A four-dimensional separation approach by offline 2D-LC/IM-TOF-MS in combination with database-driven computational peak annotation facilitating the in-depth characterization of the multicomponents from Atractylodis Macrocephalae Rhizoma (Atractylodes macrocephala)

Plant metabolites represent complex chemical system, which renders it difficult to clarify the chemical composition by conventional liquid chromatography/mass spectrometry (LC/MS) due to the limited selectivity and peak capacity. The rhizomes of Atractylodes macrocephala have been utilized as a traditional Chinese medicine Atractylodis Macrocephalae Rhizoma (Bai-Zhu), and have been reported containing multiple categories of plant metabolites. Targeting the multicomponents from A. macrocephala, an integral approach by offline two-dimensional liquid chromatography/ion mobility quadrupole time-of-flight mass spectrometry (2D-LC/IM-QTOF-MS) was established and validated. By configuring an XBridge Amide column of Hydrophilic Interaction Chromatography and an Atlantis Premier BEH C18AX column of mixed ion exchange and reversed-phase modes, the established 2D-LC/IM-QTOF-MS system showed high orthogonality up to 0.91. Dimension-enhanced, data-independent high-definition MS^E (HDMS^E) in the positive ESI mode was conducted on a Vion IM-QTOF mass spectrometer, and its hyphenation to offline 2D-LC could enable the four-dimensional separation (each dimension in 2D-LC, IM, and MS). Particularly, HDMS^E facilitated the acquisition of high-definition MS¹ and MS² spectra. In-house library-driven computational peak annotation by the bioinformatics platform UNIFI could efficiently process and annotate the HDMS^E data for the structural elucidation. By integrating reference compounds comparison, we could identify or tentatively characterize 251 components from A. macrocephala (including 115 sesquiterpenoids, 90 polyacetylenes, 11 flavonoids, 9 benzoquinones, 12 coumarins, and 14 others), which indicated large improvement in identifying those minor plant components, compared with the conventional LC/MS approach. Conclusively, offline 2D-LC/IM-QTOF-HDMS^E in combination with computational data interpretation proves to be powerful facilitating the in-depth multicomponent characterization of herbal medicine.     

Holistic quality evaluation of Saposhnikoviae Radix (Saposhnikovia divaricata) by reversed-phase ultra-high performance liquid chromatography and hydrophilic interaction chromatography coupled with ion mobility quadrupole time-of-flight mass spectrometry-based untargeted metabolomics

Untargeted metabolomics more suits the quality evaluation of TCM because of its holistic property. To assess the holistic quality difference of Saposhnikoviae Radix (the roots of Saposhnikovia divaricata), we integrate ultra-high-performance liquid chromatography coupled with ion mobility/quadrupole time-of-flight mass spectrometry (UHPLC/IM-QTOF-MS)-based untargeted metabolomics and quantitative assay. A BEH C18 column in the reversed-phase mode and a BEH Amide column in Hydrophilic Interaction Chromatography (HILIC) mode were utilized for metabolites profiling, which enabled high coverage of the non-polar to polar components in Saposhnikoviae Radix. Moreover, the application of major components knockout strategy enlarged the exposure of those minor components. Integrated use of high-definition MS^E (HDMS^E) and data-dependent acquisition (DDA) could enhance the metabolites characterization by providing reliable fragmentation information and collision cross section values. Computational in-house library-driven automated peak annotation of the HDMS^E and DDA data assisted to characterize 104 components from Saposhnikoviae Radix. Chemometric analyses of the commercial Saposhnikoviae Radix samples (64 batches collected from 11 cultivars aging from 1 to 4 years), based on the positive MS^E data, in general could indicate large discrimination between Guan-Fang-Feng (from Heilongjiang) and the others, but negligible difference among Saposhnikoviae Radix from the other ten provinces of China and with different ages. Quantitative assays of prim-O-glucosylcimifugin and 4′-O-β-D-glucosyl-5-O-methylvisamminol, by a rapid and fully validated UHPLC-UV method, could primarily deduce that Guan-Fang-Feng aging 2 and 3 years exhibited better quality. The methods established can holistically assess the quality of TCM with wide spans of plant metabolites covered.     

Ultra-high performance liquid chromatography/ion mobility time-of-flight mass spectrometry-based untargeted metabolomics combined with quantitative assay unveiled the metabolic difference among the root,leaf, and flower bud of Panax notoginseng

Despite Panax notoginseng (Sanchi: the root and rhizome) is globally popular serving as the source of food additives, health-care products, and traditional Chinese medicines (TCMs), the saponin difference between the root (PNR) and two aerial parts (leaf, PNL; flower bud, PNF) that can be vicariously used, remains unclear. Authentication of Sanchi, particular from the Chinese patent medicines (CPMs), poses great challenges. Ultra-high performance liquid chromatography/ion mobility-quadrupole time-of-flight mass spectrometry (UHPLC/IM-QTOF-MS)-based untargeted metabolomics and quantitative assay by UHPLC-UV were utilized to establish the “Identification Markers” for Sanchi. Targeted monitoring of multiple identification markers was performed for authenticating Sanchi simultaneously from 15 different CPMs. Dimension-enhanced profiling by UHPLC/IM-QTOF-MS in the negative high-definition MS^E (HDMS^E) mode and in-house library-driven peak annotation could characterize totally 328 ginsenosides (133 from PNR, 125 from PNL, and 161 from PNF). Multivariate statistical analysis of the PNR/PNL/PNF samples (45 batches) identified 27 potential markers. Five major markers (notoginsenoside R1, ginsenosides Rg1, -Rb1, -Rb2, and -Rb3) thereof were quantitatively assayed by a fully validated UHPLC-UV (detected at 203 nm) approach. The application of selective ion monitoring (SIM) of 12 differential saponins coupled with UHPLC separation could precisely identify Sanchi from 15 different CPMs (45 batches). Holistic difference in ginsenosides among three parts of P. notoginseng was unveiled, and the markers deduced may assist to identify the illicit substitution of leaf or flower as the root in the TCM compound formulae. Conclusively, the integration of untargeted metabolomics and quantitative analysis can provide reliable information enabling the precise authentication of TCM.     

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.     

Application of UPLC-QTOF-MS in MSE mode for the rapid and precise identification of alkaloids in goldenseal (Hydrastis canadensis)

Here, we describe a new application of ultra-performance liquid chromatography coupled with an electrospray ionization quadrupole time-of-flight mass spectrometry operating in MS^E mode (UPLC-QTOF-MS^E) for the sensitive, fast, and effective characterization of alkaloids in goldenseal (Hydrastis canadensis). This approach allowed identification of alkaloids using a cyclic low and high collision energy spectral acquisition mode providing simultaneous accurate precursor and fragment ion mass information. A total of 45 compounds were separated and 40 of them characterized including one new compound and 7 identified for the first time in goldenseal. The spectral data obtained using this method is comparable to those obtained by conventional LC-MSⁿ. However, the UPLC-QTOF-MS^E method offers high chromatographic resolution with structural characterization facilitated by accurate mass measurement in both MS and MS/MS modes in a single analytical run; this makes it suitable for the rapid analysis and screening of alkaloids in plant extracts.

Fast Characterization of Constituents in HuangKui Capsules Using UPLC�CQTOF-MS with Collision Energy and MassFragment Software

In this paper, ultra performance liquid chromatography (UPLC)?Cquadrupole-time-of-flight mass spectrometry (QTOF) with collision energy (MS ^E ) and MassFragment software were applied for fast separation and characterization of constituents in HuangKui Capsules (HKC). MS ^E was used for simultaneous acquisition of precursor ion information and fragment ion data at high and low collision energy in one analytical run, which facilitated the fast structural characterization of 12 constituents in HKC. When the reference compound was not available, MassFragment software was used to confirm the proposed structure, which increased our confidence in the MS fragment analysis of the proposed structure, facilitating the confirmation of the proposed structure and therefore easing the data processing procedure. In conclusion, UPLC?CQTOF-MS ^E and MassFragment software described in this paper proved to be useful for the identification of constituents in complex herbal medicines and could greatly decrease analytical time. In addition, this method may be applied to the analysis of more complex data found in Traditional Chinese Medicine.     

Large-Scale Collision Cross-Section Profiling on a Traveling Wave Ion Mobility Mass Spectrometer

Ion mobility (IM) is a gas-phase electrophoretic method that separates ions according to charge and ion-neutral collision cross-section (CCS). Herein, we attempt to apply a traveling wave (TW) IM polyalanine calibration method to shotgun proteomics and create a large peptide CCS database. Mass spectrometry methods that utilize IM, such as HDMS^E, often use high transmission voltages for sensitive analysis. However, polyalanine calibration has only been demonstrated with low voltage transmission used to prevent gas-phase activation. If polyalanine ions change conformation under higher transmission voltages used for HDMS^E, the calibration may no longer be valid. Thus, we aimed to characterize the accuracy of calibration and CCS measurement under high transmission voltages on a TW IM instrument using the polyalanine calibration method and found that the additional error was not significant. We also evaluated the potential error introduced by liquid chromatography (LC)-HDMS^E analysis, and found it to be insignificant as well, validating the calibration method. Finally, we demonstrated the utility of building a large-population peptide CCS database by investigating the effects of terminal lysine position, via LysC or LysN digestion, on the formation of two structural sub-families formed by triply charged ions.

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Characterization of phenolic compounds in green and red oak‐leaf lettuce cultivars by UHPLC‐DAD‐ESI‐QToF/MS using MSE scan mode

Lettuce (Lactuca sativa ) is one of the most popular leafy vegetables in the world and constitutes a major dietary source of phenolic compounds with health‐promoting properties. In particular, the demand for green and red oak‐leaf lettuces has considerably increased in the last years but few data on their polyphenol composition are available. Moreover, the usage of analytical edge technology can provide new structural information and allow the identification of unknown polyphenols. In the present study, the phenolic profiles of green and red oak‐leaf lettuce cultivars were exhaustively characterized by ultrahigh‐performance liquid chromatography (UHPLC) coupled online to diode array detection (DAD), electrospray ionization (ESI), and quadrupole time‐of‐flight mass spectrometry (QToF/MS), using the MS^E instrument acquisition mode for recording simultaneously exact masses of precursor and fragment ions. One hundred fifteen phenolic compounds were identified in the acidified hydromethanolic extract of freeze‐dried lettuce leaves. Forty‐eight of these compounds were tentatively identified for the first time in lettuce, and only 20 of them have been previously reported in oak‐leaf lettuce cultivars in literature. Both oak‐leaf lettuce cultivars presented similar phenolic composition, except for apigenin‐glucuronide and dihydroxybenzoic acid, only detected in the green cultivar; and for luteolin‐hydroxymalonylhexoside, an apigenin conjugate with molecular formula C₄₀H₅₄O₁₉ (monoisotopic MW = 838.3259 u ), cyanidin‐3‐O ‐glucoside, cyanidin‐3‐O ‐(3″‐O ‐malonyl)glucoside, cyanidin‐3‐O ‐(6″‐O ‐malonyl)glucoside, and cyanidin‐3‐O ‐(6″‐O ‐acetyl)glucoside, only found in the red cultivar. The UHPLC‐DAD‐ESI‐QToF/MS^E approach demonstrated to be a useful tool for the characterization of phenolic compounds in complex plant matrices.     

Systematic Screening of Chemical Constituents in the Traditional Chinese Medicine Arnebiae Radix by UHPLC-Q-Exactive Orbitrap Mass Spectrometry

Arnebiae Radix (dried root of Arnebia euchroma (Royle) Johnst.) has been used in traditional Chinese medicine (TCM) to treat macular eruptions, measles, sore throat, carbuncles, burns, skin ulcers, and inflammation. Previous studies have shown that shikonins and shikonofurans are two of their main bioactive ingredients. However, systematic investigations of their constituents have rarely been conducted. It is necessary to establish a rapid and effective method to identify the chemical constituents of Arnebiae Radix. This will help to further improve the effective resource utilization rate of this plant. In this study, a rapid and effective UHPLC-Q-Exactive Orbitrap mass spectrometry method was established to simultaneously analyze chemical ingredients in Arnebiae Radix within a short period of time. Based on the results of a full scan MS, the MS² database (mzVault and mzCloud), the diagnostic fragment ions, the retention time, and the bibliography, a total of 188 compounds were identified, with 114 of those being reported from Arnebiae Radix for the first time. The results of this study lay the foundation for obtaining a thorough understanding of the active ingredients in Arnebiae Radix and its quality control. This method may be widely used for the chemical characterization of different samples.     

MassBank: a public repository for sharing mass spectral data for life sciences

MassBank is the first public repository of mass spectra of small chemical compounds for life sciences (<3000 Da). The database contains 605 electron‐ionization mass spectrometry(EI‐MS), 137 fast atom bombardment MS and 9276 electrospray ionization (ESI)‐MSⁿ data of 2337 authentic compounds of metabolites, 11 545 EI‐MS and 834 other‐MS data of 10 286 volatile natural and synthetic compounds, and 3045 ESI‐MS² data of 679 synthetic drugs contributed by 16 research groups (January 2010). ESI‐MS² data were analyzed under nonstandardized, independent experimental conditions. MassBank is a distributed database. Each research group provides data from its own MassBank data servers distributed on the Internet. MassBank users can access either all of the MassBank data or a subset of the data by specifying one or more experimental conditions. In a spectral search to retrieve mass spectra similar to a query mass spectrum, the similarity score is calculated by a weighted cosine correlation in which weighting exponents on peak intensity and the mass‐to‐charge ratio are optimized to the ESI‐MS² data. MassBank also provides a merged spectrum for each compound prepared by merging the analyzed ESI‐MS² data on an identical compound under different collision‐induced dissociation conditions. Data merging has significantly improved the precision of the identification of a chemical compound by 21–23% at a similarity score of 0.6. Thus, MassBank is useful for the identification of chemical compounds and the publication of experimental data. Copyright © 2010 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.     

Rapid Characterization of Constituents in <Emphasis Type="Italic">Tribulus terrestris</Emphasis> from Different Habitats by UHPLC/Q-TOF MS

A strategy for rapid identification of the chemical constituents from crude extracts of Tribulus terrestris was proposed using an informatics platform for the UHPLC/Q-TOF MS^E data analyses. This strategy mainly utilizes neutral losses, characteristic fragments, and in-house library to rapidly identify the structure of the compounds. With this strategy, rapid characterization of the chemical components of T. terrestris from Beijing, China was successfully achieved. A total of 82 steroidal saponins and nine flavonoids were identified or tentatively identified from T. terrestris. Among them, 15 new components were deduced based on retention times and characteristic MS fragmentation patterns. Furthermore, the chemical components of T. terrestris, including the other two samples from Xinjiang Uygur Autonomous region, China, and Rome, Italy, were also identified with this strategy. Altogether, 141 chemical components were identified from these three samples, of which 39 components were identified or tentatively identified as new compounds, including 35 groups of isomers. It demonstrated that this strategy provided an efficient protocol for the rapid identification of chemical constituents in complex samples such as traditional Chinese medicines (TCMs) by UHPLC/Q-TOF MS^E with informatics platform.

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Preparation and Structure of (E)-1-(3′-Hydroxy-2-Furanyl)-3-(3″-Hydroxy-4″-Methoxyphenyl)-2-Propen-1-One

Abstract

A facile procedure is presented for the synthesis of (E)-1-(3′-hydroxy-2′-furanyl)-3-(3″-hydroxy-4″-methoxyphenyl)-2- propen-1-one (6). Galactosylisomaltol (1) was condensed with isovanillin (2) under strong alkaline conditions at 25 [ddot]C to form (E)-1-(3′-O-β-D-galactopyranosyloxy-2′-furanyl)-3-(3″- hydroxy-4″-methoxyphenyl)-2-propen-1-one (4). (E)-1-(3′-hydroxy-2′-furanyl)-3-(3″-hydroxy-4″-methoxyphenyl)-2-propen-1-one (6) was obtained by acid hydrolysis of 4 in a 53.9% yield. This hetero-cyclic 2-propen-1-one was characterized on the basis of spectral data (IR and ¹H NMR), physicochemical properties, and conversion to a mono-O-acetyl derivative.     

Screening of pesticides in blood with liquid chromatography–linear ion trap mass spectrometry

In clinical or forensic toxicology, general unknown screening procedures are used to identify as many xenobiotics as possible, belonging to numerous chemical classes. We present here a general unknown screening procedure based on liquid chromatography coupled with use of a single linear ion trap mass spectrometer, and focus on the identification of pesticides and/or metabolites in whole blood. After solid-phase extraction (SPE), the compounds of interest were separated using a reversed-phase column and identified by the mass spectrometer operated first in the full-scan mass spectrometry (MS) mode, in the positive and negative polarities, followed by MS² and MS³ scanning of ions selected in data-dependent acquisition. The total scan time was 2.45 s. Two mass spectral libraries (MS² and MS³), each of 450 spectra, were created for the 320 pesticides and metabolites detected after injection of pure solutions. Robustness of the spectra and matrix effects were studied and were satisfactory for the present application. Detection limits for the 320 compounds were studied by extracting 1 mL spiked blood at concentrations between 10 μg/L and 10 mg/L. If necessary, it was possible to decrease the detection limits of some compounds by 10–100-fold by scanning MS² in only one polarity, owing to a shorter total scan time. However, at the same time, the detection specificity decreased as no confirmation could be recorded in the following MS³ scan and no information could be registered in the other polarity. So, in these rare cases, confirmation by another method was required.     

Identification of triacylglycerol using automated annotation of high resolution multistage mass spectral trees

High complexity of identification for non-target triacylglycerols (TAGs) is a major challenge in lipidomics analysis. To identify non-target TAGs, a powerful tool named accurate MSⁿ spectrometry generating so-called ion trees is used. In this paper, we presented a technique for efficient structural elucidation of TAGs on MSⁿ spectral trees produced by LTQ Orbitrap MSⁿ, which was implemented as an open source software package, or TIT. The TIT software was used to support automatic annotation of non-target TAGs on MSⁿ ion trees from a self-built fragment ion database. This database includes 19108 simulate TAG molecules from a random combination of fatty acids and corresponding 500582 self-built multistage fragment ions (MS ≤ 3). Our software can identify TAGs using a “stage-by-stage elimination” strategy. By utilizing the MS¹ accurate mass and referenced RKMD, the TIT software can discriminate unique elemental composition candidates. The regiospecific isomers of fatty acyl chains will be distinguished using MS² and MS³ fragment spectra. We applied the algorithm to the selection of 45 TAG standards and demonstrated that the molecular ions could be 100% correctly assigned. Therefore, the TIT software could be applied to TAG identification in complex biological samples such as mouse plasma extracts.     

Combination of continuous digestion by peptidase and spectral similarity comparisons for peptide sequencing

Peptide sequencing is critical to the quality control of peptide drugs and functional studies of active peptides. A combination of peptidase digestion and mass spectrometry technology is common for peptide sequencing. However, such methods often cannot obtain the complete sequence of a peptide due to insufficient amino acid sequence information. Here, we developed a method of generating full peptide ladders and comparing their MS² spectral similarities. The peptide ladders, of which each component was different from the next component with one residue, were generated by continuous digestion by peptidase (carboxypeptidase Y and aminopeptidase). Then, based on the characteristics of peptide ladders, complete sequencing was realized by comparing MS² spectral similarity of the generated peptide ladders. The complete amino acid sequences of bivalirudin, adrenocorticotropic hormone, and oxytocin were determined with high accuracy. This approach is beneficial to the quality control of drug peptides as well as the identification of novel bioactive peptides.     

Targeted and nontargeted screening and identification of 50 antihypertensive adulterants in dietary supplements and herbal medicines using quadrupole–orbitrap high resolution mass spectrometry with compound database

In the present work, a novel database of drug compounds and a rapid screening method based on ultra‐high performance liquid chromatography coupled to high resolution orbitrap mass spectrometry were developed and applied in the screening and identification of targeted and nontargeted antihypertensive adulterants in dietary supplements and herbal medicines. The established screening database includes retention time, exact mass, fragments, isotopic pattern, and MS² spectra library of the target compounds and thus provides automated search and identification of the targets with a single injection. The nontargeted compounds in the samples are identified through the full MS scan and MS² data by using the Chemspider database and the data analysis in XCalibur, MassFrontier and TraceFinder software. In addition, this method possesses excellent quantitative capacity. The novel approach was applied to 65 batches of samples that are claimed as “all‐natural” products having the antihypertensive function, among which nine batches were found to be positive. Multiple targeted and nontargeted antihypertensive adulterants were detected at levels ranging from 2.8 to 27.9 mg/g. The novel database and screening method demonstrated herein will be promising and powerful tools for rapid screening of antihypertensive adulterants in dietary supplements and herbal medicines.