Application of characteristic fragment filtering with ultra high performance liquid chromatography coupled with high‐resolution mass spectrometry for comprehensive identification of components in Schisandrae chinensis Fructus

An integrated strategy of characteristic fragment filtering combined with target database screening based on ultra‐high‐performance liquid chromatography coupled with high‐resolution mass spectrometry was proposed for comprehensive profiling of components in Schisandrae chinensis Fructus. The strategy consisted of following five steps: (1) Representative standards were analyzed by ultra high performance liquid chromatography coupled with linear ion trap‐Orbitrap mass spectrometer for characteristic fragments and fragmentation rules of each structure type. (2) The raw data of 70% methanol extract was collected by ultra high performance liquid chromatography quadrupole time‐of‐flight tandem mass spectrometry. (3) The chemical components database that consisted of names, chemical formulas and structures of potential components in Schisandrae chinensis Fructus was established by summarizing previous literature to screen the collected liquid chromatography with mass spectrometry data and obtain matched compounds. (4) Characteristic fragments, literature, and reference standards were used to verify the matches. (5) Characteristic fragment filtering combined with online database querying was used to deduce potential new compounds. As a result, a total of 94 compounds were identified or characterized and 16 of them were potential new compounds. The study provided a reference for comprehensive characterization of ingredients in herbal medicine and formed the foundation for pharmacodynamic study of Schisandrae chinensis Fructus.     

Characterization of the potential new phthalides in Ligusticum chuanxiong Hort. using ultra‐performance liquid chromatography coupled with quadrupole time of flight tandem mass spectrometry

The characterization of unknown compounds is still a great challenge currently. A strategy for deduction of potential new phthalides through the characterization of isomers based on ultra‐performance liquid chromatography coupled with quadrupole time of flight tandem mass spectrometry was proposed here to characterize the unknown compounds of Ligusticum chuanxiong Hort. (Chuanxiong). This proposed strategy consisted of four steps: (1) the high resolution MS data was collected, and the peaks were screened preliminarily by UNIFI^TM platform based on the in‐house database; (2) the fragmentation patterns and the characteristic fragments were summarized based on the representative standards; (3) the target compounds were identified based on the fragmentation rules, standards comparison and false positive exclusion; (4) the unknown components were structurally characterized according to the accurate mass and fragmentation patterns analysis. This strategy was successfully applied to the identification and deduction of phthalides in Chuanxiong. A total of 81 phthalides were detected. Fifty‐five known phthalides were identified, and 26 potential new phthalides were characterized. This research enriched the material basis of Chuanxiong, and provided a liquid chromatography tandem mass spectrometry‐oriented method for the discovery of the potential new compounds.     

Characterization of polyphenol compounds from the roots and stems of Parthenocissus laetevirens by high‐performance liquid chromatography/tandem mass spectrometry

A facile method based on high‐performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry (HPLC/(–)ESI‐MSⁿ) has been established for the analyses of polyphenol compounds in the root and stems of Parthenocissus laetevirens. Two characteristic fragments [C₃O₂ (68 Da) and C₂H₂O (42 Da)] were utilized for the structural identification of polyphenols. Based on the reference standards, the fragment C₃O₂ was presented when the compound possessed a 2,3‐dihydro‐1H‐indene‐4, 6‐diol moiety. Meanwhile, the C₂H₂O fragment (42 Da) yielded from the resorcinol ring was confirmed by resveratrol and three synthesized compounds identified as (E)‐5‐styrylbenzene‐1,3‐diol, (E)‐4‐styrylphenol and (E)‐4‐(3,4,5‐trimethoxystyryl)phenol. FTICR‐MSⁿ was performed to further confirm the structures of the fragments. Overall, 15 polyphenol compounds were characterized. Three polyphenol compounds were initially and tentatively characterized from P. laetevirens for the first time, and one was proposed as a novel compound. Furthermore, a pair of stereoisomers was readily distinguished by breakdown curves, and the trans‐, cis‐isomers could be identified by HPLC/DAD‐UV spectra. Copyright © 2009 John Wiley & Sons, Ltd.     

HPLC–TOF‐MS and HPLC–MS/MS combined with multivariate analysis for the characterization and discrimination of phenolic profiles in nonfumigated and sulfur‐fumigated rhubarb

Sulfur fumigation has recently been used during the postharvest handling of rhubarb to reduce the drying duration and control pests. However, a few reports question the effect of sulfur fumigation on the bioactive components of rhubarb, which is crucial for the quality evaluation of the herbal medicine. The bottleneck limiting the study comes from the complex compounds that exist in herb samples with diverse structural features, wide concentration range and the difficulty to obtain all the reference standards. In this study, an integrated strategy based on the highly effective separation and analysis by liquid chromatography coupled with diode‐array detection and time‐of‐flight/triple‐quadruple tandem mass spectrometry combined with multivariate analysis was established. 68 phenolic compounds that exist in nonfumigated and sulfur‐fumigated herb samples of rhubarb were tentatively assigned based on their retention behavior, UV spectra, accurate molecular weight, and mass spectral fragments. Qualitative and semiquantitative comparison revealed a serious reduction of the majority of phenolic compounds in sulfur‐fumigated rhubarb. Furthermore, multivariate analysis was applied to holistically discriminate nonfumigated from sulfur‐fumigated rhubarb and explore the characteristic chemical markers. The established approach was specific and rapid for characterizing and screening sulfur‐fumigated rhubarb among commercial samples and could be applied for the quality assessment of other sulfur‐fumigated herbs.     

Rapid separation and identification of Strychnos alkaloids metabolites in rats by ultra high performance liquid chromatography with linear ion trap Orbitrap mass spectrometry

An in vivo study of Strychnos alkaloids metabolites in rats by ultra high performance liquid chromatography with linear ion trap Orbitrap MS is reported for the first time. Two major Strychnos alkaloids compounds including strychnine and brucine were investigated. To obtain optimal extraction efficiency, samples were pretreated by using an SPE plate. The structures of metabolites and their fragment ions were characterized based on the accurate mass and MSⁿ data. Forty‐seven metabolites were identified in rat urine, of which 25 were reported for the first time. Four new metabolism pathways were proposed on the basis of the identified metabolites. This study provides a practical approach for rapidly identifying complicated metabolites, a methodology that could be widely applied not only in forensic and clinically toxicological relevant cases, but also for the structural characterization of metabolites of other compounds.     

Study of 2‐Iodo‐3‐(phenylsulfinyl)‐2‐propen‐1‐ol and its Analogues by Self‐chemical Ionization Mass Spectrometry

The self‐chemical ionization (SCI) in quadrupole mass spectrometry was developed to determine the structure of (E)‐2‐iod‐3‐(phenylsulfinyl)‐2‐propen‐1‐ol and its 6 analogues. Some techniques that increase the sample quantity and heating speed and shorten vaporization time to obtain high pressure in the ion source were applied to increase the chance of ionmolecule reactions. The structures of these compounds were identified by mass spectral data of MH⁺ and some characteristic fragment ions. Compared with the mass spectra for 2‐iodo‐3‐(phenylsulfinyl)‐2‐propen‐l‐ols obtained in electron impact ionization (EI), SCI showed more information, in particular, an improvement in amount of information at the high mass area. The absence of reagent gas makes the spectrum clean and simple.     

Screening of drugs of abuse and toxic compounds in human whole blood using online solid‐phase extraction and high‐performance liquid chromatography with time‐of‐flight mass spectrometry

A novel method for the screening of 151 drugs of abuse and toxic compounds in human whole blood has been developed and validated by online solid‐phase extraction with liquid chromatography coupled to time‐of‐flight mass spectrometry. Analytes were extracted and separated by using a fully automated online solid‐phase extraction liquid chromatography system with total chromatographic run time of 26 min. Time‐of‐flight mass spectrometry screening of 151 drugs of abuse and toxic compounds was performed in a full‐scan (m/z 50–800) mode using an MS^E acquisition of molecular ions and fragment ions data at two collision energies (one was 6 eV and another one was in the range of 5–45 eV). The compounds were identified based on retention times and exact mass of molecular ions and fragment ions. The limit of detection ranged from 1 to 100 ng/mL and the recovery of the method ranged from 6.3 to 163.5%. This method is proved to be a valuable screening method allowing fast and specific identification of drugs in human whole blood.     

Sodiation‐based in‐source collision for profiling of pyranocoumarins in Radix Peucedani (Qianhu): utility of sodium adducts' stability with in‐source collision

Full scan mode of liquid chromatography‐mass spectrometry equipped with an electrospray ionization source offers a chance on global detection of complicated components; however, the scan mode carries significant challenges in rapidly capturing information of analysts. Sodiation‐based in‐source collision was proposed here, as a technique for rapid detecting untargeted analytes in full scan analysis, which was based on the stability of sodium adducts and the nonselectivity of in‐source collision. Then the technique was applied to profile of angular‐type pyranocoumarins (APs) in Radix Peucedani, with full scan analysis performed at two specific in‐source collision energy: a high energy 50 V that is tolerated by the sodium adducts of APs, and a low energy 10 V, at which abundant adducts were offered. The spectra list of two average mass spectra was exported, and stable ions were selected based on the intensity ratio of standards at the two collision energy. Then 27 plausible [M + Na]⁺ m/z values of APs were acquired after filtering the fragment ion and isotope ions and validating with [M + NH₄]⁺. Eighty‐two APs finally were tentatively identified based on their accurate spectral data of MSⁿ, fragmentation rules, and elution order regardless of their absolute configuration, which included 25 reported APs from Peucedanum praeruptorum Dunn. The technique provided a novel application of sodium adduct in qualitative analysis. And it was valuable for rapidly capturing information of analytes in full scan analysis, not only for APs but also for other compounds that could form sodium adducts. Copyright © 2017 John Wiley & Sons, Ltd.     

Efficient analysis of phytochemical constituents in the peel of Chinese wild citrus Mangshanju (Citrus reticulata Blanco) by ultra high performance liquid chromatography–quadrupole time‐of‐flight‐mass spectrometry

An efficient ultra high‐performance liquid chromatography coupled with quadrupole time‐of‐flight mass spectrometry method was developed for separation and profiling of phytochemical constituents of Chinese wild mandarin Mangshanju (Citrus reticulata Blanco). All constituents were well separated within 16 min. Based on retention times, accurate mass, MS^E fragments, and/or reference standards as well as databases, a total of 81 compounds were unambiguously identified or tentatively assigned including flavonoid glycosides, acylated flavonoid glycosides, flavones, polymethoxylated flavonoids, and limonoids as well as four other compounds. Among them, 22 polymethoxylated flavones and ten polymethoxylated flavanones/chalcones were identified in Mangshanju, more types than other citrus reported before. A basic procedure for identifying flavonoid‐O‐glycosides and the aglycones including polymethoxylated flavonoids was proposed. In addition, this method was successfully used to analyze another four mandarin germplasms, Cenxi suan ju, Xipi gousi gan, Nanfeng miju, and Or, showing that Mangshanju contained two characteristic compounds distinct from the other four citrus species. This study systematically profiled phytochemical constituents of Mangshanju, which was helpful for further utilization of Mangshanju owing to its abundant bioactive compounds.     

Profiling and analysis of multiple compounds in rhubarb decoction after processing by wine steaming using UHPLC–Q‐TOF‐MS coupled with multiple statistical strategies

Rhubarb is one of the most popular traditional Chinese medicines and has been used for thousands of years in many Asian countries. Prepared rhubarb is obtained by steaming raw rhubarb with glutinous rice wine until it turned black in appearance both inside and outside. After processing, the therapeutic effects of prepared rhubarb change a lot. To find out the exact compound changes of the chemical profile in a decoction of rhubarb after processing and to clarify the material basis of the changed therapeutic effects, an ultra‐high performance liquid chromatography with quadrupole time‐of‐flight mass spectrometry method coupled with automated data analysis software and statistical strategy was developed. As a result, 63 peaks in raw rhubarb and 54 peaks in prepared rhubarb were detected, and a total of 45 chemical compounds were identified. The analysis data were subjected to a principle component analysis and a t‐test. Based on the results, 16 peaks were found to be the main contributors to the significant difference (p < 0.05) between raw and prepared rhubarb. Compared with raw rhubarb, the content of 15 components in prepared rhubarb was lower, while only rhein (1,8‐dihydroxy‐3‐carboxy anthraquinone) showed a higher intensity.     

Unexpected detection of 3‐aroylbenzofuran side products in the preparation of 2‐arylbenzofurans: Identification,characterization, and comparison with chalcone's fragmentation patterns using EI/MSn

A gas chromatography‐mass spectrometry study of the intramolecular Wittig reaction revealed, together with the expected 2‐phenylbenzofuran, the formation of an unexpected side product that has not been reported until now. This study reports the identification of the by‐product, ie, the 3‐benzoyl‐2‐phenylbenzofuran, on the base of its mass spectrometric behaviour using a combination of electron ionization, exact mass measurement, multiple stage mass spectrometry, and labelled compounds. This study reports the common fragmentation pathways and discusses possible fragment structures of characteristic ions from a series of 3‐aroyl‐2‐arylbenzofuran derivatives obtained as by‐product under Wittig conditions. Emphasis is laid on the formation and structure investigation of the [M‐H]⁺ and [M‐OH]⁺ ions. Our results showed interesting analogies with the mass spectrometric behaviour of chalcones.     

Separation and identification of phenolic compounds in Bidens pilosa L. by ultra high performance liquid chromatography with quadrupole time‐of‐flight mass spectrometry

A validated method based on ultra‐performance liquid chromatography coupled with quadrupole time‐of‐flight mass spectrometry was established to separate and identify phenolic compounds in Bidens pilosa L. Mass spectrometry experiments were performed both in positive and negative ion modes. A total of 35 compounds were detected, and 26 phenolic compounds were unequivocally identified or tentatively assigned based on retention time, maximum UV absorption, molecular formula, and fragments. The ultra high performance liquid chromatography method was validated and showed good linearity (R² ≧ 0.9996) over the test range. The limits of detection and quantification were above 0.072 and 0.162 μg/mL, respectively. The relative standard deviations of intraday and interday precision were below 0.3 and 1.6%, respectively.     

Fragmentation of synthetic cannabinoids with an isopropyl group or a tert‐butyl group ionized by electron impact and electrospray

This study described a fragmentation pattern of 21 synthetic cannabinoids with an isopropyl group or a tert‐butyl group by electron impact ionization quadrupole mass spectrometry and electrospray ionization time‐of‐flight mass spectrometry in the positive mode. The compounds were categorized into four types according to substituted group such as a terminal amide and ester. The characteristic fragment ion in each group was obtained. The main common fragment ions for the two ionizations were formed by C–N cleavage of the amide group adjacent to the N‐hetero rings. Additionally, the fragment ions indicated the difference in the basic structure as well as substituted group, which are useful for estimating the chemical structures of unknown compounds. Copyright © 2015 John Wiley & Sons, Ltd.     

Characterization and identification of steroidal alkaloids in the Chinese herb Veratrum nigrum L. by high-performance liquid chromatography/electrospray ionization with multi-stage mass spectrometry

Electrospray ionization multi-stage mass spectrometry (ESI-MS(n)) was performed to study the fragmentation behaviour of seventeen steroidal alkaloids (4 protoverine-type alkaloids, 10 germine-type alkaloids and 3 zygadenin-type alkaloids) from the Chinese herb Veratrum nigrum L. The MS(n) spectra of the [M+H](+) ions for steroidal alkaloids provided a wealth of structural information on the substituted groups. In positive ion mode, the three types of alkaloids showed very different characteristic ions: m/z 436 or 418 for protoverine-type alkaloids; m/z 438, 420 or 402 for germine-type alkaloids; m/z 440 or 422 for zygadenin-type alkaloids. These fragments were used to deduce their mass spectral fragmentation mechanisms. Furthermore, the primary compounds in methanolic extracts of the herb of Veratrum nigrum L. were investigated by using liquid chromatography (LC)/ESI-MS(n). As a result, 21 steroidal alkaloids (5 protoverine-type alkaloids, 14 germine-type alkaloids and 2 zygadenin-type alkaloids) were selectively identified from 27 determined peaks. Eleven compounds were unambiguously identified by comparing with standard compounds and ten compounds were tentatively identified or deduced according to their MS(n) data. Two of these compounds (xingangermine and deacetyl xinganveratrine) were found to be novel steroidal alkaloids. In addition, the chemical structures of two pairs of steroidal alkaloid isomers were deduced by comparing their fragment ions. Given the important structural information of known and unknown steroidal alkaloids in crude herbal extracts, this study is useful for identifying these types of steroidal alkaloids in crude materials rapidly and selectively.     

Separation and identification of terpene-conjugated curcuminoids based on liquid chromatography–tandem mass spectrometry and their in vitro anti-inflammatory activities

Terpene-conjugated curcuminoids are conjugates of curcuminoids and bisabolanes in the rhizomes of Curcuma longa L. The fragmentation pathways of known three terpene-conjugated curcuminoids (bisabolocurcumin-ether, bisabocurcumin, and demethoxybisabolocurcumin ether) and curcumin, demethoxycurcumin, and bisdemethoxycurcumin were investigated using high-performance liquid chromatography-electrospray ionization tandem mass spectrometry in negative mode to rapidly search and discover similar unknown compounds of the acetone fraction of turmeric. Subsequently, compounds 1–3 were founded in the acetone fraction based on molecular weight and above fragmentation pathways (the characteristic fragment ions, the most and second most abundant fragment ions produced in MS² spectra). Terpecurcumin X ( 1 ) and terpecurcumin Y ( 3 ) were further separated by liquid chromatography–tandem mass spectrometry guided isolation technique to verify their structures by nuclear magnetic resonance, electrospray ionization high-resolution mass spectroscopy, ultraviolet and visible spectra and infrared spectra. Interestingly, 1 and 3 were new compounds. The results indicate the feasibility and significant advantages of liquid chromatography–tandem mass spectrometry for the rapid discovery and analysis of new constituents in traditional Chinese medicine. In vitro, Terpene-conjugated curcuminoids had better nitric oxide inhibitory activity than the other seven curcuminoids (demethoxycurcumin, bisdemethoxycurcumin, curdione, curcumenone, bisacurone, curcumenol, and germacron).     

Characteristics of the asymmetric mass distribution in proton-induced fission of actinides

To study the correlation between fragment mass distributions and shell structures of fission fragments, fragment mass and energy distributions in proton-induced fission of ²³²Th and uranium isotopes, ^233,235,238U, were precisely measured using a double-TOF method. It was found that the lighter side of the heavier wing of asymmetric mass distributions shifts to larger fragment mass number corresponding to the N/Z value of the fissioning nucleus. The results are explained qualitatively by the change of the most probable mass number of fission fragments of Z = 50 proton-shell.     

Analysis of Trace-Level Volatile Compounds in Fresh Turf Crop （Lolium perenne L.） by Gas Chromatography Quadrupole Time-of-Flight Mass Spectrometry

In the traditional research of volatile compounds, some trace-level compounds could not be identified by gas chromatography-mass spectrometry. Target and post-targeted methods were applied in the investigation of trace-level volatile compounds in fresh turf crop （Lolium perenne L.） based on gas chromatography in combination with hybrid quadrupole time-of-flight mass spectrometry. According to literatures published, a target analysis was performed by using retention index, accurate masses of characteristic ions and second-stage mass spectra （MS2 spectra）. And a series of experiments showed that low electron impact energy was beneficial to the improvement of the abundances of low abundance molecular ion peak. peak was beneficial to qualitative analysis. Totally, 60 Enhancing the abundances of low abundance molecular ion volatile compounds were identified, the great majority cornpounds of which were benzeneacetaldehyde （14.8%）, 2,5-dimethyl-pyrazine （9.6%）, and hexanal （9.3%）. Identification was complied by mass spectral search, retention index and accurate masses of characteristic ions.     

Dereplication of known pregnane glycosides and structural characterization of novel pregnanes in Marsdenia tenacissima by high‐performance liquid chromatography and electrospray ionization‐tandem mass spectrometry

In the search for novel natural products in plants, particularly those with potential bioactivity, it is important to efficiently distinguish novel compounds from previously isolated, known compounds, a process known as dereplication. In this study, electrospray ionization‐multiple stage tandem mass spectrometry (ESI‐MSⁿ) was used to study the behaviour of 12 pregnane glycosides and genins previously isolated from Marsdenia tenacissima, a traditional Chinese medicinal plant, as a basis for dereplication of compounds in a plant extract. In addition to [M + Na]⁺ and [M + NH₄]⁺ ions, a characteristic [M‐glycosyl + H]⁺ ion was observed in full‐scan mode with in‐source fragmentation. Sequential in‐trap collision‐induced dissociation of [M + Na]⁺ ions from 11,12‐diesters revealed consistent preferred losses of substituents first from C‐12, then from C‐11, followed by losses of monosaccharide fragments from the C‐3 tri‐ and tetrasaccharide substituents. A crude methanol extract of M. tenacissima stems was analysed using high‐performance liquid chromatography coupled to ESI‐MS. Several previously isolated pregnane glycosides were dereplicated, and the presence of an additional nine novel pregnane glycosides is predicted on the basis of the primary and fragment ions observed, including two with a previously unreported C₄H₇O C‐11/C‐12 substituent of pregnane glycosides. This study is the first report of prediction of the structures of novel pregnane glycosides in a crude plant extract by a combination of in‐source fragmentation and in‐trap collision‐induced dissociation and supports the usefulness of LC‐ESI‐MSⁿ not only for dereplication of active compounds in extracts of medicinal plants but also for detecting the presence of novel related compounds. Copyright © 2012 John Wiley & Sons, Ltd.     

A rapid method for simultaneous determination of 52 marker compounds in Xiao‐Qing‐Long‐Tang by ultra high performance liquid chromatography coupled with quadrupole time‐of‐flight mass spectrometry†

Xiao‐Qing‐Long‐Tang (XQLT) is a classical Chinese medicine formula. It is generally used for the treatment of common cold, bronchial asthma, and allergic rhinitis in Asia. In this study, a multicomponent quantification fingerprinting approach based on ultra high performance liquid chromatography coupled with quadrupole time‐of‐flight mass spectrometry has been developed for the analysis of compounds in XQLT in 14.5 min. A total of 52 compounds were identified by co‐chromatography of sample extract with authentic standards and comparing the retention time, UV spectra, molecular ions and characteristic fragment ions with those of authentic standards, or tentatively identified by MS^E determination along with Mass Fragment software. Moreover, the method was validated for the simultaneous quantification of 16 components in XQLT commercial products. The method is practical for comprehensive standardization of XQLT and holistic comparison of its commercial products from different manufacturers.     

Characterization and identification of saponins in Achyranthes bidentata by rapid‐resolution liquid chromatography with electrospray ionization quadrupole time‐of‐flight tandem mass spectrometry

A rapid‐resolution liquid chromatography (RRLC) method coupled with electrospray ionization quadrupole time‐of‐flight tandem mass spectrometry (Q‐TOF MS/MS) has been developed for analysis of oleanane‐type triterpenoid saponins in Achyranthes bidentata. Collision‐induced dissociation techniques were used to fragment the precursor molecular ions and the resulting product ions. A retro‐Diels‐Alder rearrangement from the oleanane aglycone skeleton in the MS/MS process yielded characteristic fragment ions in positive ion mode. These characteristic ions were helpful in predicting the aglycone structure. Losses of monosaccharide sequences, presence of sugar‐chain fragment ions, and cleavage of CO₂ were observed for important information on sugar types and attachment sequences. Fragmentation rules of three major groups of saponins from A. bidentata were summarized, and the possible fragmentation pathways were proposed. A total of 22 compounds including both the target and unknown oleanane‐type triterpenoid saponins were rapidly screened and predicted in the herbal extract by the developed method. The RRLC‐Q‐TOF MS/MS method has provided a powerful approach for rapid separation, target screening and structural elucidation of oleanane‐type saponins, and also opened perspectives for similar studies on other herbal medicines. Copyright © 2010 John Wiley & Sons, Ltd.