Online ligand screening for cytochrome C from herbal medicines through "four-in-one" measurement.

Affinity-oriented online ligand screening with LC coupled to different detectors is widely popular to capture active compounds from herbal medicines (HMs). However, false-positive extensively occurs because insufficient information is recorded for the existence and stability of ligand-protein complex. Here, efforts were made to advance the hit confidences via configuring post-column infusion-LC-energy-resolved-affinity MS (PCI-LC-ER-AMS) to achieve "four-in-one" monitoring of: 1) response decrement of potential ligands; 2) response decrement of protein; 3) ions relating to ligand-protein complexes; and 4) ligand-protein binding strength. Ligand fishing for Cyt C from HMs was conducted as a proof-of-concept. For utility justification, a mimic sample containing twelve well-defined ligands and two negative controls underwent LC separation and met Cyt C prior to Qtof-MS measurements. Compared to Cyt C- or ligand-free assay, twelve ligands instead of negative controls showed response decrements that were consistent with twelve negative peaks observed at retention times corresponding to the ligands in Cyt C ion current chromatogram. Serial ions correlating to each ligand-Cyt C complex were observed. After recording breakdown graphs, optimal collision energy (OCE) corresponding to the non-covalent bond dissociation was positively correlated with binding strength. Two HMs including Scutellariae Radix (SR) and Aconiti Lateralis Radix Preparata were investigated. Consequently, 24 compounds were merely fished from SR, and particularly, flavonoid glycosides exhibited greater OCEs and also binding strengths over aglycones. Affinity assays and cellular evaluations consolidated the significant interactions between each captured compound and Cyt C. Overall, PCI-LC-ER-AMS is eligible for confidence-enhanced online ligand screening for Cyt C from HMs through "four-in-one" measurement.

Quantitative comparison of bile acid glucuronides sub-metabolome between intrahepatic cholestasis and healthy pregnant women.

Because of the pathological indication and the physiological functions, bile acids (BAs) have occupied the research hotspot in recent decades. Although extensive efforts have been paid onto BAs sub-metabolome characterization, as the subfamily, BA glucuronides (gluA-BAs) profile is seldom concerned. Here, we made efforts to develop a LC-MS/MS program enabling quantitative gluA-BAs sub-metabolome characterization and to explore the differential species in serum between intrahepatic cholestasis of pregnancy (ICP) patients and healthy subjects. To gain as many authentic gluA-BAs as possible, liver microsomes from humans, rats, and mice were deployed to conjugate glucuronyl group to authentic BAs through in vitro incubation. Eighty gluA-BAs were captured and subsequently served as authentic compounds to correlate MS/MS spectral behaviors to structural features using squared energy-resolved MS program. Optimal collision energy (OCE) of [M-H]->[M-H-176.1]- was jointly administrated by [M-H]- mass and glucuronidation site, and identical exciting energies corresponding to 50% survival rate of 1st-generation fragment ion (EE50) were observed merely when the aglycone of a gluA-BA was consistent with the suspected structure. Through integrating high-resolution m/z, OCE, and EE50 information to identify gluA-BAs in a BAs pool, 97 ones were found and identified, and further, quantitative program was built for all annotated gluA-BAs by assigning OCEs to [M-H]->[M-H-176.1]- ion transitions. Quantitative gluA-BAs sub-metabolome of ICP was different from that of the healthy group. More GCDCA-3-G, GDCA-3-G, TCDCA-7-G, TDCA-3-G, and T-ß-MCA-3-G were distributed in the ICP group. Above all, this study not only offered a promising analytical tool for in-depth gluA-BAs sub-metabolome characterization, but also clarified gluA-BAs allowing the differentiation of ICP and healthy subjects.

Integrated Strategy Drives Direct Infusion-Tandem Mass Spectrometry as an Eligible Tool for Shotgun Pseudo-Targeted Metabolomics of Medicinal Plants.

Direct infusion (DI) has an extraordinary high-throughput advantage. Pseudo-targeted metabolomics (PTM) has been demonstrated integrating the merits of both nontargeted and targeted metabolomics. Herein, we attempted to implant DI into the PTM concept to configure a new strategy allowing shotgun PTM. First, a versatile MS/MSALL program was applied to acquire MS1 and MS2 spectra. Second, online energy-resolved MS (online ER-MS) was conducted to obtain breakdown graph as well as optimal collision energy (OCE) for each ion transition paired by precursor ion and the dominant product ion. Third, selected reaction monitoring (SRM) was responsible to output a quantitative dataset with a constant length. Moreover, breakdown graph also served as orthogonal structural evidence when matching MS2 spectra between DI-MS/MS and an in-house library to strengthen structural annotation confidence. To evaluate and illustrate the utility of the new strategy toward shotgun PTM of medicinal plants, in-depth chemome comparison was conducted within three Cistanche species, all of which are edible medicinal plants and playing essential roles for turning the deserts into the oases. A total of 185 variables participated in the quantitative measurement program. Each diagnostic ion pair was featured with an OCE. Significant species differences occurred, and echinacoside, acteoside, isoacteoside, 2'-acetyl-acteoside, tubuloside B, mannitol, sucrose, betaine, malate, as well as choline were found to be confirmative chemical markers offering primary contributions toward the species discrimination. After cross-validation with LC-MS/MS, DI-MS/MS fortified with the new strategy is an eligible tool for shotgun PTM, beyond Cistanche plants.

[Rapid chemome profiling of Cistanche salsa using DI-MS/MS~(ALL)].

The current study aims to rapidly and comprehensively profile the chemical composition of Cistanche salsa using direct infusion coupled with MS/MS~(ALL)(DI-MS/MS~(ALL)). The C. salsa extract was directly imported into electrospray ionization(ESI) source of quadrupole time-of-flight(Q-TOF) mass spectrometer with an infusion pump at a flow rate of 10 µL·min~(-1). Acquisition program was applied under negative ionization polarity to collect one MS~1 spectrum(m/z 50-1 200), followed by 1 150 MS~2 spectra with precursor isolation window(m/z 1) amongst mass range m/z 50-1 200. After each MS~2 spectrum was matched to its precursor ion, putative identification was conducted through matching mass spectral data with literature and database. A total of 31 components were identified from C. salsa, including 9 phenylethanoid glycosides, 2 iridoids, 4 saccharides, 9 organic acids, and 7 other compounds, similar to those from C. tubulosa and C. deserticola. In conclusion, DI-MS/MS~(ALL), a facile and reliable analytical tool, can be employed for qualitative analysis of chemical constituents in C. salsa. The research offers a promising strategy to achieve rapid chemome profiling of herbal medicine and provides an alternative source of Cistanches Herba.

[Rapid chemome profiling of chemical components of Lonicerae Japonicae Flos using DI-MS/MS~(ALL)].

A new method of MS/MS~(ALL) was designed to sequentially record a MS~2 spectrum at each unit mass window through gas phase fractionation concept, so as to offer an opportunity for universal MS~2 spectral recording with direct infusion(DI). As a proof-of-concept, DI-MS/MS~(ALL) was applied for rapid chemome profiling of a famous herbal medicine named Lonicerae Japonicae Flos. After each MS~2 spectrum was correlated to its precursor ion, the structural annotation was conducted by applying well-defined mass cracking rules, matching the mass spectral data with literatures and referring to those accessible databases. As a result, a total of 54 components were identified from Lonicerae Japonicae Flos extract, including 21 phenolic acids, 13 flavonoids, 12 iridoids, 4 triterpenoids and 4 other compounds. Therefore, DI-MS/MS~(ALL) is a powerful tool for comprehensive, rapid qualitative analysis of chemical profiles of traditional Chinese medicine and other chemical components of complex systems.

[Chemome profiling and comparison of three Orobanche medicinal plants].

Several Orobanche medicinal plants sometimes served as alternative sources of Cistanches Herba, attributing to the benefits such as tonifying kidney, strengthening tendons and bones. Among them, O. coerulescens, O. cernua and O. pycnostachya have been widely utilized in northern China for treatments of pains in the loins and knees, impotence, and spermatorrhea. However, their chemical profiles haven't been elucidated. In the present study, UHPLC-IT-TOF-MS was implemented to conduct in-depth chemome profiling of O. coerulescens, O. cernua and O. pycnostachya, aiming to achieve a comprehensive chemical characterization and to provide pronounced information for the quality control and clinical applications. An ACE Ultra-Core 2.5 Super C_(18)(3.0 mm×150 mm, 2.5 µm) column was deployed for chromatographic separations, and high-resolution MS~n spectra were recorded by IT-TOF-MS. Forty-eight components, in total, were observed, and thirty-eight ones were structurally annotated according to proposing mass fragmentation patterns, matching with relevant databases. Particularly, nine ones were confirmed by reference compounds. Overall, the chemical compositions of O. coerulescens and O. cernua are quite similar, and differences occur between O. pycnostachya and the prior two ones; primary chemical family is phenylethanoid glycosides, and several lignan glycosides as well as iridoid glycosides are also observed; the primary components include acteoside, isoacteoside, crenatoside and 2'-acetylacteoside, etc.

Serially coupled column liquid chromatography: An alternative separation tool.

Despite the rapid development of liquid chromatography (LC) in recent decades, it remains a challenge to achieve the desired chromatographic separation of complex matrices using a single column. Multi-column LC techniques, particularly serially coupled column LC (SCC-LC), have emerged as a promising solution to overcome this challenge. While more attention has been focused on heart-cutting or comprehensive two-dimensional LC, reviews specifically focusing on SCC-LC, which offers advantages in terms of precision and facile instrumentation, are scarce. Here, our concerns are devoted to the progress summary regarding the instrumentation and applications of SCC-LC. Emphasis is placed on column selection aiming to enlarge peak capacity, selectivity, or both through the optimization of combination types (e.g. RPLC-RPLC, -RPLC-HILIC, and achiral-chiral LC), connection devices (e.g. zero dead volume connector, tubing, and T-type connector), elution program (i.e. isocratic or gradient) and detectors (e.g. mass spectrometer, ultraviolet detector, and fluorescence detector). The application of SCC-LC in pharmaceutical, biological, environmental, and food fields is also reviewed, and future perspectives and potential directions for SCC-LC are discussed. We envision that the review can give meaningful information to analytical scientists when facing heavy chromatographic separation tasks for complicated matrices.

Two-dimensional code enables visibly mapping herbal medicine chemome: an application in Ganoderma lucidum.

BACKGROUND: Chemical profile provides the pronounced evidence for herbal medicine (HM) authentication; however, the chemome is extremely sophisticated. Fortunately, two-dimensional (2D) code, as a quick response means, is conceptually able to store abundant information, exactly fulfilling the chemical information storage demands of HMs. METHODS: We here attempted to denote both MS[Formula: see text] and MS[Formula: see text] dataset of HM with a single 2D-code chart. Measurement of Ganoderma lucidum that is one of the most famous HMs with LC-MS/MS was employed to illustrate the "coding-decoding" workflow for the conversion amongst MS/MS dataset, 2D-code, and chemical profile, and to evaluate the applicability as well. After data acquisition, and m/z value of each deprotonated molecular signal was divided into integer and decimal portions, corresponding to x and y coordinates of 2D-plot, respectively. On the other side, m/z values of all its fragment ions were exactly assigned to serial x values sharing an identical y value being equal to the precursor ion. 2D-code was thereafter produced by plotting these defined dots at a 2D-chart. Regarding a given 2D-code map, the entire chart (x coordinate: 0-600; y coordinate: 0-600) was fragmented into two regions by the line of y=x. MS[Formula: see text] spectral signals always located below the line, whereas all fragment ions lay at the left zone. After extracting information from the edges of each square frame, m/z values of both precursor ion and fragment ions could be harvested and putatively deciphered to a compound through applying some empirical mass fragmentation rules. RESULTS: The entire code of Ganoderma lucidum fruit bodies therefore corresponded exactly to a compound set. The elution program, even the employment of direct infusion, couldn't significantly impact the code, and dramatical differences occurred between different species and amongst different parts of Ganoderma lucidum as well. Not only ganoderic acid cluster but also certain primary metabolites served as the diagnostic compounds towards species differentiation. CONCLUSION: 2D-code might be a meaningful, practical visual way for rapid HM recognition because it is convenient to achieve the conversion amongst MS/MS dataset, 2D-barcode plot, and the chemome.

MS/MS fingerprint comparison between adjacent generations enables substructure identification: Flavonoid glycosides as cases.

MS/MS spectrum matching currently serves as a favored means to identify the concerned metabolites attributing to the accessibility of several famous databases. However, the rule that takes the entire structure into account frequently leads to "0 hit" when inquiring MS/MS (usually MS2) spectrum in the databases. Conjugation plays an important role for the high-level structural diversity of metabolites in all organisms, and a given conjugate usually consists of two or more substructures. If MS3 spectra participate in database retrieval, the structural annotation potential of those databases should be dramatically expanded via identifying substructures. Attributing to the ubiquitous distribution pattern, flavonoid glycosides were deployed as the representative family to justify whether the primary fragment ion termed as Y0+, resulted from neutral loss of glycosyl residue(s), generated identical MS3 spectrum with MS2 spectrum of the aglycone cation namely [A+H]+. Because of owning unique ability to measure MS/MS spectrum with the exactly desired exciting energy, linear ion trap chamber of Qtrap-MS was responsible for generating the desired MS3 and MS2 spectra. When taking both m/z and ion intensity features into consideration, the findings included: 1) glycosides sharing identical aglycones produced the same MS3 spectra for Y0+; 2) different MS3 spectra for Y0+ occurred amongst glycosides bearing distinct, even isomeric, aglycones; 3) isomeric aglycones generated different MS2 spectra; and 4) MS3 spectra for Y0+ agreed with MS2 spectra of [A+H]+ when comparing paired glycoside and aglycone. Together, fingerprint comparison between MS3 and MS2 spectra could structurally annotate the substructures and further advance MS/MS spectrum matching towards the identification of, but not limited to, aglycones for flavonoid glycosides.

Quality structural annotation for the metabolites of chlorogenic acid in rat.

Chlorogenic acid (CA) serves as a principal contributor for the health benefit spectrum of tea; however, its metabolism pattern hasn't been completely clarified. We attempted here to profile CA metabolism through fortifying isomer identification ability onto LC-MS/MS. Online energy-resolved MS was applied to configure full collision energy ramp-MS2 spectrum. Quantum structural calculation was undetaken to pursue the relationships between chemical structures and optimal collision energy/the maximum relative ion intensity. Thirty-seven metabolites were captured in biological matrices. Plausible structures were configured by applying the empirical mass fragmentation rules to m/z values and the summarized relationships were utilized to drive the plausible structures to confidence-enhanced identities. Hydrolysis, glucuronidation, sulfation, acyl migration, oxidation, and hydrogenation occupied primary metabolism channels. Together, the current study disclosed in depth the metabolism profile of CA and moreover, suggested a versatile analytical route for quality metabolite identification.