Qingqing Song

An integrated strategy to quantitatively differentiate chemome between Cistanche deserticola and C. tubulosa using high performance liquid chromatography-hybrid triple quadrupole-linear ion trap mass spectrometry.

It is important to conduct large-scale detection, identification, and quantitation of metabolites in a given sample. Herein, a practical strategy was proposed to quantitatively compare the chemome between Cistanche deserticola (CD) and C. tubulosa (CT), which have been widely believed as the ideal edible and medicinal plants for conquering the deserts. The entire workflow was implemented on high performance liquid chromatography-hybrid triple quadrupole-linear ion trap mass spectrometer and consisted of three primary steps: (1) component detection and identification, various mass spectrometric approaches were applied to globally screen the chemical constituents, and structural elucidation was achieved by comparing with authentic compounds, analyzing MS(2) spectra, and referring to the literature along with accessible databases; (2) comprehensive relative quantitation, scheduled multiple reaction monitoring algorithm was introduced for relative quantitation of all detected ingredients; and (3) chemome comparison, the quantitative dataset was subjected for multivariate statistical analysis to carry out comparative study. A total of 513 metabolites were detected and relatively quantitated, and 379 ones were annotated. Betaine, Krebs cycle intermediates, phenylethanoid glycosides, and iridoids were picked out as the chemical markers being responsible for the discrimination of the chemical profiles between CD and CT. Above all, the quantitative chemome of CD and CT were exhaustively characterized and compared, which could advance their values concerning drug development, economics, and desertification control. The proposed strategy is expected as a reliable choice for widely targeted metabolomics of plants.

Potential of hyphenated ultra-high performance liquid chromatography-scheduled multiple reaction monitoring algorithm for large-scale quantitative analysis of traditional Chinese medicines

It is a great challenge to perform quality control for traditional Chinese medicines (TCMs) that contain a great number of constituents by holistically monitoring hydrophilic and hydrophobic substances. Theoretically, the relatively low scan rate of triple quadrupole (QqQ) equipment makes it quite difficult to meet the demands of reliable quantitation of the narrow peaks generated from ultra-high performance liquid chromatography (UHPLC). Scheduled multiple reaction monitoring (sMRM) algorithm offers the potential to simultaneously monitor numerous analytes without compromising data quality, in particular for co-eluting compounds, by automatically altering the dwell time to maintain the desired cycle time on a QqQ analytical platform. In the current study, UHPLC and sMRM were hyphenated to develop a practical and robust quantitative method for as many as 133 TCM-derived components, including polar and apolar compounds. Efficient separation was achieved on a core–shell-type column (Capcell core ADME column) with adamantylethyl functional groups to generate appropriate surface polarity along with hydrophobicity in comparison with RP-C18 and HILIC columns. To verify the applicability of the developed UHPLC-sMRM method, a formula was simulated by mixing eight TCM raw materials that related to those 133 analytes. Moreover, enhanced product ion scans were triggered by sMRM to acquire MS2 spectra to enhance the confidence of peak assignment. Method validation results suggested the developed method to be accurate, precise, and reproducible. In comparison with conventional MRM, sMRM was proved to be advantageous in terms of sensitivity and precision, as well as the dependent MS2 spectral quality. Above all, our current study indicated that the integration of UHPLC and sMRM provides the potential to globally and simultaneously quantify the components in TCMs.

An integrated platform for directly widely-targeted quantitative analysis of feces part II: An application for steroids, eicosanoids, and porphyrins profiling

Steroids, especially bile acids, along with eicosanoids and porphyrins in feces play pivotal roles for the clinical diagnosis of various diseases. However, their reliable measurement is extensively obstructed by poor stability, structural diversity, broad content ranges, and tedious sample preparation protocols that account for a majority of the measurement errors. In current study, in-depth component screening was initially carried out by flexibly integrating diverse modes, such as predefined multiple reaction monitoring, stepped multiple ion monitoring, neutral loss scan, and precursor ion scan on a hybrid triple quadrupole-linear ion trap mass spectrometer, which also provided MS(2) spectra via enhanced product ion experiments. Meanwhile, a hybrid ion trap-time of flight mass spectrometer served as a complementary tool by providing accurate mass spectral information. Afterwards, because authentic compounds were unavailable for most analytes, an online optimization strategy was then proposed to optimize parameters, including precursor-to-product ion transitions and spectrometric parameters, notably collision energy. Finally, direct analysis of all detected components in feces was carried out by employing a platform integrating online pressurized liquid extraction, turbulent flow chromatography, and LC-MS/MS, and applying those optimized parameters. Seventy-one compounds, including 52 steroids and 13 eicosanoids, together with 6 porphyrins, were found and annotated in a fecal pool, and then relatively quantified in various fecal matrices. The quantitative dataset was subjected for multivariate statistical analysis and significant differences were observed among the quantitative chemome profiles of the fecal matrices from different groups. The findings obtained in the two parts demonstrated that the analytical platform in combination with the work-flow is qualified for not only directly simultaneous measurement of diverse endogenous substances, but widely targeted metabolomics of fecal matrices.

Simultaneous determination of components with wide polarity and content ranges in Cistanche tubulosa using serially coupled reverse phase-hydrophilic interaction chromatography-tandem mass spectrometry

To meet the demands from plant physiologists and pharmacognosists, sustainable efforts are being devoted by the analytical chemists from all over the world to search an approach being capable of simultaneously monitoring primary along with secondary metabolites. The key technical bottlenecks currently lie at affording satisfactory chromatographic and spectrometric performances for both hydrophilic and hydrophobic substances that span a great content range. Herein, reverse phase liquid chromatography was directly coupled with hydrophilic interaction chromatography, namely RPLC-HILIC, to integrate their merits, whereas dilution pumps were employed to tackle the mismatching for the mobile phase between them. On the other side, inferior parameters rather than the optimal ones were applied for those abundant ingredients to advance the upper limits of quantitation, such as echinacoside (1250.0μg/mL), mannitol (100.0μg/mL), and acteoside (125μg/mL), in mass spectrometer domain. As a desert parasitic plant as well as tonic materials, Cistanche tubulosa (CT) has drawn widely interests from plant physiologists and pharmacognosists regarding its quantitative metabolome. Simultaneous determination of 23 abundant and minor ingredients covering most chemical families in CT, i.e. amino acid, nucleoside, organic acid, phenylethanoid glycoside, lignan, and iridoid, was attempted to understand the physiologic patterns as well as pharmacological values of the crude materials. Although the analytes spanned wide polarity (-3.326≤cLogP≤1.421) and content (more than 5 orders of magnitude) ranges, satisfactory chromatographic and spectrometric behaviors were gained for all analytes. Reliable quantitation was demonstrated via various method validation assays, such as recovery, linearity, sensitivity, and precision. The contents of 23 hydrophilic and hydrophobic substances were quantified in 20 batches of CT. Significant variations occurred for the contents of those components. Echinacoside (1.35-387.50mg/g) was usually observed as the most abundant component, whereas ferulic acid (<0.0043mg/g) always exhibited trace distributions. Above all, the integrated equipment setup could serve as a fit-for-purpose tool for in-depth quality evaluation of CT and more importantly, for comprehensively understanding the metabolome of plants.

Integrated work-flow for quantitative metabolome profiling of plants, Peucedani Radix as a case

Universal acquisition of reliable information regarding the qualitative and quantitative properties of complicated matrices is the premise for the success of metabolomics study. Liquid chromatography-mass spectrometry (LC-MS) is now serving as a workhorse for metabolomics; however, LC-MS-based non-targeted metabolomics is suffering from some shortcomings, even some cutting-edge techniques have been introduced. Aiming to tackle, to some extent, the drawbacks of the conventional approaches, such as redundant information, detector saturation, low sensitivity, and inconstant signal number among different runs, herein, a novel and flexible work-flow consisting of three progressive steps was proposed to profile in depth the quantitative metabolome of plants. The roots of Peucedanum praeruptorum Dunn (Peucedani Radix, PR) that are rich in various coumarin isomers, were employed as a case study to verify the applicability. First, offline two dimensional LC-MS was utilized for in-depth detection of metabolites in a pooled PR extract namely universal metabolome standard (UMS). Second, mass fragmentation rules, notably concerning angular-type pyranocoumarins that are the primary chemical homologues in PR, and available databases were integrated for signal assignment and structural annotation. Third, optimum collision energy (OCE) as well as ion transition for multiple monitoring reaction measurement was online optimized with a reference compound-free strategy for each annotated component and large-scale relative quantification of all annotated components was accomplished by plotting calibration curves via serially diluting UMS. It is worthwhile to highlight that the potential of OCE for isomer discrimination was described and the linearity ranges of those primary ingredients were extended by suppressing their responses. The integrated workflow is expected to be qualified as a promising pipeline to clarify the quantitative metabolome of plants because it could not only holistically provide qualitative information, but also straightforwardly generate accurate quantitative dataset.

An integrated platform for directly widely-targeted quantitative analysis of feces part I: Platform configuration and method validation.

Direct analysis is of great importance to understand the real chemical profile of a given sample, notably biological materials, because either chemical degradation or diverse errors and uncertainties might be resulted from sophisticated protocols. In comparison with biofluids, it is still challenging for direct analysis of solid biological samples using high performance liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). Herein, a new analytical platform was configured by online hyphenating pressurized liquid extraction (PLE), turbulent flow chromatography (TFC), and LC-MS/MS. A facile, but robust PLE module was constructed based on the phenomenon that noticeable back-pressure can be generated during rapid fluid passing through a narrow tube. TFC column that is advantageous at extracting low molecular analytes from rushing fluid was employed to link at the outlet of the PLE module to capture constituents-of-interest. An electronic 6-port/2-position valve was introduced between TFC column and LC-MS/MS to fragment each measurement into extraction and elution phases, whereas LC-MS/MS took the charge of analyte separation and monitoring. As a proof of concept, simultaneous determination of 24 endogenous substances including eighteen steroids, five eicosanoids, and one porphyrin in feces was carried out in this paper. Method validation assays demonstrated the analytical platform to be qualified for directly simultaneous measurement of diverse endogenous analytes in fecal matrices. Application of this integrated platform on homolog-focused profiling of feces is discussed in a companion paper.

New instrumentation for large-scale quantitative analysis of components spanning a wide polarity range by column-switching hydrophilic interaction chromatography-turbulent flow chromatography-reversed phase liquid chromatography-tandem mass spectrometry

The achievement of satisfactory chromatographic performance for every component regardless of the polarity plays a pivotal role in large-scale targeted metabolomics of complicated matrices; however, it is almost impossible to achieve comprehensive retention of all hydrophilic and hydrophobic substances by solely employing either hydrophilic interaction chromatography (HILIC) or reversed-phase liquid chromatography (RPLC). Given the great complementarity between HILIC and RPLC, we attempted herein to find a superior instrumentation scheme for their online hyphenation. New instrumentation, namely column-switching HILIC-turbulent flow chromatography-RPLC-tandem mass spectrometry (HILIC-TFC-RPLC-MS/MS) was firstly constructed by employing five solvent pumps, two electronic 6-port/2-channel valves, three columns including an Amide-type HILIC column, an HSS T3-type RP column along with a TFC column, a hybrid triple quadrupole-linear ion trap mass spectrometer (Qtrap-MS), as well as some other essential units. Each analytical run was automatically fragmented into loading (0–4 min) and parallel elution (4–32 min) phases via switching both valves. The TFC column was in charge of trapping apolar compounds from the diluted effluent of the Amide column within the loading phase and subsequently transmitting them into the HSS T3 column according to back-flushing in the parallel elution phase. Chromatographic separations of hydrophobic substances were accomplished on the HSS T3 column, whereas the Amide column took the load of separating the other substances. Qtrap-MS always received both eluents from the HILIC and RP columns. Three existing hyphenated HILIC-RPLC schemes, such as serially coupled RPLC-HILIC, guard column-(HILIC/RPLC), and HILIC-trapping column-RPLC, were involved for comparisons. With the assignment of an optimized elution program for each scheme, HILIC-TFC-RPLC-MS/MS was slightly better than the other ones for large-scale monitoring of polar and apolar components in a mimic compound pool containing 100 components. Above all, the integrated HILIC-TFC-RPLC-MS/MS platform can serve as a feasible choice to gain a holistic view regarding both hydrophilic and hydrophobic components in complicated matrices.

Sensitive profiling of phenols, bile acids, sterols, and eicosanoids in mammalian urine by large volume direct injection-online solid phase extraction-ultra high performance liquid chromatography-polarity switching tandem mass spectrometry

Bile acids, sterols, and eicosanoids serve as important chemical categories in mammalian urine and, sometimes, as primary diagnostic markers for diverse diseases, whereas phenols, usually derived from the diet, are usually observed as annoying interferents in pharmacokinetic and metabolic profiling of phenolic derivatives. Precise analysis of these substances widely suffers from instability and trace distributions, as well as errors and uncertainties resulting from tedious sample preparation procedures. Herein, directly simultaneous determination of phenols, bile acids, sterols, and eicosanoids, in particular those trace ones, in mammalian urinary matrices was attempted using large volume direct injection-online solid phase extraction-ultra high performance liquid chromatography-polarity switching tandem mass spectrometry (LVDI-online SPE-UHPLC-psMS/MS). Large volumes (500 μL) of liquid-state samples were directly loaded onto an online SPE column via ten consecutive injections. The SPE column was responsible for accumulating targeted components at the loading phase (−4.5 to 5.0 min), while transmitting those trapped analytes into a Waters HSS T3 column at the elution phase (5.0–27.0 min). Phase switching was accomplished using an electronic 6-port/2-channel valve. Analyte detection of all analytes, 28 ones in total, was performed using both positive and negative multiple reaction monitoring modes. Various method validation assays demonstrated the developed method to be extremely sensitive (most limits of quantitation lower than 0.3 ng mL−1), precise (all RSDs of intra- and inter-day variations lower than 15%) and accurate (recoveries ramped from 80.4% to 120.0% with RSDs lower than 20%). Significant variations occurred for urine samples from different species, as well as amongst urinary matrices from different individuals within the same group. The findings proved that the developed LVDI-online SPE-UHPLC-psMS/MS method takes advantage of detecting the trace components and preserving those mutable substances from degradation, thus offering a meaningful tool for widely targeted monitoring substances in biofluids.

Qualitative and Quantitative Assessments of Aconiti Lateralis Radix Praeparata Using High-Performance Liquid Chromatography Coupled with Diode Array Detection and Hybrid Ion Trap-Time-of-Flight Mass Spectrometry.

Dual roles have been widely disclosed for Aconiti Lateralis Radix Praeparata (Chinese name: Fuzi) by acting as an effective herbal medicine accompanied with high toxicity risk and narrow therapeutic range. Aconite alkaloids, also known as diterpenoid alkaloids (DAs), have been proved to be primary therapeutic as well as toxic material basis of Fuzi. In the present study, we aim to characterize qualitative profile along with quantitative features of DAs in Fuzi, which could benefit the quality control of Fuzi as well as its safe medication in clinic. Chromatographic fingerprinting was achieved using 12 batches of crude materials collected from different habitats (similarities >0.9) by hyphenating liquid chromatography (LC) with diode array detection. Simultaneous determination of six primary DAs, namely benzoylmesaconine, benzoylaconine, benzoylhypaconine, mesaconine, aconitine and hypaconitine, was also carried out. The developed method was systematically validated and successfully applied for quantitative characterization. Significant variations were observed regarding the contents of those six analytes among different raw materials. A total of 99 DAs were detected and characterized, including 9 unambiguous identities and 77 putative assignments, as well as 13 unknown compounds, from the representative extract by LC coupled with hybrid ion trap-time-of-flight mass spectrometry. The findings obtained herein could favor in-depth understanding of the chemical composition of DAs and the quality control of Fuzi.

Direct stability characterization of aconite alkaloids in different media by autosampler-mediated incubation-online solid phase extraction-LC-MS/MS

Because degradation might occur at any step of conventional sample preparation protocols, it is of great importance to carry out direct analysis for stability characterization of drug candidates in vitro. Herein, an automated system for directly monitoring the stability of drug candidates in different media was configured by hyphenating autosampler-mediated incubation, online solid phase extraction, and high performance liquid chromatography-tandem mass spectrometry (AMI-SPE-LC-MS/MS). The thermostatic autosampler served as an incubator, and an SPE column was employed to online capture analytes after direct injection of incubated matrices. To achieve sensitive detection, a predictive multiple reaction monitoring approach was introduced to monitor the degradation products. As a proof-of-concept, this system was applied for direct stability characterization of six aconite alkaloids, including aconitine (AC), mesaconitine (MA), hypaconitine (HA), benzoylmesaconine (BMA), benzoylaconine (BAC), and benzoylhypaconine (BHA), which are the primary effective as well as toxic components in aconite-derived herbal medicines. Extensive hydrolysis to generate two degradation products was observed for AC, MA, or HA in phosphate buffer saline (PBS, pH 7.4), PBS fortified with fresh rat plasma and PBS spiked denatured rat plasma, whereas BMA, BAC, and BHA remained intact in PBS, PBS spiked rat plasma, methanol, acetonitrile (ACN), and 50% aqueous ACN. Therefore, care should be taken to maintain the stability of aconite alkaloids, in particular in alkaline media, during metabolic and pharmacokinetic evaluations. More importantly, the developed AMI-SPE-LC-MS/MS system provides a reliable tool for directly and veritably monitoring the in vitro stability of drug candidates.