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In this study, we aimed to make enantioselective chromatography more sustainable, more sensitive, and compatible with aqueous formulations analysis and ESI-MS. To achieve this, we examined the effects of transitioning from normal-phase chromatography (which uses hydrocarbon-based solvents) to reversed-phase chromatography (using mobile phases based on water) using broad-spectrum Whelk-O1 columns as a critical study. For the first time, we holistically compared the thermodynamics and kinetics of the two elution modes in order to answer the question of whether same-column chemistry can effectively separate the compounds even in reversed-phase mode and found, unexpectedly, that reversed-phase chromatography using acetonitrile as the organic modifier was competitive from a kinetic standpoint. We also evaluated the effectiveness of three organic modifiers simultaneously on a sample of 11 molecules already resolved in NP conditions with different resolutions and achieved a resolution value of 1.5 for 91% and a resolution value of 2 for 82% of cases. Finally, we separated three racemates (within a k factor of 9) using only 480 µL of solvent per chromatographic run on a millibore column of 1 mm I.D., demonstrating that our approach allows for greener chromatographic separations.

Ion-pair chromatography is the de facto standard for separating oligonucleotides and related impurities, particularly for analysis but also often for small-scale purification. Currently, there is limited understanding of the quantitative modeling of both analytical and overloaded elution profiles obtained during gradient elution in ion-pair chromatography. Here we will investigate a recently introduced gradient mode, the so-called ion-pairing reagent gradient mode, for both analytical and overloaded separations of oligonucleotides. The first part of the study demonstrates how the electrostatic theory of ion-pair chromatography can be applied for modeling gradient elution of oligonucleotides. When the ion-pair gradient mode is used in a region where the electrostatic surface potential can be linearized, a closed-form expression of retention time can be derived. A unified retention model was then derived, applicable for both ion-pair reagent gradient mode as well as co-solvent gradient mode. The model was verified for two different experimental systems and homo- and heteromeric oligonucleotides of different lengths. Quantitative modeling of overloaded chromatography using the ion-pairing reagent gradient mode was also investigated. Firstly, a unified adsorption isotherm model was developed for both gradient modes. Then, adsorption isotherms parameter of a model oligonucleotide and two major synthetic impurities were estimated using the inverse method. Secondly, the parameters of the adsorption isotherm were then used to investigate how the productivity of oligonucleotide varies with injection volume, gradient slope, and initial retention factor. Here, the productivity increased when using a shallow gradient slope combined with a low initial retention factor. Finally, experiments were conducted to confirming some of the model predictions. Comparison with the conventional co-solvent gradient mode showed that the ion-pairing reagent gradient leads to both higher yield and productivity while consuming less co-solvent.

Comprehensive ingredient research is of great significance for understanding the effective material basis of herbal medicines, but due to the diversity and complexity of their phytochemicals, such research is challenging. Here, a multifaceted strategy was proposed to analyze and identify the composition of HuangLian JieDu Decoction based on offline two-dimensional liquid chromatography combined with ultraviolet detection and high-resolution mass spectrometry. Multiple components were separated by two-dimensional liquid chromatography, which consisted of hydrophilic interaction chromatography and reversed-phase liquid chromatography, and then further characterized by high-resolution mass spectrometry with a full mass spectrometry/precursor ion list/data-dependent secondary scan data acquisition method. For data processing, database screening and molecular networking were used to identify the components in HuangLian JieDu Decoction. The offline two-dimensional liquid chromatography combined with ultraviolet detection and a high-resolution mass spectrometry system showed good orthogonality of 76.35% and a high peak capacity of 5175, effectively separating multiple components. Finally, 527 compounds, including 164 alkaloids, 133 terpenoids, 88 flavonoids, 60 phenylpropanoids, 38 organic acids, and 44 other compounds, were characterized. This integrated approach is suitable for the comprehensive characterization of herbal medicines and other complex chemical systems.

The inherent complexity of traditional Chinese medicines necessitates the application of multi-dimensional information to accomplish comprehensive profiling and confirmative identification of their chemical components. In this study, we display an enhanced strategy by integrating offline superimposed two-dimensional separation (S-2D-LC) with mass defect filter and diagnostic ion filter to comprehensively characterize the alkaloid composition of Fritillariae Pallidiflorae Bulbus (FPB). The superimposed HILIC × RP and UPCC × RP offline two-dimensional liquid chromatography system was constructed with superior orthogonality (R2=0.004 and R2=0.001) for chromatographic separation. In total, 70 fractions were collected after the first-dimensional chromatographic separation (HILIC and UPCC) and then analyzed by the second-dimensional reversed phase (RP) liquid chromatography coupled with Q-TOF/MS/MS in FAST DDA acquisition mode. A four-step interpretation strategy combining mass defect filter with diagnostic ion filter was developed to rapidly characterize alkaloids in Fritillaria species. Ultimately, a sum of 529 Fritillaria alkaloids were characterized from two botanical origins of FPB. The integrated strategy is practical to efficiently expose and comprehensively characterize more trace and isomeric components in complex herbal medicines.

Comprehensive analysis and identification of chemical components are very important to evaluate the efficacy and safety of traditional Chinese medicine (TCM). Meanwhile, the discovery of new natural compounds is of great significance for drug exploitation and development. Although two-dimensional liquid chromatography (2D LC) systems expand the peak capacity and improve selectivity and resolution, interpreting the post-processing data is tedious and time-consuming. In this study, an off-line two-dimensional liquid chromatography/ultra-high performance supercritical fluid chromatography tandem quadrupole time-of-flight mass spectrometry (2D LC/UHPSFC-Q-TOF/MS) system was established for systematic chromatographic separation and identification of bufadienolides. Subsequently, the Global Natural Product Social Molecular Networking (GNPS) was applied for dereplication of chemical components of adjacent fractions with high efficiency and accuracy. The key parameters which affected separation and detection with respect to chromatographic conditions and mass spectrometry conditions were optimized. The extract of Venenum Bufonis was fractionated into forty fractions by first-dimensional reversed phase high-performance liquid chromatography (HPLC), which were further analyzed by the second-dimensional UHPSFC-Q-TOF/MS in positive ion mode. The data of forty fractions was imported into GNPS and processed automatically within about five hours. Furthermore, the chemical components with similar featured fragments were classified into the same cluster, which was helpful for components identification. A total of 229 bufadienolides were characterized and two subclasses of compounds (bufogenins conjugated with carboxylic acid and N-heterocyclic bufogenins) were found in Venenum Bufonis for the first time. In addition, UHPSFC exhibited powerful separation ability of isomers in Venenum Bufonis. In this analysis, two new compounds were isolated and fully characterized by NMR verifying the feasibility of this combined analytical strategy. This integrated strategy can improve the efficiency in the detection of new compounds and offer greater observation of isomers from medicinal herbs and other natural sources.

The ultrahigh-performance liquid chromatography-mass spectrometry (UHPLC/MS) method was optimized and validated for the determination of oxylipins in human plasma using the targeted approach with selected reaction monitoring (SRM) in the negative-ion electrospray ionization (ESI) mode. Reversed phase UHPLC separation on an octadecylsilica column enabled the analysis of 63 oxylipins including numerous isomeric species within 12-min run time. The method was validated (calibration curve, linearity, limit of detection, limit of quantification, carry-over, precision, accuracy, recovery rate, and matrix effect) and applied to 40 human female plasma samples from breast cancer patients and age-matched healthy volunteers (control). Thirty-six oxylipins were detected in human plasma with concentrations above the limit of detection, and 21 of them were quantified with concentrations above the limit of quantitation. The concentrations determined in healthy controls are in a good agreement with previously reported data on human plasma. Quantitative data were statistically evaluated by multivariate data analysis (MDA) methods including principal component analysis (PCA) and orthogonal partial least square discriminant analysis (OPLS-DA). S-plot and box plots showed that 13-HODE, 9-HODE, 13-HOTrE, 9-HOTrE, and 12-HHTrE were the most upregulated oxylipin species in plasma of breast cancer patients.

Although far away from perfect, it is practical to assess the quality of a given herbal medicine (HM) through simultaneous determination of a panel of components. However, the confidences of the quantitative outcomes from LC-MS/MS platform risk several technical barriers, such as chemical degradation, polarity range, concentration span, and identity misrecognition. Herein, we made an attempt to circumvent these obstacles by integrating several fit-for-purpose techniques, including online extraction (OLE), serially coupled reversed phase LC-hydrophilic interaction liquid chromatography (RPLC-HILIC), tailored multiple reaction monitoring (MRM), and relative response vs. collision energy curve (RRCEC) matching. Confidence-enhanced quantitative analysis of Cistanche salsa (Csa), a well-known psammophytic species and tonic herbal medicine, was conducted as a proof-of-concept. OLE module was deployed to prohibit chemical degradation, in particular E/Z-configuration transformation for phenylethanoid glycosides. Satisfactory retention took place for each analyte regardless of polarity because of successive passing through RPLC and HILIC columns. Optimum parameters for the minor components, at the meanwhile of inferior ones for the abundant ingredients, ensured the locations of all contents in the linear ranges. The unequivocal assignment of the captured signals was achieved by matching retention times, ion transitions, and more importantly, RRCECs between authentic compounds and suspect peaks. Diverse validation assays demonstrated the newly developed method to be reliable. Particularly, the distribution of mannitol rather than galactitol was disclosed although these isomers showed identical retention time and ion transitions. The contents of 21 compounds-of-interest were definitively determined in Csa as well as two analogous species, and the quantitative patterns exerted great variations among not only different species but different Csa samples. Together, the fortification of OLE-RPLC-HILIC-tailored MRM with RRCEC matching could fully address the demands from confidence-enhanced quantitative analysis of HMs.

The quality of herbal medicines (HMs) is the prerequisite for their pronounced therapeutic outcomes in clinic, and multi-component (also known as quality markers, Q-markers) quantification has been widely emphasized as a viable means for quality evaluation. Because of the chemical diversity, the quality control practices are extensively dampened by four principal technical bottlenecks, including the lack of authentic compounds, large polarity span, extensive concentration range, and signal misrecognition for those potential Q-markers. An attempt to promote the potential of LC-MS/MS is made herein to cope with those obstacles and Chinese agarwood was employed as a case study. Firstly, a home-made fraction collector was introduced to automatically fragment the entire extract into a panel of fractions-of-interest. Secondly, quantitative 1H-NMR was deployed to offset the LC-MS/MS potential towards in-depth chemical profiling each fraction, and those well-defined fractions were then pooled and combined with some accessible authentic compounds to generate the pseudo-mixed standard solution. Thirdly, serial improvements were conducted for LC-MS/MS measurements. Reversed phase LC and hydrophilic interaction LC were serially coupled in respond to the large polarity window, and online parameter optimization, response tailoring, as well as RRCEC (relative response vs. collision energy curve) matching were integrated in MS/MS domain to advance the quantitative confidences. Simultaneous determination was conducted for 26 components, in total, in Chinese agarwood after method validation. In particular, authentic compound-free quantification was achieved for eight 2-(2-phenylethyl)chromone derivatives. Above all, the strategy is a promising solution to completely tackle with the technical barriers toward Q-marker quantification-oriented quality control of Chinese agarwood, as well as other HMs.

The development of metabolomics based on ultra-high pressure liquid chromatography coupled to high-resolution mass spectrometry (UHPLC-HRMS) now allows hundreds to thousands of metabolites to be simultaneously monitored in biological matrices. In that context, bioinformatics and multivariate data analysis (MVA) play a crucial role in the detection of relevant alteration patterns. However, sound biological interpretations must necessarily be supported by metabolite identifications to be definitive or at least have a high degree of confidence. Each compound, should be characterised by unique molecular properties. Among them, the exact mass and the chromatographic retention time are recognised as major and complementary criteria for compound identification. While the former is easily derived from the molecular structure, building generic and accurate retention time open databases still constitutes a critical issue because of the vast diversity of instruments, stationary phases and operating conditions in UHPLC-HRMS. Because several hits matching a molecular formula obtained from an exact mass and an isotopic pattern are often generated for each analyte, this methodology rarely allows a unique and unambiguous molecular identity to be gained. This work aims to provide a flexible solution to facilitate reliable compound annotation based on retention time in reversed-phase liquid chromatography (RPLC). It proposes an innovative approach based on the chromatographic linear solvent strength (LSS) theory, allowing retention times under any gradient conditions at fixed temperature, stationary phase and mobile phase type to be predicted. Starting from a subset of the Human Metabolite Database (HMDB), a new dynamic database involving LSS parameters was developed. A real case study involving steroidogenesis alterations due to forskolin exposure was conducted using the adrenal H295R OECD reference cell model for endocrine disruptor screening. The prediction of retention times was successfully achieved, facilitating steroid identification. An automated procedure which implements the compound annotation levels encouraged by the Metabolite Standard Initiative (MSI) and the Coordination of Standards in Metabolomics (COSMOS) was also developed to speed up the process and enhance the data reusability.