Bioprospecting of a Metschnikowia pulcherrima Indigenous Strain for Chasselas Winemaking in 2022 Vintage.

Interest in Metschnikowia (M.) pulcherrima is growing in the world of winemaking. M. pulcherrima is used both to protect musts from microbial spoilage and to modulate the aromatic profile of wines. Here, we describe the isolation, characterization, and use of an autochthonous strain of M. pulcherrima in the vinification of Chasselas musts from the 2022 vintage. M. pulcherrima was used in co-fermentation with Saccharomyces cerevisiae at both laboratory and experimental cellar scales. Our results showed that M. pulcherrima does not ferment sugars but has high metabolic activity, as detected by flow cytometry. Furthermore, sensory analysis showed that M. pulcherrima contributed slightly to the aromatic profile when compared to the control vinifications. The overall results suggest that our bioprospecting strategy can guide the selection of microorganisms that can be effectively used in the winemaking process.

SWATH-MS screening strategy for the determination of food dyes in spices by UHPLC-HRMS.

A multi-class wide-scope screening method for the detection and identification of artificial colours and illegal dyes in spices was developed for regulatory purposes. The screening was carried out by ultra-high performance liquid chromatography hyphenated with a quadrupole/time-of-flight mass spectrometry (UHPLC-QTOF-MS) with sequential window acquisition of all theoretical fragment-ion spectra (SWATH) and was validated with forty-one compounds by spiking experiments in curry and paprika extracts. In order to detect and identify the compounds with a high level of confidence, a home-made tandem mass spectrometry (QTOF-MS/MS) database of approximately one hundred illegal dyes and artificial colours was created. The procedure was then used to screen field samples of spices and spice blends purchased from Swiss markets. Sudan IV, Sudan I, bixin (E160b) and Ponceau 4R (E124) were all detected among the eight non-compliant samples.

Unexpected occurrence of caffeine in sleep-inducing herbal teas.

Caffeine acts as a weak psychostimulant and is known to cause trouble with sleeping. Therefore, the presence of caffeine in sleep-aid herbal teas was somewhat surprising, and confirmatory investigations were conducted to exclude any possible misidentification. The botanicals of the sedative mixtures were analysed individually by ultra performance liquid chromatography (UPLC) coupled with a time-of-flight high-resolution mass spectrometer (TOF-HRMS), and caffeine was detected in linden (Tilia spp.) extracts. The presence of caffeine was unambiguously confirmed by means of its characteristic mass spectrum acquired during direct analyses of powdered linden by thermal desorption coupled to a GC×GC-TOF-MS. Caffeine content was determined in 11 linden-based samples, with a validated UPLC-MS/MS method using two mass transitions. Concentrations were between traces and 110 mg kg(-1) in the herbal material while those in the corresponding prepared sleep-inducing hot beverages ranged from traces to 226 µg per cup.

Survey of pyrrolizidine alkaloids in teas and herbal teas on the Swiss market using HPLC-MS/MS.

Pyrrolizidine alkaloids (PAs) are a large class of natural compounds amongst which the esterified 1,2-unsaturated necine base is toxic for humans and livestock. In the present study, a method was developed and validated for the screening and quantification of nine PAs and one PA N-oxide in teas (Camellia sinensis (L.) O. Kuntze) and herbal teas (camomile, fennel, linden, mint, rooibos, verbena). Samples were analysed by HPLC on a RP-column, packed with sub-2 µm core-shell particles, and quantified using tandem mass spectrometry operating in the positive electrospray ionisation mode. These PAs and some of their isomers were detected in a majority of the analysed beverages (50/70 samples). In 24 samples, PA concentrations were above the limit of quantification and the sum of the nine targeted PAs was between 0.021 and 0.954 µg per cup of tea. Thus, in some cases, total concentrations exceed the maximum daily intake recommended by the German Federal Institute for Risk Assessment and the UK's Committee On Toxicity (i.e. 0.007 µg kg(-1) bw).

Hydrophilic interaction chromatography versus reversed phase liquid chromatography coupled to mass spectrometry: effect of electrospray ionization source geometry on sensitivity.

In this study, the influence of electrospray ionization (ESI) source design on the overall sensitivity achieved in hydrophilic interaction chromatography (HILIC) and reversed phase liquid chromatography (RPLC), was investigated. State-of-the-art triple quadrupole mass analyzers from AB Sciex, Agilent Technologies and Waters equipped with brand specific source geometries were tested with various mobile phase pH on 53 pharmaceutical compounds. The design of the ESI source showed to strongly influence the gain in sensitivity that can be achieved in HILIC compared to RPLC mode. The 6460 Triple Quadrupole LC/MS system from Agilent Technologies was particularly affected by mobile phase settings. Indeed, compared to RPLC conditions, 92% of the compounds had an increased signal-to-noise ratio at a flow rate of 300 µL/min in HILIC mode at pH 6, while this percentage dropped to only 7% at 1000 µL/min and pH 3. In contrast, the influence of flow rate and mobile phase pH on the gain in sensitivity between RPLC and HILIC was found very limited with the API 5000 LC/MS/MS system from AB Sciex, as only 15 to 36% of the tested compounds showed an enhanced sensitivity in HILIC mode. With the Xevo TQ-S instrument from Waters, superior sensitivity in HILIC was noticed for 85% of the compounds with optimal conditions (i.e., pH 3 and 1000 µL/min), whereas at sub-optimal conditions (i.e. pH 6 and 300 µL/min), it represented less than 50%. The gain in sensitivity observed in HILIC was found less significant with the recent LC-MS platforms used in this study than for old-generation instruments. Indeed, the improved ESI sources equipping the recent mass analyzers allow for enhanced evaporation efficiency, mainly for RPLC mobile phases containing high proportion of water and this even at high flow rates.

Screening and determination of sibutramine in adulterated herbal slimming supplements by HPTLC-UV densitometry.

The adulteration of herbal supplements is of growing importance, especially when they contain undeclared compounds like sibutramine that are unsafe drugs. Sibutramine was withdrawn from US and European markets in 2010. In this study, an HPTLC-UV densitometric method was developed for the quantification of sibutramine in herbal diet foods. Sample extracts were directly applied onto HPTLC silica gel plates and separated with a mobile phase made of a toluene-methanol mixture. Sibutramine was quantified at 225 nm and its unequivocal identification was confirmed by MS using a TLC-MS interface. During two surveys, 52 weight loss supplements obtained via the Internet were screened. Half of those were adulterated with sibutramine at amounts reaching up to 35 mg per capsule. The results of this validated HPTLC method were compared with those obtained by HPLC-UV and HPLC-MS/MS. The results were not significantly different with the three methods.

Analysis of the neurotoxin anisatin in star anise by LC-MS/MS.

The aim of this work was to develop an analytical method capable of determining the presence of anisatin in star anise. This neurotoxin may induce severe side effects such as epileptic convulsions. It is therefore of prime importance to have rapid and accurate analytical methods able to detect and quantify anisatin in samples that are purportedly edible star anise. The sample preparation combined an automated accelerated solvent extraction with a solid-supported liquid-liquid purification step on EXtrelut®. Samples were analysed on a porous graphitic carbon HPLC column and quantified by tandem mass spectrometry operating in the negative ionisation mode. The quantification range of anisatin was between 0.2 and 8 mg kg⁻¹. The applicability of this validated method was demonstrated by the analysis of several Illicium species and star anise samples purchased on the Swiss market. High levels of anisatin were measured in Illicium lanceolatum, I. majus and I. anisatum, which may cause health concerns if they are misidentified or mixed with edible Illicium verum.

Targeted multidimensional gas chromatography using a heart-cutting device and cryogenic focusing for the determination of benzophenone derivatives in foodstuffs.

Photoinitiators are used to promote the polymerization process during the curing of varnishes or inks on cartonboard packaging. Depending on storage conditions and shelf life, these substances are able to migrate through the packaging layer into the foodstuff. This type of contamination phenomenon is therefore becoming a critical issue in terms of food safety. In order to tackle this problem, a fast and selective method was developed for the determination of benzophenone and three methylbenzophenone isomers in cereal-based foodstuffs and their cardboard packaging. Food samples or packages were efficiently extracted by pressurized liquid extraction using acetonitrile, and the extracts were directly injected onto the analytical system. The analysis was performed by multidimensional gas chromatography-mass spectrometry using a heart-cutting approach to reduce the background noise from complex matrices. The strategy employed two distinct cuts each containing its proper deuterated internal standard leading to accurate quantification. By integrating a cryofocusing effect, an enhancement in signal/noise ratio was achieved by a factor >10, which markedly decreased the sensitivity threshold. Moreover, baseline separation of the critical isomers allowed their unequivocal determination. The method was fully validated on cereal-based foodstuffs based upon an analysis of variance, and excellent performances were obtained at the decision limit making this method well suited for official food controls.

Characterization of positional and configurational tropane alkaloid isomers by combining GC with NPD, MS and FTIR.

A purified stem bark extract from Schizanthus grahamii was analyzed by combining capillary gas chromatography with a selective nitrogen phosphorus detector, mass spectrometry and Fourier transform infrared spectroscopy. Several positional and configurational tropane isomers were detected and their structural elucidation was tentatively determined based on mass spectra and confirmed by vapor phase infrared spectra. Electron impact mass spectra of the isomeric alkaloids were virtually superimposable, whereas the corresponding infrared spectra differed markedly. Retention indices were established under isothermal and temperature program conditions for further peak assignment and identification. In order to point out possible artifacts in the hot and surface-active injection port, different sample introduction techniques were evaluated, namely split, splitless and on-column injections.

High-precision heteronuclear 2D NMR experiments using 10-ppm spectral window to resolve carbon overlap.

The acquisition of a complementary heteronuclear 2D NMR experiment with 10-ppm carbon window allows chemists to improve by a factor 20-25 the spectral resolution and determine carbon chemical shifts with five figures from 2D spectra.

Dual-injection system with multiple injections for determining bidimensional retention indexes in comprehensive two-dimensional gas chromatography.

A new instrumental approach for collection of retention index data in the first (1D) and second (2D) dimensions of a comprehensive two-dimensional (2D) gas chromatography (GCxGC) experiment has been developed. First-dimension indexes were determined under conventional linear programmed temperature conditions (Van den Dool indexes). To remove the effect that the short secondary column imposes on derived 1D indexes, as well as to avoid handling of pulsed GCxGC peaks, the proposed approach uses a flow splitter to divert part of the primary column flow to a supplementary detector to simultaneously generate a conventional 1D chromatogram, along with the GCxGC chromatogram. The critical 2D indexes (KovAts indexes) are based upon isovolatility curves of normal alkanes in 2D space, providing a reference scale against which to correlate each individual target peak throughout the entire GCxGC run. This requires the alkanes to bracket the analytes in order to allow retention interpolation. Exponential curves produced in the 2D separation space require a novel approach for delivery of alkane standards into the 2D column by using careful solvent-free solid-phase microextraction (SPME) sampling. Sequential introduction of alkane mixtures during GCxGC runs was performed by thermal desorption in a second injector which was directly coupled through a short transfer line to the entrance of the secondary column, just prior to the modulator so that they do not have to travel through the 1D column. Thus, each alkane mixture injection was quantitatively focused by the cryogenic trap, then launched at predetermined times onto the 2D column. The system permitted construction of an alkane retention map upon which bidimensional indexes of a 25-perfume ingredient mixture could be derived. Comparison of results with indexes determined in temperature-variable one-dimensional (1D) GC showed good correlation. Plotting of the separation power in the second dimension was possible by mapping Trennzahl values throughout the 2D space. The methodology was applied to the separation of a standard mixture composed of 25 analytes (very diverse in polarity and structure) suspected to be allergens in perfume samples. The method will allow straightforward determination of temperature-variable retention indexes of target analytes.

Fast comprehensive two-dimensional gas chromatography with cryogenic modulation.

The fast separation of a mixture of 29 compounds by using comprehensive two-dimensional gas chromatography is reported. Capillary column sets with shorter lengths and smaller inner diameter in both the first and second dimensions have been tested, for both fast chiral and achiral separations. Fast chiral separations, which included enantiomer separations of limonene, linalool, citronellol, and alpha-isomethylionone, were achieved within 23 min, which corresponds to approximately 2-fold faster than analyses under conditions previously considered as normal. Fast achiral separations, which do not have the restriction of requiring a minimum quality of chiral resolution, were obtained within 5 min, which is markedly faster than separations on the normal column set under conditions more commonly employed. The achiral fast GC x GC method used a 5 m x 0.1 mm i.d. first dimension column, interfaced to a 0.3 m x 0.05 mm i.d. second column, with temperature program rate of 35 degrees C.min-1; a modulation period of 1 s was employed. Peak widths at baseline on the first column were a little over 1 s, while modulated peak widths at half-height recorded with a flame ionization detector operating at 200 Hz were approximately 30 ms. The benefits and limitations of GC x GC for fast chiral and achiral separations are reported and discussed.

Generating multiple independent retention index data in dual-secondary column comprehensive two-dimensional gas chromatography.

A method producing simultaneously three retention indexes for compounds has been developed for comprehensive two-dimensional gas chromatography by using a dual secondary column approach (GC x 2GC). For this purpose, the primary flow of the first dimension column was equally diverted into two secondary microbore columns of identical geometry by means of a three-way flow splitter positioned after the longitudinally modulated cryogenic system. This configuration produced a pair of comprehensive two-dimensional chromatograms and generated retention data on three different stationary phases in a single run. First dimension retention indexes were determined on a polar SolGel-Wax column under linear programmed-temperature conditions according to the van den Dool approach using primary alcohol homologues as the reference scale. Calculation of pseudoisothermal retention indexes in both second dimensions was performed on low-polarity 5% phenyl equivalent polysilphenylene/siloxane (BPX5) and 14% cyanopropylphenyl/86% dimethylpolysiloxane (BP10) columns. To construct a retention correlation map in the second dimension separation space upon which KovAts indexes can be derived, two methods exploiting "isovolatility" relationships of alkanes were developed. The first involved 15 sequential headspace samplings of selected n-alkanes by solid-phase microextraction (SPME), with each sampling followed by their injection into the GC at predetermined times during the chromatographic run. The second method extended the second dimension retention map and consisted of repetitive introduction of SPME-sampled alkane mixtures at various isothermal conditions incremented over the temperature program range. Calculated second dimension retention indexes were compared with experimental values obtained in conventional one-dimensional GC. A case study mixture including 24 suspected allergens (i.e., fragrance ingredients) was used to demonstrate the feasibility and potential of retention index information in comprehensive 2D-GC.

Evaluation of solid-phase microextraction desorption parameters for fast GC analysis of cocaine in coca leaves.

By its simplicity and rapidity, solid-phase microextraction (SPME) appears as an interesting alternative for sample introduction in fast gas chromatography (fast GC). This combination depends on numerous parameters affecting the desorption step (i.e., the release of compounds from the SPME fiber coating to the GC column). In this study, different liner diameters, injection temperatures, and gas flow rates are evaluated to accelerate the thermal desorption process in the injection port. This process is followed with real-time direct coupling a split/splitless injector to a mass spectrometer by means of a short capillary. It is shown that an effective, quantitative, and rapid transfer of cocaine (COC) and cocaethylene (CE) is performed with a 0.75-mm i.d. liner, at 280 degrees C and 4 mL/min gas flow rate. The 7-microm polydimethylsiloxane (PDMS) coating is selected for combination with fast GC because the 100-microm PDMS fiber presents some limitations caused by fiber bleeding. Finally, the developed SPME-fast GC method is applied to perform in less than 5 min, the quantitation of COC extracted from coca leaves by focused microwave-assisted extraction. An amount of 7.6 +/- 0.5 mg of COC per gram of dry mass is found, which is in good agreement with previously published results.

Identification of isomeric tropane alkaloids from Schizanthus grahamii by HPLC-NMR with loop storage and HPLC-UV-MS/SPE-NMR using a cryogenic flow probe.

Two fully automated HPLC-NMR methods are reported and compared for the structure elucidation of four isomeric tropane alkaloids from the stem-bark of an endemic Chilean plant, Schizanthus grahamii Gill. (Solanaceae). The first approach interfaced a conventional HPLC column to NMR by means of a loop storage unit. After elution with a mobile phase consisting of deuterated water and standard protonated organic solvents, the separated analytes were momentarily stored in a loop cassette and then transferred one-at-a-time to the NMR flow probe for measurements. The second strategy combined HPLC with parallel ion-trap MS detection and NMR spectroscopy using an integrated solid-phase extraction (SPE) unit for post-column analyte trapping. The SPE cartridges were dried under a gentle stream of nitrogen and analytes were sequentially eluted and directed to a cryogenically cooled flow-probe with an NMR-friendly solvent. The structures of the four isomeric alkaloids, 3alpha-senecioyloxy-7beta-hydroxytropane, 3alpha-hydroxy-7beta-angeloyloxytropane, 3alpha-hydroxy-7beta-tigloyloxytropane and 3alpha-hydroxy-7beta-senecioyloxytropane, were unambiguously determined by combining NMR assignments with MS data.

Analysis of isomeric tropane alkaloids from Schizanthus grahamii by very fast gas chromatography.

This study presents a very fast GC analysis applied for the baseline separation of isomeric tropane alkaloids extracted from the stem-bark of Schizanthus grahamii (Solanaceae). The work provided a challenging application where isothermal analysis in conjunction with very short narrow bore columns (3 m x 100 microm ID and 1.5 m x 50 microm ID) was particularly suited for the speeding up. Experimental parameters were used in the optimisation steps, including selection of stationary phase, temperature, internal column diameter and optimal practicable gas velocity. Some considerations about sample injection in fast isothermal analysis are also briefly presented. Finally, the investigated approach allowed a very fast baseline separation of four positional and configurational isomers in less than 9 s.

In vitro shoot and root organogenesis, plant regeneration and production of tropane alkaloids in some species of Schizanthus.

A rapid in vitro propagation system leading to formation of shoots from callus, roots, and plantlets was developed for Schizanthus hookeri Gill. (Solanaceae), an endemic Chilean plant. The genus Schizanthus is of particular interest due to the presence of several tropane alkaloids. So far, in vitro propagation of species of this genus has not been reported. Propagation of S. hookeri consisted of two phases, the first one for callus initiation and shoot formation and the second for rhizogenesis and plantlet regeneration. From a single callus that rapidly increased in cell biomass (from approximately 50 mg to approximately 460 mg/culture tube [25 x 130 mm] in 60 days) in the presence of 2.69 microM NAA and 2.22 microM BA, more than 10 shoots/callus explant were formed. From the latter, approx. twenty plantlets formed after 90-110 days shoot subculture in medium devoid of growth regulators that favored root formation. Ten alkaloids ranging from simple pyrrolidine derivatives to tropane esters derived from angelic, tiglic, senecioic or methylmesaconic acids were obtained from in vitro regenerated plantlets. One of them, 3alpha-methylmesaconyloxytropane, was not previously described. The same growth conditions, as well as other growth regulator levels tested, were required to induce callus and root formation in S. grahamii Gill. Root organogenesis occurred despite a high level of BA vs. NAA used, (i.e., 4.44 microM BA and 0.54 microM NAA); however no shoot formation was achieved. In the case of S. tricolor Grau et Gronbach, only callus formation was obtained in the presence of various growth regulators.

Use of porous graphitic carbon column for the separation of natural isomeric tropane alkaloids by capillary LC and mass spectrometry.

This study presents the outstanding chromatographic selectivity of a porous graphitic carbon support for the separation of four isomeric tropane alkaloids from the stem-bark of Schizanthus grahamii (Solanaceae). Capillary liquid chromatography coupled to mass spectrometry was studied after the appropriate selection of mobile phase composition, temperature, nature and concentration of the acidic modifier. Fragmentation behaviour by in-source collision-induced dissociation (CID) on a single quadrupole mass spectrometer, or MSn with an ion-trap, was investigated for structural identification. The ability to differentiate the isomers by in-source CID was demonstrated and a fragmentation pathway, based on MSn sequences together with accurate mass experiments, was proposed.