Biflavanones, diterpenes, and coumarins from the roots of Stellera chamaejasme L

A new biflavanone (1) with a C-3/C-3" linkage, a new daphnane-type diterpene (2) acylated by an unsaturated fatty acid, and a new coumarin glycoside (3), along with six lignans, two phenylpropanoids, five flavonoids, two diterpenes, and three coumarins were isolated from the roots of Stellera chamaejasme L. (Thymelaeaceae). Elucidation of these secondary metabolites of S. chamaejasme L. supplied strong chemical verification of the close taxonomic relationships among the genera Stellera, Daphne, and Wikstroemia, all of which belong to the family Thymelaeaceae.     

A New Biflavonoid from Stellera chamaejasme L.

A new biflavonoid, stelleranol, was isolated from the roots of Stellera chamaejasme L..Its structure was determined by the analysis of MS and NMR data, especially 2D NMR spectra.     

Identification and determination of the major constituents in Kai‐Xin‐San by UPLC‐Q/TOF MS and UFLC‐MS/MS method

In order to have overall chemical material information of Kai‐Xin‐San (KXS), the reliable ultra‐high‐performance liquid chromatography quadrupole time‐of‐flight mass spectrometer (UHPLC–Q‐TOF‐MS) and ultra‐fast liquid chromatography mass spectrometer (UFLC‐MS/MS) methods were developed for the identification and determination of the major constituents in KXS. Moreover, the UHPLC–Q‐TOF‐MS method was also applied to screen for multiple absorbed components in rat plasma after oral administration of KXS. The UHPLC–Q‐TOF‐MS method was achieved on Agilent 6520 Q‐TOF mass and operated in the negative ion mode. Good separation was performed on a ZORBAX Eclipse Plus C18 column with a gradient elution at a flow rate of 0.2 ml/min. A total of 92 compounds in KXS were identified or tentatively characterized based on their exact molecular weights, fragmentation patterns, and literature data. A total of 26 compounds including 23 prototype components and three metabolites were identified in rat plasma after oral administration of KXS. Then, 16 major bioactive constituents were chosen as the benchmark substances to evaluate the quality of KXS. Their quantitative analyses were performed by a triple quadrupole tandem mass spectrometer (MS/MS) operating in multiple‐reaction monitoring mode(MRM). The analysis was completed with a gradient elution at a flow rate of 0.4 ml/min within 35 min. The simple and fast method was validated and showed good linearity, precision, and recovery. Furthermore, the method was successful applied for the determination of 16 compounds in KXS. All results would provide essential data for identification and quality control of active chemical constituents in KXS. Copyright © 2016 John Wiley & Sons, Ltd.     

Rapid and global detection and characterization of the constituents in ShengMai San by ultra-performance liquid chromatography-high-definition mass spectrometry

An ultra-performance liquid chromatography-high definition mass spectrometry (UPLC-HDMS) method was developed for detection and characterization of the chemical constituents in ShengMai San (SMS), a traditional Chinese medical formula (TCMF). The full-scan LC-MS/MS data sets combined with extra mass were acquired within 14 min using UPLC-HDMS in the MS(E) mode in a single injection. As a result, 92 compounds were identified by comparing the accurate mass and fragments information with that of the authentic standards as well as by MS analysis and the correlative references data. These constituents included ginsenosides, lignans, steroidal saponins and homoisoflavanones. Among them, 25-hydroxyginsenosides were discovered in SMS for the first time. Compare with the previous studies, our research detected more compounds and presented more rapid by applying UPLC-HDMS. It is concluded that a rapid and effective method has been established based on UPLC-HDMS with the utilization of MS(E) , which shows high sensitivity and resolution that is suitable for identifying the constituents of SMS, and this method could be applied to other TCMF.     

An effective method for determining the ingredients of Shuanghuanglian formula in blood samples using high‐resolution LC–MS coupled with background subtraction and a multiple data processing approach

Shuanghuanglian formula (SF) is a combination of Flos lonicerae japonicae, Radix scutellariae, and Fructus forsythiae, commonly used to treat viral or bacterial infections. However, the constituents absorbed into the blood after oral administration of SF are difficult to determine and thus remain unclear. Here, we report the application of an accurate background subtraction and multiple data processing approach (Bs‐Mpa) for the comprehensive detection of compounds of SF in vivo. A sensitive and reliable ultra‐performance LC coupled with ESI quadrupole TOF MS (UPLC–ESI‐Q‐TOF‐MS) approach coupled with Bs‐Mpa, which is implemented in the Strip tool from UPLC to remove nonrelated ion signals from accurate mass LC–MS data, was established to characterize the chemical constituents and rat metabolites of SF. In the loading plot of the principal component analysis, 68 ions of interest were extracted from blood samples, among them, 39 absorbed prototype components of SF and 29 metabolites were identified in vivo. It is concluded that the integrative Bs‐Mpa method can be successfully applied for the rapid discovery of multiple components from a traditional Chinese medicine. The above challenge was addressed by using the proposed Bs‐Mpa method and it was particularly suitable for applying to the global characterization of the constituents or metabolites in rat blood after oral administration of other well‐known formulae.     

A UPLC‐MS/MS method for simultaneous determination of five flavonoids from Stellera chamaejasme L. in rat plasma and its application to a pharmacokinetic study

Stellera chamaejasme L. has been used as a traditional Chinese medicine for the treatment of scabies, tinea, stubborn skin ulcers, chronic tracheitis, cancer and tuberculosis. A sensitive and selective ultra‐high liquid chromatography–tandem mass spectrometry (UPLC‐MS/MS) method was developed and validated for the simultaneous determination of five flavonoids (stelleranol, chamaechromone, neochamaejasmin A, chamaejasmine and isochamaejasmin) of S. chamaejasme L. in rat plasma. Chromatographic separation was accomplished on an Agilent Poroshell 120 EC‐C₁₈ column (2.1 × 100 mm, 2.7 μm) with gradient elution at a flow rate of 0.4 mL/min and the total analysis time was 7 min. The analytes were detected using multiple reaction monitoring in positive ionization mode. The samples were prepared by liquid–liquid extraction with ethyl acetate. The UPLC‐MS/MS method was validated for specificity, linearity, sensitivity, accuracy and precision, recovery, matrix effect and stability. The validated method exhibited good linearity (r ≥ 0.9956), and the lower limits of quantification ranged from 0.51 to 0.64 ng/mL for five flavonoids. The intra‐ and inter‐day precision were both <10.2%, and the accuracy ranged from −11.79 to 9.21%. This method was successfully applied to a pharmacokinetic study of five flavonoids in rats after oral administration of ethyl acetate extract of S. chamaejasme L.     

Purification and identification of carvacrol from the root of Stellera chamaejasme and research on its insecticidal activity

The root powder of Stellera chamaejasme was extracted with petroleum ether. The crude extract obtained was chromatographed repeatedly with silica gel columns. One pure natural product was obtained, which was identified as 5-isopropyl-2-methylphenol (carvacrol), separated for the first time from the root of S. chamaejasme. The results of a laboratory bioassay showed that it had good insecticidal activity against Aphis craccivora and Leucania separata. Its median lethal concentrations (LC(50)) (24 h after treatment) were 16.8 and 12.7 mg L(-1), respectively. This is the first report of the insecticidal activity of carvacrol against A. craccivora and L. separata.     

A New Chromone Derivative from Stellera chamaejasme L.

A new chromone derivative,3-[1-(2,4,6-trihydroxyphenyl)3-di-(4-hydroxyphenyl) 1-propanone-2-yl]5,7-dihydroxy-8-di(4-hydroxyphenyl)methyl-4H-1-benzopyran-4-one,named as isomohsenone was isolated from the roots of Stellera chamaejasme L.together with known chamaechromone. Its structure was determined by the analysis of MS and NMR data,especially 2D NMR spectra.     

Simultaneous determination nucleosides in marine organisms using ultrasound-assisted extraction followed by hydrophilic interaction liquid chromatography-electrospray ionization time-of-flight mass spectrometry

A new method has been developed based on ultrasound-assisted extraction (UAE) followed by hydrophilic interaction chromatography (HILIC) and electrospray ionization time-of-flight mass spectrometry (ESI-TOF/MS) for the simultaneous determination of 16 nucleosides and nucleobases in medicinal extracts of various marine organisms. The separation was achieved on a Venusil HILIC column (250×4.6 mm id, 5 μm) and gradient elution using a solution of acetonitrile and buffer (0.20% formic acid and 20 mmol/L ammonium acetate) as the mobile phase. Identification of the 16 target nucleosides and nucleobases was based on the retention time, UV spectra, and mass measurements of the protonated molecules ([M+H](+)) and main fragment ions (ESI-TOF/MS). In addition, non-target compounds of 2'-deoxyinosine and four other amino acids were also tentatively identified by ESI-TOF/MS. The 16 target compounds were quantified by HILIC-ESI-TOF/MS under optimized mass conditions. All calibration curves showed good linearity (r(2)>0.9951). The recoveries were 84.72-124.10%, and the limits of detection of the 16 target compounds were 0.6-130.0 ng/mL. The developed method was applied to quantify the target compounds in 15 batches of various marine organisms. The method has potential applicability for the identification and determination of highly polar and low-concentration active compounds in marine organisms.     

高效液相色谱-电喷雾飞行时间质谱分析娑罗子中皂苷类成分

采用高效液相色谱/电喷雾飞行时间质谱联用技术(HPLC/ESI-TOF/MS),研究4种七叶皂苷的分子结构与裂解规律间的关系,并对娑罗子中的七叶皂苷类化合物进行鉴定。实验采用反相C18色谱柱,以乙腈-0.2%乙酸溶液为流动相,二元线性梯度洗脱,通过与电喷雾飞行时间质谱联用获得娑罗子中各皂苷成分的精确分子量和分子式;采用质谱碰撞诱导解离技术获得各化合物碎片裂解信息,结合文献对娑罗子中的14种皂苷类化合物进行了初步鉴定。研究表明,高效液相色谱-电喷雾飞行时间质谱联用技术是娑罗子中皂苷类化合物鉴别的有效工具。     

高效液相色谱-串联质谱法同时测定保健品及中成药中非法添加的17种壮阳类化学药

建立了高效液相色谱-串联质谱(LC-MS/MS)同时测定补肾壮阳类保健品及中成药中非法添加的17种壮阳化学药的定性、定量分析方法。实验优化了前处理方法,并针对几对同分异构体成分优化了分离条件和质谱参数。样品经甲醇超声萃取,提取液经Aglient Extend C18色谱柱(100 mm×2.1 mm, 3.5 μm)分离,流动相为乙腈和水(含有10 mmol/L乙酸铵),梯度洗脱,流速为0.25 mL/min,以电喷雾离子源正离子多反应监测(MRM)模式进行MS/MS检测。该方法能很好地分离并定量17种壮阳类化学药以及其中的3组同分异构体。该方法简便、快速、准确可靠,已经应用于补肾壮阳类保健品及中成药中非法添加壮阳类化学药的筛查及检测。统计近3年检测的样品,发现补肾壮阳类产品中非法添加成分检出率高,需引起相关监管部门的重视。     

HPLC‐Q‐TOF‐MS/MS for analysis of major chemical constituents of Yinchen–Zhizi herb pair extract

The Yinchen–Zhizi herb pair (YZHP) consists of Herba Artemisiae Scopariae (Yinchen in Chinese) and Fructus Gardeniae (Zhizi in Chinese), and is mainly used to treat icteric hepatitis, itching skin and eczema. However, the bioactive constituents responsible for the pharmacological effects of YZHP are still unclear to date. In this work, a rapid and sensitive method was established to comprehensively study the constituents in YZHP extract by HPLC‐Q‐TOF MS/MS. The analysis was performed on an HPLC system equipped with an Agilent poroshell 120 EC‐C₁₈ column (100 × 2.1 mm, 2.7 mm) working in a gradient elution program coupled to quadrupole‐time‐of‐flight mass spectrometry operating in the negative ion mode. As a result, a total of 46 compounds including 17 from Herba Artemisiae Scopariae and 36 from Fructus Gardeniae were detected and tentatively identified in YZHP extract by comparing the retention time and mass spectrometry and retrieving the reference literature. More importantly, a series of constituents, such as many iridoid glycosides, were reported for the first time in this formula. The HPLC‐Q‐TOF MS/MS method was developed and utilized successfully to identify the major constituents in YZHP extract and would be helpful for further metabolism and pharmacology research on YZHP. Copyright © 2013 John Wiley & Sons, Ltd.     

Isolation and identification of 2-isopropyl-5-methylphenol from Stellera chamaejasme and its insecticidal activity against Aphis craccivora and Pieris rapae

In this study, the root powder of Stellera chamaejasme was extracted with chloroform. The crude extract thus obtained was chromatographed several times with silica gel columns and finally one pure natural product was obtained. This product was identified as 2-isopropyl-5-methylphenol (thymol), which was separated for the first time from S. chamaejasme root. The results of a laboratorial bioassay showed that it had good insecticidal activity against Aphis craccivora and Pieris rapae. Its median lethal concentrations (LC50) (24 h after treatment) were 14.81 and 47.68 mg L(-1), respectively. This is the first time that the insecticidal activity of thymol against A. craccivora and P. rapae has been reported.     

Fragmentation patterns study of iridoid glycosides in Fructus Gardeniae by HPLC‐Q/TOF‐MS/MS

Iridoid glycosides (IGs), the major constituents in Fructus Gardeniae, have demonstrated various pharmacological activities, but there is no systematic chemical profile of IGs in Fructus Gardeniae in the published literature until now. Therefore, it is imperative that a rapid and sensitive high‐performance liquid chromatography coupled with quadrupole time‐of‐flight tandem mass spectrometry (HPLC‐Q/TOF‐MS/MS) method is established for comprehensive characterization of IGs in Fructus Gardeniae. Firstly, the fragmentation patterns of six known IGs were investigated and proposed and further concluded the diagnostic fragment ions and characteristic fragmentation pathways. Then, based on the summarized fragmentation patterns and the known compounds in the literatures, the other IGs in Fructus Gardeniae were identified successively. As a result, a total of 20 IGs were identified, of which three pairs of epimers were structurally characterized and differentiated. More importantly, one compound, the isoshanzhiside methyl ester, was tentatively identified as a new compound. The results of this study demonstrate the superiority of HPLC‐MS with a high‐resolution mass spectrometer for the rapid and sensitive structural elucidation of the multiple groups of constituents in Fructus Gardeniae. Copyright © 2014 John Wiley & Sons, Ltd.     

瑞香狼毒中灭蚜活性物质的结构鉴定

报道从瑞香狼毒(StellerachamaejasmeL.)根中发现分离到2个显示高农药活性化合物，能有效杀灭农业重要害虫蚜虫，经波谱及元素分析，确定其结构为1,5-二苯基-1-戊酮(1)和1,5-二苯基-2-烯-1-戊酮(2)。     

Fast quantification of cyclophosfamide and its N‐dechloroethylated metabolite 2‐dechloroethylcyclophosphamide in human plasma by UHPLC‐MS/MS

A rapid, novel and reliable UHPLC‐MS/MS method was developed and validated for simultaneous determination of cyclophosphamide (CP) and its dechloroethylated metabolite, 2‐dechloroethylcyclosphamide (2‐DCECP) in human plasma. The plasma samples were conducted by protein precipitation with 3‐fold acetonitrile, containing 0.1% formic acid. Mass spectrometric detection was performed using electrospray positive ionization with multiple reaction monitoring mode, using tinidazole as internal standard (IS). Chromatographic separation was performed on an Agilent poroshell 120 SB‐C₁₈ column (2.1 × 75 mm, 2.7 µm) using gradient elution of acetonitrile and 0.1% formic acid at a flow rate of 0.5 mL/min, the total run time was 2.5 min. The limit of quantification (LOQ) was 20 ng/mL for both CP and 2‐DCECP. Accuracies and precisions were <15% at LOQ and below 10% at quality control concentration levels. This UHPLC‐MS/MS method was successfully applied for the estimation of CP and 2‐DCECP in human plasma, which was also useful for clinical toxicology studies and therapeutic drug monitoring of CP. Copyright © 2014 John Wiley & Sons, Ltd.     

Qualitative and quantitative analysis of the chemical constituents in Mahuang‐Fuzi‐Xixin decoction based on high performance liquid chromatography combined with time‐of‐flight mass spectrometry and triple quadrupole mass spectrometers

High‐performance liquid chromatography coupled with time‐of‐flight mass spectrometry (HPLC‐TOF/MS) and high‐performance liquid chromatography–triple quadrupole mass spectrometry (HPLC‐QQQ/MS/MS) were utilized to clarify the chemical constituents of Mahuang‐Fuzi‐Xixin Decoction. There are 52 compounds, including alkaloids, amino acids and organic acids were identified or tentatively characterized by their characteristic high resolution mass data by HPLC‐QQQ/MS/MS. In the subsequent quantitative analysis, 10 constituents, including methyl ephedrine, aconine, songrine, fuziline, neoline, talatisamine, chasmanine, benzoylmesaconine, benzoylaconine and benzoylhypaconine were simultaneously determined by HPLC‐QQQ/MS/MS with multiple reaction monitoring mode. Satisfactory linearity was achieved with wide linear range and fine determination coefficient (r > 0.9992). The relative standard deviations (RSD) of inter‐ and intra‐day precisions were <3%. This method was also validated by repeatability, stability and recovery with RSD <3% respectively. A highly sensitive and efficient method was established for chemical constituents studying, including identification and quantification of Mahuang‐Fuzi‐Xixin decoction.     

高效液相色谱–串联质谱法检测环境水体中18种全氟化合物

建立了快速检测环境水体中18种全氟化合物的高效液相-串联质谱方法。环境水样经大体积PEP固相萃取柱快速提取、富集和净化后,过0.22μm微孔滤膜,再利用Agilent Zorbax Eclipse XDB-C18反相色谱柱分离,以5 mmol/L乙酸铵溶液和甲醇为流动相梯度洗脱,最终进入电喷雾离子源负离子扫描,在多反应监测模式下定性及定量分析。所有目标化合物的质量浓度在1~20μg/L范围内与色谱峰面积线性良好,相关系数(r2)大于0.999,方法检出限为0.18~0.48 ng/L。18种全氟化合物的加标回收率为90.1%~107.3%,相对标准偏差为0.3%~8.2%(n=6)。该方法操作简单、快速,灵敏度高,可满足环境水体中全氟化合物的检测。