Characterization of gallotannins from Astronium species by flow injection analysis- electrospray ionization-ion trap-tandem mass spectrometry and matrix-assisted laser desorption/ionization time-of- flight mass spectrometry

The species Astronium urundeuva (Allemao) Engl. and Astronium graveolens Jacq., which are used in Brazilian folk medicine to treat allergies, inflammation, diarrhea and ulcers, were investigated for their composition. The aim of this study was to define a rapid and reliable analytical approach, based on the flow-injection analysis-electrospray ionization-ion trap-tandem mass spectrometry (FIA-ESI-IT-MS-MS) and matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-ToF-MS), to investigate the full range of hydrolyzable tannins present in the extracts of these Astronium species. The MALDI-ToF-MS analysis allowed us to ascertain the presence of hydrolysable tannins in both Astronium species as a series of gallotannins with degrees of polymerization of 7 to 13 galloyl units. Moreover, the analysis by FIA-ESI-IT-MS-MS, as well as confirming this result and chemically defining gallotannins as galloylglucose compounds, highlighted the presence of further classes of hydrolysable tannins, such as hexahydrodiphenoyl esters of glucose and some gallic acid derivatives, providing information about their structure by a careful study of their fragmentation patterns. Finally, the evaluation of the number of positional isomers of gallotannins occurring in both Astronium species was obtained by high-performance liquid chromatography-electrospray ionization-ion trap mass spectrometry (HPLC/ESI-IT-MS). This is the first mass spectrometric evidence relating to the existence of gallotannins in Astronium genus.     

Identification of autoxidation oligomers of flavan‐3‐ols in model solutions by HPLC‐MS/MS

Autoxidation of flavan‐3‐ols was carried out in aqueous/methanol model solutions under mildly acidic conditions (pH 6.0), and these autoxidation products were analyzed by using high performance liquid chromatography (HPLC) coupled with tandem mass spectrometry (MS/MS). The results showed that (+)‐catechins and (?)‐epicatechins generated autoxidation reaction with each other to form a series of oligomers that had the same [M ? H]^? molecular ions (MS¹) as those of natural procyanidins, but had completely different fragment ions (MS²). According to MS/MS analysis, the major fragments of these oligomers were derived not only from the retro‐Diels–Alder (RDA) dissociations on the C‐rings of the flavan‐3‐ol units, but also from the quinone‐methide (QM) cleavage of the interflavan linkages (IFL), and thus they were identified as B‐type dehydrodicatechins, B‐type dehydrotricatechins and A‐type dehydrotricatechins, respectively. The potential structures of their [M ? H]^? molecular ions and partial fragment ions were deduced on the basis of the MS/MS characterization and the oxidation of flavan‐3‐ols in previous reports. Some specific fragment ions were found to be very useful for identifying the autoxidation oligomers (the B‐type dehydrodicatechins at m/z 393, the B‐type dehydrotricatechins at m/z 681 and the A‐type dehydrotricatechins at m/z 725). Copyright © 2008 John Wiley & Sons, Ltd.     

UPLC‐QTOF‐MSE‐based diagnostic product ion filtering to unveil unstable C6‐C2 glucoside conjugates in Forsythia suspensa

Forsythia suspensa contains C₆‐C₂ glucoside conjugates (CCGCs) that are chemically unstable, thereby hindering their isolation and purification. In the present study, ultra‐performance liquid chromatography‐quadrupole time‐of‐flight mass spectrometry (UPLC‐QTOF) was utilized to screen and identify unstable CCGCs in the fruits and leaves of F. suspensa without any tedious isolation and purified process based on independent information acquisition (also called MS^E) and individual MS/MS experiments. Diagnostic product ion filtering (DPIF) was further applied to mine unknown analogs in MS^E high energy levels based on characteristic m/z of key substructures. A modified nomenclature for CCGCs is hereby proposed to facilitate discussions. Possible fragmentation pathways of major types of known CCGCs were proposed and used for deducing their structures. A total of 8 potentially new CCGCs were discovered and initially identified. The accuracy of their identification was further verified by structural elucidation of 3 unstable CCGCs isolated from the fruits of F. suspensa using 1D and 2D‐NMR spectroscopy. The established UPLC‐QTOF‐MS^E‐based DPIF technique facilitates the rapid discovery and direct identification of unstable CCGCs in fruits and leaves of F. suspensa .     

Semi‐quantitative and structural metabolic phenotyping by direct infusion ion trap mass spectrometry and its application in genetical metabolomics

The identification of quantitative trait loci (QTL) for plant metabolites requires the quantitation of these metabolites across a large range of progeny. We developed a rapid metabolic profiling method using both untargeted and targeted direct infusion tandem mass spectrometry (DIMSMS) with a linear ion trap mass spectrometer yielding sufficient precision and accuracy for the quantification of a large number of metabolites in a high‐throughput environment. The untargeted DIMSMS method uses top‐down data‐dependent fragmentation yielding MS² and MS³ spectra. We have developed software tools to assess the structural homogeneity of the MS² and MS³ spectra hence their utility for phenotyping and genetical metabolomics. In addition we used a targeted DIMS(MS) method for rapid quantitation of specific compounds. This method was compared with targeted LC/MS/MS methods for these compounds. The DIMSMS methods showed sufficient precision and accuracy for QTL discovery. We phenotyped 200 individual Lolium perenne genotypes from a mapping population harvested in two consecutive years. Computational and statistical analyses identified 246 nominal m/z bins with sufficient precision and homogeneity for QTL discovery. Comparison of the data for specific metabolites obtained by DIMSMS with the results from targeted LC/MS/MS analysis showed that quantitation by this metabolic profiling method is reasonably accurate. Of the top 100 MS¹ bins, 22 ions gave one or more reproducible QTL across the 2 years. Copyright © 2009 John Wiley & Sons, Ltd.     

Top–down glycolipidomics: fragmentation analysis of ganglioside oligosaccharide core and ceramide moiety by chip‐nanoelectrospray collision‐induced dissociation MS2–MS6

We developed a straightforward approach for high‐throughput top–down glycolipidomics based on fully automated chip‐nanoelectrospray (nanoESI) high‐capacity ion trap (HCT) multistage mass spectrometry (MSⁿ) by collision‐induced dissociation (CID) in the negative ion mode. The method was optimized and tested on a polysialylated ganglioside fraction (GT1b), which was profiled by MS¹ and sequenced in tandem MS up to MS⁶ in the same experiment. Screening of the fraction in the MS¹ mode indicated the occurrence of six [M ? 2H]^2? ions which, according to calculation, support 13 GT1 variants differing in their relative molecular mass due to dissimilar ceramide (Cer) constitutions. By stepwise CID MS²–MS⁵ on the doubly charged ion at m/z 1077.20 corresponding to a ubiquitous GT1b structure, the complete characterization of its oligosaccharide core including the identification of sialylation sites was achieved. Structure of the lipid moiety was further elucidated by CID MS⁶ analysis carried out using the Y₀ fragment ion, detected in MS⁵, as a precursor. MS⁶ fragmentation resulted in a pattern supporting a single ceramide form having the less common (d20 : 1/18 : 0) configuration. The entire top–down experiment was performed in a high‐throughput regime in less than 3 min of measurement, with an analysis sensitivity situated in the subpicomolar range. Copyright © 2009 John Wiley & Sons, Ltd.     

Pharmacokinetics of 2,3,5,4′‐tetrahydroxystilbene‐2‐O‐β‐D‐glucoside in rat using ultra‐performance LC‐quadrupole TOF‐MS

2,3,5,4′‐Tetrahydroxystilbene‐2‐O‐β‐D‐glucoside (THSG) from Polygoni multiflori has been demonstrated to possess a variety of pharmacological activities, including antioxidant, anti‐inflammatory and hepatoprotective activities. Ultra‐performance LC‐quadrupole TOF‐MS with MS Elevated Energy data collection technique and rapid resolution LC with diode array detection and ESI multistage MSⁿ methods were developed for the pharmacokinetics, tissue distribution, metabolism, and excretion studies of THSG in rats following a single intravenous or oral dose. The three metabolites were identified by rapid resolution LC‐MSⁿ. The concentrations of the THSG in rat plasma, bile, urine, feces, or tissue samples were determined by ultra‐performance LC‐MS. The results showed that THSG was rapidly distributed and eliminated from rat plasma. After the intravenous administration, THSG was mainly distributing in the liver, heart, and lung. For the rat, the major distribution tissues after oral administration were heart, kidney, liver, and lung. There was no long‐term storage of THSG in rat tissues. Total recoveries of THSG within 24 h were low (0.1% in bile, 0.007% in urine, and 0.063% in feces) and THSG was excreted mainly in the forms of metabolites, which may resulted from biotransformation in the liver.     

A comprehensive library‐based,automated screening procedure for 46 synthetic cannabinoids in serum employing liquid chromatography‐quadrupole ion trap mass spectrometry with high‐temperature electrospray ionization

Considering the vast variety of synthetic cannabinoids and herbal mixtures – commonly known as ‘Spice’ or ‘K2’ – on the market and the resulting increase of severe intoxications related to their consumption, there is a need in clinical and forensic toxicology for comprehensive up‐to‐date screening methods. The focus of this project aimed at developing and implementing an automated screening procedure for the detection of synthetic cannabinoids in serum using a liquid chromatography‐ion trap‐MS (LC‐MSⁿ) system and a spectra library‐based approach, currently including 46 synthetic cannabinoids and 8 isotope labelled analogues. In the process of method development, a high‐temperature ESI source (IonBooster^TM, Bruker Daltonik) and its effects on the ionization efficiency of the investigated synthetic cannabinoids were evaluated and compared to a conventional ESI source. Despite their structural diversity, all investigated synthetic cannabinoids benefitted from high‐temperature ionization by showing remarkably higher MS intensities compared to conventional ESI. The employed search algorithm matches retention time, MS and MS²/MS³ spectra. With the utilization of the ionBooster source, limits for the automated detection comparable to cut‐off values of routine MRM methods were achieved for the majority of analytes. Even compounds not identified when using a conventional ESI source were detected using the ionBooster‐source. LODs in serum range from 0.1 ng/ml to 0.5 ng/ml. The use of parent compounds as analytical targets offers the possibility of instantly adding new emerging compounds to the library and immediately applying the updated method to serum samples, allowing the rapid adaptation of the screening method to ongoing forensic or clinical requirements. The presented approach can also be applied to other specimens, such as oral fluid or hair, and herbal mixtures and was successfully applied to authentic serum samples. Quantitative MRM results of samples with analyte concentrations above the determined LOD were confirmed as positive findings by the presented method. Copyright © 2014 John Wiley & Sons, Ltd.     

New cathinone‐derived designer drugs 3‐bromomethcathinone and 3‐fluoromethcathinone: studies on their metabolism in rat urine and human liver microsomes using GC–MS and LC–high‐resolution MS and their detectability in urine

3‐Bromomethcathinone (3‐BMC) and 3‐Fluoromethcathinone (3‐FMC) are two new designer drugs, which were seized in Israel during 2009 and had also appeared on the illicit drug market in Germany. These two compounds were sold via the Internet as so‐called “bath salts” or “plant feeders.” The aim of the present study was to identify for the first time the 3‐BMC and 3‐FMC Phase I and II metabolites in rat urine and human liver microsomes using GC–MS and LC–high‐resolution MS (HR‐MS) and to test for their detectability by established urine screening approaches using GC–MS or LC–MS. Furthermore, the human cytochrome‐P450 (CYP) isoenzymes responsible for the main metabolic steps were studied to highlight possible risks of consumption due to drug–drug interaction or genetic variations. For the first aim, rat urine samples were extracted after and without enzymatic cleavage of conjugates. The metabolites were separated and identified by GC–MS and by LC–HR‐MS. The main metabolic steps were N‐demethylation, reduction of the keto group to the corresponding alcohol, hydroxylation of the aromatic system and combinations of these steps. The elemental composition of the metabolites identified by GC–MS could be confirmed by LC–HR‐MS. Furthermore, corresponding Phase II metabolites were identified using the LC–HR‐MS approach. For both compounds, detection in rat urine was possible within the authors' systematic toxicological analysis using both GC–MS and LC–MSⁿ after a suspected recreational users dose. Following CYP enzyme kinetic studies, CYP2B6 was the most relevant enzyme for both the N‐demethylation of 3‐BMC and 3‐FMC after in vitro–in vivo extrapolation. Copyright © 2012 John Wiley & Sons, Ltd.     

Approach to the study of flavone di‐C‐glycosides by high performance liquid chromatography‐tandem ion trap mass spectrometry and its application to characterization of flavonoid composition in Viola yedoensis

The mass spectrometric (MS) analysis of flavone di‐C‐glycosides has been a difficult task due to pure standards being unavailable commercially and to that the reported relative intensities of some diagnostic ions varied with MS instruments. In this study, five flavone di‐C‐glycoside standards from Viola yedoensis have been systematically studied by high performance liquid chromatography‐electrospray ionization‐tandem ion trap mass spectrometry (HPLC‐ESI‐IT‐MSⁿ) in the negative ion mode to analyze their fragmentation patterns. A new MS² and MS³ hierarchical fragmentation for the identification of the sugar nature (hexoses or pentoses) at C‐6 and C‐8 is presented based on previously established rules of fragmentation. Here, for the first time, we report that the MS² and MS³ structure‐diagnostic fragments about the glycosylation types and positions are highly dependent on the configuration of the sugars at C‐6 and C‐8. The base peak (^0,2X₁^0,2X₂^? ion) in MS³ spectra of di‐C‐glycosides could be used as a diagnostic ion for flavone aglycones. These newly proposed fragmentation behaviors have been successfully applied to the characterization of flavone di‐C‐glycosides found in V. yedoensis. A total of 35 flavonoid glycosides, including 1 flavone mono‐C‐hexoside, 2 flavone 6,8‐di‐C‐hexosides, 11 flavone 6,8‐di‐C‐pentosides, 13 flavone 6,8‐C‐hexosyl‐C‐pentosides, 5 acetylated flavone C‐glycosides and 3 flavonol O‐glycosides, were identified or tentatively identified on the base of their UV profiles, MS and MSⁿ (n = 5) data, or by comparing with reference substances. Among these, the acetylated flavone C‐glycosides were reported from V. yedoensis for the first time. Copyright © 2014 John Wiley & Sons, Ltd.     

Structural elucidation of rat biliary metabolites of corynoxeine and their quantification using LC‐MSn

Corynoxeine (COR) is one of 4 bioactive oxindole alkaloids in Uncaria species. In this work two phase I metabolites, namely 11‐hydroxycorynoxeine (M1) and 10‐hydroxycorynoxeine (M2), and two phase II metabolites, namely 11‐hydroxycorynoxeine 11‐O‐β‐d ‐glucuronide (M3) and 10‐hydroxycorynoxeine 10‐O‐β‐d ‐glucuronide (M4), were detected in rat bile after oral dose of COR (0.105 mmol/kg), by optimized high‐performance liquid chromatography–tandem mass spectrometry (LC‐MSⁿ) with electrospray ionization in positive ion mode. Structures of M1–4 were determined by LC‐MSⁿ, nuclear magnetic resonance, circular dichroism and high‐resolution MS spectra. COR and its metabolites in rat bile were quantified by LC‐MSⁿ. The LC‐MSⁿ quantification methods for COR and its metabolites yielded a linearity with coefficient of determination ≥0.995 from 5.0 × 10^?10 to 5.0 × 10^?7 m . The recoveries of stability tests varied from 96.80 to 103.10%. Accuracy ranged from 91.00 to 105.20%. Relative standard deviation for intra‐day and inter‐day assay was <5.0%. After the oral dose 0.14% of COR was detected in rat bile from 0 to 8 h, in which in total 97.8% COR biotransformed into M1–4. M1 and M2 yielded 48.1 and 49.7%, which successively glucuronidated to M3 and M4 at 47.2 and 43.8%, respectively. Copyright © 2014 John Wiley & Sons, Ltd.     

Regioisomer characterization of triacylglycerols by non‐aqueous reversed‐phase liquid chromatography/electrospray ionization mass spectrometry using silver nitrate as a postcolumn reagent

Triacylglycerols (TAGs) provide a challenge for mass spectrometry (MS) analysis because of their complexity. In particular, for dietary, nutritional and metabolic purposes, the positional placement of fatty acids on the glycerol backbone of TAGs is a crucial aspect. To solve this problem, we have investigated the TAGs' fragmentation patterns using an ion trap mass spectrometer. A series of pure regioisomeric pairs of TAGs (POP/PPO, POO/OPO and OSO/SOO) were cationized by Ag⁺ after their separation by non‐aqueous reversed‐phase liquid chromatography (NARP‐LC) before MS to improve MS sensitivity. Electrospray ionization–MS (ESI‐MS) conditions were optimized in order to produce characteristic [M + Ag + AgNO₃]⁺ ions from each TAG, which were then fragmented to produce MS/MS spectra and then fragmented further to produce up to MS⁵ spectra. The observation of ions produced by LC‐MS⁵ of on‐line Ag⁺‐cationized TAG provided unambiguous information on the fatty acid distribution on the glycerol backbone. These strategies of MS to MS⁵ experiments were applied to identify components and to determine the regiospecificity of TAG within a complex mixture of lipids in natural oils. Copyright © 2010 John Wiley & Sons, Ltd.     

Characterization of additives in plastics: From MS to MS10 multistep mass analysis and theoretical calculations of tris(2,4‐di‐tert‐butylphenyl)phosphate

In the analysis by electrospray (+) of an extract of hemp sprouts put in a polypropylene vial, we found a large contamination of a plastic additive. It was characterized by multiple‐stage MSⁿ experiments (MS ÷ MS¹⁰) and identified as tris(2,4‐di‐tert‐butylphenyl)phosphate, also known with the synonyms F32IRS6B46, oxidized Naugard 524, and others. The MS² ÷ MS⁷ spectra are characterized by consecutive eliminations of six isobutene molecules from the tert‐butyl moieties, some of them also occurring in the ion source. The first three are calculated to occur preferentially from the ortho positions, whereas eliminations from the para positions are estimated to be less favored at about 5–6 kcal/mol in each step. Once the first three isobutene molecules are eliminated, the remaining three are lost from the tert‐butyl moieties in para positions (MS⁵ ÷ MS⁷), yielding protonated triphenylphosphate, whose structure has been confirmed by the MS² spectrum of triphenylphosphate standard: the latter spectrum is almost superimposable with the MS⁸ spectrum of the analyte under investigation. MS⁸ and MS⁹ spectra show main losses of water and C₆H₄ molecules. The MS¹⁰ spectrum of precursor ions at m/z 215 shows the gas‐phase addition of water and methanol and ions at m/z 168, attributable to the loss of a phosphorus oxide radical. Density functional theory (DFT) calculations (Becke 3LYP [B3LYP] 6‐311+G(2d,2p)) have been used to evaluate structure and stability of different ionic and neutral species involved in the decomposition pathways and to calculate thermochemical data of the decomposition reactions. This multistep mass analysis combined with theoretical calculations resulted to be particularly useful and effective, yielding chemical, thermochemical, and mechanistic data of significant utility in the structural characterization and identification of the unknown analyte as well as to define its gas‐phase reactivity under a multistep low‐energy collision‐induced dissociation regime.     

Analysis of low‐density lipoprotein‐associated proteins using the method of digitized native protein mapping

The method of digitized native protein mapping, combining nondenaturing micro 2DE, grid gel‐cutting, and quantitative LC‐MS/MS (in data‐independent acquisition mode, or MS^E), was improved by using a new MS/MS mode, ion mobility separation enhanced‐MS^E (HDMS^E), and applied to analyze the area of human plasma low‐density lipoprotein (LDL). An 18 mm × 4.8 mm rectangular area which included LDL on a nondenaturing micro 2D gel of human plasma was grid‐cut into 72 square gel pieces and subjected to quantitative LC‐MS/MS. Compared with MS^E, HDMS^E showed significantly higher performance, by assigning 50% more proteins and detecting each protein in more squares. A total of 253 proteins were assigned with LC‐HDMS^E and the quantity distribution of each was reconstructed as a native protein map. The maps showed that Apo B‐100 was the most abundant protein in the grid‐cut area, concentrated at pI ca. 5.4–6.1 and apparent mass ca. 1000 kDa, which corresponded to four gel pieces, squares 39–42. An Excel macro was prepared to search protein maps which showed protein quantity peaks localized within this concentrated region of Apo B‐100. Twenty‐two proteins out of the 252 matched this criterion, in which 19 proteins have been reported to be associated with LDL. This method only requires several microliters of a plasma sample and the principle of the protein separation is totally different from the commonly used ultracentrifugation. The results obtained by this method would provide new insights on the structure and function of LDL.     

Identification and structural characterization of novel O‐ and N‐glycoforms in the urine of a Schindler disease patient by Orbitrap mass spectrometry

Schindler disease is an inherited metabolic disorder caused by the deficient activity of α‐N‐acetylgalactosaminidase enzyme. An accurate diagnosis requires, besides clinical examination, complex and costly biochemical and molecular genetic tests. In the last years, mass spectrometry (MS) based on nanofluidics and high‐resolution instruments has become a successful alternative for disease diagnosis based on the investigation of O‐glycopeptides in patient urine. A complex mixture of glycoforms extracted from the urine of a 3‐year‐old patient was investigated by Orbitrap MS equipped with Nanospray Flex Ion Source in the negative ion mode. For structural characterization of several molecular species, collision‐induced dissociation MS²–MS³ was carried out using collision energy values within 20–60 eV range. By our approach, 39 novel species associated to this condition were identified, among which O‐glycopeptides, free O‐glycans and one structure corresponding to an N‐glycan never characterized in the context of Schindler disease. The experiments conducted at a resolution of 60 000 allowed the discrimination and identification of a total number of 69 different species with an average mass accuracy of 9.87 ppm, an in‐run reproducibility of almost 100%, an experiment‐to‐experiment and day‐to‐day reproducibility of about 95%. This study brings contributions in the diagnosis of Schindler disease through the elucidation of potential biomarker species in urine. Our multistage MS results completed with 39 new glycoforms the inventory of potential biomarker structures associated to Schindler disease. For the first time, an N‐glycan was identified and structurally characterized in Schindler patient urine, which opens new research directions in the field. Copyright © 2015 John Wiley & Sons, Ltd.     

In‐time and in‐space tandem mass spectrometry to determine the metabolic profiling of flavonoids in a typical sweet cherry (Prunus avium L.) cultivar from Southern Italy

This paper presents a comprehensive analytical methodology, based on ‘in‐time’ and ‘in‐space’ tandem mass spectrometry (MS) techniques, to identify and quantify flavonoid compounds in a typical Italian sweet cherry cultivar (cv. Ferrovia). Five anthocyanins, four flavan‐3‐ols and nine flavonols were determined by means of hyphenated high‐performance liquid chromatography – multi‐stage MS (HPLC‐MSⁿ) analyses (MSⁿ up to MS⁴), among which quercetin‐3‐O‐rutinoside‐7‐O‐glucoside, kaempferol‐3‐O‐rutinoside‐7‐O‐glucoside, quercetin‐3‐O‐galactosyl‐rhamnoside and quercetin‐3‐O‐coumaroylglucoside were tentatively identified in sweet cherries for the first time. Ultrafast HPLC and tandem MS (UHPLC‐MS/MS) analyses through multiple reaction monitoring experiments showed that cyanidin‐3‐O‐rutinoside and cyanidin‐3‐O‐glucoside were the main anthocyanins of cv. Ferrovia at maturity. Moreover, consistent levels of catechin and epicatechin as well as quercetin‐3‐O‐rutinoside and kaempferol‐3‐O‐rutinoside were also found. Because flavonoids have been ascribed as potential health‐promoting compounds, gathered findings provide new insight into the knowledge of the quali‐quantitative profile of these phytochemicals into a widespread fruit such as sweet cherry. Copyright © 2014 John Wiley & Sons, Ltd.     

Ultra‐performance liquid chromatography/tandem mass spectrometry (UPLC/MS/MS) and UPLC/MSE analysis of RNA oligonucleotides

Fast and efficient ultra‐performance liquid chromatography/tandem mass spectrometry (UPLC/MS/MS) analysis of short interfering RNA oligonucleotides was used for identity confirmation of the target sequence‐related impurities. Multiple truncated oligonucleotides and metabolites were identified based on the accurate mass, and their presumed sequence was confirmed by MS/MS and MS^E (alternating low and elevated collision energy scanning modes) methods. Based on the resulting fragmentation of native and chemically modified oligonucleotides, it was found that the MS^E technique is as efficient as the traditional MS/MS method, yet MS^E is more general, faster, and capable of producing higher signal intensities of fragment ions. Fragmentation patterns of modified oligonucleotides were investigated using RNA 2′‐ribose substitutions, phosphorothioate RNA, and LNA modifications. The developed sequence confirmation method that uses the MS^E approach was applied to the analysis of in vitro hydrolyzed RNA oligonucleotide. The target RNA and metabolites, including the structural isomers, were resolved by UPLC, and their identity was confirmed by MS^E. Simultaneous RNA truncations from both termini were observed. The UPLC quadrupole time‐of‐flight (QTOF) MS/MS and MS^E methods were shown to be an effective tool for the analysis and sequence confirmation of complex oligonucleotide mixtures. Copyright © 2010 John Wiley & Sons, Ltd.     

Energy‐resolved technique for discovery and identification of malonyl‐triterpene saponins in Caulophyllum robustum by UHPLC‐electrospray Fourier transform mass spectrometry

Malonyl‐triterpene saponins (MTSs) attract scientific attentions because of their structural diversities and valuable bioactivities. However, its thermal instability brings a huge amount of challenges for isolation and purification of this class of compounds. To our best knowledge, there has been no report on isolation and analysis of MTSs from genus Caulophyllum. In this study, a strategy combining data acquisition using an energy‐resolved technique and the narrow widow extracted ion chromatograms as data mining method was developed for discovery and identification of MTSs in Caulophyllum robustum hair roots by ultra high liquid chromatography coupled to electrospray ionization Fourier transform mass spectrometry. The method was performed at an independent MS full scan using our bottom‐up energies by in‐source collision induced dissociations with 0, 25, 50 and 100 eV in both positive and negative modes. Precursor ion as well as fragment ion information was simultaneously collected from four energy‐resolved MS spectra in a single run of 18 min. The fragmentation pathways of intact deprotonated, protonated and sodium ions of MTSs were proposed for the structural elucidation of Caulophyllum MTSs. A flowchart involving a stepwise procedure based on key fragments from ESI^?/ESI⁺‐FT‐MS^{(1, 1)} to MS^{(1, 4)} spectra was constructed for the identification of structural elements in the MTSs. As a result, a total of 23 MTSs were discovered and tentatively identified, which had not been reported from Caulophyllum species before. All of these were potentially new compounds. This study provides an excellent example for discovery and identification of MTSs in herb medicines. Copyright © 2016 John Wiley & Sons, Ltd.     

Electrospray multistage mass spectrometry in the negative ion mode for the unambiguous molecular and structural characterization of acidic hydrolysates from 4‐O‐methylglucuronoxylan generated by endoxylanases

A rapid analytical methodology is proposed to answer the two questions about the molecular and structural features of the acidic xylo‐oligosaccharides (XOSs) formed upon the enzymatic hydrolysis of 4‐O‐methylglucuronoxylan. The shortest acidic XOSs carrying a methylglucuronic acid moiety and the possible distribution of larger products (molecular feature) are instantly found by electrospray ionization mass spectrometry (ESI‐MS) in the negative ion mode, which filters the unwanted neutral XOS. The acidic moiety is then unambiguously localized along the xylose backbone (structural feature) by ESI‐MSⁿ in the negative ion mode via the selection/activation/dissociation of the product ions formed upon the one‐way and stepwise glycosidic bond cleavage at the reducing end. Using the shortest acidic XOS with a known shape generated by glycoside hydrolase family (GH) 10 and GH11 xylanases as a proof of principle, pairs of diagnostic ions are proposed to instantly interpret the MSⁿ fingerprints and localize the acidic moiety along the xylose chain of the activated ion. The original structure of the acidic XOS is then reconstructed by adding as many xylose units at the reducing end as MSⁿ steps. Relying on pairs of ions, the methodology is robust enough to highlight the presence of isomeric products. Mass spectra reported in the present article will be conveniently used as reference data for the forthcoming analysis of acidic XOS generated by new classes of enzymes using this multistage mass spectrometry methodology.     

L‐Valine assisted distinction between the stereo‐isomers of D‐hexoses by positive ion ESI tandem mass spectrometry

The binary mixtures of 7 hexoses and 20 amino acids were investigated by electrospray ionization ion trap mass spectrometry (ESI‐ITMS). The adduct ions of the amino acid and the hexose were detected for 12 amino acids but not for the other 8 amino acids which are basic acidic amino acids and amides. The ions of amino acid–hexose complexes were further investigated by tandem mass spectrometry (MS/MS), and some of them just split easily into two parts whereas the others gave rich fragmentation, such as the complex ions of isoleucine, phenylalanie, tyrosine, and valine. We found that hexoses could be complexed by two molecules of valine but only by one molecule of the other amino acids. Among seven kinds of valine–hexose complexes coordinated by potassium ion, the MS² spectra of the ion at m/z 453 yielded unambiguous differentiation. And the fragmentation ions are sensitive to the stereochemical differences at the carbon‐4 of hexoses in the complexes, as proved by the MS². Copyright © 2010 John Wiley & Sons, Ltd.