Quinovic acid glycosides from Uncaria guianensis.

From the bark of Uncaria guianensis, two new quinovic acid glycosides, quinovic acid 3 beta-O-beta-D-quinovopyranoside and quinovic acid 3 beta-O-beta-D-fucopyranosyl-(27----1)-beta-D-glucopyranosylester, have been isolated, in addition to known quinovic acid 3 beta-O-[beta-D-glucopyranosyl-(1----3)-beta-D-fucopyranosyl]-(27----1)- beta-D-glucopyranosylester and quinovic acid 3 beta-O-beta-D-fucopyranoside. Their structures were elucidated by spectral and chemical studies.     

Profile of native N‐linked glycan structures from human serum using high performance liquid chromatography on a microfluidic chip and time‐of‐flight mass spectrometry

Protein glycosylation involves the addition of monosaccharides in a stepwise process requiring no glycan template. Therefore, identifying the numerous glycoforms, including isomers, can help elucidate the biological function(s) of particular glycans. A method to assess the diversity of the N‐linked oligosaccharides released from human serum without derivatization has been developed using on‐line nanoLC and high resolution TOF MS. The N‐linked oligosaccharides were analyzed with MALDI FT‐ICR MS and microchip LC MS (HPLC–Chip/TOF MS). Two microfluidic chips were employed, the glycan chip (40 nL enrichment column, 43×0.075 mm² i.d. analytical column) and the high capacity chip (160 nL enrichment column, 140×0.075 mm² i.d. analytical column), both with graphitized carbon as the stationary phase. Both chips offered good sensitivity and reproducibility in separating a heterogeneous mixture of neutral and anionic oligosaccharides between injections. Increasing the length and volume of the enrichment and the analytical columns improved resolution of the peaks. Complex type N‐linked oligosaccharides were the most abundant oligosaccharides in human serum accounting for ∼96% of the total glycans identified, while hybrid and high mannose type oligosaccharides comprise the remaining ∼4%.     

27-Nor-triterpenoid glycosides from Mitragyna inermis

From the bark of Mitragyna inermis, two 27-nor-triterpenoid glycosides, named inermiside I (1) and II (2), were isolated and their structures determined based on extensive 2D-NMR and MS spectral analysis as 6-deoxy-beta-D-glucopyranosyl-[3-O-beta-D-glucopyranosyl-(1-->6)-beta-D-glucopyranosyl]-pyrocincholate and 6-deoxy-beta-D-glucopyranosyl-pyrocincholate, respectively. In addition, the known quinovic acid (6), 3-O-[beta-D-glucopyranosyl-(1-->4)-alpha-L-rhamnopyranosyl]-quinovoic acid (3),beta-D-glucopyranosyl-[3-O-(beta-D-glucopyranosyl)]-quinoviate (4) and cytotoxic 3-O-(beta-D-6-deoxy-glucopyranosyl)-quinovic acid (5) were also isolated.     

Separation of Organoarsenicals by Means of Zwitterionic Hydrophilic Interaction Chromatography (ZIC®‐HILIC) and Parallel ICP‐MS/ESI‐MS Detection

An analytical scheme was developed for the separation and detection of organoarsenicals using a zwitterionic stationary phase of hydrophilic interaction chromatography (ZIC^®‐HILIC) coupled in parallel to electrospray ionization mass spectrometry (ESI‐MS) and to inductively coupled plasma mass spectroscopy (ICP‐MS). The optimization of separation and detection for organoarsenicals was mainly focused on the influence of the percentage of acetonitrile (MeCN) used as a major component of the mobile phase. Isocratic and gradient elution was applied by varying the MeCN percentage from 78 % to 70 % MeCN and 22 % to 30 % of an aqueous solution of ammonium acetate (125 mM NH₄Ac; pH 8.3) on a ZIC^®‐HILIC column (150 × 2.1 mm id, 3.5 μm), to allow for the separation and successful detection of nine organoarsenicals (i.e., 3‐nitro‐4‐hydroxyphenylarsonic acid (roxarsone, Rox), phenylarsonic acid (PAA), p‐arsanilic acid (p‐ASA), phenylarsine oxide (PAO), dimethylarsinate (DMA), methylarsonate (MMA), arsenobetaine (AsB), arsenocholine (AsC) and trimethylarsine oxide (TMAO)) within 45 min. All analytes were prepared in the mobile phase. The flow rate of the mobile phase, the splitting ratio between ICP‐MS and ESI‐MS detection, and the oxygen addition were adapted to ensure that there appeared a stably burning inductively coupled plasma. Furthermore, the analytical method was evaluated by the identification and quantification of AsB in the reference material DORM‐2 (dogfish muscle) resulting in a 95‐% recovery with respect to the AsB concentration in the extract.     

Validation of a quantitative assay of arbutin using gas chromatography in Origanum majorana and Arctostaphylos uva‐ursi extracts

Introduction – Arbutin is a skin‐whitening agent that occurs naturally in the bark and leaves of various plants. It is commonly quantified in plant extracts and skin‐whitening products by HPLC. Objective – To develop an alternative gas chromatographic method for the separation and quantification of arbutin in Origanum majorana and Arctostaphylos uva‐ursi extracts. Methodology – N,O‐Bis(trimethylsilyl)acetamide and trimethylchlorosilane were used as silylation reagents, and the gas chromatographic separation of silylated extracts and standards was performed using a DB‐5 narrow bore column. GC‐MS was used for the compound identification, and the quantification was carried out by GC‐FID. The quantitative results were compared with those of HPLC analysis. Results – The developed method gave a good sensitivity with linearity in the range 0.33–500 mg/mL and recovery >98%, allowing the quantification of arbutin in O. majorana and A. uva‐ursi extracts. The relative standard deviations (RSD) relating to intra‐day and inter‐day precision were <0.002% and <4.8%, respectively. The GC results correlated well with those obtained by HPLC analysis. Conclusion – The analysis of marjoram and bearberry samples showed that the established GC method was rapid, selective, and demonstrated that arbutin could be screened alternatively by gas chromatography. Copyright © 2009 John Wiley & Sons, Ltd.     

A Comprehensive Characterisation of Rosemary tea Obtained from Rosmarinus officinalis L. Collected in a sub‐Humid Area of Tunisia

Introduction

Rosemary (Rosmarinus officinalis L.) is an aromatic plant common in Tunisia and it is widely consumed as a tea in traditional cuisine and in folk medicine to treat various illnesses. Currently, most research efforts have been focused on rosemary essential oil, alcoholic and aqueous extracts, however, little is reported on rosemary infusion composition.

Objective

To investigate compounds present in rosemary tea obtained from Rosmarinus officinalis L. collected in a sub‐humid area of Tunisia in order to assess whether the traditional rosemary tea preparation method could be considered as a reference method for rosemary's compounds extraction.

Methodology

Qualitative characterisation of Rosmarinus officinalis tea obtained after rosemary infusion in boiled water was determined by high performance liquid chromatography coupled with electrospray ionisation quadrupole time‐of‐flight mass spectrometry (HPLC‐ESI‐QTOF‐MS). Quantitative analysis relies on high performance liquid chromatography with diode array detector (HPLC‐DAD).

Results

Forty‐nine compounds belonging to six families, namely flavonoids, phenolic acids, phenolic terpenes, jasmonate, phenolic glycosides, and lignans were identified. To the best of the authors' knowledge eucommin A is characterised for the first time in rosemary. Rosmarinic acid (158.13 μg/g dried rosemary) was the main compound followed then by feruloylnepitrin (100.87 μg/g) and luteolin‐3′‐O‐(2″‐O‐acetyl)‐β‐d ‐glucuronide (44.04 μg/g). Among quantified compounds, luteolin‐7‐O‐rutinoside was the compound with the lowest concentration.

Conclusion

The infusion method allows several polyphenols present in rosemary tea to be extracted, therefore it could be a reference method for rosemary's compounds extraction. Moreover, traditional Tunisian Rosmarinus officinalis tea consumption is of interest for its rich phenolic content. Copyright © 2017 John Wiley & Sons, Ltd.     

New insights into the catalytic mechanism of human glycine N‐acyltransferase

Even though the glycine conjugation pathway was one of the first metabolic pathways to be discovered, this pathway remains very poorly characterized. The bi‐substrate kinetic parameters of a recombinant human glycine N‐acyltransferase (GLYAT, E.C. 2.3.1.13) were determined using the traditional colorimetric method and a newly developed HPLC–ESI‐MS/MS method. Previous studies analyzing the kinetic parameters of GLYAT, indicated a random Bi–Bi and/or ping‐pong mechanism. In this study, the hippuric acid concentrations produced by the GLYAT enzyme reaction were analyzed using the allosteric sigmoidal enzyme kinetic module. Analyses of the initial rate (v) against substrate concentration plots, produced a sigmoidal curve (substrate activation) when the benzoyl‐CoA concentrations was kept constant, whereas the plot with glycine concentrations kept constant, passed through a maximum (substrate inhibition). Thus, human GLYAT exhibits mechanistic kinetic cooperativity as described by the Ferdinand enzyme mechanism rather than the previously assumed Michaelis–Menten reaction mechanism.     

Identification by HPLC-PAD-MS and quantification by HPLC-PAD of phenylethanoid glycosides of five Phlomis species

The qualitative and quantitative determination of phenylethanoid glycosides in methanolic extracts of five species of the genus Phlomis (Lamiaceae) has been investigated using a new reversed-phase HPLC method combined with photodiode-array detection and electrospray/MS analysis. Forsythoside B, verbascoside, samioside, alyssonoside, isoverbascoside, leucosceptosides A and B and martynoside were detected. The quantification of the above constituents was performed using echinacoside as internal standard. Knowledge of the content of phenylethanoid glycosides contributes to the chemotaxonomy of the genus.     

Simultaneous quantitation of five flavonoid glycosides in Herba Epimedii by high-performance liquid chromatography-tandem mass spectrometry

A rapid and accurate reversed-phase liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method has been developed and validated for the quantitative determination of five flavonoid glycosides, icariin, epimedin A, epimedin B, epimedin C and hyperin in Herba Epimedii. Chromatographic separations were performed using a C(18) narrow-bore HPLC column; a mixture of an aqueous solution of ammonium formate (pH 4.0) and acetonitrile was used as the mobile phase, with compounds detected in the positive ion mode with multiple-reaction monitoring using a triple-quadrupole mass spectrometer equipped with an electrospray ionisation interface. This method for the determination of the reported flavonoid glycosides was accurate and reproducible, with a lower limit of quantication of 0.5 microg/mL. The standard calibration curves for the above-mentioned compounds were linear (r(2) > 0.998) over the concentration range 0.5-10.0 microg/mL. The relative standard deviations for intra- and inter-day precision over the concentration range for the flavonoid glycosides were lower than 7.8% with accuracy between 90.1 and 111.0%. The established method was successfully applied to the quality assessment of samples of Herba Epimedii collected from Korea and China.     

Quinovic acid glycosides from Guettarda angelica

Two new triterpene glycosides isolated from the root bark Guettarda angelica were proven to be quinovic acid-3β-O-[β-d-glucopyranosyl-(1 → 3)-α-l-rhamnopyranoside] and quinovic acid-3β-O-β-d-glucopyranosyl-(28 → 1)-β-d-glucopyranosyl ester. In addition quinovic acid and two known glycoside derivatives (quinovic acid-3β-O-β-d-glucopyranoside and quinovic acid-3β-O-α-l-rhamnopyranoside) were isolated. The structures were elucidated by spectroscopic analysis of the peracetyl methyl ester derivatives.     

New Acylated Flavonol Glycosides and a Phenolic Profile of Pritzelago alpina,a Forgotten Edible Alpine Plant

Thirteen acylated flavonoid glycosides, 1 – 13 , including eleven new congeners, 3 – 13 , were isolated from the aerial parts of Pritzelago alpina (Brassicaceae) by a combination of column chromatography on Sephadex LH‐20, and preparative and semi‐preparative HPLC. The structures were established by extensive NMR and MS experiments in combination with acid hydrolysis and sugar analysis by GC/MS. The new compounds were shown to be kaempferol and quercetin glycosides acylated for most of them by a branched short chain fatty acid or a hydroxycinnamic acid residue on the sugar portion. As shown by a HPLC‐DAD analysis of a MeOH extract, these compounds are the main phenolic constituents in the aerial parts of the plant.     

Identification of some Bioactive Metabolites in a Fractionated Methanol Extract from Ipomoea aquatica (Aerial Parts) through TLC,HPLC, UPLC‐ESI‐QTOF‐MS and LC‐SPE‐NMR Fingerprints Analyses

Introduction

The plant species Ipomoea aquatica contains various bioactive constituents, e.g. phenols and flavonoids, which have several medical uses. All previous studies were executed in Asia; however, no reports are available from Africa, and the secondary metabolites of this plant species from Africa are still unknown.

Objective

The present study aims finding suitable conditions to identify the bioactive compounds from different fractions.

Methodology

Chromatographic fingerprint profiles of different fractions were developed using high‐performance liquid chromatography (HPLC) and then these conditions were transferred to thin‐layer chromatography (TLC). Subsequently, the chemical structure of some bioactive compounds was elucidated using ultra‐performance liquid chromatography‐quadrupole time of flight‐tandem mass spectrometry (UPLC‐QTOF‐MS) and liquid chromatography‐solid phase extraction‐nuclear magnetic resonance (LC‐SPE‐NMR) spectroscopy.

Results

The HPLC fingerprints, developed on two coupled Chromolith RP‐18e columns, using a gradient mobile phase (methanol/water/trifluoroacetic acid, 5:95:0.05, v/v/v), showed more peaks than the TLC profile. The TLC fingerprint allows the identification of the types of chemical constituents, e.g. flavonoids. Two flavonoids (nicotiflorin and ramnazin‐3‐O‐rutinoside) and two phenolic compounds (dihydroxybenzoic acid pentoside and di‐pentoside) were tentatively identified by QTOF‐MS, while NMR confirmed the structure of rutin and nicotiflorin.

Conclusion

The HPLC and TLC results showed that HPLC fingerprints give more and better separated peaks, but TLC helped in determining the class of the active compounds in some fractions. Bioactive constituents were identified as well using MS and NMR analyses. Two flavonoids and two phenolic compounds were tentatively identified in this species for the first time, to the best of our knowledge. Copyright © 2017 John Wiley & Sons, Ltd.     

Analytical characterisation of crude extracts from an African Ancistrocladus species using high-performance liquid chromatography and capillary electrophoresis coupled to ion trap mass spectrometry

The analysis by HPLC, CE and CE-MS/MS of root bark extracts of a, so far undescribed, Central-African Ancistrocladus species (family Ancistrocladaceae) is described. Owing to the complexity of the extract, the application of reversed-phase HPLC resulted in a partially incomplete separation of the naphthylisoquinoline alkaloids, whilst CE using a non-aqueous buffer proved to be a very valuable complementary method for a first characterisation of the crude extract. By performing additional CE-MS/MS experiments, in combination with parallel isolation studies and structural elucidation using conventional methods, six alkaloidal substances present in the plant could be identified.     

A label‐free internal standard method for the differential analysis of bioactive lupin proteins using nano HPLC‐Chip coupled with Ion Trap mass spectrometry

Quantitative proteomics based on MS is useful for pointing out the differences in some food proteomes relevant to human nutrition. Stable isotope label‐free (SIF) techniques are suitable for comparing an unlimited number of samples by the use of relatively simple experimental workflows. We have developed an internal standard label‐free method based on the intensities of peptide precursor ions from MS/MS spectra, collected in data dependent runs, for the simultaneous qualitative characterization and relative quantification of storage proteins of Lupinus albus seeds in protein extracts of four lupin cultivars (cv Adam, Arés, Lucky, Multitalia). The use of an innovative microfluidic system, the HPLC‐Chip, coupled with a classical IT mass spectrometer, has allowed a complete qualitative characterization of all proteins. In particular, the homology search mode has permitted to identify single amino acid substitutions in the sequences of vicilins (β‐conglutin precursor and vicilin‐like protein). The MS/MS sequencing of substituted peptides confirms the high heterogeneity of vicilins according to the peculiar characteristics of the vicilin‐encoding gene family. Two suitable bioinformatics parameters were optimized for the differential analyses of the main bioactive proteins: the “normalized protein average of common reproducible peptides” (N‐ACRP) for γ‐conglutin, which is a homogeneous protein, and the “normalized protein mean peptide spectral intensity” (N‐MEAN) for the highly heterogenous class of the vicilins.     

Quantitative analysis of major dibenzocyclooctane lignans in Schisandrae fructus by online TLC-DART-MS

Introduction – Direct analysis in real time (DART) ion source is a powerful ionising technique for the quick and easy detection of various organic molecules without any sample preparation steps, but the lack of quantitation capacity limits its extensive use in the field of phytochemical analysis. Objective – To improvise a new system which utilize DART‐MS as a hyphenated detector for quantitation. Methodology – A total extract of Schisandra chinensis fruit was analyzed on a TLC plate and three major lignan compounds were quantitated by three different methods of UV densitometry, TLC‐DART‐MS and HPLC‐UV to compare the efficiency of each method. To introduce the TLC plate into the DART ion source at a constant velocity, a syringe pump was employed. The DART‐MS total ion current chromatogram was recorded for the entire TLC plate. The concentration of each lignan compound was calculated from the calibration curve established with standard compound. Results – Gomisin A, gomisin N and schisandrin were well separated on a silica‐coated TLC plate and the specific ion current chromatograms were successfully acquired from the TLC‐DART‐MS system. The TLC‐DART‐MS system for the quantitation of natural products showed better linearity and specificity than TLC densitometry, and consumed less time and solvent than conventional HPLC method. Conclusion – A hyphenated system for the quantitation of phytochemicals from crude herbal drugs was successfully established. This system was shown to have a powerful analytical capacity for the prompt and efficient quantitation of natural products from crude drugs. Copyright © 2010 John Wiley & Sons, Ltd.     

Simultaneous determination of five characteristic stilbene glycosides in root bark of Morus albus L. (Cortex Mori) using high-performance liquid chromatography

Introduction – Cortex Mori, one of the well‐known traditional Chinese herbal medicines, is derived from the root bark of Morus alba L. according to the China Pharmacopeia. Stilbene glycosides are the main components isolated from aqueous extracts of Morus alba and their content varies depending on where Cortex Mori was collected. We have established a qualitative and quantitative method based on the bioactive stilbene glycosides for control of the quality of Cortex Mori from different sources. Objective – To develop a high‐performance liquid chromatography coupled with ultraviolet absorption detection for simultaneous quantitative determination of five major characteristic stilbene glycosides in 34 samples of the root bark of Morus alba L. (Cortex Mori) from different sources. Methodology – The analysis was performed on an ODS column using methanol‐water‐acetic acid (18: 82: 0.1, v/v/v) as the mobile phase and the peaks were monitored at 320 nm. Results – All calibration curves showed good linearity (r ≥ 0.9991) within test ranges. This method showed good repeatability for the quantification of these five components in Cortex Mori with intra‐ and inter‐day standard deviations less than 2.19% and 1.45%, respectively. Conclusion – The validated method was successfully applied to quantify the five investigated components, including a pair of cis‐trans‐isomers 1 and 2 and a pair of isomers 4 and 5 in 34 samples of Cortex Mori from different sources. Copyright © 2010 John Wiley & Sons, Ltd.     

Stable isotopic labeling‐based quantitative targeted glycomics (i‐QTaG)

Mass spectrometry (MS) analysis combined with stable isotopic labeling is a promising method for the relative quantification of aberrant glycosylation in diseases and disorders. We developed a stable isotopic labeling‐based quantitative targeted glycomics (i‐QTaG) technique for the comparative and quantitative analysis of total N‐glycans using matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOF MS). We established the analytical procedure with the chemical derivatizations (i.e., sialic acid neutralization and stable isotopic labeling) of N‐glycans using a model glycoprotein (bovine fetuin). Moreover, the i‐QTaG using MALDI‐TOF MS was evaluated with various molar ratios (1:1, 1:2, 1:5) of ¹³C₆/¹²C₆‐2‐aminobenzoic acid‐labeled glycans from normal human serum. Finally, this method was applied to direct comparison of the total N‐glycan profiles between normal human sera (n = 8) and prostate cancer patient sera (n = 17). The intensities of the N‐glycan peaks from i‐QTaG method showed a good linearity (R² > 0.99) with the amount of the bovine fetuin glycoproteins. The ratios of relative intensity between the isotopically 2‐AA labeled N‐glycans were close to the theoretical molar ratios (1:1, 1:2, 1:5). We also demonstrated that the up‐regulation of the Lewis antigen (～82%) in sera from prostate cancer patients. In this proof‐of‐concept study, we demonstrated that the i‐QTaG method, which enables to achieve a reliable comparative quantitation of total N‐glycans via MALDI‐TOF MS analysis, has the potential to diagnose and monitor alterations in glycosylation associated with disease states or biotherapeutics. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:840–848, 2015     

Comprehensive Study of Mediterranean (Croatian) Propolis Peculiarity: Headspace,Volatiles, Anti‐Varroa‐Treatment Residue,Phenolics, and Antioxidant Properties

Eight propolis samples from Croatia were analyzed in detail, to study the headspace, volatiles, anti‐Varroa‐treatment residue, phenolics, and antioxidant properties. The samples exhibited high qualitative/quantitative variability of the chemical profiles, total phenolic content (1,589.3–14,398.3 mg GAE (gallic acid equivalent)/l EtOH extract), and antioxidant activity (11.1–133.5 mmol Fe²⁺/l extract and 6.2–65.3 mmol TEAC (Trolox^® equivalent antioxidant capacity)/l extract). The main phenolics quantified by HPLC‐DAD at 280 and 360 nm were vanillin, p‐coumaric acid, ferulic acid, chrysin, galangin, and caffeic acid phenethyl ester. The major compounds identified by headspace solid‐phase microextraction (HS‐SPME), simultaneous distillation extraction (SDE), and subsequent GC‐FID and GC/MS analyses were α‐eudesmol (up to 19.9%), β‐eudesmol (up to 12.6%), γ‐eudesmol (up to 10.5%), benzyl benzoate (up to 28.5%), and 4‐vinyl‐2‐methoxyphenol (up to 18.1%). Vanillin was determined as minor constituent by SDE/GC‐FID/MS and HPLC‐DAD. The identified acaricide residue thymol was ca. three times more abundant by HS‐SPME/GC‐FID/MS than by SDE/GC‐FID/MS and was not detected by HPLC‐DAD.     

Bottom‐up proteome analysis of E. coli using capillary zone electrophoresis‐tandem mass spectrometry with an electrokinetic sheath‐flow electrospray interface

The Escherichia coli proteome was digested with trypsin and fractionated using SPE on a C18 SPE column. Seven fractions were collected and analyzed by CZE‐ESI‐MS/MS. The separation was performed in a 60‐cm‐long linear polyacrylamide‐coated capillary with a 0.1% v/v formic acid separation buffer. An electrokinetic sheath‐flow electrospray interface was used to couple the separation capillary with an Orbitrap‐Velos operating in higher‐energy collisional dissociation mode. Each CZE‐ESI‐MS/MS run lasted 50 min and total MS time was 350 min. A total of 23 706 peptide spectra matches, 4902 peptide IDs, and 871 protein group IDs were generated using MASCOT with false discovery rate less than 1% on the peptide level. The total mass spectrometer analysis time was less than 6 h, the sample identification rate (145 proteins/h) was more than two times higher than previous studies of the E. coli proteome, and the amount of sample consumed (<1 μg) was roughly fourfold less than previous studies. These results demonstrate that CZE is a useful tool for the bottom‐up analysis of prokaryote proteomes.