real time (DART) mass spectrometry

A method is described for the rapid identification of biogenic, volatile organic compounds (VOCs) emitted by plants, including the analysis of the temperature dependence of those emissions. Direct analysis in real time (DART) enabled ionization of VOCs from stem and leaf of several eucalyptus species including E. cinerea, E. citriodora, E. nicholii and E. sideroxylon. Plant tissues were placed directly in the gap between the DART ionization source skimmer and the capillary inlet of the time‐of‐flight (TOF) mass spectrometer. Temperature‐dependent emission of VOCs was achieved by adjusting the temperature of the helium gas into the DART ionization source at 50, 100, 200 and 300°C, which enabled direct evaporation of compounds, up to the onset of pyrolysis of plant fibres (i.e. cellulose and lignin). Accurate mass measurements facilitated by TOF mass spectrometry provided elemental compositions for the VOCs. A wide range of compounds was detected from simple organic compounds (i.e. methanol and acetone) to a series of monoterpenes (i.e. pinene, camphene, cymene, eucalyptol) common to many plant species, as well as several less abundant sesquiterpenes and flavonoids (i.e. naringenin, spathulenol, eucalyptin) with antioxidant and antimicrobial properties. The leaf and stem tissues for all four eucalypt species showed similar compounds. The relative abundances of methanol and ethanol were greater in stem wood than in leaf tissue suggesting that DART could be used to investigate the tissue‐specific transport and emissions of VOCs. Copyright © 2009 John Wiley & Sons, Ltd.     

ToF‐SIMS study of 1‐dodecanethiol adsorption on Au,Ag, Cu and Pt surfaces

Time‐of‐flight secondary ion mass spectrometry (ToF‐SIMS) has been used to perform a chemical analysis of long‐chain thiol (CH₃(CH₂)₁₁SH)‐treated gold, silver, copper and platinum surfaces. All the mass peaks from positive and negative ion spectra within the range m/z = 0–2000 u are studied. ToF‐SIMS data revealed that on gold, silver and copper substrates 1‐dodecanethiol form dense standing‐up phases, but on platinum being a catalytically active substrate, we were able to identify also surface‐aligned parallel lying molecules in addition to a standing thiolate layer. Our study shows that when ToF‐SIMS spectra are analyzed, not only the existence of oligomers but also metal + hydrocarbon fragments give information about the order of SAM. Copyright © 2008 John Wiley & Sons, Ltd.     

MALDI‐based intact spore mass spectrometry of downy and powdery mildews

Fast and easy identification of fungal phytopathogens is of great importance in agriculture. In this context, matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOF MS) has emerged as a powerful tool for analyzing microorganisms. This study deals with a methodology for MALDI‐TOF MS‐based identification of downy and powdery mildews representing obligate biotrophic parasites of crop plants. Experimental approaches for the MS analyses were optimized using Bremia lactucae, cause of lettuce downy mildew, and Oidium neolycopersici, cause of tomato powdery mildew. This involved determining a suitable concentration of spores in the sample, selection of a proper MALDI matrix, looking for the optimal solvent composition, and evaluation of different sample preparation methods. Furthermore, using different MALDI target materials and surfaces (stainless steel vs polymer‐based) and applying various conditions for sample exposure to the acidic MALDI matrix system were investigated. The dried droplet method involving solvent evaporation at room temperature was found to be the most suitable for the deposition of spores and MALDI matrix on the target and the subsequent crystallization. The concentration of spore suspension was optimal between 2 and 5 × 10⁹ spores per ml. The best peptide/protein profiles (in terms of signal‐to‐noise ratio and number of peaks) were obtained by combining ferulic and sinapinic acids as a mixed MALDI matrix. A pretreatment of the spore cell wall with hydrolases was successfully introduced prior to MS measurements to obtain more pronounced signals. Finally, a novel procedure was developed for direct mass spectra acquisition from infected plant leaves. Copyright © 2012 John Wiley & Sons, Ltd.     

Molecular identification of Asian lacquers from different trees using Py‐GC/MS and ToF‐SIMS

Traditional Asian lacquers are natural products with highly valued properties, including beauty, gloss, and durability. Pyrolysis‐gas chromatography/mass spectrometry is the technique of choice to study insoluble polymeric lacquer films. In the present study, pyrolysis‐gas chromatography/mass spectrometry results showed that the pyrolysis products of lacquer films were different for all of the studied trees, with urushiol derivatives detected in Toxicodendron vernicifluum from China, Japan, and Korea; laccol in Toxicodendron succedaneum from Vietnam; and thitsiol in Gluta usitata from Myanmar. Time‐of‐flight secondary ion mass spectrometry (ToF‐SIMS) was also used to characterize the Asian lacquers, avoiding the time‐consuming and destructive processes of other techniques. The ToF‐SIMS spectra provided structural characterization of a series of urushiol, laccol, and thitsiol derivatives for T vernicifluum from China, Japan, and Korea; T succedaneum from Vietnam; and G usitata from Myanmar, respectively. To differentiate the ToF‐SIMS results for the different Asian lacquer films, principal component analysis was used because it can extract differences in the spectra and indicate what peaks are responsible for these differences. The results indicate that lacquer films from different lacquer trees can be very different. Therefore, ToF‐SIMS with principal component analysis is suitable for the characterization and differentiation of Asian lacquer films in cultural heritage applications.     

Mitigating dead‐time effects during multivariate analysis of ToF‐SIMS spectral images

ToF‐SIMS spectra are formed by bombarding a surface with a pulse of primary ions and detecting the resultant ionized surface species using a time‐of‐flight mass spectrometer. Typically, the detector is a time‐to‐digital converter. Once an ion is detected using such detectors, the detector becomes insensitive to the arrival of additional ions for a period termed as the (detector) dead‐time. Under commonly used ToF‐SIMS data acquisition conditions, the time interval over which ions arising from a single chemical species reach the detector is on the order of the detector dead‐time. Thus, only the first ion reaching the detector at any given mass is counted. The event registered by the data acquisition system, then, is the arrival of one or more ions at the detector. This behavior causes ToF‐SIMS data to violate, in the general case, the assumption of linear additivity that underlies many multivariate statistical analysis techniques. In this article, we show that high‐mass‐resolution ToF‐SIMS spectral‐image data follow a generalized linear model, and we propose a data transformation and scaling procedure that enables such data sets to be successfully analyzed using standard methods of multivariate image analysis. Copyright © 2008 John Wiley & Sons, Ltd.     

Comparison of direct mass spectrometry methods for the on‐line analysis of volatile compounds in foods

For the on‐line monitoring of flavour compound release, atmospheric pressure chemical ionization (APCI) and proton transfer reaction (PTR) combined to mass spectrometry (MS) are the most often used ionization technologies. APCI‐MS was questioned for the quantification of volatiles in complex mixtures, but direct comparisons of APCI and PTR techniques applied on the same samples remain scarce. The aim of this work was to compare the potentialities of both techniques for the study of in vitro and in vivo flavour release. Aroma release from flavoured aqueous solutions (in vitro measurements in Teflon bags and glass vials) or flavoured candies (in vivo measurements on six panellists) was studied using APCI‐ and PTR‐MS. Very similar results were obtained with both techniques. Their sensitivities, expressed as limit of detection of 2,5‐dimethylpyrazine, were found equivalent at 12 ng/l air. Analyses of Teflon bag headspace revealed a poor repeatability and important ionization competitions with both APCI‐ and PTR‐MS, particularly between an ester and a secondary alcohol. These phenomena were attributed to dependency on moisture content, gas/liquid volume ratio, proton affinities and product ion distribution, together with inherent drawbacks of Teflon bags (adsorption, condensation of water and polar molecules). Concerning the analyses of vial headspace and in vivo analyses, similar results were obtained with both techniques, revealing no competition phenomena. This study highlighted the equivalent performances of APCI‐MS and PTR‐MS for in vitro and in vivo flavour release investigations and provided useful data on the problematic use of sample bags for headspace analyses. Copyright © 2013 John Wiley & Sons, Ltd.     

Sulfides: chemical ionization induced fragmentation studied with Proton Transfer Reaction‐Mass Spectrometry and density functional calculations

We report the energy‐dependent fragmentation patterns upon protonation of eight sulfides (organosulfur compounds) in Proton Transfer Reaction‐Mass Spectrometry (PTR‐MS). Studies were carried out, both, experimentally with PTR‐MS, and with theoretical quantum‐chemical methods. Charge retention usually occurred at the sulfur‐containing fragment for short chain sulfides. An exception to this is found in the unsaturated monosulfide allylmethyl sulfide (AMS), which preferentially fragmented to a carbo‐cation at m/z 41, C₃H₅⁺. Quantum chemical calculations (DFT with the M062X functional 6‐31G(d,p) basis sets) for the fragmentation reaction pathways of AMS indicated that the most stable protonated AMS cation at m/z 89 is a protonated (cyclic) thiirane, and that the fragmentation reaction pathways of AMS in the drift tube are kinetically controlled. The protonated parent ion MH⁺ is the predominant product in PTR‐MS, except for diethyl disulfide at high collisional energies. The saturated monosulfides R‐S‐R’ (with R<R’) have little or no fragmentation, at the same time the most abundant fragment ion is the smaller R‐S⁺ fragment. The saturated disulfides R‐S‐S‐R display more fragmentation than the saturated monosulfides, the most common fragments are disulfide containing fragments or long‐chain carbo‐cations. The results rationalize fragmentation data for saturated monosulfides and disulfides and represent a detailed analysis of the fragmentation of an unsaturated sulfide. Apart from the theoretical interest, the results are in support of the quantitative analysis of sulfides with PTR‐MS, all the more so as PTR‐MS is one of a few techniques that allow for ultra‐low quantitative analysis of sulfides. Copyright © 2013 John Wiley & Sons, Ltd.     

Rapidly differentiating grape seeds from different sources based on characteristic fingerprints using direct analysis in real time coupled with time‐of‐flight mass spectrometry combined with chemometrics

The seeds of grapevine (Vitis vinifera) are a byproduct of wine production. To examine the potential value of grape seeds, grape seeds from seven sources were subjected to fingerprinting using direct analysis in real time coupled with time‐of‐flight mass spectrometry combined with chemometrics. Firstly, we listed all reported components (56 components) from grape seeds and calculated the precise m/z values of the deprotonated ions [M–H]^–. Secondly, the experimental conditions were systematically optimized based on the peak areas of total ion chromatograms of the samples. Thirdly, the seven grape seed samples were examined using the optimized method. Information about 20 grape seed components was utilized to represent characteristic fingerprints. Finally, hierarchical clustering analysis and principal component analysis were performed to analyze the data. Grape seeds from seven different sources were classified into two clusters; hierarchical clustering analysis and principal component analysis yielded similar results. The results of this study lay the foundation for appropriate utilization and exploitation of grape seed samples. Due to the absence of complicated sample preparation methods and chromatographic separation, the method developed in this study represents one of the simplest and least time‐consuming methods for grape seed fingerprinting.     

Capillary electrophoresis for fast detection of heterogeneous population in colistin‐resistant Gram‐negative bacteria

It has been shown that diverse strains of bacteria can be separated according to their characteristic surface properties by means of CE. We employed here this analytical technique to the study of colistin‐resistance in Gram‐negative bacteria, which involves the selection of mutants with modified outer membrane composition resulting in changes of surface cell properties. In the same way as with molecular entities, we performed firstly the validation of an ITP‐based CE method for three common pathogenic Gram‐negative bacteria namely Escherichia coli, Pseudomonas aeruginosa, and Klebsiella pneumoniae. Secondly, we compared the electrophoretic profiles of bacterial samples from a colistin‐susceptible clinical isolate of K. pneumoniae and from the corresponding colistin‐resistant derivative. By a simple CE run taking a few minutes, the coexistence of several bacterial subpopulations in the colistin‐resistant derivative was clearly evidenced. This work encourages further research that would allow applications of CE in clinical laboratory for a daily monitoring of bacterial population in cared patients when “last‐chance” colistin treatment is initiated against multidrug‐resistant bacteria.     

On‐line process monitoring of coffee roasting by resonant laser ionisation time‐of‐flight mass spectrometry: bridging the gap from industrial batch roasting to flavour formation inside an individual coffee bean

Resonance‐enhanced multiphoton ionisation time‐of‐flight mass spectrometry (REMPI‐TOFMS) enables the fast and sensitive on‐line monitoring of volatile organic compounds (VOC) formed during coffee roasting. On the one hand, REMPI‐TOFMS was applied to monitor roasting gases of an industrial roaster (1500 kg/h capacity), with the aim of determining the roast degree in real‐time from the transient chemical signature of VOCs. On the other hand, a previously developed μ‐probe sampling device was used to analyse roasting gases from individual coffee beans. The aim was to explore fundamental processes at the individual bean level and link these to phenomena at the batch level. The pioneering single‐bean experiments were conducted in two configurations: (1) VOCs formed inside a bean were sampled in situ, i.e. via a drilled μ‐hole, from the interior, using a μ‐probe (inside). (2) VOCs were sampled on‐line in close vicinity of a single coffee bean's surface (outside). The focus was on VOCs originating from hydrolysis and pyrolytic degradation of chlorogenic acids, like feruloyl quinic acid and caffeoyl quinic acid. The single bean experiments revealed interesting phenomena. First, differences in time–intensity profiles between inside versus outside (time shift of maximum) were observed and tentatively linked to the permeability of the bean's cell walls material. Second, sharp bursts of some VOCs were observed, while others did exhibit smooth release curves. It is believed that these reflect a direct observation of bean popping during roasting. Finally, discrimination between Coffea arabica and Coffea canephora was demonstrated based on high‐mass volatile markers, exclusively present in spectra of Coffea arabica. Copyright © 2013 John Wiley & Sons, Ltd.