Metabolic profiling of roots of liquorice (Glycyrrhiza glabra) from different geographical areas by ESI/MS/MS and determination of major metabolites by LC-ESI/MS and LC-ESI/MS/MS

Liquid chromatography electrospray mass spectrometry (LC-ESI/MS) has been applied to the full characterization of saponins and phenolics in hydroalcoholic extracts of roots of liquorice (Glycyrrhiza glabra). Relative quantitative analyses of the samples with respect to the phenolic constituents and to a group of saponins related to glycyrrhizic acid were performed using LC-ESI/MS. For the saponin constituents, full scan LC-MS/MS fragmentation of the protonated (positive ion mode) or deprotonated (negative ion mode) molecular species generated diagnostic fragment ions that provided information concerning the triterpene skeleton and the number and nature of the substituents. On the basis of the specific fragmentation of glycyrrhizic acid, an LC-MS/MS method was developed in order to quantify the analyte in the liquorice root samples. Chinese G. glabra roots contained the highest levels of glycyrrhizic acid, followed by those from Italy (Calabria).     

Determination of phenolic compounds in Yucca gloriosa bark and root by LC-MS/MS

On the basis of the biological activities shown by yuccaols and gloriosaols from Yucca schidigera and Yucca gloriosa, the content of yuccaols and gloriosaols in two different parts of Y. gloriosa (roots and bark), was determined for each single compound, and compared with phenolic determination in Y. schidigera bark, concluding that Y. gloriosa bark and roots are rich sources of phenolic derivatives structurally related to resveratrol. LC/ESIMS (liquid chromatography coupled to electrospray mass spectrometry) qualitative and an LC/ESIMS/MS (liquid chromatography coupled to tandem electrospray mass spectrometry) quantitative studies of the phenolic fraction of Y. gloriosa were performed. LC/ESIMS/MS multiple reaction monitoring (MRM) method previously described for yuccaols in Y. schidigera was applied and optimised for separation and determination of gloriosaols and yuccaols in Y. gloriosa. Due to the sensitivity and the repeatability of the assay, we suggest this method as suitable for industrial quality control of raw materials and final products.     

Determination of steroidal glycosides in Yucca gloriosa flowers by LC/MS/MS

An high-performance liquid chromatography (HPLC) coupled with tandem mass spectrometry (MS/MS) method, was developed for the quantitative analysis of the steroidal glycosides occurring in Yucca gloriosa flowers. The HPLC experiments were performed by means of an octadecyl-modified reversed-phase C-18 column and a binary mobile phase system under gradient elution conditions. The fragmentation patterns of steroidal saponins were analyzed by ESI–MSⁿ in positive ion mode and a specific multiple reaction monitoring MS/MS detection was developed for their quantitative determination. The described method provides high sensitivity and specificity for quantitative determination of the steroidal glycosides in Y. gloriosa flowers. Quantification was performed against an external calibration line obtained using each pure steroidal glycoside. Short- and long-term repeatabilities of the methods were better than 3 and 6%, respectively. The method was validated according to EMEA guidelines and applied to real samples.     

Mutagenic activity promoted by amentoflavone and methanolic extract of Byrsonima crassa Niedenzu

Byrsonima crassa is a plant pertaining to the Brazilian central savannah-like belt of vegetation and popularly used for the treatment of gastric dysfunctions and diarrhoea. The methanol extract contains catechin, tannins, terpenes and flavonoids; both mutagenic potential and antioxidant properties have been ascribed to flavonoids. The mutagenicity of some flavonoids is believed to be associated with the formation of reactive oxygen species and seems to depend on the number and position of hydroxyl groups. In the present study the mutagenic activity of the methanol, chloroform and 80% aqueous methanol extracts, as well as acetate and aqueous sub-fractions, of this medicinal plant were evaluated by Salmonella typhimurium assay, using strains TA100, TA98, TA102 and TA97a, and in mouse reticulocytes. The results showed mutagenic activity of the methanolic extract in the TA98 strain without S9, but no mutagenicity to mouse cells in any of the extracts. The acetate fraction showed strong signs of mutagenicity without S9, suggesting that in this enriched fraction were concentrated the compounds that induced mutagenic activity. The aqueous fraction showed no mutagenic activity. The TLC and HSCCC analyses of the acetate fraction with some standard compounds permitted the isolation of the quercetin-3-O-beta-D-galactopyranoside, quercetin-3-O-alpha-L-arabinopyranoside, amentoflavone, methyl gallate and (+)-catechin, of which only the amentoflavone exhibited positive mutagenicity to TA98 (+S9, -S9).     

Phenolic composition and biological activities of Juniperus drupacea Labill. berries from Turkey

The present study was designed to define the phenolic profile and the biological potential of berries methanol extract of Juniperus drupacea Labill. from Turkey.The total phenolic content (Folin-Ciocalteau assay) was 48.06 ± 0.99 mg GAE/g extract. The HPLC-DAD-ESI-MS analysis allowed the determination of the complete phenolic profile of J. drupacea berries. Phenolic acids represented more than 60% of the total phenolics, and tyrosol was the major one (1324 ± 0.64 μg/g extract); within the flavonoids amentoflavone was detected as the main constituent (927 ± 0.35 μg/g extract).The extract exhibited good antioxidant properties, as determined by different in vitro models: DPPH test (IC₅₀ 0.38 ± 0.02 mg/mL), reducing power (12.63 ± 0.14 ASE/mL), Fe²⁺ chelating ability (IC₅₀ 2.26 ± 0.06 mg/mL), and TBA test (IC₅₀ 2.47 ± 1.13 μg/mL).Cytotoxicity against Artemia salina was highlighted (LC₅₀ 489.47 ± 27.8 μg/mL), and a significant decrease (p ? 0.05; p ? 0.01) in HepG2 cells viability was observed at the higher concentrations (5-10 μg/mL).The extract displayed good antibacterial activity towards Gram-positive bacteria and in particular Staphylococcus aureus was the most susceptible strain (MIC 78.12 μg/mL).     

Simultaneous analysis of flavonoids from Hypericum japonicum Thunb.ex Murray (Hypericaceae) by HPLC-DAD-ESI/MS

A novel and sensitive HPLC-UV method has been developed for the simultaneous determination of five major flavonoids in Hypericum japonicum hydroalcoholic extract. The chemical profile of five flavonoids, including taxfolin-7-O-alpha-L-rhamnoside (1), isoquercitrin (2), quercitrin (3), quercetin-7-O-alpha-L-rhamnoside (4) and quercetin (5) was acquired by using high-performance liquid chromatography-diode array detector coupled to an electrospray tandem mass spectrometer (HPLC-DAD-ESI/MS). The analysis was performed on a ZORBAX SB-C18 analytical column (5 microm, 250 mm x 4.6 mm, i.d.) with a gradient solvent system of acetonitrile-0.5% aqueous formic acid. The validation was carried out and the linearities (r(2)>0.9997) and recoveries (ranged from 98.4% to 99.8%) were acceptable. The limits of detection (LOD) of these flavonoids ranged from 0.5 to 7.5 ng. The results indicated that the contents of investigated flavonoids in H. japonicum varied significantly from habitat to habitat with contents ranging from 2.00 to 34.18 mg/g. The proposed method is simple, effective and suitable for the quality control of this traditional Chinese medicine (TCM).     

Phenolic compounds, organic acids profiles and antioxidative properties of beefsteak fungus (Fistulina hepatica).

The phenolic compounds and the organic acids composition of the edible beefsteak fungus Fistulina hepatica was determined by HPLC/DAD and HPLC/UV, respectively. The results showed a profile composed by five phenolic compounds (caffeic, p-coumaric and ellagic acids, hyperoside and quercetin) and six organic acids (oxalic, aconitic, citric, malic, ascorbic and fumaric acids). The quantification of the identified compounds revealed that ellagic acid (ca. 49.7%) and malic acid (ca. 57.9%) are the main compounds in this species. In a general way the phenolic profile revealed to be more constant than the organic acids one and could be more useful for the quality control of the species. Beefsteak fungus was also investigated for its capacity to act as a scavenger of DPPH() radical and reactive oxygen species (superoxide radical, hydroxyl radical and hypochlorous acid). Good results were obtained against DPPH in a concentration-dependent manner. Beefsteak fungus also displayed good activity against superoxide radical, achieved by its capacity to act as both scavenger and xanthine oxidase inhibitor. A prooxidant effect was noticed for hydroxyl radical, which may be due to its capacity for iron ions reduction. Little ability for iron chelation was also observed. Beefsteak fungus showed a weak protective effect against hypochlorous acid.     

UPLC-MS/MS同时测定不同产地石仙桃中30种酚类成分的含量

目的 建立超高效液相色谱串联质谱(UPLC-MS/MS)同时测定石仙桃中30种酚类成分含量的方法,并对不同产地的石仙桃药材进行质量评价.方法 采用Welch Xtimate UHPLC C18(2.1 mm×100 mm,1.8μm)色谱柱,以0.1％甲酸水溶液-乙腈为流动相进行梯度洗脱,流速为0.2 mL·min-1...     

Screening method for stimulants in urine by UHPLC‐MS/MS: identification of isomeric compounds

A fast screening method for the detection of more than 60 stimulants in urine was developed. The method consisted of a dilution of the urine (1:5 v/v) and analysis by ultra high performance liquid chromatography coupled to tandem mass spectrometry, using a C18 column (1.7 µm particle size), a mobile phase containing deionized water and acetonitrile with formic acid, and gradient elution. The chromatographic run time was 5 min. The detection was performed in positive mode electrospray ionization, monitoring one or two specific ion transitions for each analyte. Appropriate repeatability was obtained, with relative standard deviation (RSD) values below 25% for most of the analytes. Regarding intermediate precision, estimated during routine work, higher RSDs were obtained, probably due to between‐day differences in the status of the mass spectrometer and in the chromatographic system. Matrix effect ranged from 60 to 255% with RSD lower than 35% for the majority of compounds. Despite the matrix effect observed, the signal/noise ratio of the analytes spiked at 50 ng/mL was greater than three in all tested samples, allowing a correct detection of all substances at the minimum required performance levels required by the World Anti‐Doping Agency and demonstrating the suitability of the method. The method was tested in administration study samples and satisfactorily in operation for more than one year with routine doping samples. The presence of isomeric stimulants with closely similar chromatographic and/or mass spectrometric properties did not allow the unequivocal identification of these compounds after the first analysis. Different possibilities for separation and identification of isomeric compounds are presented. Copyright © 2015 John Wiley & Sons, Ltd.     

Qualitative and quantitative analysis of steroidal saponins in crude extracts from Paris polyphylla var. yunnanensis and P. polyphylla var. chinensis by high performance liquid chromatography coupled with mass spectrometry

High performance liquid chromatography coupled with electrospray ionization multi-stage tandem mass spectrometry (HPLC–ESI-MSⁿ) and triple quadrupole mass spectrometric detection (HPLC–ESI-MS/MS), respectively, had been employed for the simultaneous identification and quantification of steroidal saponins in the rhizomes of Parispolyphylla var. yunnanensis and P. polyphylla var. chinensis, which are the qualified plants of “Chonglou” in Chinese. The HPLC experiments were performed by means of a reversed-phase C-18 column and a binary mobile phase system consisting of 0.1% aqueous formic acid and acetonitrile under gradient elution conditions. The characteristic fragmentation patterns of diosgenin- and pennogenin-type steroidal saponins were investigated using ESI-MSⁿ in negative ion mode. The MSⁿ data of the [M−H]⁻ ions provided structural information on the sugar sequence of the oligosaccharide chains and the aglycones of steroidal saponins. As a result, ten and seven saponins were determined in P. polyphylla var. yunnanensis and P. polyphylla var. chinensis, respectively, including four unknown compounds. One unknown compound was tentatively identified as diosgenin-3-O-α-l-rhamnopyranosyl(1 → 4) [α-l-rhamnopyranosyl(1 → 2)]-β-d-glucopyranoside and the aglycones of the other three new compounds were reported from Chonglou for the first time. The developed HPLC–ESI-MS/MS method was validated and found to be satisfactorily linear, selective and robust. The limits of detection (LODs) and quantitation (LOQs) ranged, respectively, from 0.5 to 10 ng/mL and 2 to 34 ng/mL depending on six various compounds. The intra- and inter-day precisions of the method were evaluated and were less than 5.0%. Recoveries ranged from 92% to 104% for all compounds. The established quality evaluation method was successfully used for simultaneous quantification of six predominant steroidal saponins in the rhizomes of these two Paris species.     

Determination of pinostrobin in rat plasma by LC-MS/MS: application to pharmacokinetics

A rapid and sensitive method for the determination of pinostrobin in rat plasma was developed using liquid chromatography tandem mass spectrometry (LC-MS/MS) for the first time. Isoliquiritigenin was used as an internal standard in rat plasma. Chromatographic separation was performed on an HiQ Sil C₁₈ column with isocratic elution at a flow rate of 1 mL/min. The mobile phase consisted of water and methanol (9:91, v/v) containing 0.1% formic acid. The quantification limit was 10 ng/mL within a linear range of 10-1000 ng/mL (R = 0.9984). The intra- and inter-day assay precision ranged from 3.8-5.3% to 3.2-5.2%, respectively, and the intra- and inter-day assay accuracy was between 93.2-95.1% and 95.5-104.3%, respectively. Our results indicated that the LC-MS/MS method is effective for pharmacokinetic study of pinostrobin in rat plasma.     

Characterization of bioactive compounds from raw and ripe Mangifera indica L. peel extracts

Mango is one of the important tropical fruits in the world. As it is a seasonal fruit, it is processed for various products. During its processing, peel is one of the major byproducts, which is being wasted. Bioactive conserves were extracted using 80% acetone from peels of raw and ripe mango fruits and subjected to acid hydrolysis. The prominent phenolic compounds identified by HPLC were protocatechuic acid, gentisic acid and gallic acid. The phenolic acid derivatives present in acetone extracts of raw and ripe peels were tentatively identified by LC–MS. Gallic acid, syringic acid, mangiferin, ellagic acid, gentisyl-protocatechuic acid, quercetin were the phenolic compounds identified in both raw and ripe peels, while raw peel showed the presence of glycosylated iriflophenone and maclurin derivatives also. β-Carotene was the major carotenoid followed by violaxanthin and lutein. Thus, both raw and ripe mango peel extracts have different phenolic compounds and carotenoids, which will have various pharmaceutical applications.     

Determination of phenolic compounds in Prunella L. by liquid chromatography-diode array detection

Four species of Prunella L. (Prunella vulgaris L., Prunella laciniata L., Prunella grandiflora L. and Prunella orientalis Bornm.) belong to the family of Lamiaceae and representing popular Western and Chinese herbal medicine were examined for the content of phenolic compounds. Phenolic acids (rosmarinic acid, caffeic acid, ferulic acid, chlorogenic acid, protocatechuic acid), flavonoids (rutin, quercetin) in different quantitative proportions depending on extracts were determined by the rapid, selective and accurate method combining solvent/acid hydrolysis extraction and high performance liquid chromatography-diode array detection (HPLC-DAD). Water, methanol, butanol, acetonitrile, ethyl acetate, hexane and their acidic solutions were used to examine the efficiency of different solvent systems for the extraction of phenolic compounds. Acid hydrolysis extraction was established as the most suitable extraction method for phenolic compounds.     

Moving pieces in a taxonomic puzzle: venom 2D-LC/MS and data clustering analyses to infer phylogenetic relationships in some scorpions from the Buthidae family (Scorpiones).

The Buthidae is the most clinically important scorpion family, with over 500 species distributed worldwide. Taxonomical positions and phylogenetic relationships concerning the representative genera and species of this family have been mostly inferred based upon comparisons between morphological characters. Yet, some authors have performed such inferences by comparing some structural properties of a few selected molecules found in the venoms from these scorpions. Here, we propose a novel methodology pipeline designed to address these issues. We have analyzed the whole venoms from some species that exemplify peculiar cases in the Buthidae family (Tityus stigmurus, Tityus serrulatus, Tityus bahiensis, Leiurus quinquestriatus quinquestriatus and Leiurus quinquestriatus hebraeus), by means of a proteomic approach using a 2D-LC/MS technique. The molecules found in these venoms were clustered according to their physicochemical properties (molecular mass and hydrophobicity), by using the machine learning-based Weka software. The clusters assessment, along with the number of molecules found in a given cluster for each scorpion, which assigns for the venom and structural family complexities, respectively, was used to generate a phenetic correlation tree for positioning these species. Our results were in accordance with the classical taxonomy viewpoint, which places T. serrulatus and T. stigmurus as very close species, T. bahiensis as a less related species in the Tityus genus and L. q. quinquestriatus and L. q. hebraeus with small differences within the same species (L. quinquestriatus). Therefore, we believe that this is a well-suited method to determine venom complexities that reflect the scorpions' evolutionary history, which can be crucial to reconstruct their phylogeny through the molecular evolution of their venoms.     

Determination of phenolic compounds in fennel by HPLC and HPLC-MS using a monolithic reversed-phase column

A reversed-phase high-performance liquid chromatography (HPLC) method for analyzing phenolic compounds in fennel (Foeniculum vulgare) has been developed. The use of a monolithic column with short dimensions in combination with optimized chromatographic conditions allows over 100 samples per day to be analyzed. Chromatographic parameters such as column temperature and injection volume, were found to be crucial in obtaining adequate selectivity and resolution, consequently allowing short run times. The method was validated for the major phenolic compounds present in fennel plant material: 3-O-caffeoylquinic acid (3-CQA), chlorogenic acid, 4-O-caffeoylquinic acid (4-CQA), eriocitrin, rutin, miquelianin, 1,3-O-dicaffeoylquinic acid (1,3-diCQA), 1,5-O-dicaffeoylquinic acid (1,5-diCQA), 1,4-O-dicaffeoylquinic acid (1,4-diCQA) and rosmarinic acid. The limits of detection (LOD) and the limits of quantitation (LOQ) ranged from 0.05 to 1.0 microg/mL and from 0.15 to 2.5 microg/mL, respectively. With some adaptation, the extraction procedure could be even less invasive, which is useful in screening work.     

LC-MS/MS as a tool for identification of bioactive compounds in marine sponge Spongosorites halichondriodes (Dendy 1905)

The sensitivity, specificity and selectivity of liquid chromatography/mass spectrometry tandem mass spectrometry (LC-MS/MS) make it an essential tool for the characterization and identification of low molecular compounds such as fatty acids, sterols, cholastane derivatives, nucleosides etc. In the current work, the marine sponge Spongosorites halichondriodes (order Halichondrida, Family Halichondriidae); a particularly rich source of cytotoxic compounds is studied for the initial characterization of bioactive compounds. The composition of ethyl acetate and butanol extracts were subjected to LC-MS and LC-MS/MS. Many novel sterol derivatives compounds which were not reported in any marine sponge mainly belonged to the group of C₂₅-C₂₈ saturated and unsaturated esters like 3β, 4β, 7α, 12α-tetrahydroxy-5β-cholan-24-oic acid methyl ester, 7 α, 12 β-dihydroxy-5 β-cholan24-oic acid methyl ester, novel isocoumarin citrinolactone A, a triterpenoid glycyrrhetinic acid as well as other unknown compounds in this species such as nucleoside inosine was identified. Other compound investigated was 3β, 6β, 7α-trihydroxy-5β-cholan-24-oic acid methyl ester. All the sterol ester derivatives are reported here for the first time in marine sponge belonging to family Halichondriidae. However, the literature report supports the occurrence of 3β-hydroxy sterols which is considered as a biomarker for this family.     

Advanced glycation end-products/peptides: a preliminary investigation by LC and LC/MS

An investigation on AGE-peptides, originating by proteolysis of in vitro glycated proteins, was carried out by LC methods with different detection applied to the mixture produced by proteinase K digestion of in vitro glycated human serum albumin (HSA). Classical approaches, like spectroscopic (UV, fluorescence) and mass spectrometric methods (MALDI, LC/ESI/MS), show that the digestion mixture is highly complex. However, there are clearcut differences between the digestion mixtures of glycated and unglycated HSA, in the former case allowing identification of possible glycated peptides belonging to the AGE-peptide class. MS/ MS experiments on selected species seem to be promising as regards structural information.     

Metabolic profiling and biological capacity of Pieris brassicae fed with kale (Brassica oleracea L. var. acephala)

Phenolic and organic acid profiles of aqueous extracts from Pieris brassicae material and the host kale (Brassica oleracea L. var. acephala) leaves were determined by HPLC/UV–DAD/MSⁿ-ESI and HPLC–UV, respectively. The identified phenolics included acylated and nonacylated flavonoid glycosides, hydroxycinnamic acyl gentiobiosides, and sulphate phenolics. Kale exhibited the highest content (11 g/kg lyophilized extract), while no phenolics were identified in the butterflies or exuviae. Nine different organic acids were characterized in the materials, with kale showing the highest amount (112 g/kg lyophilized extract). With the exception of the exuviae extract, the rest were screened for bioactivity. Using spectrophotometric microassays, all exhibited antiradical capacity against DPPH and NO in a concentration-dependent way, whereas only kale and excrement extracts were active against superoxide. All displayed activity on intestinal smooth muscle, albeit with distinct relaxation–contraction profiles. Larvae and butterfly extracts were more efficacious for intestinal relaxation than was kale extract, whereas excrement extract evoked only contractions, thus evidencing their different compositions. Collectively, these results show that P. brassicae sequesters and metabolizes kale’s phenolic compounds. Moreover, the extract’s bioactivities suggest that they may constitute an interesting source of bioactive compounds whose complex chemical structures preclude either synthesis or isolation.     

Ficus carica L.: Metabolic and biological screening

Ficus carica L. is one of the earliest cultivated fruit trees. In this work, metabolite profiling was performed on the leaves, pulps and peels of two Portuguese white varieties of F. carica (Pingo de Mel and Branca Tradicional). Phenolics and organic acids profiles were determined by HPLC/DAD and HPLC/UV, respectively. All samples presented a similar phenolic profile composed by 3-O- and 5-O-caffeoylquinic acids, ferulic acid, quercetin-3-O-glucoside, quercetin-3-O-rutinoside, psoralen and bergapten. 3-O-Caffeoylquinic acid and quercetin-3-O-glucoside are described for the first time in this species. Leaves’ organic acids profile presented oxalic, citric, malic, quinic, shikimic and fumaric acids, while in pulps and peels quinic acid was absent. The antioxidant potential of the different plant parts was checked. All materials exhibited activity against DPPH and nitric oxide radicals in a concentration-dependent way. However, only the leaves presented capacity to scavenge superoxide radical. Leaves were always the most effective part, which seems to be related with phenolics compounds. Additionally, acetylcholinesterase inhibitory capacity was evaluated, but no effect was observed. Antimicrobial potential was also assessed against several bacterial species, although no activity was noticed. This is the first study comparing the chemical composition and biological potential of F. carica pulps, peels and leaves.     

Qualitative and quantitative determination of ten alkaloids in traditional Chinese medicine Corydalis yanhusuo W.T. Wang by LC-MS/MS and LC-DAD

An analytical method incorporating high-performance liquid chromatography (HPLC) with MS and UV-detection was developed for the qualitative and quantitative determination of alkaloids in Corydalis yanhusuo. Ten alkaloids, including seven tertiary alkaloids and three quaternary alkaloids, were identified by comparing their retention times, UV and MS spectra with those of authentic compounds. Furthermore, the collision-induced dissociations of the [M+H](+) and [M](+) ions were studied to clarify the MS behavior of the different types of alkaloids. In positive ion electrospray ionization tandem mass spectrometry (ESI-MS) all the tertiary alkaloids yielded prominent [M+H](+) ions and quaternary alkaloids yielded prominent [M](+) ions in the first order mass spectra. Fragments involving losses of H, CH(3), CO, H(2)O and OCH(3) were observed in the MS/MS spectra. In addition, quantification of the 10 alkaloids in Corydalis yanhusuo from methanol and ethyl acetate extract of different origins were performed by this method, which provides a new tool for the assessment of quality of Corydalis yanhusuo preparations. The method provides the best sensitivity and specificity for characterization and quantitative determination of the alkaloids in Corydalis yanhusuo so far.