Optimization of elution conditions and comparison of emerging biocompatible columns on the resolving power and detection sensitivity of oligonucleotides by ion-pairing reversed-phase liquid chromatography mass spectrometry.

A generic performance comparison strategy has been developed to evaluate the impact of mobile-phase additives (ion-pairing agent / counter ion systems), distinct stationary phases on resulting resolving power, and MS detectability of oligonucleotides and their critical impurities in gradient IP-RPLC. Stationary-phase considerations included particle type (core-shell vs. fully porous particles), particle diameter, and pore size. Separations were carried out at 60°C to optimize mass transfer (C-term). The incorporation of an active column preheater mitigated thermal mismatches, leading to narrower peaks and overcoming peak splitting. Acetonitrile as organic modifier outweighed methanol in terms of peak-capacity generation and yielded a 30% lower back pressure. Performance screening experiments were conducted varying ion-pairing agents and counter ions, while adjusting gradient span achieved an equivalent effective retention window. Hexafluoromethylisopropanol yielded superior chromatographic resolution, whereas hexafluoroisopropanol yielded significantly higher MS detection sensitivity. The 1.7 µm core-shell particle columns with 100 Å pores provided maximum resolving power for small (15-35 mers) oligonucleotides. Sub-min analysis for 15-35 polyT ladders was achieved operating a 50 mm long column at the kinetic performance limits. High-resolution separations between a 21-mer modified RNA sequence oligonucleotides and its related (shortmer and phosphodiester) impurities and complementary strand were obtained using a coupled column set-up with a total length of 450 mm.

A Critical Evaluation of In Vitro Hesperidin 2S Bioavailability in a Model Combining Luminal (Microbial) Digestion and Caco-2 Cell Absorption in Comparison to a Randomized Controlled Human Trial.

SCOPE: Bioavailability strongly determines polyphenol bioactivity, and is strongly influenced by food matrix, enzymatic and microbial degradation, and gastrointestinal absorption. To avoid human trials for pre-screening of polyphenol bioavailability, studies have focused on in vitro model development. Nevertheless, their predictive value for bioavailability can be questioned. METHOD AND RESULTS: We used the orange flavonoid hesperidin 2S to validate a model combining digestion in the simulator of the human intestinal microbial ecosystem (SHIME) and Caco-2 cell transport, with a human intervention study. In vitro, hesperidin was resistant to degradation in the stomach and small intestine, but was rapidly deconjugated on reaching the proximal colon. Extensive and colon-region-specific degradation to smaller phenolics was observed. Hydrocaffeic and dihydroisoferulic acid accumulated in proximal, and hydroferulic acid in distal colon. Caco-2 transport was the highest for dihydroisoferulic acid. In humans, plasma and urine hesperetin-glucuronide levels increased significantly, whereas the impact on small phenolics was limited. CONCLUSIONS: In the combined in vitro model, smaller phenolics strongly accumulated, whereas in humans, hesperetin conjugates were the main bioavailable compounds. Future in vitro model development should focus on simulating faster polyphenol absorption and elimination of smaller phenolics to improve their predictive value of in vivo polyphenol bioavailability.

A First Step in the Quest for the Active Constituents in Filipendula ulmaria (Meadowsweet): Comprehensive Phytochemical Identification by Liquid Chromatography Coupled to Quadrupole-Orbitrap Mass Spectrometry.

Filipendula ulmaria (meadowsweet) is traditionally used for the treatment of inflammatory diseases and as a diuretic and antirheumatic. Extracts of Filipendulae herba are on the market in the European Union as food supplements. Nevertheless, its active constituents remain to be revealed. During this study, the phytochemical composition of Filipendulae Ulmariae Herba was comprehensively characterised for the first time with two complementary generic ultrahigh-performance liquid chromatography-photodiode array-accurate mass mass spectrometry methods. Selective ion fragmentation experiments with a hybrid quadrupole-orbital trap mass spectrometer significantly contributed to compound identification: a total of 119 compounds were tentatively identified, 69 new to F. ulmaria. A rich diversity of phenolic constituents was detected and only a few non-phenolic phytochemicals were observed. Metabolisation and pharmacological studies should be conducted to investigate which of these constituents or metabolites there of contribute to the activity of F. ulmaria after oral intake.

Pilot-scale production of cloudy juice from low-quality pear fruit under low-oxygen conditions.

In this study, a process for the production of premium quality yellowish, cloudy pear juice from low-quality fruit under low-oxygen conditions was developed. The production process consisted of (1) shredding, (2) pressing with spiral-filter technology including a vacuumised extraction cell, (3) holding in an inert gas buffer tank, (4) pasteurisation, (5) and refrigerated storage. First, the system parameters of a spiral-filter press were optimised with the aim of producing a yellowish, cloudy pear juice with the highest possible juice yield. A maximum juice yield of 78% could be obtained. Enzymatic browning during juice extraction could be suppressed as a result of the fast processing and the low air (oxygen) levels in the extraction chamber of the spiral-filter press. Furthermore, we observed that instantaneous pasteurisation at 107 °C for 6s, subsequent aluminium laminate packaging and cold storage had only a minimum effect on the phenolic composition.

A comparative study between spiral-filter press and belt press implemented in a cloudy apple juice production process.

In this study, advantages and disadvantages of the innovative, low-oxygen spiral-filter press system were studied in comparison with the belt press, commonly applied in small and medium size enterprises for the production of cloudy apple juice. On the basis of equivalent throughput, a higher juice yield could be achieved with spiral-filter press. Also a more turbid juice with a higher content of suspended solids could be produced. The avoidance of enzymatic browning during juice extraction led to an attractive yellowish juice with an elevated phenolic content. Moreover, it was found that juice produced with spiral-filter press demonstrates a higher retention of phenolic compounds during the downstream processing steps and storage. The results demonstrates the advantage of the use of a spiral-filter press in comparison with belt press in the production of a high quality cloudy apple juice rich in phenolic compounds, without the use of oxidation inhibiting additives.

Automated analytical standard production with supercritical fluid chromatography for the quantification of bioactive C17-polyacetylenes: a case study on food processing waste.

Food processing enterprises produce enormous amounts of organic waste that contains valuable phytochemicals (e.g. C17-polyacetylenes). Knowledge on the phytochemicals content is a first step towards valorisation. Quantification of C17-polyacetylenes is however often hampered by the lack of commercially available standards or by tedious multistep in-house standard production procedures. In the current study, a new and straightforward supercritical fluid chromatography purification procedure is described for the simultaneous production of 2 analytical C17-polyacetylene standards. Respectively, 5 and 6 mg of falcarinol and falcarindiol were purified in 17 h on analytical scale. After confirming the identity and quality (97% purity) by Nuclear Magnetic Resonance, accurate mass-Mass Spectrometry (am-MS) and Photo Diode Array (PDA) detection the C17-polyacetylene standards were used for the analysis of industrial vegetable waste with Liquid Chromatography coupled to PDA and am-MS detection. Measurements showed varying concentrations of C17-polyacetylenes in the organic waste depending on its nature and origin.

Tackling the challenge of selective analytical clean-up of complex natural extracts: the curious case of chlorophyll removal.

Alkaline saponification is often used to remove interfering chlorophylls and lipids during carotenoids analysis. However, saponification also hydrolyses esterified carotenoids and is known to induce artifacts. To avoid carotenoid artifact formation during saponification, Larsen and Christensen (2005) developed a gentler and simpler analytical clean-up procedure involving the use of a strong basic resin (Ambersep 900 OH). They hypothesised a saponification mechanism based on their Liquid Chromatography-Photodiode Array (LC-PDA) data. In the present study, we show with LC-PDA-accurate mass-Mass Spectrometry that the main chlorophyll removal mechanism is not based on saponification, apolar adsorption or anion exchange, but most probably an adsorption mechanism caused by H-bonds and dipole-dipole interactions. We showed experimentally that esterified carotenoids and glycerolipids were not removed, indicating a much more selective mechanism than initially hypothesised. This opens new research opportunities towards a much wider scope of applications (e.g. the refinement of oils rich in phytochemical content).

Generic characterization of apolar metabolites in red chili peppers (Capsicum frutescens L.) by orbitrap mass spectrometry.

The aim of the present study was to develop a generic analytical method for the identification and quantitation of apolar plant metabolites in biomass using liquid chromatography-photodiode array-accurate mass mass spectrometry (LC-PDA-amMS). During this study, a single generic sample preparation protocol was applied to extract apolar plant metabolites. Compound identification was performed using a single generic screening method for apolar compounds without the need for dedicated fractionation. Such a generic approach renders vast amounts of information and is virtually limited by only the solubility and detector response of the metabolites of interest. Method validation confirmed that this approach is applicable for quantitative purposes. Furthermore, an identification-quantitation strategy based on amMS and molar extinction coefficients was used for carotenoids, eliminating the need for reference standards for each carotenoid. To challenge the validated method, chili peppers (Capsicum frutescens L.) were analyzed to unravel their complex phytochemical composition (carotenoids, glycolipids, glycerolipids, capsaicinoids, lipid-soluble vitamins).

Improving Method Reliability in Carotenoid Analysis through Selective Removal of Glycerolipid Interferences by Lipase Treatment.

Saponification is most often used to hydrolyze glycerolipid interferences during carotenoid analysis. Ester bonds of other plant metabolites such as carotenoids are, however, also hydrolyzed during saponification, thus altering the natural carotenoid composition. A straightforward and selective cleanup procedure was therefore developed involving the enzymatic hydrolysis of matrix glycerolipids. The optimized procedure (100 µL of extracted vegetable or algal oil in 20 mL of 50:50 phosphate buffer/methanol with 25 µL of sodium n-octyl sulfate, 30 mg of bile salts, and 250 µL of NaCl solution (5 mM), magnetic stirring for 2 h at 40 °C with 1 mL of Lipozyme TL 100 L and 1 mL of Lipozyme CALB L) removed the greater part of triglycerides (94.8-100.0%) and diglycerides (88.2-99.8%) while preserving the natural carotenoid composition.

Ultra high performance liquid chromatography versus high performance liquid chromatography: stationary phase selectivity for generic carotenoid screening.

Aim of study was to find the most suitable LC column for generic carotenoid screening. To represent the diversity of carotenoids in nature and to optimize chromatographic separation, a set of carotenoid standards was carefully chosen to account for the various classes of carotenoids. The HPLC C30 column has since long been the 'golden standard' in the chromatographic separation of carotenoids. Since approximately one decade, new UHPLC technology has led to much shorter analysis times, smaller peak widths and higher chromatographic resolution. However, there are currently no UHPLC columns on the market containing the specific stationary phase chemistry of the HPLC C30 column. Therefore during this study, we investigated the separation of carotenoids on a set of UHPLC columns and compared it to their separation on the HPLC C30 column. Comparison of carotenoids separations on the different stationary phases with objective column comparison parameters clearly indicated that the HPLC C30 column is an overall better performer in the separation of carotenoids. This is due to the lack of UHPLC column chemistries that are adapted for carotenoid analysis. However, analysis time on the HPLC C30 column takes about four times longer compared to UHPLC analysis. Therefore, with the range of columns that are commercially available nowadays, a choice has to be made between very high selectivity (HPLC C30 column) and analysis times that are adapted to modern laboratory requirements (UHPLC technology). Therefore, carotenoid separations would be even more performing if an appropriate UHPLC C30 column would be available.

Detection of flavonoids in microalgae from different evolutionary lineages.

Flavonoids are important secondary plant metabolites believed to be present mainly in land plants. As phenolics were detected previously in microalgae using photometric assays, we wanted to investigate the nature of these phenolics and verify whether flavonoids are present. Therefore, in this study, we used state-of-the-art ultra-high performance liquid chromatography-two-dimensional mass spectrometry (UHPLC-MS/MS) technology to investigate whether microalgae also contain flavonoids. For this, representative microalgal biomass samples from divergent evolutionary lineages (Cyanobacteria, Rhodophyta, Chlorophyta, Haptophyta, Ochrophyta) were screened for a set of carefully selected precursors, intermediates, and end products of the flavonoid biosynthesis pathways. Our data unequivocally showed that microalgae contain a wide range of flavonoids and thus must possess the enzyme pool required for their biosynthesis. Further, some of the microalgae displayed an intricate flavonoid pattern that is compatible with the established basic flavonoid pathway as observed in higher plants. This implies that the flavonoid biosynthesis pathway arose much earlier in evolution compared to what is generally accepted.

Total uncertainty budget calculation for the determination of mercury in incineration ash (BCR 176R) by atomic fluorescence spectrometry.

The mercury mass fraction has been determined by atomic fluorescence spectrometry (AFS) in the framework of the project "Certification of a reference material (trace elements in fly ash) in replacement of BCR CRM 176". Calculation of the uncertainty budget, as described in this manuscript, emphasizes a practical and realistic approach to estimation of uncertainty components on the basis of statistical assumptions. GUM Workbench software was used, and resulted in a mercury mass fraction of 1.58+/-0.11 mg kg(-1) (with coverage factor k=2.2, 95% probability) related to dry mass, submitted in the certification exercise. The calculated total uncertainty budget applies to analogous samples analyzed by this procedure.