Mass spectrometry in the study of molecular complexes between 5-fluorouracil and catechins.

5-Fluorouracil (5FU) is a widely employed antineoplastic agent that acts as antimetabolite. However, 5FU activity is strongly reduced against a subset of cancer cells called cancer stem cells (CSCs), which are believed to be responsible for chemoresistance and tumour recurrence. It was found that epigallocatechin-3-gallate (EGCG), the most abundant catechin present in green tea extract, suppresses CSCs grown in various cancers. This chemosensitizing effect of EGCG was investigated in 5FU-resistant (5FUR) CRC cells, showing that EGCG enhances 5FU-induced cytotoxicity. However, the real mechanism of an improved 5FU chemosensitivity in the presence of EGCG was not evaluated. Considering the capability of catechins to form bimolecular noncovalent complexes, in the present study, the interaction of catechins and 5FU was studied by different mass spectrometric approaches. The ESI(+) and ESI(-) spectra of [5FU-catechin] mixtures were studied, showing the formation of protonated and deprotonated bimolecular complexes, whose nature was confirmed by MS/MS experiments (product and precursor ion scans). To exclude the possible origin of these species as ESI artefacts, a further series of experiments were performed by high-resolution liquid chromatography-mass spectrometry. By this approach, bimolecular complexes have been detected at retention times different from those of free 5FU and catechins, proving their presence in the original solution. Analogous studies were performed on 5FU-green tea extract mixtures, showing that 5FU leads to complexes not only with EGCG but also with other catechins. These molecular species, differently to free 5FU drug alone, would in principle possess a new biological activity and could be an explanation of the described activity cited above.

MASS SPECTROMETRY FOR A HOLISTIC VIEW OF NATURAL EXTRACTS OF PHYTOTHERAPEUTIC INTEREST.

In the study of natural products new strategies which favor a holistic approach, integrating the traditional reductionist methods usually employed, have been proposed. In this frame, the studies carried out by us in the last decade show that fingerprints, mainly obtained by electrospray ionization mass spectrometry (ESI-MS), lead to the characterization of natural extracts from different botanical species but also of phytotherapeutic products constituted by mixtures of extracts from different plants. Laser desorption ionization and matrix-assisted laser desorption ionization techniques were also employed and by the use of different matrices some complementary results were achieved. Results obtained by standard spectrophotometric and liquid chromatography methods were compared with those achieved by direct infusion of the extract in ESI-MS conditions, indicating an excellent agreement between the two approaches. The findings of these researches were considered in the frame of complex systems theory, investigating how relationships between a system's parts can give rise to its collective behaviors and how the system interacts and forms relationships with its environment. In this view, the peculiar pharmacological behavior of biologically active natural compounds can be justified by the occurrence of molecular interactions due to the high complexity of the natural matrix. Some of these interactions have been widely studied in the case of green tea extracts (GTEs) proving unequivocally the presence of caffeine/catechin complexes in GTE samples. The presence of bimolecular complexes has been observed also in the case of Ceylon tea and Mate extracts. These data indicate that the formation of complexes in natural extracts is a common behavior and their presence must be considered in the description of natural extracts and, consequently, in their biological activity. ©2020 John Wiley & Sons Ltd. Mass Spec Rev.

Evidence of noncovalent complexes in some natural extracts: Ceylon tea and mate extracts.

Considering the high complexity of natural extracts, because of the presence of organic molecules of different chemical nature, the possibility of formation of noncovalent complexes should be taken into account. In a previous investigation, the formation of bimolecular complexes between caffeine and catechins in green tea extracts (GTE) has been experimentally proven by means of mass spectrometric and 1 H nuclear magnetic resonance experiments. The same approaches have been employed in the present study to evaluate the presence of bimolecular complexes in Ceylon tea and mate extracts. The obtained results show that in the case of Ceylon tea extracts, protonated theaflavin is detectable, together with theaflavin/caffein complexes, while caffeine/catechin complexes, already detected in green tea, are still present but at lower concentration. This aspect is evidenced by the comparison of precursor ion scans performed on protonated caffeine for the two extracts. The spectra obtained in these conditions for GTE and Ceylon tea show that the complexes of caffeine with epigallocatechin (EGC), epicatechin gallate (ECG), and epigallocatechin gallate (EGCG), highy abundant in the case of GTE (signal-to-chemical noise ratio in the range 50-100), are negligible (signal-to-chemical noise ratio in the range 2-3) in the case of Ceylon tea. Mate extracts show the formation of bimolecular complexes involving caffeine but not catechins, and chlorogenic acid becomes responsible for other complex formation. Under positive ion and negative ion conditions, accurate mass measurements allow the identification of malealdehyde, chlorogenic acid, caffeine, two isomers of dicaffeoylquinic acid, rutin, and kaempferol-3-O-rutinoside. These data indicate that the formation of complexes in natural extracts is a common behavior, and their presence must be considered in the description of natural extracts and, consequently, in their biological activity.

Experimental Evidence of the Presence of Bimolecular Caffeine/Catechin Complexes in Green Tea Extracts.

A hypothesis on the peculiar pharmacological behavior of biologically active natural compounds is based on the occurrence of molecular interactions originating from the high complexity of the natural matrix, following the rules of supramolecular chemistry. In this context, some investigations were performed to establish unequivocally the presence of caffeine/catechin complexes in green tea extracts (GTEs). 1H NMR spectroscopy was utilized to compare profiles from GTEs with caffeine/catechin mixtures in different molar ratios, showing that peaks related to caffeine in GTEs are generally upfield shifted compared to those of free caffeine. On the other hand, ESIMS experiments performed on GTE, by means of precursor ion scan and neutral loss scan experiments, proved unequivocally the presence of caffeine/catechin complexes. Further investigations were performed by an LC-MS method operating at high-resolution conditions. The reconstructed ion chromatograms of the exact mass ions corresponding to caffeine/catechin species have been obtained, showing the presence of complexes of caffeine with gallate-type catechins. Furthermore, this last approach evidenced the presence of the same complex with different structures, consequently exhibiting different retention times. Both MSE and product ion MS/MS methods confirm the nature of caffeine/catechin complexes of the detected ions, showing the formation of protonated caffeine.

An integrated approach to the evaluation of a metabolomic fingerprint for a phytocomplex. Focus on artichoke [Cynara cardunculus subsp. scolymus] leaf.

The availability of reliable herbal formulations is essential in order to assure the maximal activity and to limit unwanted side-effects. The correct concentration of declared components of herbal products is a matter of health legislation and regulation, but is still a topic under debate in the field of quality control assessment. In the present work specific constituents of artichoke leaf extracts, considered as a test herbal product, were measured by standard spectrophotometric and HPLC methods (for quantitative determination of some components only), and results were correlated with the ESI-MS (showing the full metabolomic fingerprint). Phytocomplex stability over time was also investigated in batches submitted to different storage conditions. The results indicated excellent agreement between the two approaches in the measurement of total caffeoylquinic acids and chlorogenic acid contents, but the metabolomic ESI-MS method approach provides a more complete evaluation and monitoring of the composition of a herbal product, without focusing only on a single/few compound measurements. Therefore, the ESI-MS method can be proposed for the evaluation of the quality of complex matrices, such as those in a phytocomplex. Another aspect lies in the possibility to obtain a broad-spectrum stability control of herbal formulations, requiring minimal sample pre-processing procedures.

A rapid and highly specific method to evaluate the presence of pyrrolizidine alkaloids in Borago officinalis seed oil.

Pyrrolizidine alkaloids (PAs) are complex molecules, present in plants as free bases and N-oxides. They are known for their hepatotoxicity, and consequently there is a health risk associated with the use of medicinal herbs that contain PAs. Unfortunately, there is no international regulation of PAs in foods, unlike those for herbs and medicines: in particular, for herbal preparation or herbal extracts, the total PA content must not exceed 1 µg/kg or 1 µg/l, respectively. Borago officinalis seed oil is a source of γ-linolenic acid, and its use is increased in both pharmaceutical and health food industries. Even if studies based on gas chromatography and TLC methods showed that PAs are not co-extracted with oil, the development of a rapid and sensitive method able to evaluate the presence of PAs in commercially available products is surely of interest. The presence of PAs in a commercially available Borago officinalis seed oil was tested either in the oil sample diluted with tetrahydrofuran/methanol (MeOH)/H2 O (85/10/5 v:v:v) or after extraction with MeOH/H2 O (50/50 v:v) solution The samples were analysed by electrospray ionization in positive ion mode and in high mass resolution (60,000) conditions. In both cases to evaluate the effectiveness of the method, spiking experiments were performed adding known amount of two PA standards to the borage seed oil. A limit of detection in the order of 200 ppt was determined for these two compounds, strongly analogous to Borago officinalis seed oil PAs. Consequently, if present, PAs level in Borago officinalis seed oil must lower than 200 ppt.

Seed oil triglyceride profiling of thirty-two hybrid grape varieties.

Triglyceride profile of seed oil samples from 32 hybrid grape varieties not studied before was investigated. A new method for the analysis of triacylglycerols (TAGs) has been developed based on the direct infusion in the electrospray ionization (ESI) source and employing tetrahydrofuran/methanol/water (85:10:5 v|v|v) as solvent; the formation of [M + Na](+) ions in high yield has been observed. TAGs were identified by ESI-tandem mass spectrometry analysis, and the matrix-assisted-laser-desorption-ionization and time-of-flight profile of samples was determined. Six were the principal TAGs identified in seed oil: trilinolein (LLL) was the most abundant (43%), followed by dilinoleoyl-oleoylglycerol (LOL, 23%), and dilinoleoyl-palmitoylglycerol (LPL, 15%). Compounds present in lower concentration were LSL and LOO (11%), LOP (6%), and LSP (2%). Compared with seed oils produced from V. Vinifera grapes, some significant differences in the relative abundances of TAGs were found, in particular hybrid grape seed oils showed higher LOL and lower LPL content, respectively. Among the samples studied, a particularly high content of LLL (rich in unsaturated fatty acids) was found in seed oils from two red varieties.

The protein profile of Theobroma cacao L. seeds as obtained by matrix-assisted laser desorption/ionization mass spectrometry.

The water-soluble protein profile of the seeds of green, red, and yellow Theobroma cacao L. fruits has been determined by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-ToF-MS). The seeds were powdered under liquid nitrogen and defatted. The residues were dialyzed and lyophilized. The obtained samples were suspended in the matrix solution of sinapinic acid. The obtained MALDI mass spectra showed the presence of a wide number of proteins with molecular weight ranging from 8000 to 13,000 Da and a cluster of peaks centered at 21,000 Da that were attributed to albumin. The abundance of this peak was found to depend on the different portion of the seed (husk, apical and cortical parts); however, the MALDI mass spectra obtained from the different varieties of cocoa were practically superimposable. Changes in the protein profiles were also observed after the cocoa seeds were treated by fermentation and roasting, which are processes usually employed for the commercial production of cocoa.

Can crude oils be distinguished by different component distribution obtained by laser desorption ionization mass spectrometry and evaluated by chemometrics?

Fourteen different crude oil samples were analyzed as collected by mass spectrometry. For two of them the results obtained by means of different techniques, i.e. electrospray ionization, atmospheric pressure chemical ionization, laser desorption/ionization, were compared. The latter technique leads to the best results: even if unable to give specific information on heteroatom-containing components, it allows a general view to be obtained of the panorama of the oil composition in terms of molecular weight distribution. The statistical evaluation of the mass spectrometry data by multivariate techniques, such as cluster analysis (Average Linkage) and correspondence analysis, allows evidence for the differences and similarities among the crude oils under study.

Differentiation of Vitis vinifera varieties by MALDI-MS analysis of the grape seed proteins.

Until now the study of pathogenic related proteins in grape juice and wine, performed by ESI-MS, LC/ESI-MS, and MALDI/MS, has been proposed for differentiation of varieties. In fact, chitinases and thaumatin-like proteins persist through the vinification process and cause hazes and sediments in bottled wines. An additional instrument, potentially suitable for the grape varieties differentiation, has been developed by MALDI/MS for the grape seed protein analysis. The hydrosoluble protein profiles of seeds extract from three different Vitis vinifera grape (red and white) varieties were analyzed and compared. In order to evaluate the environmental conditions and harvest effects, the seed protein profiles of one grape variety from different locations and harvests were studied.

A rapid liquid chromatography electrospray ionization mass spectrometry(n) method for evaluation of synephrine in Citrus aurantium L. samples.

Immature bitter orange fruit and its extracts have been introduced into the market as an alternative to Ephedra in weight loss products. However, the safety of the immature bitter orange fruit and its extracts is a debated argument due to the presence of synephrine, a constituent known as a sympathomimetic agent. In this paper, we describe the development of a new, rapid, and simple liquid chromatography-electrospray ionization-tandem mass spectrometry method devoted to the quantitative determination of synephrine in bitter orange samples, containing a high quantity of synephrine, and sweet orange samples, known to contain a low level of synephrine but at the same time being one of the main synephrine sources in a normal human diet. Two bitter orange dry extracts containing 5 and 6% sSynephrine and 10 sweet orange samples have been analyzed. Between the sweet orange samples, six were fresh oranges and four were fresh-squeezed juices; in these samples, the synephrine levels ranged from 0.00128 to 0.00349%.

Classification of green coffee beans by differences in protein composition obtained by matrix-assisted laser desorption/ionization mass spectrometry.

It is well known that proteins and peptides play an important role in the flavour of roasted coffee, but little is reported in the literature about their characterization. In view of the potential of matrix-assisted laser desorption/ionization mass spectrometry in the analysis of proteins in complex mixtures, two varieties of coffee green beans, Arabicas and Robustas, were analyzed by this technique, in order to obtain fingerprints of their native proteins. Differences were observed between Arabicas and Robustas green beans, and cluster analysis allows differentiation of samples of the same variety from different plantations.