David Arráez-Román

HPLC-DAD-ESI-MS/MS screening of bioactive components from Rhus coriaria L. (Sumac) fruits

Rhus coriaria L. (sumac) is an important crop widely used in the Mediterranean basin as a food spice, and also in folk medicine, due to its health-promoting properties. Phytochemicals present in plant foods are in part responsible for these consequent health benefits. Nevertheless, detailed information on these bioactive compounds is still scarce. Therefore, the present work was aimed at investigating the phytochemical components of sumac fruit epicarp using HPLC-DAD-ESI-MS/MS in two different ionisation modes. The proposed method provided tentative identification of 211 phenolic and other phyto-constituents, most of which have not been described so far in R. coriaria fruits. More than 180 phytochemicals (tannins, (iso)flavonoids, terpenoids, etc.) are reported herein in sumac fruits for the first time. The obtained results highlight the importance of R. coriaria as a promising source of functional ingredients, and boost its potential use in the food and nutraceutical industries.

Use of advanced techniques for the extraction of phenolic compounds from Tunisian olive leaves: Phenolic composition and cytotoxicity against human breast cancer cells

A comparison among different advanced extraction techniques such as microwave-assisted extraction (MAE), supercritical fluid extraction (SFE) and pressurized liquid extraction (PLE), together with traditional solid-liquid extraction, was performed to test their efficiency towards the extraction of phenolic compounds from leaves of six Tunisian olive varieties. Extractions were carried out at the best selected conditions for each technique; the obtained extracts were chemically characterized using high-performance liquid chromatography (HPLC) coupled to electrospray time-of-flight mass spectrometry (ESI-TOF-MS) and electrospray ion trap tandem mass spectrometry (ESI-IT-MS(2)). As expected, higher extraction yields were obtained for PLE while phenolic profiles were mainly influenced by the solvent used as optimum in the different extraction methods. A larger number of phenolic compounds, mostly of a polar character, were found in the extracts obtained by using MAE. Best extraction yields do not correlate with highest cytotoxic activity against breast cancer cells, indicating that cytotoxicity is highly dependent on the presence of certain compounds in the extracts, although not exclusively on a single compound. Therefore, a multifactorial behavior is proposed for the anticancer activity of olive leaf compounds.

Comparison of different extraction procedures for the comprehensive characterization of bioactive phenolic compounds in Rosmarinus officinalis by reversed-phase high-performance liquid chromatography with diode array detection coupled to electrospray time-of-flight mass spectrometry

In the present work, a comparative study between two environmentally friendly and selective extraction techniques, such as supercritical fluid extraction (SFE) and pressurized liquid extraction (PLE) have been carried out focusing in the bioactive phenolic compounds present in Rosmarinus officinalis. For the analysis of the SFE and PLE extracts, a new methodology for qualitative characterization has been developed, based on the use of reversed-phase high-performance liquid chromatography (RP-HPLC), equipped with two different detection systems coupled in series: diode array detector (DAD) and time of flight mass spectrometry (TOF-MS) detector connected via an electrospray ionization interface (ESI). The use of a small particle size C(18) column (1.8 μm) provided a great resolution and made possible the separation of several isomers. Moreover, UV-visible spectrophotometry is a valuable tool for identifying the class of phenolic compounds, whereas MS data enabled to structurally characterize the compounds present in the extracts. The applied methodology was useful for the determination of many well-known phenolic compounds present in R. officinalis, such as carnosol, carnosic acid, rosmadial, rosmanol, genkwanin, homoplantaginin, scutellarein, cirsimaritin and rosmarinic acid, as well as other phenolic compounds present in other species belonging to Lamiaceae family.

Reversed-phase ultra-high-performance liquid chromatography coupled to electrospray ionization-quadrupole-time-of-flight mass spectrometry as a powerful tool for metabolic profiling of vegetables: Lactuca sativa as an example of its application

Lettuce (Lactuca sativa), a leafy vegetal widely consumed worldwide, fresh cut or minimally processed, constitutes a major dietary source of natural antioxidants and bioactive compounds. In this study, reversed-phase ultra-high-performance liquid chromatography (RP-UHPLC) coupled to electrospray ionization-quadrupole-time-of-flight mass spectrometry (ESI-QTOF-MS) was applied for the comprehensive profiling of polar and semi-polar metabolites from three lettuce cultivars (baby, romaine, and iceberg). The UHPLC systems allowed the use of a small-particle-size C18 column (1.8 μm), with very fine resolution for the separation of up to seven isomers, and the QTOF mass analyzer enabled sensitive detection with high mass resolution and accuracy in full scan. Thus, a total of 171 compounds were tentatively identified by matching their accurate mass signals and suggested molecular formula with those previously reported in family Asteraceae. Afterwards, their structures were also corroborated by the MS/MS data provided by the QTOF analyzer. Well-known amino acids, organic acids, sesquiterpene lactones, phenolic acids and flavonoids were characterized, e.g. lactucin, lactucopicrin, caftaric acid, chlorogenic acid, caffeoylmalic acid, chicoric acid, isochlorogenic acid A, luteolin, and quercetin glycosides. For this plant species, this is the first available report of several isomeric forms of the latter polyphenols and other types of components such as nucleosides, peptides, and tryptophan-derived alkaloids. Remarkably, 10 novel structures formed by the conjugation of known amino acids and sesquiterpene lactones were also proposed. Thus, the methodology applied is a useful option to develop an exhaustive metabolic profiling of plants that helps to explain their potential biological activities and folk uses.

Rosmarinus Officinalis Leaves as a Natural Source of Bioactive Compounds

In an extensive search for bioactive compounds from plant sources, the composition of different extracts of rosemary leaves collected from different geographical zones of Serbia was studied. The qualitative and quantitative characterization of 20 rosemary (Rosmarinus officinalis) samples, obtained by microwave-assisted extraction (MAE), was determined by high performance liquid chromatography coupled to electrospray quadrupole-time of flight mass spectrometry (HPLC–ESI-QTOF-MS). The high mass accuracy and true isotopic pattern in both MS and MS/MS spectra provided by the QTOF-MS analyzer enabled the characterization of a wide range of phenolic compounds in the extracts, including flavonoids, phenolic diterpenes and abietan-type triterpenoids, among others. According to the data compiled, rosemary samples from Sokobanja presented the highest levels in flavonoids and other compounds such as carnosol, rosmaridiphenol, rosmadial, rosmarinic acid, and carnosic acid. On the other hand, higher contents in triterpenes were found in the extracts of rosemary from Gložan (Vojvodina).

Extensive characterisation of bioactive phenolic constituents from globe artichoke (Cynara scolymus L.) by HPLC–DAD-ESI-QTOF-MS

The aim of this work was to characterise the phenolic compounds in artichoke (hearts) by using HPLC coupled to DAD-ESI-QTOF-MS, which proved useful in characterising 61 phenolic and other polar compounds. Notably, of the 61 compounds characterised, 34 new phenolic compounds with their isomers have been tentatively characterised in artichoke for the first time, namely: 3 hydroxybenzoic acids, 17 hydroxycinnamic acids, 4 lignans, 7 flavones, 2 flavonols, and 1 phenol derivative. Moreover, a total of 28 isomers of previously described phenolics have also been detected. The data compiled from the qualitative polyphenol characterisation indicate that the artichoke extract analysed (Blanca de Tudela variety) could be regarded as a bioactive functional food and also as a promising source of antioxidant phenolic compounds.

Optimization of Microwave-Assisted Extraction for the Characterization of Olive Leaf Phenolic Compounds by Using HPLC-ESI-TOF-MS/IT-MS2

In the present work, a simple and rapid method for the extraction of phenolic compounds from olive leaves, using microwave-assisted extraction (MAE) technique, has been developed. The experimental variables that affect the MAE process, such as the solvent type and composition, microwave temperature, and extraction time, were optimized using a univariate method. The obtained extracts were analyzed by using high-performance liquid chromatography (HPLC) coupled to electrospray time-of-flight mass spectrometry (ESI-TOF-MS) and electrospray ion trap tandem mass spectrometry (ESI-IT-MS(2)) to prove the MAE extraction efficiency. The optimal MAE conditions were methanol:water (80:20, v/v) as extracting solvent, at a temperature equal to 80 °C for 6 min. Under these conditions, several phenolic compounds could be characterized by HPLC-ESI-MS/MS(2). As compared to the conventional method, MAE can be used as an alternative extraction method for the characterization of phenolic compounds from olive leaves due to its efficiency and speed.

Characterization of isomers of oleuropein aglycon in olive oils by rapid-resolution liquid chromatography coupled to electrospray time-of-flight and ion trap tandem mass spectrometry.

In this work, rapid-resolution liquid chromatography (RRLC) coupled to electrospray ionization time-of-flight mass spectrometry (ESI-TOF-MS) and ion trap multiple mass spectrometry (IT-MS(n)) has been applied to separate and characterize eleven isomers of oleuropein aglycon in fourteen Spanish extra-virgin olive oils. After the extra-virgin olive oil sample had been dissolved in hexane and cleaned up by a diol-bonded phase solid-phase extraction (SPE) cartridge, the eluting extract was resolved in methanol and analyzed on an Angilent 1200 system with a 4.6 x 150 mm, 1.8 microm Zorbax Eclipse plus C18 column. Mass spectrometry was carried out on a Bruker Daltonics microTOF mass spectrometer and a Bruker Daltonics ion trap mass spectrometer. The characterization of isomers of oleuropein aglycon was based on accurate mass data and the isotope function of characteristic fragment ions in the studied compounds by TOF-MS, and the fragment ions were further confirmed by IT-MS(n). The fragmentation pathway of oleuropein aglycon was successfully elucidated and all possible transformations among isomers of oleuropein aglycon were suggested.

Tentative Characterization of Novel Phenolic Compounds in Extra Virgin Olive Oils by Rapid-Resolution Liquid Chromatography Coupled with Mass Spectrometry

Rapid-resolution liquid chromatography (RRLC) coupled with electrospray time-of-flight mass spectrometry (ESI-TOF-MS) and electrospray ion-trap multiple mass spectrometry (ESI-IT-MS(n)) has been applied to separate and characterize the phenolic compounds in five Spanish extra virgin olive oils. The phenolic compounds were characterized on the basis of accurate mass data according to studies by TOF-MS, and the fragmentation ions were further confirmed by IT-MS(n) whenever possible. Eight compounds, hydroxytyrosol (m/z 153), tyrosol (m/z 137), deacetoxy oleuropein aglycon (m/z 319), luteolin (m/z 285), pinoresinol (m/z 357), acetoxypinoresinol (m/z 415), apigenin (m/z 269), and deacetoxy ligstroside aglycon (m/z 303), all normally found in olive oil, were characterized in all five samples. Nevertheless, 30 uncommon phenolic compounds, including 10 ligstroside aglycon isomers (m/z 361) and two methyl oleuropein aglycon isomers (m/z 391), were also characterized in some of the five samples, and their fragmentation pathways were successfully elucidated.

Phytochemical Characterisation of Green Beans (Phaseolus vulgaris L.) by Using High-performance Liquid Chromatography Coupled with Time-of-flight Mass Spectrometry

INTRODUCTION Although green beans (Phaseolus vulgaris L.) constitute a major agro-industrial crop worldwide and involve many bean varieties, the phytochemical composition and how this fluctuates among varieties is not well known. OBJECTIVE The purpose of this work was to conduct a comprehensive characterisation of the phytochemical compounds found in three varieties of green bean. METHODOLOGY Hydromethanol extracts from green beans were analysed by high-performance liquid chromatography (HPLC) coupled with electrospray time-of-flight mass spectrometry (ESI-TOF-MS) in order to characterise phytochemical compounds from different varieties of P. vulgaris L. The compounds were characterised based on interpreting their mass spectrum provided by the TOF-MS as well as by comparison with information from the literature (some compounds have been described previously in Fabaceae). RESULTS In this work, 72 phytochemical compounds were tentatively characterised by HPLC-ESI-TOF-MS. These compounds were classified as, 10 phenolic acids, 59 flavonoids, two lignans and an iridoid. Notably, out of the 72 compounds, 54 are new and their isomers have been characterised for the first time in green beans. CONCLUSION The phytochemical composition of three different varieties of P. vulgaris L have been characterised using HPLC-ESI-TOF-MS. A total of 72 phytochemical compounds were characterised, 54 being reported in green beans for the first time. Among these were the main flavonoids detected. These results highlight the influence that variety can exert on the quality of phytochemicals. Given that new phytochemical compounds have been characterised, this study offers a useful approach for improving and updating the food-composition tables.