Multidimensional Gas Chromatography of Organosulfur Compounds in Coffee and Structure-Odor Analysis of 2-Methyltetrahydrothiophen-3-one.

Organosulfur compounds (OSCs) in coffee remain challenging to analyze by conventional gas chromatography (GC) due to their low concentrations amid coffee's complex matrix and susceptibility to chiral-odor influences. In this study, multidimensional GC (MDGC) methods were developed to profile OSCs in coffee. Conventional GC was compared to comprehensive GC (GC×GC) for untargeted OSC analysis in eight specialty coffees, and GC×GC was found to improve the fingerprinting of OSCs in coffee (50 vs 16 OSCs identified). Of the 50 OSCs, 2-methyltetrahydrothiophen-3-one (2-MTHT) was of high interest due to its chirality and known aroma contribution. Following that, a heart-cutting method for chiral GC (GC-GC) was developed, validated, and applied to the coffees. The mean enantiomer ratio of 2-MTHT was observed to be 1.56 (R/S) in brewed coffees. Overall, MDGC techniques allowed for more comprehensive analyses of coffee OSCs, from which (R)-2-MTHT was found to be the predominant enantiomer with the lower odor threshold.

A high-throughput analysis of volatile compounds with various polarities using headspace stir bar sorptive extraction.

Long sample extraction time is usually necessary in the analysis of volatile flavour compounds to achieve high extraction efficiency. However, the long extraction time reduces sample throughput, which results in waste of labour and energy. Therefore, in this study, an improved headspace-stir bar sorptive extraction was developed to extract volatile compounds with varying polarities in a short time. With the aim of achieving high throughput, extraction conditions were selected and optimised based on the combinations of different extraction temperatures (80-160 °C), extraction times (1-61 min), and sample volumes (50-850 µL) through the response surface methodology with Box-Behnken design. After obtaining the preliminary optimal conditions (160 °C, 25 min, and 850 µL), the effect of cold stir bars with shorter extraction time on the extraction efficiency was evaluated. The cold stir bar improved the overall extraction efficiency with better repeatability, and the extraction time was further shortened to 1 min. Then, the effects of different ethanol concentrations and salt additions (sodium chloride or sodium sulfate) were studied, and 10% ethanol concentration with no salt addition provided the highest extraction efficiency for most compounds. Finally, it was verified that the high-throughput extraction condition was feasible for the volatile compounds spiked in a honeybush infusion.

Characterisation of catechins and their oxidised derivatives in Ceylon tea using multi-dimensional liquid chromatography and high-resolution mass spectrometry.

Tea is a complex food matrix comprising of many structurally diverse compounds, of which catechins and their oxidised derivatives are of particular interest due to their nutritional functionality. However, these catechins and derivatives exist in various isomeric forms with few or no pure standards available, rendering their analysis challenging. A method combining multi-dimensional liquid chromatography (MDLC) and high-resolution mass spectrometry (HRMS) was developed for the characterisation of these compounds using Ceylon tea as a model. Based on a Plackett-Burman (PB) design, flow rate and initial methanol percentage were identified as the most significant factors (p < 0.05) affecting chromatogram coverage and resolution (Rs) for comprehensive two-dimensional LC (LCxLC) and heart-cutting two-dimensional LC (LC-LC) respectively. Central composite design (CCD) was then applied using these parameters for method optimisation and to identify second-order relationships between screened parameters. The optimised LCxLC (flow rate: 2.18 mL/min and initial methanol percentage: 28.0%) and LC-LC (flow rate: 0.86 mL/min and initial methanol percentage for different cuts: A- 10.0%; B- 15.8%; and C- 18.7%) methods were applied to the analysis of Ceylon tea samples from seven regions of Sri Lanka and demonstrated an improved separation of co-eluting isomeric compounds. Finally, with the mass spectral information from HRMS, a total of 31 compounds (eight monomers, 17 dimers, five trimers and one tetramer) were detected and putatively identified in Ceylon tea.

Biovalorization of spent Konacha tea leaves via single-culture fermentation involving wine yeasts and lactic acid bacteria.

AIMS: The objective of this study was to explore the potential of fermentation as a biovalorization strategy for spent tea leaves (STL), a major agrifood waste generated from the tea extraction industry. Fermentation by wine yeasts or lactic acid bacteria (LAB) has shown promising results in previous studies across various substrates. METHODS AND RESULTS: Konacha (green tea) STL slurries were inoculated with single strains of wine yeasts or LAB respectively. After a 48-h fermentation, changes in selected nonvolatile and volatile compositions were evaluated. Fermentation by LAB increased organic acid content by 5- to 7-fold (except Lactobacillus fermentum) and modulated the composition of major tea catechins, whereas wine yeast fermentation resulted in a 30% increase in amino acid content. Strain-specific production of specific volatile compounds was also observed such as butanoic acid (L. fermentum), isoamyl acetate (Pichia kluyveri) and 4-ethylphenol (L. plantarum). CONCLUSIONS: Both volatile and nonvolatile compound compositions of Konacha STL were successfully modified via wine yeast and LAB fermentation. SIGNIFICANCE AND IMPACT OF STUDY: Our findings indicate that Konacha STL is a suitable medium for biovalorization by wine yeasts or LAB via the generation of commercially useful volatile and nonvolatile compounds. Future optimizations could further render fermentation an economically viable strategy for the upcycling of STL.

Recent advances in analytical strategies for coffee volatile studies: Opportunities and challenges.

Coffee has attracted significant research interest owing to its complex volatile composition and aroma, which imparts a pleasant sensorial experience that remains challenging to analyse and interpret. This review summarises analytical challenges associated with coffee's volatile and matrix complexity, and recent developments in instrumental techniques to resolve them. The benefits of state-of-the-art analytical techniques applied to coffee volatile analysis from experimental design to sample preparation, separation, detection, and data analysis are evaluated. Complementary method selection coupled with progressive experimental design and data analysis are vital to unravel the increasing comprehensiveness of coffee volatile datasets. Considering this, analytical workflows for conventional, targeted, and untargeted coffee volatile analyses are thus proposed considering the trends towards sorptive extraction, multidimensional gas chromatography, and high-resolution mass spectrometry. In conclusion, no single analytical method addresses coffee's complexity in its entirely, and volatile analysis must be tailored to the key objectives and concerns of the analyst.

Distinguishing citrus varieties based on genetic and compositional analyses.

Simple sequence repeats (SSR) markers and secondary metabolite composition were used in combination to study seven varieties of citrus for the first time. With reference to established accessions of citrus, two of the varieties (Chanh Giay and Ma Nao Pan) were predicted to be Mexican key limes, while three were mandarin hybrids (Nagpur, Pontianak and Dalandan) and the remaining two (Qicheng and Mosambi) were related to the sweet orange. Notably, Dalandan was genetically more like a mandarin despite often referred to as an orange locally, whereas Mosambi was more likely to be a sweet orange hybrid although it has also been called a sweet lime due to its green peel and small size. Several key secondary metabolites such as polymethoxyflavones (sinensetin, tangeretin etc.), furanocoumarins (bergapten, citropten etc.) and volatiles (citronellol, α-sinensal etc.) were identified to be potential biomarkers for separation of citrus species. However, despite having similar genetic profiles, variations in the volatile profile of the two limes were observed; similarly, there were differences in the secondary metabolite profiles of the three mandarin hybrids despite having a common ancestral parent, highlighting the usefulness of genetic and compositional analyses in combination for revealing both origins and flavour profiles especially in citrus hybrids. This knowledge would be crucial for variety screening and selection for use in flavour or fragrance creation and application.

Sequential combination of solid-phase sorbents to enhance the selectivity of organosulfur compounds for flavour analysis.

Flavour analysis remains challenging due to the range of selectivity demands, from the extraction of multiclass volatile compounds to the purification of low-concentration odourants (e.g. organosulfur compounds) amidst the high food matrix noise. In this study, the varying selectivities of solid-phase extraction (SPE) were leveraged upon for both multiclass and organosulfur compound analysis, using coffee as a model matrix. Polymeric SPE (Bond Elut ENV) was screened for significant (p < 0.05) parameters affecting the recovery of 37 multiclass compounds, and the most influential parameters were optimised using a Box-Behnken design (elution solvent of 67:33 dichloromethane:ethyl acetate, loading pH of 4.8, and wash solvent of water). Following this, low-concentration organosulfur compounds which were challenging to detect in the complex coffee matrix were purified by adding a sequential SPE step for selectivity. A silver-based ligand-exchange SPE step (MetaSEP IC-Ag) was optimised for organosulfur compound recovery (wash solvents of dichloromethane and 43% acetonitrile in dichloromethane, elution solvent of 90 mmol/L 1,4-dithiothreitol in dichloromethane). This was found to be complementary to polymeric SPE's matrix clean-up effect (which improved average organosulfur compound recovery by 5.45 times). Finally, both multiclass and sequential organosulfur extraction techniques demonstrated good performance in terms of reproducibility (1.0-9.0%) and linearity (R2 > 0.995), allowing for the detection of 3-mercapto-3-methylbutyl formate (3.741 ± 0.387 ng/mL) in coffee samples. In conclusion, this study highlights the potential for SPE to address a variety of complex flavour analysis demands with the appropriate selection and combination of solid-phases.

Neutral loss scan in complement with high-resolution MS/MS: Combination of detection methods for flavonoid and limonoid glycosides analysis.

In this study, neutral loss scan and high-resolution MS/MS were used in combination to detect and tentatively identify various flavonoid and limonoid glycosides in navel orange albedo, juice, peel and pulp. These compound classes are of research interest due to their flavour and bioactive properties, and although flavonoid glycosides have been previously studied in other food matrices, to the best of our knowledge, neutral loss scans have not been used for the elucidation of limonoid glycosides. Neutral loss masses of 120, 162 and 308 Da were selected for the detection of hexose, rutinose and neohesperidose-substituted flavonoids, whereas 197 Da was explored for limonoid glycosides due to their tendency to form ammonium adducts. Fragmentation patterns obtained from targeted MS/MS were then used to differentiate rutinose and neohesperidose substituents as well as flavonoid subclasses of flavones, flavanones and flavonols. Additionally, high-resolution MS/MS was also used for the identification of aglycones by accurate mass (to four decimal places), allowing for the differentiation of aglycones with similar unit masses but different chemical formulas. In total, 19 flavonoid glycosides and six limonoid glycosides were detected. This workflow allows for a rapid screening of flavonoid and limonoid glycosides in citrus, which can be further extended to other food products such as tea.

Effect of three milling processes (cyclone-, bead- and stone-millings) on the quality of matcha: Physical properties, taste and aroma.

Analysis of three matcha (cyclone-, bead- and stone-milled) revealed differences in their sizes and surface morphologies. Using liquid chromatography, 4 sugars, 6 organic acids, 18 amino acids and 9 polyphenols were detected in all matcha samples and shown to present in different amount. Moreover, 108 volatile compounds were detected and 30 potential flavour-contributing compounds were quantified by gas chromatography time-of-flight mass spectrometry using headspace-stir bar sorptive extraction-thin-film solid-phase microextraction (HS-SBSE-TFSPME). Sensory evaluation by a trained panel found that the matcha samples possess different notes (cyclone-milled: leafy; bead-milled: fishy; and stone-milled: roasty) which is supported by the volatile compound analysis. Finally, the three matcha were differentiated based on non-volatile and volatile components using principal component analysis, and the correlation between chemical composition and sensory evaluation data was carried out using partial least square regression. In conclusion, milling processes clearly affected the physical, chemical and sensory qualities of matcha.

Combination of solid phase microextraction and low energy electron ionisation gas chromatography-quadrupole time-of-flight mass spectrometry to meet the challenges of flavour analysis.

The flavour analysis of volatile compounds remains challenging not only because of their diversity in properties and dynamic range, but also due to the high background noise from food matrix constituents. To improve sensitivity and specificity for a multiclass range of compounds, a combination of solid phase micro-extraction (SPME) devices and low energy electron ionisation (LE-EI) was proposed for the analysis of 36 volatile compounds, using coffee as a model matrix. From a pre-evaluation of devices and extraction modes, the combined use of direct immersion-stir bar sorptive extraction and headspace-thin-film SPME (SBSE-TFSPME) was selected to increase compound recovery, and further optimised for extraction temperature (88 °C) and time (110 min). Furthermore, to complement sample preparation by improving method specificity, a LE-EI technique was developed by evaluating the effect of ionisation energy, source temperature, and emission current on the formation of the diagnostic molecular ions and their preservation. This LE-EI method (15 eV, 150 °C, 0.3 µA) was validated with SBSE-TFSPME as a complete workflow in coffee matrices, and was found to possess good repeatability (intra-day RSD: 1.6-7.3 %), intermediate precision (inter-day RSD: 4.1-12.2 %), and linearity (R2 > 0.98). Even for complex coffee samples, the method detection limit reached the pg/mL range (e.g. 2,4,5-trimethylthiazole was detected at 15 pg/mL). In conclusion, this study provided insights on the potential of SPME and LE-EI to improve the sensitivity and specificity of analysis for a range of volatile compounds from food and other complex matrices.

Investigation of changes in non-traditional indices of maturation in Navel orange peel and juice using GC-MS and LC-QTOF/MS.

Changes in non-traditional indices of maturity, such as flavonoids and volatile compounds, during maturation were studied in Navel orange. Navel oranges were obtained at four stages of maturation, and non-volatile and volatile compounds in the peel and juice were analysed using liquid chromatography coupled with a quadrupole time-of-flight detector (LC-QTOF/MS) and gas chromatography with mass spectrometry and a flame ionisation detector (GC-MS/FID), respectively. Twenty-eight non-volatile and 62 volatile compounds in the peel as well as 22 non-volatile and 11 volatile compounds in the juice were found to have significant changes (p < 0.05) in abundances during maturation. Notably, most flavonoids (e.g. narirutin) and limonoids (e.g. nomilin) showed decreasing abundances during maturation. For volatile compounds, majority of detected alcohols peaked in abundances during middle maturation stages, while almost all detected aldehydes peaked at full maturity. Most terpenes peaked at earlier maturation stages in juice extracts compared to peel oil extracts. This comprehensive study could facilitate selection of Navel oranges for the extraction of valuable bioactive or flavour contributing compounds that are of interest to fragrance, flavour and nutraceutical industries.

Enhanced extraction using a combination of stir bar sorptive extraction and thin film-solid phase microextraction.

Sorptive extraction techniques have experienced increased popularity, but they face limitations in dynamic range and sensitivity. In this study, a new method combining stir bar sorptive extraction (SBSE) and thin-film solid-phase microextraction (TFSPME) was developed, and optimization for extraction temperature (70 °C) and time (120 min) was carried out. Polydimethylsiloxane (PDMS)-coated SBSE and PDMS/carboxen (PDMS/CAR)-coated TFSPME were used, and both headspace and direct immersion extraction modes were also studied. Using 40 selected volatile compounds, the combined method generally gave a wider linearity range with lower minimum limits (2 to 3 orders), satisfactory coefficient of determination (R2>0.980), and improved sensitivity when compared to SBSE-only or TFSPME-only techniques. Furthermore, despite the combined use of two extraction devices, the repeatability (<13.1 %) and reproducibility (<13.4 %) of the combined method were comparable to SBSE-only or TFSPME-only results. Higher recoveries of up to 20% were also achieved by the combined method. Compared to the conventional SBSE method, the new method provided superior performance in terms of dynamic range and sensitivity for compounds of various polarities. In conclusion, this study provided insights on the suitability of the various extraction methods for compounds of different chemical properties which could aid in future applications for volatiles analysis in food, biological, and environmental sectors.

Identification of key odorants in honeysuckle by headspace-solid phase microextraction and solvent-assisted flavour evaporation with gas chromatography-mass spectrometry and gas chromatograph-olfactometry in combination with chemometrics.

At present, the identification of honeysuckle aroma depends on experienced tasters, which results in inconsistencies due to human error. The key odorants have the potential to distinguish the different species and evaluate the quality of honeysuckle. Hence, in this study, a more scientific approach was applied to distinguish various honeysuckles. The volatile compounds of different species and parts of honeysuckle were separately extracted by headspace-solid phase microextraction (HS-SPME) and solvent assisted flavor evaporation (SAFE). Compounds with greater volatility such as aldehydes, limonene, γ-terpinene, and terpinolene were preferentially extracted by HS-SPME. As a complementary extraction method to HS-SPME, SAFE was found to recover comparatively more polar compounds such as eugenol, decanoic acid, and vanillin. Subsequently, key odorants with the highest flavour dilution (FD) factors were detected by aroma extract dilution analysis (AEDA). These were benzaldehyde, 4-ethylphenol, decanoic acid, vanillin, 3-methyl-2-butenal, and ß-ionone in honeysuckle flowers and γ-octalactone, 4-ethyl phenol, and vanillin in honeysuckle stem. Finally, principal component analysis (PCA) was conducted to analyze not only the key odorants of species and parts of honeysuckle but also their different origins. The results of PCA suggested that the species of honeysuckle contributed much more to variations in aroma rather than their origins. In conclusion, the application of the key odorants combined with PCA was demonstrated as a valid approach to differentiate species, origins, and parts of honeysuckle.

Proteomic analysis of human placental syncytiotrophoblast microvesicles in preeclampsia.

BACKGROUND: Placental syncytiotrophoblast microvesicles (STBM) are shed into the maternal circulation during normal pregnancy. STBM circulate in significantly increased amounts in preeclampsia (PE) and are considered to be among contributors to the exaggerated proinflammatory, procoagulant state of PE. However, protein composition of STBM in normal pregnancy and PE remains unknown. We therefore sought to determine the protein components of STBM and whether STBM protein expressions differ in preeclamptic and normal pregnancies. Patients with PE (n = 3) and normal pregnant controls (n = 6) were recruited. STBM were prepared from placental explant culture supernatant. STBM proteins were analyzed by a combination of 1D Gel-LC-MS/MS. Protein expressions levels were quantified using spectral counts and validated by immunohistochemistry. RESULTS: Over 400 proteins were identified in the STBM samples. Among these, 25 proteins were found to be differentially expressed in preeclampsia compared to healthy pregnant controls, including integrins, annexins and histones. CONCLUSION: STBM proteins include those that are implicated in immune response, coagulation, oxidative stress, apoptosis as well as lipid metabolism pathways. Differential protein expressions of STBM suggest their pathophysiological relevance in PE.

Automated dynamic liquid-liquid-liquid microextraction followed by high-performance liquid chromatography-ultraviolet detection for the determination of phenoxy acid herbicides in environmental waters.

Automated dynamic liquid-liquid-liquid microextraction (D-LLLME) controlled by a programmable syringe pump and combined with HPLC-UV was investigated for the extraction and determination of 5 phenoxy acid herbicides in aqueous samples. In the extraction procedure, the acceptor phase was repeatedly withdrawn into and discharged from the hollow fiber by the syringe pump. The repetitive movement of acceptor phase into and out of the hollow fiber channel facilitated the transfer of analytes into donor phase, from the organic phase held in the pore of the fiber. Parameters such as the organic solvent, concentrations of the donor and acceptor phases, plunger movement pattern, speed of agitation and ionic strength of donor phase were evaluated. Good linearity of analytes was achieved in the range of 0.5-500 ng/ml with coefficients of determination, r2 > 0.9994. Good repeatabilities of extraction performance were obtained with relative standard deviations lower than 7.5%. The method provided up-to 490-fold enrichment within 13 min. In addition, the limits of detection (LODs) ranged from 0.1 to 0.4 ng/mL (S/N = 3). D-LLLME was successfully applied for the analysis of phenoxy acid herbicides from real environmental water samples.

West Nile virus core protein; tetramer structure and ribbon formation.

We have determined the crystal structure of the core (C) protein from the Kunjin subtype of West Nile virus (WNV), closely related to the NY99 strain of WNV, currently a major health threat in the U.S. WNV is a member of the Flaviviridae family of enveloped RNA viruses that contains many important human pathogens. The C protein is associated with the RNA genome and forms the internal core which is surrounded by the envelope in the virion. The C protein structure contains four alpha helices and forms dimers that are organized into tetramers. The tetramers form extended filamentous ribbons resembling the stacked alpha helices seen in HEAT protein structures.

Analysis of phenoxy herbicides in bovine milk by means of liquid-liquid-liquid microextraction with a hollow-fiber membrane.

A preconcentration technique, which involves liquid-liquid-liquid microextraction, was developed to determine phenoxy herbicides in bovine milk. A layer of organic phase was impregnated into the pores of a 3.5 cm long porous hollow fiber, while the internal volume of the fiber was filled with NaOH solution (the acceptor solution) that was connected directly to the needle of a microsyringe. The fiber was then immersed into 8 ml of acidified milk sample. When the sample solution was stirred, acidic analytes were extracted into the organic phase and back extracted simultaneously into the alkaline acceptor medium as the analytes were protonated at low pH and deprotonated at high pH. After extracting for a prescribed time, 5 microl acceptor solution was taken back into the syringe and injected directly into a HPLC system for quantification. The analytes were extracted quantitatively from the sample solution into the acceptor solution with a large enrichment factor of 900. Due to its low cost, the hollow-fiber extraction device was disposed of after a single extraction that eliminated the possibility of carry over effects. In addition, because a small volume of organic solvent was required and little waste is generated, the procedure is environmentally friendly, and is compatible with the "green chemistry" concept.