Zingiberis Rhizoma (ZR) is a homologous plant with pungent tastes and aromas, which has unique nutritional value and tremendous application potentiality. Zingiberis Rhizoma Praeparatum (ZRP) and Carbonised Ginger (CG) are processed products of ZR through different processing methods, and they are commonly used ingredients in food supplements. This study used ZR, ZRP and CG from different batches to further understand composition differences after processing. Additionally, we performed non-targeted metabolomics-based profiling of gingerols by ultra-high-performance liquid chromatography coupled with hybrid triple quadrupole time-of flight mass spectrometry (UHPLC-Q-TOF/MS) in combination with multivariate analysis and compounds identification. In which, we developed a comprehensive SWATH-IDA bi-directionally verified (SIBDV) method integrating the advantages of Sequential Windowed Acquisition of all Theoretical fragment ions (SWATH (TM)) and traditional information-dependent acquisition (IDA) mode for characterization of gingerols. Potential chemical markers were selected by principal component analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA) of chemometrics methods. After that, the threshold variable importance in projection (VIP) value and P value were employed to screen the valuable MS features for discriminating ZR, ZRP and CG. In total, 59 gingerols in the different samples were structurally identified. Results allowed the selection of 33 gingerols, which are nominated as novel markers for materials authentication in ZR, ZRP and CG. The analysis of the study showed that the content of gingerols showed a downward trend after processing, but shogaols and gingerone compounds had an upward trend, resulting in differences in application and pharmacodynamic efficacy. These findings provide promising perspectives in the quality control of ZR, ZRP and CG, as well as for laying the foundation in food design and development.

Betulin, as a new type of natural food preservative, is widely used in various kinds of meat products. However, its detailed mechanism of action and metabolism have not been clarified. In this study, for further gain insight of the mechanism of betulin as a preservative, an efficient method has been applied for measuring the antioxidant capacity of betulin, based on the absorbance of the DPPH• and ABTS• radical cation. When the concentration of betulin was more than 2.0 mg/mL, the scavenging rate of ABTS and DPPH radical reached over 90%, which was equivalent to the antioxidant capacity of Trolox. It is indicated that betulin has significant DPPH and ABTS free radical scavenging ability. This should be one of the important mechanisms for betulin as a preservative. A sensitive method using UHPLC-Q-TOF-MS/MS was established to determine the metabolite profile in vivo and in vitro of betulin. 32 phase I and 2 phase II metabolites were structurally characterized. This study will provide theoretical support for the safety and effectiveness of betulin in the field of preservatives and provide theoretical basis for the further study of betulin and the other natural preservatives. This research also contributes to the development of the food industry.

Pulsatilla Adans (PSA) herbs (Ranunculaceae) have been widely used in traditional medicine in China and other countries. However, the authentication and quality control of PSA herbs have always been a challenging task due to their similar morphological characteristics and the diversity of the multiple components that exist in the complicated matrix. Herein, a novel integrated strategy combining UHPLC/Q-Orbitrap-MS techniques with chemometrics analysis is proposed for the discrimination of PSA materials. We developed a comprehensive method integrating a nontargeted bidirectionally screened (NTBDS) MS data set and a targeted extraction peak area analysis for the characterization of triterpenoid saponins of PSA from different species. After that, partial least-squares discriminant analysis (PLS-DA) was performed on the obtained MS data set and the parameter variable importance for the projection (VIP) value and P value were employed to screen the valuable MS features to discriminate PSA from different species. In addition, the receiver operating characteristic (ROC) curve is used to verify the reliability of MS features. Finally, heatmap visualization was employed to clarify the distribution of the identified triterpenoid saponins, and four medicinal species of PSA were successfully differentiated. Additionally, 34 constituents were reported in PSAs for the first time, 81 triterpenoid saponins were identified as differential components, and 12 chemical ingredients were characterized as potential chemical markers to differentiate the four officinal PSA herbs. This is the first time that the differences in different PSA herbs have been observed systematically at the chemical level. The results suggested that using the identified characteristic components as chemical markers to identify different PSA herbs was effective and viable. This method provides promising perspectives in the analysis and identification of the ingredients of Chinese herbal medicines, and the identification of similar herbs from the same species.

INTRODUCTION Triterpenoid saponins are the major bioactive constituents of Pulsatilla chinensis, playing an important role in various biological activities such as anti-tumour, cognition-enhancing, anti-biosis, anti-inflammatory, hypoglycemic and immunological adjuvant. OBJECTIVE To establish a systematic strategy based on ultra-high-performance liquid chromatography-quadrupole time-of-flight tandem mass spectrometry (UPLC-Q-TOF-MS/MS) for the efficient characterisation and identification of triterpenoid saponins in crude extracts from Pulsatilla chinensis. METHODOLOGY In this work, the strategy includes two aspects: (1) positive mode: by target screening, we can deduce the aglycone type and the composition of sugar moiety according to the fragment ions; untargeted screening includes four steps, find unknown, formula finder, ChemSpider search and MS/MS identification; (2) negative mode: according to the MS/MS spectra, the composition of sugar chain bonded to C-28 is inferred reasonably. The extract of Pulsatilla chinensis was separated within 60 min on a C18 column and eluted with methanol and water both containing 0.1% formic acid. RESULTS As a result, a total of 22 triterpenoid saponins (11 pairs of isomers) with four aglycone skeletons were tentatively identified or elucidated in crude extracts from Pulsatilla chinensis based on their retention times, the mass spectrometric fragmentation patterns, and MS and MS/MS data. CONCLUSION This study provides an efficient analysis strategy to rapidly identify the triterpenoid saponins in Pulsatilla species even in traditional Chinese medicines.

Betulinic acid is a triterpenoid organic acid with remarkable antitumor properties and is naturally present in many fruits, condiments and traditional Chinese medicines. Currently, a strategy was developed for the identification of metabolites following the in vivo and in vitro biotransformation of Betulinic acid with rat intestinal bacteria utilizing ultra high performance liquid chromatography with time-of-flight mass spectrometry with polymeric solid-phase extraction. As a result, 46 metabolites were structurally characterized. The results demonstrated that Betulinic acid is universally metabolized in vivo and in vitro, and Betulinic acid could undergo general metabolic reactions, including oxidation, methylation, desaturation, loss of O and loss of CH2 . Additionally, the main metabolic pathways in vivo and in vitro were determined by calculating the relative content of each metabolite. This is the first study of Betulinic acid metabolism in vivo, whose results provide novel and useful data for better understanding of the safety and efficacy of Betulinic acid.

A tissue-smashing based ultra-rapid extraction coupled with ultra-performance liquid chromatography tandem-mass spectrometry (UPLC-MS/MS) method was developed to determine 10 major triterpenoid saponins from Pulsatilla herbs. Compound 4 was characterized as betulinic acid glycoside 3-O-α-arabinopyranosyl-28-O-β-glucopyranosyl-23-hydroxy with HR-ESI-MS, 1H-NMR and 13C-NMR experiment. The MS spectra result showed that the ionization of compound 4 was more efficient in the positive mode. Meanwhile, the ions at m/z 789.6 and m/z 627.5 were selected as precursor and product ion for the determination, respectively. The chromatographic separation was carried out on a Phenomenex Kinetex C18 column using a gradient mobile phase system composed of 0.1% formic acid both in methanol and water at a flow rate of 0.4 mL/min. The detection was performed by multiple reaction monitoring mode, using electrospray ionization in the positive and negative mode. The total run time was 6 min. The calibration curves possessed good linearity with all coefficients higher than 0.9987. The intra- and interday precisions were no more than 4.9%, and the average recoveries were from 97.6% to 103.4% with RSD <4.7%. Moreover, hierarchical cluster analysis was performed to compare and discriminate the Pulsatilla herbs based on the quantitative data. The hierarchical cluster analysis results demonstrated that Pulsatilla chinensis, Pulsatilla cernua, Pulsatilla dahurica, Pulsatilla turczainovii samples could be easily discriminated from each other based on the contents of triterpenoid saponins and the established method is feasible for quality control of Pulsatilla herbs.

Betulin is an active natural pentacyclic triterpene ingredient with valuable anti-cancer and anti-HIV efficacies. In the present study, an efficient approach was developed to screening and identification of metabolites and to assess the metabolic profiles of betulin in vivo using UHPLC-Q-TOF-MS/MS system based on multiple mass defect filter data acquisition (MMDF) and multiple data processing techniques. Based on the proposed method, 56 phase I and 6 phase II metabolites were detected in rats after oral administration of betulin. The main biotransformation routes of betulin were identified as demethylation, dehydroxylation, deoxidization, dehydration. Conjugation with sulfate, taurine, cysteine and N-acetylcysteine groups produced 6 phase II metabolites. This study not only provided useful information for further study of the pharmacology and mechanism of betulin in vivo, but also provided essential data for further pharmaceutical studies of other pentacyclic triterpenes.

Ultra high performance liquid chromatography coupled with mass spectrometry and combining a tissue-smashing extraction technique was developed for the simultaneous quantitative analysis of 12 compounds in the roots of Pulsatilla chinensis. Among them, compound 6 was characterized and accurately quantified in this herb for the first time. The parameters of extraction condition were simultaneously optimized with a Box-Behnken design and Derringer's function. The optimized conditions were as follows: sample quantity of 0.5 g, ethanol concentration of 70%, and extraction time of 200 s. Multiple-reaction monitoring scanning was employed for the quantification between positive and negative mode in a single run of 6 min. Full validation of the method was carried out, and the results indicated that the method was rapid, specific, and reliable. The developed method was successfully applied to quantify the 12 compounds in 33 batches of P. chinensis from different provinces. Moreover, the principal component analysis was performed to compare the P. chinensis collected from different provinces of China based on quantitative data and the results indicated that the content of compounds could be used to differentiate the origins of P. chinensis. These results demonstrated that this method is feasible and reliable for the quality control of P. chinensis.

The detection of drug metabolites, particularly for minor metabolites, continues to be a challenge owing to the complexity of biological samples. Imperatorin is an active natural furocoumarin ingredient originating from many traditional Chinese herbal medicines. In the present study, the metabolites of imperatorin after oral administration were qualitatively investigated, and possible metabolic pathways of it were subsequently proposed. Bile samples were collected after oral administration and pretreated by the application of Waters Ostro. The QTOF-MS/MS data was acquired using ultra high performance liquid chromatography coupled to quadrupole time flight spectrometry (UPLC-QTOF-MS). Based on this analytical strategy, 32 metabolites (23 phase I and 9 phase II metabolites) were identified in rat bile. The results demonstrated that C5H8 could be easily eliminated from imperatorin forming the metabolite M1. It also indicated that imperatorin and M1 underwent extensive metabolic reactions including oxidation, hydrolysis, methylation, glucuronide conjugation, C2H5NO2S conjugation and C3H5NO2S conjugation. This is the first study of imperatorin metabolism in bile samples. The proposed metabolic pathways in this research will provide essential data for further pharmaceutical studies of other linear-type furocoumarins.

目的:建立同时测定白蒲黄片中7种五环三萜皂苷类成分白头翁皂苷A3、白头翁皂苷B4、23-羟基白桦脂酸、白头翁皂苷B、白头翁皂苷C、刺人参苷S和齐墩果酸的HPLC-MS法.方法:采用Diamonsil ODS C18(250 mm×4.6 mm,5μm)色谱柱,流动相为含0.1％甲酸的水(A)-含0.1％甲酸的甲醇(B),梯度洗脱程序为25％ A(0 min),5％A(0～6 min),5％A(6 ～14min),25％A(保持6 min),流速0.8 mL·min-1,柱温30 ℃,进样量10 μL;采用正离子和负离子模式同时监测,正离子和负离子监测模式下源喷射电压分别为5.5 kV和-4.5 kV,离子源温度为650℃,雾化气(Gas 1)344 kPa,加热气(Gas 2)412 kPa,接口持续加热,帘气172 kPa,全程氮气通入状态.结果:在14 min内白蒲黄片中7种有效成分白头翁皂苷A3、白头翁皂苷B4、23-羟基白桦脂酸、白头翁皂苷B、白头翁皂苷C、刺人参苷S和齐墩果酸被完全分离;峰面积与其浓度呈良好的线性;平均回收率范围为95.11％ ～ 107.3％,RSD为0.86％ ～ 3.26％.结论:本文建立的方法经验证简便、重现性好、专属性高,可为白蒲黄片质量控制提供参考.

随着医学模式由“生物医学”模式向“生物—心理—社会医学”模式的转变，人文社会学科与医学的交叉渗透形成了许多新兴的交叉学科，越来越多的人文社会学科的理论与方法被医学领域所应用并带来了医学的革命性变化，尤其在医学创新人才培养上，突出人文精神的培养具有深刻的影响。本文秉承“以人文精神培养为主线，以职业精神培养为目标，以创新精神、创新思维和创新能力为根本”的思想，把人文社会学科的理论与方法贯穿于整个医学创新人才的培养全过程之中，把医学生人文精神的塑造作为创新医学人才培养的目标。经过多年的研究与实践，取得了可喜的成绩。

The aim of this study was to develop a liquid chromatography-tandem mass spectrometry (HPLC–MS/MS) method for the simultaneous quantitative determination of the following triterpenoidal saponins: anemoside A3, anemoside B4, 23-hydroxybetulinic acid, pulsatilloside B, pulsatilloside C, and cirenshenoside S in rat urine. The chromatographic separation was performed using a Sapphire C18 column (250 mm × 4.6 mm, 5 μm) and gradient elution was used during the analysis. Anemoside A3, anemoside B4, and 23-hydroxybetulinic acid were detected with the mass spectrometer in negative ion mode monitoring at m/z 749.6/471.2, 1,219.7/749.4, 471.4/471.4 and pulsatilloside B, pulsatilloside C, and cirenshenoside S were in positive ion mode monitoring at m/z 819.4/347.2, 965.5/493.2, and 1,097.9/493.1, respectively. The calibration curves were indicative of good linearity (r 2 ≥ 0.9965) in the range of interest for each analyte. Intra-day and inter-day precision (CV %) was less than 9.1 and 8.7 %, respectively, and accuracy was between −8.4 and 8.3 %. Recovery was 81.02–102.9 %. The method is very rapid, simple, and reliable, and suitable for excretion studies. It can be routinely used for simultaneous determination of six triterpenoidal saponins in rat urine. These results indicated that the newly developed method can also be applied to studies after administration of extraction of saponins from Pulsatilla chinensis to rats.

Objective:To develop an HPLC-MS method for the determination of ginkgolide A(GA),ginkgolide B(GB),ginkgolide C(GC),bilobalide(BB),quercetin(QCT),isorhamnetin(ISR)and kaempferol(KAE)in Shuxuening injection.Methods:The samples were separated on a Dikma Diamonsil C18 column(150 mm×4.6 mm,5 μm)with a mobile phrase of acetonitrile and water containing 0.05% formic acid in a gradient elution at a flow rate of 0.8 mL·min-1,and the injection volume was 10 μL.The column temperature was set at 30 ℃.Multiple-reaction monitoring(MRM)scanning was employed for quantification with switching electrospray ion source polarity in negative mode.The ion spray voltage was set to-4.5 kV and the turbo spray temperature was maintained at 650℃.The precursor-to-product ion pairs of seven analytes were m/z 453.1/351.2 for GA,m/z 423.1/367.0 for GB,m/z 439.0/383.0 for GC,m/z 325.0/163.1 for BB,m/z 301.0/150.9 for QCT,m/z 315.1/300.0 for ISR and m/z 284.9/93.0 for KAE,respectively.Results:The complete separation was obtained within 7 min for the seven compounds.The regression equations showing good linear relationships between peak areas and contents of each compound were obtained.The average recoveries of the compounds ranged from 97.7% to 101.5% and the precision in terms of RSD was in the range of 0.57%-1.20%.Conclusion:The method can detect the contents of 7 components in Shuxuening injection with good repeatability,recovery and precision,and can be used for the quality control of Shuxuening injection.

OBJECTIVE To develop a method for the determination of arbutin,gallic acid,bergenin,protocatechuic acid,chlorogenic acid,catechin,epicatechin,epicatechin gallate,and ferulic acid in Bergenia purpurascens by HPLC-MS/MS.METHODS The samples were separated on a Diamonsil C18 column(4.6 mm×150 mm,5 μm) by gradient elution using methanol and 0.2‰(V/V) formic acid aqueous solution as the mobile phase at a flow rate of 0.8 mL·min-1.The column temperature was room temperature.Multiple-reaction monitoring(MRM) scanning was employed for quantification with switching electrospray ion source polarity in negative mode.RESULTS Complete separation was achieved within 10 min for the nine compounds(arbutin,gallic acid,bergenin,protocatechuic acid,chlorogenic acid,catechin,epicatechin,epicatechin gallate,ferulic acid).The nine regression equations showed linear relationships between peak areas and contents of each compound.The average recoveries(n=9) of the compounds listed above were 100.2%,100.0%,99.8%,99.9%,97.9%,101.2%,99.8%,101.7%,102.4% and RSD were 0.80%,3.06%,2.72%,3.28%,3.58%,1.44%,0.95%,0.96%,0.48%,respectively.CONCLUSION The method is simple,accurate and has good reproducibility,thus can be used for the determination of the nine compounds in Bergenia purpurascens.

The Er-Mu preparation (EMP) is a well-known traditional Chinese prescription that has been clinically employed for the treatment of asthma and bronchial inflammation for hundreds of years. Neomangiferin, mangiferin, peimine, peiminine, timosaponin BII and timosaponin AIII are the major active ingredients of EMP for their anti-inflammatory or anti-asthmatic effects. The aim of this study was to investigate the pharmacokinetics of the target compounds from the recipe of EMP and the single herb extracts of Anemarrhenae asphodeloides Bge. (ARR) and Fritillariae cirrhosae D.Don (FCB), and the influence of compatibility on the pharmacokinetics of the main active ingredients. The rats were randomly assigned to three groups and orally administered with the recipe of EMP and the single herb extracts of ARR and FCB, respectively. The concentrations of the target compounds in rat plasma were determined by an optimal liquid chromatography-electrospray ionization mass spectrometry (HPLC-ESI-MS) and multiple reaction monitoring (MRM) with a multi-switching monitoring mode coupled with simple protein precipitation method, and the main pharmacokinetic parameters were estimated. Significant differences (p<0.05) were found in the pharmacokinetic parameters of neomangiferin, mangiferin, peimine and peiminine between the single ARR or FCB extract and the combination treatment (p<0.05). The developed HPLC-ESI-MS method by switching positive and negative ESI sources in a single run was successfully applied to study the pharmacokinetics of six compounds in SD rat, which was powerful in terms of sensitivity, selectivity, time savings and solvent consumption in the quantitative analysis of complex herbal medicines. It was surmised that formula compatibility could significantly influence the pharmacokinetics of EMP and our study has preliminarily elucidated the priority in the compatible administration of EMP based on pharmacokinetic studies.

Objective To develop a method for determining the plasma protein binding rate of betulinic acid in rat plasma,human plasma and bovine serum albumin(BSA).Methods The plasma protein binding rate of betulinic acid was determined by using equilibrium dialysis.The concentration of plasma betulinic acid was determined by using HPLC.The plasma protein binding rate of betulinic acid was determined and compared in different serum samples.The HPLC procedure was performed on DiamonsilTM C18 column at 30 ℃.The mobile phase was acetonitrile-0.2% acetic acid(75∶ 25) in gradient elution,flow velocity was 1.0 mL/min and detection wavelength was 203 nm.Results When the concentration was from 50 mg/L to 100 mg/L,the concentration of betulinic acid had no significant influence on plasma protein binding rate.There was a significant difference between the plasma protein binding rate of betulinic acid in rat plasma and that in human plasma.The plasma protein binding rate of betulinic acid in human plasma was higher than that in rat plasma.Conclusion Betulic acid has a higher binding intension with plasma protein.HPLC for determining the plasma protein binding rate of betulinic acid is simple,rapid and sensitive with a higher selectivity.

A novel quantitative method using high-performance liquid chromatography coupled to electrospray ionization tandem mass spectrometry was developed for simultaneous determination of the important active constituents including four steroidal saponins, two xanthone glycosides, two isoflavonoids, and one anthraquinone in different parts of Anemarrhena asphodeloides from different habitats. Hierarchical clustering analysis and principal components analysis were performed to differentiate and classify the samples. The separation was performed on a C(18) column with acidified aqueous acetonitrile gradients. Quantification of the analytes was achieved by use of a hybrid quadrupole linear ion-trap mass spectrometer. Multiple-reaction monitoring scanning was employed with switching electrospray ion source polarity between positive and negative modes in a single run. The validation results of the method indicated that the method was simple, rapid, specific, and reliable. The results demonstrated that the quantitative difference in content of nine active compounds was useful not only for chemotaxonomy of many samples from different sources but also for the standardization and differentiation of many similar samples. Simultaneous quantification of bioactive components by HPLC-ESI-MS coupled with chemometric techniques would be a well-acceptable strategy to comprehensively control the quality of A. asphodeloides.

A simple, fast and sensitive method for the simultaneous determination of cnidilin and its two metabolites (M1 and M2) in rat bile and stool using HPLC coupled with electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS) has been developed. The sample pretreatment was simple, because methanol was the only additive used for dilution of bile and ultrasound of stool. Pimpinellin was used as internal standard (IS). The separation was performed on a reverse phase C18 column with gradient elution consisting of 0.5‰ aqueous formic acid and methanol (containing 0.5‰ formic acid). The detection was in the multiple-reaction monitoring mode within 7 min. All the analytes were in accordance with the requirement of the validation of the method in vivo (linearity, precision, accuracy, limit of detection and limit of quantification). After oral administrating 24 mg/kg of the prototype drug cnidilin, M1 and M2 were determined in bile within 36 h, and in stool within 60 h. Cnidilin in bile was completely excreted in 24 h, and the main excretive amount of cnidilin was 80% in the first 6 h, but the drug recovery in bile within 24 h was <1.95%. In stool, the main excretive amount of cnidilin was 95.8% in the first 24 h, and the drug recovery within 48 h was lower than 1.48%.

OBJECTIVE To establish an HPLC method for content determination of glimepiride in Complex metformin hydrochloride-glimepiride capsules.METHODS The chromatographic separation was performed on a Diamonsil C_18 column(250 mm×4.6 mm,5 μm).The mobile phase consisted of 0.05 mol·L~-1 ammonium formate(pH4)and acetonitrile(40:60) at a flow rate of 1.0 mL·min~-1,with detection wavelength of 280 nm.RESULTS The calibration curves showed a good linearity within the range of 1.014-20.280 μg·mL~-1 for glimepiride(r=0.9999).The mean recovery was 99.5% with the RSD of 1.0 %.CONCLUSION It is a simple,accurate and specific method with no interference between metformin hydrochloride and glimepiride.It should be a referred method for determination of the content of glimepiride in the complex formulations containing glimepiride.

In this study, a new liquid chromatography–tandem mass spectrometry (LC–MS/MS) method has been developed and validated for the determination of six flavonoids including sophoricoside, genistin, genistein, rutin, quercetin and kaempferol in rat plasma after oral administration of Fructus Sophorae extract using sulfamethalazole as internal standard (IS). The plasma samples were pretreated and extracted by liquid–liquid extraction. Chromatographic separation was accomplished on a C18 column with a simple linear gradient elution. The detection was accomplished by multiple-reaction monitoring (MRM) scanning after electrospray ionization (ESI) source operating in the negative ionization mode. The optimized mass transition ion pairs (m/z) for quantitation were 431.1/267.9 for sophoricoside and genistin, 269.0/133.0 for genistein, 609.2/300.0 for rutin, 301.0/150.9 for quercetin, 284.9/93.0 for kaempferol and 252.0/155.9 for IS. The total run time was 8.0min. Full validation of the assay was implemented including specificity, linearity, accuracy, precision, recovery and matrix effect. This is the first report on determination of the major flavones in rat plasma after oral administration of Fructus Sophorae extract. The results provided a meaningful basis for the clinical application of this herb.