HPLC ESI-MSn Research Articles

Possible key intermediates in arsenic biochemistry: Synthesis and identification by liquid chromatography electrospray ionization mass spectrometry and high resolution mass spectrometry

Arsenic is a type 1 carcinogen and its toxicity is critically dependent on chemical speciation. However, after decades of research, the biogenesis of at least fifty naturally occurring arsenic species is still not well understood.Here, based on experimental work, it is proposed a set of pathways for the formation of multiple arsenic species that might help to clarify the present situation.These are focused on the thiol protein arsenic bond and on its interaction with reactive metabolites. In fact, arsenic bound to glutathione interacting with sulfur adenosyl methionine (SAM), MethylCB12 and AdoCB12, forms a number of complexes that might be key intermediates in arsenic biochemistry. These include dimethylarsino glutathione (DMAG) m/z 412 [M+H]+, synthesized non-enzymatically from glutathione and cacodylate. Trimethylarsonio glutathione (TMAG) m/z 426 [M]+ synthesized from DMA, GSH and SAM, apparently by a classical Challenger methylcarbonium attack. Tetramethyl arsonium ion m/z 135 [M]+ is formed in a third step, apparently by carbanion methylation. The presence of trimethylarsine oxide (TMAO) m/z 137 [M+H]+ is attributed to the hydrolysis of TMAG or TMA, or to carbanion methylation of dimethylarsinoyl glutathione (m/z 428 [M]+) formed from cacodylate and GSH. Cantoni type attacks of DMAG on SAM were unsuccessful, eventually due to competition of the trivalent S+ atom of SAM for the AsIII atom attack. The presence of dimethylarsonio diglutathione (DMADG m/z 717 [M]+), is suggested to result from a GS- attack on dimethylarsenoyl glutathione (m/z 428 [M+H]+). The presence of dimethylarsenoyladenosine (m/z 372 [M+H]+), trimethylarsenosugar adenine (m/z 370 [M]+), and dimethylthioarsenosugar adenine (m/z 388 [M+H]+), is explained by the synthesis of the pecursor dimethylarsonio-adenosine glutathione DMAAG (m/z 661 [M]+), a likely source of oxo-and trimethylated arsenosugars, as well as of thio-arsenosugars by the cleavage of its S-C bond. The results gathered suggest that cell vacuoles might play a major role in arsenic metabolism, and that the dominance of oxo-As sugars, in algae extracts, may be supported by a mechanism of synthesis independent of DMAAG (m/z 661).They also offer an explanation for the reason why arsenobetaine, and tetramethylarsonium are loosely bound to biotic tissues. Four arsenic species new to science, to the best of our knowledge, and a number of known arsenic compounds were synthesized in this work, identified by HPLC–ESI-MSn and FTICR–ESI-MS, and suggestions regarding their mechanisms of synthesis were advanced. These results provide a framework for arsenic biochemistry which may explain the origin of a significant part of arsenic known metabolites.

Quantitative analysis of triterpenoids in different parts of Ilex hainanensis, Ilex stewardii and Ilex pubescens using HPLC–ELSD and HPLC–MSn and antibacterial activity

An HPLC–ELSD method was first developed for the quantitative analysis of principle triterpenoids in Ilex hainanensis, Ilex stewardii and Ilex pubescens. The established method, with excellent precision, repeatability and recovery, was successfully applied to determine four triterpenoids in 24 samples from different species and medicinal parts of Ilex, and the changing trend was discussed. HPLC–ESI–MSn was used for the identification of constituents in samples. The proposed method was simple, effective and suitable for investigations of these plants. Furthermore, the ethanol extracts from leaves of Ilex species, as well as their main components, were assessed for their antibacterial activities. The results indicated that the extracts of I. hainanensis, I. stewardii and I. pubescens could inhibit the growth of the tested oral pathogens, Streptococcus mutans and Actinomyces viscosu. Particularly, ilexgenin A was the most effective with MIC values of 7.8 and ⩽3.9μg/ml, respectively.

Metabolite fingerprint of “capim dourado” (Syngonanthus nitens), a basis of Brazilian handcrafts

Syngonanthus nitens is a grass-like species, whose flower stems assume a beautiful golden colour when dried, hence their common name “capim dourado”. This plant represents an important source of income for rural communities, especially in Jalapão region, being the scapes used in the craft industry to make, together with buriti palm strips, traditional handcrafts from their coils. Therefore, considering that scapes and not flowerheads are used, we carried out an analytical study to define the metabolite fingerprint of both S. nitens parts, with the aim, on one hand, to identify the molecules responsible for the characteristic golden colour of the capim dourado herb and, on the other hand, to identify the occurrence of interesting constituents in S. nitens flowerheads to increase the value of this part of the plant, considered a waste matter of golden grass handcrafts. Therefore an HPLC–ESI-MSn method, based on high-performance liquid chromatography coupled to electrospray negative ionization multistage ion trap mass spectrometry, was developed to rapidly identify and guide the isolation of the secondary metabolites occurring in flowers and scapes. On the basis of the on-line HPLC–ESI-MSn screening, 17 compounds, including 6 new molecules, were isolated, and their structures were unambiguously elucidated by NMR spectroscopic data.

In vitro absorption and antiproliferative activities of monomeric and polymeric anthocyanin fractions from açai fruit (Euterpe oleracea Mart.)

Abstract Anthocyanins are among the most important and widely consumed natural pigments in foods, and have attracted increased attention as natural food colourants and potent bioactive agents. However, anthocyanins are generally unstable and may undergo chemical changes that include oxidative and polymerisation reactions during processing and storage. The role of anthocyanin polymerisation reactions on in vitro intestinal absorption and anti-cancer properties has not been assessed. This study investigated the chemical composition, antioxidant properties, antiproliferative activity, and in vitro absorption of monomeric and polymeric anthocyanin fractions from acai fruit (Euterpe oleracea Mart.). Cyanidin-3-rutinoside (58.5 ± 4.6%) and cyanidin-3-glucoside (41.5 ± 1.1%) were the predominant compounds found in monomeric fractions, while a mixture of anthocyanin adducts were found in polymeric fractions and characterised using HPLC–ESI-MSn analyses. Monomeric fractions (0.5–100 μg cyanidin-3-glucoside equivalents/ml) inhibited HT-29 colon cancer cell proliferation by up to 95.2% while polymeric anthocyanin fractions (0.5–100 μg cyanidin-3-glucoside equivalents/ml) induced up to 92.3% inhibition. In vitro absorption trials using Caco-2 intestinal cell monolayers demonstrated that cyanidin-3-glucoside and cyanidin-3-rutinoside were similarly transported from the apical to the basolateral side of the cell monolayers (0.5–4.9% efficiency), while no polymeric anthocyanins were transported following incubation for up to 2 h. The addition of polymeric anthocyanin fractions also decreased monomeric anthocyanin transport by up to 40.3 ± 2.8%. Results from this study provide novel information regarding the relative size, absorption, and bioactive properties of anthocyanin monomers and polymer adducts.

Simultaneous Determination of Six Steroidal Alkaloids of Veratrum dahuricum by HPLC–ELSD and HPLC–MSn

A high performance liquid chromatography coupled with evaporative light scattering detection (HPLC–ELSD) and electrospray ionization multistage mass spectrometry (HPLC–ESI–MSn), respectively, has been performed for the simultaneous determination of six steroidal alkaloids, including pseudojervine, veratrosine, jervine, veratramine, 3-veratroylzygadenine, 3-angeloylzygadenine, in Veratrum dahuricum collected in different seasons. The plants were soaked in methanol and extracted ultrasonically. The six steroidal alkaloids were obtained by silica gel column chromatography, eluting with gradient petroleum and acetyl acetate. The intra-day and inter-day precisions of the method were evaluated and were less than 1.4%. The content of steroidal alkaloids in the plant varied significantly from spring to autumn, confirming the necessity to control the quality of V. dahuricum during its preparation and application.

Rapid structural determination of new trace cassaine‐type diterpenoid amides in fractions from Erythrophleum fordii by liquid chromatography‐diode‐array detection/electrospray ionization tandem mass spectrometry and liquid chromatography/nuclear magnetic resonance

Electrospray ionization multi-stage tandem mass spectrometry (ESI-MSn), and combination with HPLC (HPLC/ESI-MSn), have been extensively applied to on-line analysis of natural products. Hyphenation of liquid chromatography to nuclear magnetic resonance spectroscopy (HPLC/NMR) has been developed in the last decade, which is utilized for the analysis of metabolites and drug impurities. In the study reported here, the fragmentation behaviors of eight cassaine-type diterpenoid amides from Erythrophleum fordii were investigated by ESI-MSn. The fragmentation rules and NMR spectral characteristics are summarized, and the relationship among the rules, characteristics and the structures is described. According to the fragmentation rules and NMR spectral characteristics, seven trace constituents and two formerly obtained compounds of cassaine-type diterpenoid amides in the fractions from E. fordii were structurally characterized on the basis of HPLC/HRMS, HPLC-DAD/ESI-MSn, HPLC/1H NMR and HPLC/1H-1H COSY rapidly. Among them, constituents 1-5 are new compounds, and 6 and 7 are reported from E. fordii for the first time. The aim is to develop an effective analytical method for structural identification of new trace natural products in plants.

Isolation and identification of 5-carboxy-pyranoanthocyanins in beverages from cherry (Prunus cerasus L.)

Abstract Novel pyranoanthocyanins were identified by HPLC–ESI-MSn in fermented and unfermented fruit juices from cherry (Prunus cerasus L.). The two major compounds, namely 5-carboxy-pyranocyanidin 3-O-(2G-glucosylrutinoside) and 5-carboxy-pyranocyanidin 3-O-rutinoside were isolated by a combination of high-speed countercurrent chromatography with semi-preparative HPLC. Their structures were fully elucidated by means of NMR spectroscopy and mass spectrometry. As minor compounds 5-carboxy-pyranocyanidin 3-glucoside, 5-carboxy-pyranopeonidin-3-glucoside, and 5-carboxy-pyranopeonidin 3-rutinoside could be identified. These compounds are formed during storage of the juice through the reaction of pyruvic acid with the respective genuine anthocyanins. Industrial relevance For the consumers the color of fruit juices is one of the most important quality markers. However due to a fast degradation of anthocyanins, changes in color from purple/red hues to orange/brown hues can be observed during storage. The main objective of this study was the isolation, structural characterization and evaluation of pigments that are formed during aging of fruit juices. Pyranoanthocyanins were reported to possess a color stabilizing effect. In the present study it could be demonstrated that the color contribution of these newly formed pigments to the overall color of fruit juice was overestimated. Instead, the formation of polymeric pigments yields the highest impact (> 80%) on the color of aged fruit juices.

Studies on the flavones using liquid chromatography–electrospray ionization tandem mass spectrometry

Fragmentation pathways of nine flavone compounds have been studied by using electrospray ionization multi-stage tandem mass spectrometry (ESI-MSn). Analyzing the product ion spectra of flavonoids and aglycones, we observed some diagnostic neutral losses, such as CH3, H2O, residue of glucose and gluconic acid, which are very useful for the identification of the functional groups in the structures. Furthermore, specific retro Diels-Alder (RDA) fragments for flavones with different hydroxyl substitution have also been discussed. The information is helpful for the rapid identification of the location site of hydroxyl substitution on flavones. Fragmentation pathways of C-glycosidic flavonoid have also been discussed using ESI-MSn, demonstrating ions [MH-60]−, [MH-90]−, [MH-120]− are characteristic ions of C-glycosidic flavonoid. According to the fragmentation mechanism of mass spectrometry and HPLC–MS data, the structures of seven flavones in Scutellaria baicalensis Georgi have been identified on-line without time-consuming isolation. The HPLC–ESI-MSn method for analyzing constituents in the Scutellaria baicalensis Georgi has been established.

Acknowledgment of editorial board members and guest reviewers

Avocado (Persea americana) is an important tropical fruit, but little is known about their antioxidant capacities and phytochemical composition. The objective of this research was to determine antioxidant capacities, total phenolic content and identify and quantify major antioxidant compounds in avocados of different strains and cultivars. Ripe Florida avocados from seven cultivars (Slimcado, Booth 7, Booth 8, Choquette, Loretta, Simmonds, and Tonnage) of West Indian or Guatemalan strains were separated into seeds, peels and pulp, and freeze dried. Hass avocado of Mexican strain was chosen for comparison. Samples were extracted with acetone/water/acetic acid solvent and analysed using the Folin–Ciocalteu assay for total phenolic content. Antioxidant capacities were determined by oxygen radical absorbance capacity (ORAC) and DPPH assays. Procyanidins were identified and quantified using HPLC-MSn. Antioxidant pigments (chlorophylls and carotenoids) were estimated spectrophotometrically. For all varieties, seeds contained the highest antioxidant capacities, phenolic content, and procyanidins, whereas the pulp had the lowest. Procyanidins, including catechin, epicatechin, A- and B-type dimers, A- and B-type trimers, tetramers, pentamers and hexamers, were identified in peels and seeds using normal-phase HPLC–ESI-MSn. Antioxidant capacities, phenolic contents and procyanidins in avocados were highly correlated, suggesting that procyanidins were the major phenolic compounds that contributed to antioxidant capacities. Carotenoids and chlorophylls were found to be concentrated in avocado peels but did not correlate with antioxidant capacities. This study suggested that avocado seeds and peels, industrial wastes of avocado processing, can be exploited as source of antioxidants.