Characterization of protostane triterpenoids in Alisma orientalis by ultra‐performance liquid chromatography coupled with quadrupole time‐of‐flight mass spectrometry

A reliable and sensitive ultra‐performance liquid chromatography/quadrupole time‐of‐flight mass spectrometry (UPLC/Q‐TOF‐MS) method has been optimized and established for analysis of protostane triterpenoids in a commonly used traditional Chinese herbal medicine Alisma orientalis (Sam.) Juzep. The separation of crude extract of A. orientalis was achieved on a Waters ACQUITY HSS T3 column (100 mm × 2.1 mm, 1.8 µm) eluting with 0.1% (v/v) formic acid/acetonitrile. A total of 20 protostane triterpenoids including 19 known compounds and a new one were well separated within 7 min. The collision‐induced dissociation (CID) tandem mass spectrometric (MS/MS) fragmentation patterns of protostane triterpenoids was firstly reported in this study. The hydrogen rearrangement at the C‐23‐OH leads to dissociation of the bond between C‐23 and C‐24 in the protostane triterpenoid skeleton during the CID process. This dissociation was the characteristic CID fragmentation pathway of this class of triterpenoids, and was useful for further differentiation of some positional isomers which contain an acetyl unit on the C‐23 or C‐24 position. The identities of isolated compounds were identified by comparing their retention times and CID fragmentation behaviors with those of reference standards or tentatively assigned by matching the empirical molecular formulae with those reported in the literature. It is concluded that this newly established UPLC/Q‐TOF‐MS method is a powerful approach for structural elucidation of protostane triterpenoids isolated from A. orientalis. Copyright © 2010 John Wiley & Sons, Ltd.     

Gas‐fragmentation study of the novel synthetic zwitterionic drug 3‐methyl‐9‐(2‐oxa‐2λ5‐2H‐1,3,2‐oxazaphosphorine‐2‐cyclohexyl)‐3,6,9‐triazaspiro[5,5]undecane chloride (SLXM‐2) by electrospray ionization tandem mass spectrometry

The zwitterionic drug 3‐methyl‐9‐(2‐oxa‐2λ5‐2H‐1,3,2‐oxazaphosphorine‐2‐cyclohexyl)‐3,6,9‐triazaspiro[5,5]undecane chloride (SLXM‐2) is a novel synthetic compound which has shown anticancer activity and low toxicity in vivo. In this study, the various gas‐phase fragmentation routes were analyzed by electrospray ionization mass spectrometry (positive ion mode) in conjunction with tandem mass spectrometry (ESI‐MSⁿ) for the first time. In ESI‐MS the fragment ion at m/z 289 (base peak) was formed by loss of the chlorine anion from the zwitterionic precursor SLXM‐2. The fragment ion at m/z 232 was formed from the ion at m/z 289 by loss of 1‐methylaziridine. The detailed gas‐phase collision‐induced dissociation (CID) fragmentation mechanisms obtained from the various precursor ions extracted from the zwitterionic SLXM‐2 drug was obtained by tandem mass spectrometry analyses. Copyright © 2010 John Wiley & Sons, Ltd.     

Liquid chromatography–mass spectrometry for analysis of a novel β2‐adrenoceptor agonist trantinterol and its metabolites in beagle dog urine

A liquid chromatography–tandem mass spectrometry method was developed for the identification of metabolites of trantinterol, a novel β₂‐adrenoceptor agonist, in beagle dog urine. The separation of metabolites was performed on a reversed‐phase C₈ column using 0.1% formic acid in water and methanol (70 : 30, v/v) as the mobile phase. The structural information and elemental information of metabolites were acquired by an electrospray ionization tandem mass spectrometer and a quadrupole time‐of‐flight mass spectrometer, respectively. A total of 13 metabolites were detected and characterized on the basis of their tandem MS/MS fragmentation patterns. The accurate masses of nine metabolites were determined and two metabolites were further confirmed by comparing with reference standards. The metabolic pathways of trantinterol in beagle dog are proposed. Copyright © 2009 John Wiley & Sons, Ltd.     

Poly[(2‐(acryloyloxy) ethyl]trimethylammonium chloride‐co‐ethylene dimethacrylate monolith on‐line solid‐phase extraction coupled with liquid chromatography and tandem mass spectrometry for the fast determination of salicylic acid in foodstuffs

We report the fabrication of an anion‐exchange monolithic column in a stainless‐steel chromatographic column (10 mm × 2.1 mm i.d.) using [2‐(acryloyloxy) ethyl]trimethylammonium chloride as the monomer and ethylene dimethacrylate as the crosslinker. The prepared monolith was developed as the adsorbent for the on‐line solid‐phase extraction of salicylic acid in various animal‐origin foodstuffs combined with liquid chromatography and tandem mass spectrometry. The monolith was characterized by using Fourier transform infrared spectroscopy, scanning electron microscopy, nitrogen adsorption analysis, and elemental analysis. Potential factors affecting the on‐line solid‐phase extraction and liquid chromatography with tandem mass spectrometry analysis were studied in detail. Under the optimized conditions, the total analysis time including cleanup and liquid chromatography with tandem mass spectrometry separation was 17 min. The developed method gave the linear range of 15–750 μg/kg, detection limits (S/N = 3) of 5 μg/kg, and quantification limits (S/N = 10) of 15 μg/kg. The recoveries obtained by spiking 10, 20, and 100 μg/kg of salicylic acid in the animal‐origin food samples were in the range of 85.2–98.4%. In addition, the monolith was stable enough for 550 extraction cycles with the precision of peak area ≤11.6%.     

Characterisation of oxazepam degradation products by high‐performance liquid chromatography/electrospray ionisation mass spectrometry and electrospray ionisation quadrupole time‐of‐flight tandem mass spectrometry

Oxazepam has been subjected to controlled degradation at 100°C for 3 h in 0.5 M HCl and 0.5 M NaOH. Following neutralisation of the degradation mixture and removal of salts by solid‐phase extraction (SPE), isocratic high‐performance liquid chromatography/mass spectrometry (HPLC/MS) using water/methanol (25:75 v/v) as the mobile phase was carried out using a flow diverter to collect fractions prior to their characterisation by electrospray ionisation multi‐stage mass spectrometry (ESI‐MSⁿ) and proposal of the corresponding fragmentation patterns. The elemental compositions of the degradation products and their MS fragments were evaluated using electrospray ionisation quadrupole time‐of‐flight tandem mass spectrometry (ESI‐QTOF‐MS/MS) which was then used to support the proposed fragmentation patterns. Copyright © 2010 John Wiley & Sons, Ltd.     

Simultaneous determination of aurantio‐obtusin,chrysoobtusin, obtusin and 1‐desmethylobtusin in rat plasma by UHPLC‐MS/MS

A sensitive and reliable ultra‐high‐performance liquid chromatography–electrospray ionization–tandem mass spectrometry (UHPLC‐MS/MS) method was developed and validated for the simultaneous determination of four active components of Semen Cassiae extract (aurantio‐obtusin, chrysoobtusin, obtusin and 1‐desmethylobtusin) in rat plasma after oral administration. Chromatographic separation was achieved on an Agilent Poroshell 120 C₁₈ column with gradient elution using a mobile phase that consisted of acetonitrile‐ammonium acetate in water (30 mm ) at a flow rate of 0.4 mL/min. Detection was performed by a triple‐quadrupole tandem mass spectrometer in multiple reaction monitoring mode. The calibration curve was linear over a range of 3.24–1296 ng/mL for aurantio‐obtusin, 0.77–618 ng/mL for chrysoobtusin, 34.55–1818 ng/mL for obtusin and 1.86–1485 ng/mL for 1‐desmethylobtusin. Inter‐ and intra‐day assay variation was <15%. All analytes were shown to be stable during all sample storage and analysis procedures. Copyright © 2013 John Wiley & Sons, Ltd.     

Structure and end‐group analysis of complex hexanediol‐neopentylglycol‐adipic acid copolyesters by matrix‐assisted laser desorption/ionization collision‐induced dissociation tandem mass spectrometry

Sequences and end groups of complex copolyesters were determined by fragmentation analysis by means of matrix‐assisted laser desorption/ionization collision‐induced dissociation tandem mass spectrometry (MALDI CID MS/MS). The complexity of the crude copolyester mixture was reduced by a chromatographic separation followed by a MALDI time‐of‐flight (TOF) investigation of fractions. Due to overlapping compositional and end‐group information a clear assignment of end groups was very difficult. However, the fragmentation of suitable precursor ions resulted in typical fragment ion patterns and, therefore, enabled a fast and unambiguous determination of the end groups and composition of this important class of polymers. Copyright © 2009 John Wiley & Sons, Ltd.     

Simultaneous determination of neochlorogenic acid,chlorogenic acid,cryptochlorogenic acid and geniposide in rat plasma by UPLC‐MS/MS and its application to a pharmacokinetic study after administration of Reduning injection

A simple, specific and sensitive ultra‐performance liquid chromatography tandem mass spectrometry (UPLC‐MS/MS) method was established and validated for simultaneous determination of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid and geniposide in rat plasma using puerarin as an internal standard (IS). Plasma samples were pretreated by a one‐step direct protein precipitation procedure with acetonitrile after acidified using as little as 50 μL plasma. Chromatographic separation was performed on an Acquity BEH C₁₈ column (100 × 2.1 mm, 1.7 µm) at a flow rate of 0.2 mL/min by a gradient elution, using 0.2% acetic acid–methanol as mobile phase. The detection was performed on a triple quadrupole tandem mass spectrometer by multiple reaction monitoring via electrospray ionization source with negative ion mode. Calibration curves showed good linearity (r > 0.995) over wide concentration ranges. The intra‐ and inter‐day precisions were <15%, and the accuracy was within ±8.0%. The validated method was successfully applied to a pharmacokinetic study of the four bioactive components in rats after intravenous administration of Reduning injection. Copyright © 2014 John Wiley & Sons, Ltd.     

Gas‐phase fragmentation of protonated piplartine and its fungal metabolites using tandem mass spectrometry and computational chemistry

Piplartine, an alkaloid produced by plants in the genus Piper , displays promising anticancer activity. Understanding the gas‐phase fragmentation of piplartine by electrospray ionization tandem mass spectrometry can be a useful tool to characterize biotransformed compounds produced by in vitro and in vivo metabolism studies. As part of our efforts to understand natural product fragmentation in electrospray ionization tandem mass spectrometry, the gas‐phase fragmentation of piplartine and its two metabolites 3,4‐dihydropiplartine and 8,9‐dihydropiplartine, produced by the endophytic fungus Penicillium crustosum VR4 biotransformation, were systematically investigated. Proposed fragmentation reactions were supported by ESI‐MS/MS data and computational thermochemistry. Cleavage of the C‐7 and N‐amide bond, followed by the formation of an acylium ion, were characteristic fragmentation reactions of piplartine and its analogs. The production of the acylium ion was followed by three consecutive and competitive reactions that involved methyl and methoxyl radical eliminations and neutral CO elimination, followed by the formation of a four‐member ring with a stabilized tertiary carbocation. The absence of a double bond between carbons C‐8 and C‐9 in 8,9‐dihydropiplartine destabilized the acylium ion and resulted in a fragmentation pathway not observed for piplartine and 3,4‐dihydropiplartine. These results contribute to the further understanding of alkaloid gas‐phase fragmentation and the future identification of piplartine metabolites and analogs using tandem mass spectrometry techniques. Copyright © 2017 John Wiley & Sons, Ltd.     

Ultra‐performance liquid chromatography tandem mass spectrometry for the rapid,simultaneous analysis of ziprasidone and its impurities

The separation and characterization of the unknown degradation product of second‐generation antipsychotic drug ziprasidone are essential for defining the genotoxic potential of the compound. The aim of this study was to develop a simple UHPLC method coupled with tandem mass spectrometry (MS/MS) for chemical characterization of an unknown degradant, and the separation and quantification of ziprasidone and its five main impurities (I–V) in the raw material and pharmaceuticals. Chromatographic conditions were optimized by experimental design. The MS/MS fragmentation conditions were optimized individually for each compound in order to obtain both specific fragments and high signal intensity. A rapid and sensitive UHPLC–MS/MS method was developed. All seven analytes were eluted within the 7 min run time. The best separation was obtained on the Acquity UPLC BEH C₁₈ (50 × 2.1 mm × 1.7 μm) column in gradient mode with ammonium‐formate buffer (10 mm ; pH 4.7) and acetonitrile as mobile phase, with the flow rate of 0.3 mL min^?1 and at the column temperature of 30°C. The new UHPLC–MS/MS method was fully validated and all validation parameters were confirmed. The fragmentation pathways and chemical characterization of an unknown degradant were proposed and it was confirmed that there are no structural alerts concerning genotoxicity.     

GC‐MS of amaryllidaceous galanthamine‐type alkaloids

Galanthamine‐type alkaloids produced by plants of the Amaryllidaceae family are potent acetylcholinesterase inhibitors. One of them, galanthamine, has been marketed as a hydrobromide salt for the treatment of Alzheimer's disease. In the present work, gas chromatography with electron impact mass spectrometry (GC‐EIMS) fragmentation of 12 reference compounds isolated from various amaryllidaceous plants and identified by spectroscopic methods (1D and 2D nuclear magnetic resonance, circular dichroism, high‐resolution MS (HRMS) and EIMS) was studied by tandem mass spectrometry (GC‐MS/MS) and accurate mass measurements (GC‐HRMS). The studied compounds showed good peak shape and efficient GC separation with a GC‐MS fragmentation pattern similar to that obtained by direct insertion probe. With the exception of galanthamine‐N‐oxide and N‐formylnorgalanthamine, the galanthamine‐type compounds showed abundant [M]^+. and [M‐H]⁺ ions. A typical fragmentation pattern was also observed, depending on the substituents of the skeleton. Based on the fragmentation pathways of reference compounds, three other galanthamine‐type alkaloids, including 3‐O‐(2′‐butenoyl)sanguinine, which possesses a previously unelucidated structure, were identified in Leucojum aestivum ssp. pulchelum, a species endemic to the Balearic islands. GC‐MS can be successfully applied to Amaryllidaceae plant samples in the routine screening for potentially new or known bioactive molecules, chemotaxonomy, biodiversity and identification of impurities in pharmaceutical substances. Copyright © 2012 John Wiley & Sons, Ltd.     

Determination of ecliptasaponin A in rat plasma and tissues by liquid chromatography‐tandem mass spectrometry

A sensitive, rapid and specific high‐performance liquid chromatography tandem mass spectrometry method (HPLC‐MS/MS) was developed to determine ecliptasaponin A in rat plasma and tissues after oral administration. Ginsenoside Rg1 was used as the internal standard (IS). The plasma and tissues samples were prepared by liquid‐liquid extraction with ethyl acetate and separated on an Eclipse Plus C₁₈ column (2.1 mm × 150 mm, 5 µm) at a flow rate of 0.4 mL/min using acetonitrile and water (containing 0.05% acetic acid) as the mobile phase. The tandem mass detection was carried out with eletrospray ionization in negative mode. Quantification was performed by using multiple reaction monitoring (MRM), which monitored the fragmentation of m/z 633.4→587.2 for ecliptasaponin A and m/z 859.4→637.4 for the IS. The calibration curves obtained were linear in different matrices, and the lower limit of quantification (LLOQ) achieved was 0.5 ng/mL both for rat plasma and tissues. The intra‐ and inter‐day precisions were below 15%. This method was successfully applied to pharmacokinetic study of ecliptasaponin A in rat plasma and tissues after oral administration. Copyright © 2015 John Wiley & Sons, Ltd.     

High‐efficiency sample preparation approach to determine acrylamide levels in high‐fat foods

An improved sample preparation method was developed to enhance acrylamide recovery in high‐fat foods. Prior to concentration, distilled deionized water was added to protect acrylamide from degradation, resulting in a higher acrylamide recovery rate from fried potato chips. A Chrome‐Matrix C₁₈ column (2.6 μm, 2.1 × 100 mm) was used for the first time to analyze acrylamide levels using ultra high performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry, displaying good separation of acrylamide from interference. A solid‐phase extraction procedure was avoided, and an average recovery of >89.00% was achieved from different food matrices for three different acrylamide spiking levels. Good reproducibility was observed, with an intraday relative standard deviation of 0.04–2.38%, and an interday relative standard deviation of 2.34–3.26%. Thus, combining the improved sample preparation method for acrylamide analysis with the separation on a Chrome‐Matrix C₁₈ column (2.6 μm, 2.1 × 100 mm) using ultra high performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry is highly useful for analyzing acrylamide levels in complex food matrices.     

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.     

Rapid separation and identification of major constituents in Pseudolarix kaempferi by ultra‐performance liquid chromatography coupled with electrospray and quadrupole time‐of‐flight tandem mass spectrometry

A rapid and reliable method based on ultra‐performance liquid chromatography (UPLC) coupled with photodiode‐array detection (PDA) and electrospray ionization quadrupole time‐of‐flight tandem mass spectrometry (ESI‐Q‐TOF‐MS/MS) has been developed for separation and identification of major constituents in extracts of root bark of Pseudolarix kaempferi Gordon (PKG). Identification of the constituents was carried out by interpretation of their retention times, UV absorption spectra, MS and MS/MS spectra, as well as the data provided by authentic standards and literatures. A total of 20 components were separated in only 8.0 min on a small particle size C₁₈ column (1.7 µm). These components included nine diterpene acids, seven glycosides and four triterpenoids, among which pseudolaric acid C‐O‐β‐D‐glucopyranoside and pseudolaric acid C₂‐O‐β‐D‐glucopyranoside were separated and identified for the first time in this study. Furthermore, the fragmentation patterns of the three types of compounds were elucidated for the first time. This established UPLC‐PDA/Q‐TOF‐MS/MS method is reliable and effective for the separation and identification of the 20 compounds and will be useful for quality control of the crude materials of Pseudolarix kaempferi Gordon and their related preparations. Copyright © 2009 John Wiley & Sons, Ltd.     

Determination of triclosan metabolites by using in‐source fragmentation from high‐performance liquid chromatography/negative atmospheric pressure chemical ionization ion trap mass spectrometry

Triclosan is a widely used broad‐spectrum antibacterial agent that acts by specifically inhibiting enoyl–acyl carrier protein reductase. An in vitro metabolic study of triclosan was performed by using Sprague‐Dawley (SD) rat liver S9 and microsome, while the in vivo metabolism was investigated on SD rats. Twelve metabolites were identified by using in‐source fragmentation from high‐performance liquid chromatography/negative atmospheric pressure chemical ionization ion trap mass spectrometry (HPLC/APCI‐ITMS) analysis. Compared to electrospray ionization mass spectrometry (ESI‐MS) and tandem mass spectrometry (MS/MS) that gave little fragmentation for triclosan and its metabolites, the in‐source fragmentation under APCI provided intensive fragmentations for the structural identifications. The in vitro metabolic rate of triclosan was quantitatively determined by using HPLC/ESI‐ITMS with the monitoring of the selected triclosan molecular ion. The metabolism results indicated that glucuronidation and sulfonation were the major pathways of phase II metabolism and the hydroxylated products were the major phase I metabolites. Moreover, glucose, mercapturic acid and cysteine conjugates of triclosan were also observed in the urine samples of rats orally administrated with triclosan. Copyright © 2010 John Wiley & Sons, Ltd.     

Characterization of different poly(2‐oxazoline) block copolymers by MALDI‐TOF MS/MS and ESI‐Q‐TOF MS/MS

A complete library of poly(2‐oxazoline) block copolymers was synthesized via cationic ring opening polymerization for the characterization by two different soft ionization techniques, namely matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOF MS) and electrospray ionization quadrupole time‐of‐flight mass spectrometry (ESI‐Q‐TOF MS). In addition, a detailed characterization was performed by tandem MS to gain more structural information about the block copolymer composition and its fragmentation behavior. The fragmentation of the poly(2‐oxazoline) block copolymers revealed the desired polymer structure and possible side reactions, which could be explained by different mechanisms, like 1,4‐ethylene or hydrogen elimination and the McLafferty +1 rearrangement. Polymers with aryl side groups showed less fragmentation due to their higher stability compared to polymers with alkyl side groups. These insights represent a further step toward the construction of a library with fragments and their fragmentation pathways for synthetic polymers, following the successful examples in proteomics. © 2010 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem, 2010     

The determination of capreomycin in human plasma by LC–MS/MS using ion‐pairing chromatography and solid‐phase extraction

A bioanalytical method was developed and validated for the quantification of capreomycin (Cm) analogs, Cm IA and Cm IB, in human plasma. This implemented ion‐pairing solid phase extraction, followed by ion‐pairing high‐performance liquid chromatography, with tandem mass spectrometry detection. Chromatographic separation was achieved using a Discovery C₁₈, 5 μm, 4.6 × 50 mm analytical column. An isocratic mobile phase consisting of water and acetonitrile with 0.1% formic acid and 4mm heptafluorobutyric acid (80:20; v/v) was used at a flow‐rate of 500 μL/min. An AB Sciex API 3000 mass spectrometer at unit resolution, in multiple reaction monitoring mode, was used for detection. Electrospray ionization was used for ion production. The method was successfully validated for the range 469–30,000 ng/mL for Cm IA and for Cm IB, with cefotaxime as the internal standard. The within‐ and between‐day precision determinations for Cm IA and IB, expressed as the percentage coefficient of variation, were < 20.0% at the lower limit of quantification (LLOQ) and < 8.2% at all other test concentrations. Recovery of both analogs was > 72.3% and reproducible at the low, medium and high end of the calibration range. No significant matrix effects were observed for the analyte. The assay performed well when applied to clinical samples generated from children in a clinical multidrug resistant tuberculosis research study in South Africa.