Zhixin Zhang

Global chemical profiling based quality evaluation approach of rhubarb using ultra performance liquid chromatography with tandem quadrupole time‐of‐flight mass spectrometry

A global chemical profiling based quality evaluation approach using ultra performance liquid chromatography with tandem quadrupole time-of-flight mass spectrometry was developed for the quality evaluation of three rhubarb species, including Rheum palmatum L., Rheum tanguticum Maxim. ex Balf., and Rheum officinale Baill. Considering that comprehensive detection of chemical components is crucial for the global profile, a systemic column performance evaluation method was developed. Based on this, a Cortecs column was used to acquire the chemical profile, and Chempattern software was employed to conduct similarity evaluation and hierarchical cluster analysis. The results showed R. tanguticum could be differentiated from R. palmatum and R. officinale at the similarity value 0.65, but R. palmatum and R. officinale could not be distinguished effectively. Therefore, a common pattern based on three rhubarb species was developed to conduct the quality evaluation, and the similarity value 0.50 was set as an appropriate threshold to control the quality of rhubarb. A total of 88 common peaks were identified by their accurate mass and fragmentation, and partially verified by reference standards. Through the verification, the newly developed method could be successfully used for evaluating the holistic quality of rhubarb. It would provide a reference for the quality control of other herbal medicines.

A metabolomic strategy to screen the prototype components and metabolites of Qingkailing injection in rat urine by high‐performance liquid chromatography with tandem mass spectrometry

Xenobiotic metabolome identification of Chinese herbal formula in biological systems is a very challenging task. Qingkailing injection is a typical Chinese herbal injection, which is wildly used clinically in China. However, the holistic metabolic fate of the ingredient from Qingkailing injection remains unclear. In this work, a metabolomic strategy for comprehensively elucidating Qingkailing injection derived prototype components and metabolites in rat urine conducted by hybrid linear ion trap high-resolution mass spectrometry was developed. High-performance liquid chromatography coupled with hybrid linear ion trap high-resolution mass spectrometry was developed to obtain the urine profiling between the control group and Qingkailing injection treated group. Orthogonal partial least squares discriminate analysis was applied to distinguish the exogenous and the endogenous. In the S-plot, 37 xenobiotics derived from Qingkailing injection were found in urine, including 18 prototype compounds and 19 metabolites. The characterization of the prototype components and metabolites in rats urine provided essential data for further pharmacological studies of Qingkailing injection. Our results indicated that the metabolomic approach was an effective tool to discover, screen, and analyze the multiple prototype components and their metabolites from complicated traditional Chinese preparations in vivo.

Pharmacokinetic-Pharmacodynamic Modeling to Study the Antipyretic Effect of Qingkailing Injection on Pyrexia Model Rats.

Qingkailing injection (QKLI) is a modern Chinese medicine preparation derived from a well-known classical formulation, An-Gong-Niu-Huang Wan. Although the clinical efficacy of QKLI has been well defined, its severe adverse drug reactions (ADRs) were extensively increased. Through thorough attempts to reduce ADR rates, it was realized that the effect-based rational use plays the key role in clinical practices. Hence, the pharmacokinetic-pharmacodynamic (PK-PD) model was introduced in the present study, aiming to link the pharmacokinetic profiles with the therapeutic outcomes of QKLI, and subsequently to provide valuable guidelines for the rational use of QKLI in clinical settings. The PK properties of the six dominant ingredients in QKLI were compared between the normal treated group (NTG) and the pyrexia model group (MTG). Rectal temperatures were measured in parallel with blood sampling for NTG, MTG, model control group (MCG), and normal control group (NCG). Baicalin and geniposide exhibited appropriate PK parameters, and were selected as the PK markers to map the antipyretic effect of QKLI. Then, a PK-PD model was constructed upon the bacalin and geniposide plasma concentrations vs. the rectal temperature variation values, by a two-compartment PK model with a Sigmoid Emax PD model to explain the time delay between the drug plasma concentration of PK markers and the antipyretic effect after a single dose administration of QKLI. The findings obtained would provide fundamental information to propose a more reasonable dosage regimen and improve the level of individualized drug therapy in clinical settings.

Hypothalamus metabolomic profiling to elucidate the tissue-targeted biochemical basis of febrile response in yeast-induced pyrexia rats

In the previous reports regarding thermoregulation, the hypothalamus is thought to be the primary centre in the central nervous system for controlling the body temperature. However, to date, there has not been sufficient evidence to reveal its thermoregulatory mechanism. In the current study, we utilised a tissue-targeted metabolomics strategy to elucidate the underlying biochemical mechanisms of thermoregulation in the fever process by analysing the global metabolic profile of the hypothalamus in yeast-induced pyrexia rats. Data acquisition was completed using the HPLC-LTQ-Orbitrap/MS in both positive and negative ion mode. Principal component analysis was used to observe the cluster characteristics between the control group and the pyrexia group. Potential biomarkers were screened using orthogonal partial least-squares-discriminant analysis. Seventeen potential biomarkers were identified in the hypothalamus samples to discriminate the control and pyrexia groups, including amino acids, nucleic acids, vitamins, carbohydrates, and phospholipids. As a result, purine metabolism was enhanced pronouncedly, and perturbation of lipid metabolism was also observed. Meanwhile, amino acid metabolism and energy metabolism were also activated significantly. In conclusion, the study indicated that hypothalamus-targeted metabolomics could provide a powerful tool to further understand the pathogenesis of febrile response.

A Metabolomic Strategy to Screen the Prototype Components and Metabolites of Shuang-Huang-Lian Injection in Human Serum by Ultra Performance Liquid Chromatography Coupled with Quadrupole Time-of-Flight Mass Spectrometry.

Shuang-huang-lian injection (SHLI) is a famous Chinese patent medicine, which has been wildly used in clinic to treat acute respiratory tract infection, pneumonia, influenza, and so forth. Despite the widespread clinical application, the prototype components and metabolites of SHLI have not been fully elucidated, especially in human body. To discover and screen the constituents or metabolites of Chinese medicine in biofluids tends to be more and more difficult due to the complexity of chemical compositions, metabolic reactions and matrix effects. In this work, a metabolomic strategy to comprehensively elucidate the prototype components and metabolites of SHLI in human serum conducted by UPLC-Q-TOF/MS was developed. Orthogonal partial least squared discriminant analysis (OPLS-DA) was applied to distinguish the exogenous, namely, drug-induced constituents, from endogenous in human serum. In the S-plot, 35 drug-induced constituents were found, including 23 prototype compounds and 12 metabolites which indicated that SHLI in human body mainly caused phase II metabolite reactions. It was concluded that the metabolomic strategy for identification of herbal constituents and metabolites in biological samples was successfully developed. This identification and structural elucidation of the chemical compounds provided essential data for further pharmacological and pharmacokinetics study of SHLI.

One Single Amino Acid for Estimation the Content of Total Free Amino Acids in Qingkailing Injection Using High-Performance Liquid Chromatography-Diode Array Detection

Qingkailing injection (QKLI), a modern traditional Chinese medicine preparation, has been widely used in clinics due to its fast and significant efficacy in treatment of high fever. The free amino acids (AAs) were considered to be the most abundant active ingredients indisputably. So developing an accurate and simple determination method to measure the contents of total free AAs in QKLI is very crucial. In current study, the accurate and simple method of using one single standard AA for simultaneous quantification of multiple AAs (One for M) in QKLI was developed. Particularly, the calculation methods and the robustness of relative correction factors (RCFs) were investigated systematically. No statistically significant difference between these two quantification methods of One for M and classic regression equation was found by the t-test (P  =  95%, P > 0.05). The results showed that the precision (RSD < 4.88%), the robustness (RSD < 4.04%), and the average recoveries (94.11%–107.94%) of this newly proposed method all met the requirements for content determination. This One for M method will provide a scientific reference for the quantitative determination of AAs in other traditional Chinese medicines and their preparations owing to its accuracy and simplicity.

Applying characteristic fragment filtering for rapid detection and identification of ingredients in rhubarb by HPLC coupled with linear ion trap-Orbitrap mass spectrometry

Chemical characteristic fragment filtering in MSn chromatograms was proposed to detect and identify the components in rhubarb rapidly using high-performance liquid chromatography coupled with linear ion trap-Orbitrap mass spectrometry. Characteristic fragments consist of diagnostic ions and neutral loss fragments. Characteristic fragment filtering is a postacquisition data mining method for the targeted screening of groups with specific structures, including three steps: first, in order to comprehensively summarize characteristic fragments for global identification of the ingredients in rhubarb, representative authentic standards of dominant chemical categories contained in rhubarb were chosen, from which fragmentation rules and a characteristic fragments schedule were proposed; second, characteristic fragment filtering was used to rapidly recognize analogous skeletons; finally, combined with retention time, accurate mass, characteristic fragments, and previous literature, the structures of the filtered compounds were identified or tentatively characterized. As a result, a total of 271 compounds were detected and identified in rhubarb, including 34 anthraquinones, 83 anthrones, 46 tannins, 17 stilbenes, 24 phenylbutanones, 26 acylglucosides, 26 chromones, and 15 other compounds, 69 of which are potentially new compounds. The proposed characteristic fragment filtering strategy would be a reference for the large-scale detection and identification of the ingredients of herbal medicines.

Delipidation‐based solid‐phase extraction pretreatment technique for plasma broad‐coverage metabolomic profiling to reveal the potential pathogenesis of yeast‐induced fever in rats

During the process of metabolomics profiling by using ultra high performance liquid chromatography coupled with time-of flight mass spectrometry, blood sample pretreatment is a crucial step for biomarker discovery. Herein, in order to prevent the potential loss of metabolites and ion suppression phenomena caused by the proteins and phospholipids contained in blood fluids, a delipidation-based solid-phase extraction pretreatment technique for plasma broad-coverage metabolomic profiling was performed. This technique can be summarized as a single extraction, a single elution of solid-phase extraction plate, followed by four times measuring with electrospray ionization in positive and negative ion mode, respectively. This approach significantly increased the number of features detected in plasma, and 1572 features in positive mode and 1352 features in negative mode were detected, respectively. Besides, the stability and repeatability of the approach were greatly improved. For these advantages, the approach was employed to elucidate the potential pathogenesis of yeast-induced fever in rats. The biomarkers associated with the pathogenesis of fever were shown to be related to amino acids metabolism and lipid metabolism. The delipidation-based solid-phase extraction pretreatment approach can provide a useful tool to reveal the pathological mechanisms of such systemic pathological process.

An integrated strategy by using target tissue metabolomics biomarkers as pharmacodynamic surrogate indices to screen antipyretic components of Qingkaikling injection

Traditional Chinese medicine (TCM) treatment can be valuable therapeutic strategies. However, the active components and action mechanisms that account for its therapeutic effects remain elusive. Based on the hypothesis that the components of a formula which exert effect would be measurable in target tissue, a target tissue metabolomics-based strategy was proposed for screening of antipyretic components in Qingkaikling injection (QKLI). First, we detected the components of QKLI which could reach its target tissue (hypothalamus) by determining the hypothalamus microdialysate and discovered that only baicalin and geniposide could be detected. Then, by conducting hypothalamus metabolomics studies, 14 metabolites were screened as the potential biomarkers that related to the antipyretic mechanisms of QKLI and were used as its pharmacodynamic surrogate indices. Subsequently, the dynamic concentration of baicalin and geniposide in hypothalamus microdialysates and biomarkers in hypothalamus were measured and correlated with each other. The results indicated that only baicalin shown a good correlation with these biomarkers. Finally, a network pharmacology approach was established to validate the antipyretic activity of baicalin and the results elucidated its antipyretic mechanisms as well. The integrated strategy proposed here provided a powerful means for identifying active components and mechanisms contributing to pharmacological effects of TCM.

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 UNIFITM 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.