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 degrees 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 mu m core-shell particle columns with 100 & Aring; 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.

Small interfering RNAs (siRNAs) have emerged as a highly promising class of therapeutics, capable of effectively treating a wide range of indications, including previously challenging targets. To correctly characterize the duplex content of siRNA therapeutics, a careful design of the analytical conditions is required. This is due to the weak interactions governing the duplex formation and thermal stability of these double-stranded oligonucleotides. In this study, we demonstrate that the reliability of duplex content analyses can be compromised by denaturation or hybridization artifacts caused by environmental factors related with sample preparation or with the ‘non-denaturing’ chromatographic analysis method. To address this issue, we propose to characterize the siRNA duplex in various analytical media with unbiased techniques such as circular dichroism spectrophotometry and use the results to evaluate potential artifacts in the ‘non-denaturing’ method, developed to determine the duplex content. Through this approach, one can optimize the sample preparation and develop ‘non-denaturing’ method conditions to minimize the influence of environmental factors on the duplex content, and thereby determine the assay of siRNA duplex with no bias.

SCOPEBioavailability 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 RESULTSWe 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.CONCLUSIONSIn 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.

Apple pomace can be used as resource for the extraction of phenolic compounds with antioxidant properties. Pressing of apple in juice and pomace at lab scale in open air (aerobic) and under N-2 atmosphere (anaerobic) showed a recovery of phenolic compounds of 85% in juice and pomace after anaerobic pressing, compared to 43% after aerobic pressing, indicating loss of phenolic compounds by oxidation and the advantage of anaerobic over aerobic pressing. After a membrane screening and concentration test at lab scale, the commercial nanofiltration membrane NFX was selected to concentrate phenolic compounds in an ethanol:water extract of apple pomace. At pilot scale, the concencentration of 10 selected phenolic compounds and quinic acid increased from 59.5mg/L in the ethanol:water extract to 1256.1mg/L in the final retentate, that is, by a factor 21.1. The volume of the crude extract was reduced by a factor of 28.5 during the filtration, indicating some loss of phenolic compounds during pilot testing due to membrane fouling or oxidation of polyphenols. The pilot concentration test using a spiral-wound membrane module showed good flux and concentration of phenolic compounds, indicating the technical feasibility of membrane technology for efficient concentration of polyphenols in an ethanol:water extraction solvent. Unfortunately, the extraction and concentration process was not economically feasible under the assumptions made. Practical applicationsThe valorization of food waste for the production of high-added value products is an increasingly hot topic. Phytochemicals are present in relatively low concentration in the fruit matrix, and concentration in the extraction solvent is necessary to develop an industrially relevant process. In this study, membrane filtration was selected for concentration due to its low energy consumption and mild processing conditions compared to other technologies. Membrane screening and testing at lab and pilot scale with techno-economic assessment can be used by food and nutraceutical industries to evaluate membrane technology for concentration of phytochemicals extracted from agroindustrial by-products.

During the post-certification stability monitoring of the certified reference material (CRM) BCR-551 (DNPH derivatives dissolved in acetonitrile), a decreased concentration of one of the analytes of this CRM, the DNPH-formaldehyde derivative, was detected in reference samples (stored at −70 °C), while the concentration of normal “on-sale” samples (stored at −20 °C) remained stable. This behaviour is contrary to the expectation of better stability at lower temperatures. Apparently, the DNPH-formaldehyde derivative reacts with dinitrophenylhydrazine (DNPH) remaining from the synthesis phase to produce two new substances. These substances have been identified as C13H12N8O8 (substance 1) and C20H16N12O12 (substance 2) which, based on their structure, are suggested to be produced consecutively: DNPH + DNPH-formaldehyde derivative → substance 1 and substance 1 + DNPH-formaldehyde derivative → substance 2. Since acetonitrile freezes at −45 °C, reference samples are frozen at −70 °C, while normal samples are still liquid at −20 °C. We believe that this leads to a cryo-concentration of the solutes above the eutectic point and thus to an increased reaction rate in the reference samples. This case demonstrates that care should be taken when extrapolating stability results towards conditions that never have been tested, especially if phase transitions are involved, even at temperature as low as −70 °C. Furthermore, a slower degradation rate at lower temperatures can be overcompensated by a higher concentration due to cryo-concentration above the eutectic temperature.

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.

The objective of this study was to explore the utility of the phenolic pattern (1) to differentiate or associate between cultivars with a different or same genetic background respectively and (2) to assess some important quality traits (color, disease resistance, and browning) in apple fruit. A unique set of cultivars/candivars occurring in Belgium were selected for this survey. It was possible to segregate the studied apple cultivars/candivars into three clusters: red-flesh apple candivars, classic/new cultivars and candivars and heritage apple cultivars. It was found that the heritage apple varieties selected for their disease resistance and cultivated under organic agricultural practice demonstrate a remarkable different phenolic profile. Furthermore, it was found that the phenolic makeup of the peel could suggest the genetic relationships among the apple cultivars. In conclusion, the knowledge of the phenolic profile could be a useful instrument in breeding programs to identify apples with better quality traits, processing characteristics and health-benefiting effects.

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.

Cyclic peptides have found numerous and wide ranging applications that include drug molecules, nanomaterials, and chiral chromatography stationary phases. However, in the crucial cyclization step, high dilution conditions are often required, resulting in large volumes of solvent being consumed to prepare relatively small quantities of product. This paper demonstrates the synthesis of a cyclic nonapeptide with in-line solvent recycling via organic solvent nanofiltration (OSN) resulting in a significant reduction in the solvent load of the reaction and concomitant improvement in process mass intensification (PMI). The membrane was used to remove the reaction product from the reaction vessel, as the cyclic peptide product shows limited stability in the presence of an excess of reaction reagent. In comparison to the standard batch reaction, no loss in yield or product purity was observed for the OSN process tested. The proof-of-concept study outlined in this paper was performed on a real active pharmaceutical ingredient (API), and the technique used is widely applicable and flexible.

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.

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.

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).

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.

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.

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.

Although conventional thermal processing is still the most commonly used preservation technique in cloudy apple juice production, detailed knowledge on phenolic compound degradation during thermal treatment is still limited. To evaluate the extent of thermal degradation as a function of time and temperature, apple juice samples were isothermally treated during 7,200s over a temperature range of 80-145 °C. An untargeted metabolomics approach based on liquid chromatography-high resolution mass spectrometry was developed and applied with the aim to find out the most heat labile phenolic constituents in cloudy apple juice. By the use of a high resolution mass spectrometer, the high degree of in-source fragmentation, the quality of deconvolution and the employed custom-made database, it was possible to achieve a high degree of structural elucidation for the thermolabile phenolic constituents. Procyanidin subclass representatives were discovered as the most heat labile phenolic compounds of cloudy apple juice.

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).

Phthalates, which are ubiquitous in the environment, are readily metabolized in human bodies to their respective monoesters. These phthalate monoesters are non-persistent with short half-lives, which make them the ideal biomarkers of human exposure to phthalates. In this study a direct analysis method without preconcentration was developed and validated for the following phthalate ester metabolites in urine: mono-(2-ethylhexyl) phthalate, mono-(2-ethyl-5-hydroxyhexyl) phthalate, mono-(2-ethyl-5-oxohexyl)phthalate, monobenzyl phthalate, mono-isobutylphthalate, mono-n-butyl phthalate and monoethyl phthalate. The recovery of the phthalate ester metabolites varied between 97% and 104%. The intraday precision for the replicate analysis (n = 10) of a urine sample did not exceed 5% for most of the compounds. The coefficient of variance amounted to 2-3%. The limit of quantification was set equal to 0.5 mu g/L for the majority of the compounds. A comparison between the direct analysis method and a foregoing solid phase extraction (SPE) of the urine sample was made. Finally, the applicability of the direct analysis method was tested in three interlaboratory comparisons. (C) 2013 Elsevier B.V. All rights reserved.

Thirty-nine phenolic compounds were analysed using ultra high performance liquid chromatography (UHPLC) coupled with diode array and accurate mass spectrometry detection using electrospray ionisation (DAD/ESI-am-MS). Instrumental parameters such as scan speed, resolution, and mass accuracy were optimised to establish accurate mass measurements. The method was fully validated in terms of model deviation (r2 > 0.9990), range (typically 10–3500 ng g−1), intra/inter-day precision (<6% and <8%, respectively) and accuracy (typically 100 ± 10%). The mass accuracy of each selected phenolic compound was below 1.5 ppm. The results confirmed that the UHPLC-DAD/ESI-am-MS method developed here was convenient and reliable for the determination of phenolic compounds in apple extracts.