Bart Noten

An improved mass spectrometric method for identification and quantification of phenolic compounds in apple fruits

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 (r(2)>0.9990), range (typically 10-3500 ngg(-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.

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.

Thermal degradation of cloudy apple juice phenolic constituents

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.

Direct analysis of phthalate ester biomarkers in urine without preconcentration: method validation and monitoring.

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 0.5μg/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.

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).

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.

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.

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.

Variability of the phenolic profiles in the fruits from old, recent and new apple cultivars cultivated in Belgium

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.

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).