应用高效液相色谱和电喷雾质谱联用技术分析鉴定车前草中的苯乙醇苷化合物

本文采用高效液相色谱与电喷雾质谱联用技术在线分析鉴定了车前草提取物中的三种苯乙醇苷化合物。实验采用反相C18色谱柱,0.2%的醋酸水溶液和乙腈梯度洗脱,车前草中的苯乙醇苷化合物得到很好的分离。在电喷雾质谱负离子条件下,获得了三种苯乙醇苷化合物的分子离子峰,分子量信息,进一步通过质谱的源内CID技术得到相应化合物的结构信息。通过得到的这些信息与文献中的已知化合物或标准品对照从而推断出化合物的结构。     

Direct characterization of aqueous extract of Hibiscus sabdariffa using HPLC with diode array detection coupled to ESI and ion trap MS

The phenolic fraction and other polar compounds of the Hibiscus sabdariffa were separated and identified by HPLC with diode array detection coupled to electrospray TOF and IT tandem MS (DAD‐HPLC‐ESI‐TOF‐MS and IT‐MS). The H. sabdariffa aqueous extract was filtered and directly injected into the LC system. The analysis of the compounds was carried out by RP HPLC coupled to DAD and TOF‐MS in order to obtain molecular formula and exact mass. Posterior analyses with IT‐MS were performed and the fragmentation pattern and confirmation of the structures were achieved. The H. sabdariffa samples were successfully analyzed in positive and negative ionization modes with two optimized linear gradients. In positive mode, the two most representative anthocyanins and other compounds were identified whereas the phenolic fraction, hydroxycitric acid and its lactone were identified using the negative ionization mode.     

Comprehensive identification and structural characterization of target components from Gelsemium elegans by high‐performance liquid chromatography coupled with quadrupole time‐of‐flight mass spectrometry based on accurate mass databases combined with MS/MS spectra

This study reports an applicable analytical strategy of comprehensive identification and structure characterization of target components from Gelsemium elegans by using high‐performance liquid chromatography quadrupole time‐of‐flight mass spectrometry (LC‐QqTOF MS) based on the use of accurate mass databases combined with MS/MS spectra. The databases created included accurate masses and elemental compositions of 204 components from Gelsemium and their structural data. The accurate MS and MS/MS spectra were acquired through data‐dependent auto MS/MS mode followed by an extraction of the potential compounds from the LC‐QqTOF MS raw data of the sample. The same was matched using the databases to search for targeted components in the sample. The structures for detected components were tentatively characterized by manually interpreting the accurate MS/MS spectra for the first time. A total of 57 components have been successfully detected and structurally characterized from the crude extracts of G. elegans , but has failed to differentiate some isomers. This analytical strategy is generic and efficient, avoids isolation and purification procedures, enables a comprehensive structure characterization of target components of Gelsemium and would be widely applicable for complicated mixtures that are derived from Gelsemium preparations. Copyright © 2017 John Wiley & Sons, Ltd.     

Development of a tandem time‐of‐flight mass spectrometer with an electrospray ionization ion source

A new tandem time‐of‐flight mass spectrometer with an electrospray ionization ion source ‘ESI‐TOF/quadTOF’ was designed and constructed to achieve the desired aim of structural elucidation via high‐energy collision‐induced dissociation (CID), and the simultaneous detection of all fragment ions. The instrument consists of an orthogonal acceleration‐type ESI ion source, a linear TOF mass spectrometer, a collision cell, a quadratic‐field ion mirror and a microchannel plate detector. High‐energy CID spectra of doubly protonated angiotensin II and bradykinin were obtained. Several fragment ions such as a‐, d‐, v‐ and w‐type ions, characteristic of high‐energy CID, were clearly observed in these spectra. These high‐energy CID fragment ions enabled confirmation of the complete sequence, including leucine–isoleucine determinations. It was demonstrated that high‐energy CID of multiply protonated peptides could be achieved in the ESI‐TOF/quadTOF. Copyright © 2010 John Wiley & Sons, Ltd.     

Analysis of phenolic compounds from different morphological parts of Helichrysum devium by liquid chromatography with on‐line UV and electrospray ionization mass spectrometric detection

A simple and rapid method has been used for the screening and identification of the main phenolic compounds from Helichrysum devium using high‐performance liquid chromatography with on‐line UV and electrospray ionization mass spectrometric detection (LC‐DAD/ESI‐MSⁿ). The total aerial parts and different morphological parts of the plant, namely leaves, flowers and stems, were analyzed separately. A total of 34 compounds present in the methanolic extract from Helichrysum devium were identified or tentatively characterized based on their UV and mass spectra and retention times. Three of these compounds were positively identified by comparison with reference standards. The phenolic compounds included derivatives of quinic acid, O‐glycosylated flavonoids, a caffeic acid derivative and a protocatechuic acid derivative. The characteristic loss of 206 Da from malonylcaffeoyl quinic acid was used to confirm the malonyl linkage to the caffeoyl group. This contribution presents one of the first reports on the analysis of phenolic compounds from Helichrysum devium using LC‐DAD/ESI‐MSⁿ and highlights the prominence of quinic acid derivatives as the main group of phenolic compounds present in these extracts. We also provide evidence that the methanolic extract from the flowers was significantly more complex when compared to that of other morphological parts. Copyright © 2009 John Wiley & Sons, Ltd.     

Qualitative and quantitative characterization of the arsenic‐binding behaviour of sulfur‐containing peptides and proteins by the coupling of reversed phase liquid chromatography to electrospray ionization mass spectrometry

Phenylarsenic‐substituted cysteine‐containing peptides and proteins were completely differentiated from their unbound original forms by the coupling of reversed phase liquid chromatography with electrospray ionization mass spectrometry. The analysis of biomolecules possessing structure‐stabilizing disulfide bridges after reduction provides new insights into requirements concerning the accessibility of cysteine residues for reducing agents as well as for arsenic compounds in a spatial protein structure. Complementary binding studies performed using direct ESI‐MS without chromatographic coupling in different solvent systems demonstrated that more than one binding site were activated for aprotinin and lysozyme in denaturing solvents because of a stronger defolding. From the intensities of the different charge states occurring in the mass spectra as well as from the LC elution behaviour, it can be deduced that the folding state of the arsenic‐bound protein species resembles the native, oxidized conformation. In contrast, although the milk protein α‐lactalbumin has several disulfide bridges, only one phenylarsenic moiety was bound under strongly denaturing conditions. Because of the charge state distribution in the ESI mass spectra, a conformational change to a molten globule structure is assumed. For the second considered milk protein ß‐lactoglobulin, a noncovalent interaction with phenylarsine oxide was detected. In general, smaller apparent binding constants for the condensation reactions of the biomolecules with phenylarsine oxide leading to covalent arsenic–sulfur bindings were determined from direct injection ESI‐MS measurements than from LC‐ESI‐MS coupling. The following order of binding affinities for one phenylarsenic group can be assumed from both ESI‐MS and LC‐ESI‐MS: nonapeptide vasopressin > nonapeptide vasotocin > lysozyme > aprotinin > α‐lactalbumin > thioredoxin. Kinetic investigations by LC‐ESI‐MS yielded a partial reaction order of 2 for vasopressin, Lys and α‐lactalbumin and corresponding half‐lives of 0.93, 2.56 and 123.5 min, respectively. Copyright © 2012 John Wiley & Sons, Ltd.     

Characterization of N‐Boc/Fmoc/Z‐N′‐formyl‐gem‐diaminoalkyl derivatives using electrospray ionization multi‐stage mass spectrometry

N‐Boc/Fmoc/Z‐N′‐formyl‐gem‐diaminoalkyl derivatives, intermediates particularly useful in the synthesis of partially modified retro‐inverso peptides, have been characterized by both positive and negative ion electrospray ionization (ESI) ion‐trap multi‐stage mass spectrometry (MSⁿ). The MS² collision induced dissociation (CID) spectra of the sodium adduct of the formamides derived from the corresponding N‐Fmoc/Z‐amino acids, dipeptide and tripeptide acids show the [M + Na‐NH₂CHO]⁺ ion, arising from the loss of formamide, as the base peak. Differently, the MS² CID spectra of [M + Na]⁺ ion of all the N‐Boc derivatives yield the abundant [M + Na‐C₄H₈]⁺ and [M + Na‐Boc + H]⁺ ions because of the loss of isobutylene and CO₂ from the Boc protecting function. Useful information on the type of amino acids and their sequence in the N‐protected dipeptidyl and tripeptidyl‐N′‐formamides is provided by MS² and subsequent MSⁿ experiments on the respective precursor ions. The negative ion ESI mass spectra of these oligomers show, in addition to [M‐H]^?, [M + HCOO]^? and [M + Cl]^? ions, the presence of in‐source CID fragment ions deriving from the involvement of the N‐protecting group. Furthermore, MSⁿ spectra of [M + Cl]^? ion of N‐protected dipeptide and tripeptide derivatives show characteristic fragmentations that are useful for determining the nature of the C‐terminal gem‐diamino residue. The present paper represents an initial attempt to study the ESI‐MS behavior of these important intermediates and lays the groundwork for structural‐based studies on more complex partially modified retro‐inverso peptides. Copyright © 2013 John Wiley & Sons, Ltd.     

Study of active naphtodianthrone St John's Wort compounds by electrospray ionization Fourier transform ion cyclotron resonance and multi‐stage mass spectrometry in sustained off‐resonance irradiation collision‐induced dissociation and infrared multiphoton dissociation modes

Five well‐known active naphtodianthrone constituents of Hypericum perforatum (St John's Wort) extracts have been investigated by electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (ESI‐FTICRMS) and ESI‐FTICRMSⁿ. The studied compounds were hypericin, pseudohypericin, protohypericin, protopseudohypericin (biosynthetic precursors of the two former compounds, respectively) and isopseudohypericin (alkaline degradation product of pseudohypericin). Dissociation mass spectrometry measurements performed on the [M–H]^? ion presented a variable efficiency as a function of the used activation mode. Sustained off‐resonance irradiation collision‐induced dissociation (SORI–CID) only led to a restricted number of fragment ions. In contrast, IRMPD ensured the detection of numerous product ions. Ions detected in ESI‐FTICRMS and ESI‐FTICRMSⁿ experiments were measured with a very high mass accuracy (typically mass error is lower than 0.5 mDa at m/z close to 500) that allowed unambiguous formulae to be assigned to each signal observed in a mass spectrum. In spite of similar structures, specific fragmentation patterns were observed for the different compounds investigated. This study may be useful in the future to characterize in natural extracts these compounds (or derivatives of these compounds) by liquid chromatography/tandem mass spectrometry (LC/MS/MS) experiments by considering the MS/MS transitions highlighted in this paper. Copyright © 2009 John Wiley & Sons, Ltd.     

Differentiation of lisinopril and its RSS diastereomer by liquid chromatography combined with collision‐induced dissociation mass spectrometry

A simple and sensitive liquid chromatography tandem multiple‐stage mass spectrometry (HPLC/MS/MS) method suitable for bulk lisinopril analysis was developed, by which lisinopril and its RSS isomer were separated and differentiated. In the collision‐induced dissociation (CID) mass spectra of the [M + H]⁺ ions, the abundance of the fragment ion of m/z 246 for lisinopril was about two times higher than the ion of m/z 245; however, the former fragment ion was noted to be a little lower than the latter for RSS isomer at all collision energies. In the CID mass spectra of the [M + Li]⁺ ion, the abundance of the rearrangement ion of m/z 315 for the RSS isomer was about three times higher than that for lisinopril. Furthermore, the difference was supported by the results of energy‐resolved mass spectrometry (ERMS) in the test range of collision energies. Similar differences were also observed between the CID mass spectra of lisinopril and RSS isomer methylester, which indicated that the RSS isomer could be rapidly characterized by the CID mass spectra of both the protonated and lithium adduct ion. Elemental compositions of all the ions were confirmed by Fourier Transform ion cyclotron resonance ESI mass spectrometry (FT‐ICR‐ESI/MS). In addition, theoretical computations were carried out to support the experimental results. Copyright © 2009 John Wiley & Sons, Ltd.     

Characterization and quantification of 10‐hydroxycamptothecine in Camptotheca acuminate and its medicinal preparation by liquid chromatography–ion trap mass spectrometry

A rapid and sensitive method for the identification and quantification of 10‐hydroxycamptothecine (HCPT) in Camptotheca acuminata Decne is described. The HCPT standard solution was directly infused into the ion trap mass spectrometers (IT/MS) for collecting the MSⁿ spectra. The electrospray ionization (ESI) mass spectral fragmentation pathway of HCPT was proposed and the ESI‐MSⁿ fragmentation behavior of HCPT was deduced in detail. The major fragment ions of HCPT were confirmed by MSⁿ in both negative ion and positive ion mode. The possible main cleavage pathway of fragment ions was studied. Quantification of HCPT was assigned in negative‐ion mode at a product ion at m/z 363 → 319 by LC‐MS. The LC‐MS method was validated for linearity, sensitivity, accuracy and precision, and then used to determine the content of the HCPT. Lastly, the LC‐MS method was successfully applied to determine HCPT in real samples of Camptotheca acuminate Decne and its medicinal preparation in the first time. Copyright © 2013 John Wiley & Sons, Ltd.     

Combined mass spectrometric and chromatographic methods for in‐depth analysis of phenolic secondary metabolites in barley leaves

Structural analysis via HPLC‐ESI‐MSn, UPLC‐HESI‐MS/MS and NMR reported 152 phenolic secondary metabolites in spring barley seedlings (Hordeum vulgare L.). Flavonoids with various patterns of glycosylation and acylation, as well as hydroxycinnamic acid glycosides, esters and amides, were identified in methanolic extracts from leaves of nine varieties of barley originating from different regions of the world. Hordatines derivatives, flavones acylated directly on the aglycone, and hydroxyferulic acid derivatives deserve special attention. Preparative chromatography enabled characterization of a number of compounds at trace levels with the 6‐C‐[6″‐O‐glycosyl]‐glycosides and the 6‐C‐[2″,6″‐di‐O‐glycosides]‐glucoside structure of flavones. Derivatives of flavonols, quercetin and isorhamnetin were observed only in Syrian varieties. The ultra performance liquid chromatography profiles of UV‐absorbing secondary metabolites were used for chemotaxonomic comparison between nine varieties of barley from different climatic conditions. The hierarchical clustering of bred lines from the Fertile Crescent and European and American varieties indicates a great diversity of chemical phenotypes within barley species. Copyright © 2015 John Wiley & Sons, Ltd.     

Effect of pH on the Metabolism of Aconitine under Rat Intestinal Bacteria and Analysis of Metabolites Using HPLC/MS-MSn Technique

A semi‐quantitative method of mass spectrometry (MS) has been described for the analysis of metabolites of aconitine by rat intestinal bacteria at different pH. At pH 7.0, the rat intestinal bacteria exhibit optimal activity for the metabolism of aconitine. A high‐performance liquid chromatography‐electrospray ionization multiple‐stage mass spectrometry (HPLC/ESI‐MSⁿ) method has been applied to investigate the characteristic product ions of metabolites. Then, the logical fragmentation pathways of metabolites have been proposed. By comparing the retention time (t_R) of HPLC and the ESI‐MSⁿ data with the data of standard compounds and reports from literature, ten metabolites have been identified and a distinctive metabolite (15‐deoxyaconitine) has been deduced first time. The experimental results demonstrate that HPLC/ESI‐MSⁿ is a specific and useful method for the identification of metabolites of aconitine. Also, in the present paper, the HPLC‐MS method was introduced to determine the synthetical metabolite prior to the study of the toxicity by the method of Bliss.     

Challenges in the identification process of phenidate analogues in LC‐ESI‐MS/MS analysis: Information enhancement by derivatization with isobutyl chloroformate

A new analytical technique for the structural elucidation of four representative phenidate analogues possessing a secondary amine residue, which leads to a major/single amine‐representative fragment/product ion at m/z 84 both in their GC‐EI‐MS and LC‐ESI‐MS/MS spectra, making their identification ambiguous, was developed. The method is based on “in vial” chemical derivatization with isobutyl chloroformate in both aqueous and organic solutions, followed by liquid chromatography‐electrospray ionization mass spectrometry (LC‐ESI‐MS/MS). The resulting carbamate derivatives promote rich fragmentation patterns with full coverage of all substructures of the molecule, enabling detailed structural elucidation and unambiguous identification of the original compounds at low ng/mL levels.     

Quality assessment of Cortex Phellodendri by high‐performance liquid chromatography coupled with electrospray ionization mass spectrometry

A simple method based on liquid chromatography coupled with diode array detection and electrospray ionization mass spectrometry (LC‐DAD‐ESI‐MS) was developed for the quality assessment of Cortex Phellodendri (CP), which was mainly derived from two species of Phellodendron chinense Schneid and Phellodendron amurense Rupr. Total 41 compounds, including 14 phenols, 24 alkaloids and three liminoidal triterpenes were identified or tentatively characterized from the 75% methanol extract of CP samples by online ESI‐MSⁿ fragmentation and UV spectra analysis. Among them, two phenols and six alkaloids were simultaneously quantified using HPLC‐DAD method. The validated HPLC‐DAD method showed a good linearity, precision, repeatability and accuracy for the quantification of eight marker compounds. Furthermore, the plausible fragmentation pathway of the representative compounds were proposed in the present study. The differences of the chemical constituents content and the comprehensive HPLC profiles between the two CP species using LC‐DAD‐ESI‐MS method are reported for the first time, indicating that the CP drugs from different resources should be used separately in the clinic. Copyright © 2009 John Wiley & Sons, Ltd.     

Distinguishing of 2‐MAPB and 6‐MAPB: Solution of the problem

Differentiation of new psychoactive substance (NPS), 6‐(2‐methylaminopropyl)benzofuran (6‐MAPB), and its positional isomer, 2‐(2‐methylaminopropyl)benzofuran (2‐MAPB), by means of gas chromatography/mass spectrometry (GC/MS) with quadrupole detection is ambiguous. Reliable distinguishing of the two isomers could be achieved by MS/MS spectra recorded after collision‐induced dissociation (CID) of precursor ions. Both electron ionization (EI) and electrospray ionization (ESI) methods could be used for these purposes.     

Global detection and identification of components from crude and processed traditional Chinese medicine by liquid chromatography connected with hybrid ion trap and time‐of‐flight‐mass spectrometry

We herein present a chemical profiling method to efficiently process the information acquired by ultra fast liquid chromatography (UFLC)‐electrospray ionization source in combination with hybrid ion trap and high‐resolution time‐of‐flight mass spectrometry (UFLC‐(ESI)‐IT‐TOF/MS), facilitating the structural determination of serial components contained in crude or processed traditional Chinese medicine (TCM). Under the optimized UFLC and IT‐TOF‐MSⁿ conditions, over 39 compounds were separated and detected in crude or processed Fructus corni within 25 min. The components were identified by comparing the mass spectra and retention time with reference compounds, or tentatively assigned by elucidating low‐energy collision‐induced dissociation (CID) fragment ions and matching empirical molecular formula with that of the published compounds. Several factors in the processing procedure were examined. The experimental results demonstrate that the chemical reactions that occurred in the processing procedure can be used to elucidate the processed mechanism of F. corni, which is regularly affected by the processing conditions. This study provides a novel approach and methodology to identify the complicated components from various complex mixtures such as crude TCM, processed TCM, and biological samples. It can be used as a valid analytical method for further understanding the processing mechanism of TCM, along with the intrinsic quality control of TCM and its processed product.     

Analysis of fungal cyclopentenone derivatives from Hygrophorus spp. by liquid chromatography/electrospray-tandem mass spectrometry

Fruitbodies of the genus Hygrophorus (Basidiomycetes) contain a series of anti-biologically active compounds. These substances named hygrophorones possess a cyclopentenone skeleton. LC/ESI-MS/MS presents a valuable tool for the identification of such compounds. The mass spectral behaviour of typical selected members of this group under positive and negative ion electrospray conditions is discussed. Using the ESI collision-induced dissociation (CID) mass spectra of the [M + H]+ and [M - H]- ions, respectively, the compounds can be classified with respect to the substitution pattern at the cyclopentenone ring and the type of oxygenation at C-6 (hydroxy/acetoxy or oxo function) of the side chain. The elemental composition of the fragment ions was determined by ESI-QqTOF measurements. Thus, in case of the negative ion CID mass spectra an unusual loss of CO2 from the deprotonated molecular ions could be observed.     

Liquid chromatography–tandem mass spectrometry of porphyrins and porphyrinogens in biological materials: separation and identification of interfering poly(ethylene) glycol by travelling wave ion mobility spectrometry/tandem mass spectrometry

Biological and clinical samples for porphyrin and porphyrinogen analyses by liquid chromatography–tandem mass spectrometry (LC‐MS/MS) are often contaminated with poly(ethylene)glycol (PEG), which complicates the interpretation of mass spectra and characterisation of new porphyrin metabolites. Two contaminating PEG molecules (m/z 833 and m/z 835) were completely separated from uroporphyrin I (m/z 831) by travelling wave ion mobility spectrometry and characterised by tandem mass spectrometry. One of the PEG species (m/z 835) also co‐eluted with uroporphyrinogen I (m/z 837) and was unresolvable by travelling wave ion mobility spectrometry/MS, therefore contaminating the MS/MS mass spectra owing to isotope distribution. These PEG species, with the [M + H]⁺ ions at m/z at 833 and/or m/z 835, co‐eluted with uroporphyrin I and uroporphyrinogen I by LC‐MS/MS and could be wrongly identified as uroporphomethenes. Copyright © 2013 John Wiley & Sons, Ltd.     

A simultaneous determination method for 5‐fluorouracil and its metabolites in human plasma with linear range adjusted by in‐source collision‐induced dissociation using hydrophilic interaction liquid chromatography–electrospray ionization–tandem mass spectrometry

We applied a new technique for quantitative linear range shift using in‐source collision‐induced dissociation (CID) to complex biological fluids to demonstrate its utility. The technique was used in a simultaneous quantitative determination method of 5‐fluorouracil (5‐FU), an anticancer drug for various solid tumors, and its metabolites in human plasma by liquid chromatography–electrospray ionization–tandem mass spectrometry (LC/ESI‐MS/MS). To control adverse effects after administration of 5‐FU, it is important to monitor the plasma concentration of 5‐FU and its metabolites; however, no simultaneous determination method has yet been reported because of vastly different physical and chemical properties of compounds. We developed a new analytical method for simultaneously determining 5‐FU and its metabolites in human plasma by LC/ESI‐MS/MS coupled with the technique for quantitative linear range shift using in‐source CID. Hydrophilic interaction liquid chromatography using a stationary phase with zwitterionic functional groups, phosphorylcholine, was suitable for separation of 5‐FU from its nucleoside and interfering endogenous materials. The addition of glycerin into acetonitrile‐rich eluent after LC separation improved the ESI‐MS response of high polar analytes. Based on the validation results, linear range shifts by in‐source CID is the reliable technique even with complex biological samples such as plasma. Copyright © 2016 John Wiley & Sons Ltd.