Fast profiling of chemical constituents in Yiqing Capsule by ultra-performance liquid chromatography coupled to electrospray ionization tandem mass spectrometry

An ultra-performance liquid chromatography/electrospray ionization tandem mass spectrometry (UPLC-ESI-MS(n)) has been developed for structural characterization and identification of multi-constituents in Yiqing Capsule, a well-known combined herbal remedy prepared from the extract mixtures of Rhizoma Coptidis, Radix et Rhizoma Rhei and Radix Scutellariae. The UPLC analysis was performed on an Agilent ZorBax SB-C(18) column (4.6 mm×50 mm, 1.8 μm) and gradient elution of 0.1% formic acid solution and acetonitrile in 16 min. Based on their retention times and mass spectra in comparison with the data from standards or references, a total of 29 compounds including 3 phenolic acids and 4 anthraquinones from Radix et Rhizoma Rhei, 8 alkaloids from Rhizoma Coptidis and 14 flavonoids from Radix Scutellariae were unambiguously identified or tentatively characterized in the complex system. The MS data and fragmentation information of two isomers of feruloylquinic acid were first reported in Radix et Rhizoma Rhei and in Yiqing Capsules. This study is expected to be accepted as an effective and reliable pattern for comprehensive and systematic characterization of this commonly used Chinese herbal preparation.     

Simultaneous quantification of Echinacea species, Flos Lonicerae, Radix Scutellaria and Fructus Forsythiae combinations by rapid resolution liquid chromatography

Echinacea angustifolia and E. purpurea are commonly used in North America for their anti-bacterial effects. Flos Lonicerae, Radix Scutellaria and Fructus Forsythiae are traditional Chinese medicinal herbs commonly used for the treatment of complaints such as pneumonia, acute upper respiratory tract infection, and acute bronchitis. A reproducible, simple, and reliable rapid resolution liquid chromatographic (RRLC) method has been developed to analyze extracts of products formulated containing E. angustifolia, E. purpurea, Flos Lonicerae, Radix Scutellariae and Fructus Forsythiae simultaneously in one run in less than 6 minutes. The method uses a C18-HST column, a mobile phase consisting of 0.1% aqueous phosphoric acid solution and acetonitrile, and UV detection at 327 nm and 229 nm. A stability test was performed that revealed that chlorogenic acid is more stable in acidic pH, and hence it is best to keep the extract of E. augustifolia, E. purpurea, Flos Lonicerae, Radix Scutellariae and Fructus Forsythiae in mild acidic conditions at approximately pH 5.     

Identification and quantification of free radical scavengers in the flower buds of Lonicera species by online HPLC-DPPH assay coupled with electrospray ionization quadrupole time-of-flight tandem mass spectrometry

Flos Lonicerae, derived from the flower buds of several medicinal Lonicera species, is a commonly used herbal medicine with multiple pharmacological activities, one of the major ones being antioxidant activity. In this study, free radical scavengers in the flower buds of six Lonicera species were screened, identified and quantified by online HPLC-DPPH (1,1-diphenyl-2-picrylhydrazyl) assay coupled with LC quadrupole time-of-flight tandem mass spectrometry (Q-TOF MS/MS). The antioxidants were firstly screened from the complex plant matrix by the online HPLC-DPPH assay. Then the active compounds were identified by LC Q-TOF MS/MS, and the possible fragmentation pathways were proposed. The reactivity of antioxidants available was investigated using an internal standard method by online LC assay. The contents of 12 antioxidants were also determined or estimated by HPLC coupled with diode array detector. The total antioxidant capability determined by the online method was used as the marker to evaluate the quality of Flos Lonicerae. The results were important to clarify the material basis and therapeutic mechanism of Flos Lonicerae.     

微波辅助萃取/高效液相色谱串联质谱法对山银花中活性成分含量的测定

建立了微波辅助萃取/高效液相色谱串联质谱法(MAE/HPLC-MS/MS)同时测定山银花中10种活性成分含量的方法。山银花药材采用MAE萃取,萃取溶剂为乙醇-水(7∶3),固液比1∶30,萃取温度70℃,萃取时间10 min。采用HPLC-MS/MS测定萃取液中活性成分的含量,色谱柱采用Agilent Poroshell120 SB-C18(100 mm×2.1 mm,2.7μm),以0.5%甲酸-乙腈为流动相进行梯度洗脱,负离子多重反应离子监测模式检测。在优化条件下,10种成分的定量分析在10 min内完成。结果表明,10种活性成分的线性范围为0.05～500 mg/L,相关系数(r)不低于0.996 9,检出限和定量下限分别在69～4 413μg/kg和231～14709μg/kg范围,回收率为94%～105%。采用该方法检测6个不同产地的山银花样品,10种活性成分的含量在3.98～14 356.31 mg/kg范围。该方法快速、准确,可有效地用于山银花药材的质量控制。     

Qualitative and quantitative analysis of Radix Astragali products by fast high-performance liquid chromatography-diode array detection coupled with time-of-flight mass spectrometry through dynamic adjustment of fragmentor voltage

A novel fast high-performance liquid chromatography (HPLC) method coupled with diode array detection (DAD) and time-of-flight mass spectrometry (TOF/MS) was developed for qualitative and quantitative analysis of Radix Astragali products. The potential of fast HPLC on 1.8-microm particles was compared with the performance of HPLC on conventional 5-microm particles columns. Significant advantages of fast HPLC include high-speed chromatographic separation, four times faster than HPLC with conventional columns, and great enhancement in sensitivity with limits of detection low to 0.001 ng. With dynamic adjustment of fragmentor voltage in TOF/MS, an efficient transmission of the ions was achieved to obtain the best sensitivity and abundant fragmentation. By accurate mass measurements within 5 ppm error for each molecular ion and subsequent fragment ions, a reliable identification and differentiation of six major saponins including two groups of isomers and twelve main isoflavonoids was described here for the first time. For quantitative analysis by fast HPLC-TOF/MS, linearity of response over two orders of magnitude was demonstrated (r(2)>0.99) for all analytes. Intra-day reproducibility was below 3% RSD and inter-day values were below 5% RSD. A good correlation (slope=1.1108, r(2)=0.9853) was observed for accuracy test. It is concluded that the fast and sensitive HPLC-DAD-TOF/MS is powerful in qualitative and quantitative analysis of complex herbal medicines in terms of time savings, sensitivity, selectivity, precision, accuracy as well as increasing sample throughout and lowering solvent consumption.     

Simultaneous determination of the bioactive components in rat plasma by UPLC‐MS/MS and application in pharmacokinetic studies after oral administration of radix Scutellariae extract

A highly sensitive and rapid ultraperformance liquid chromatography–tandem mass spectrometry (UPLC‐MS/MS) method has been developed and validated for simultaneous quantification of the four main bioactive compounds, i.e. baicalin, baicalein, wogonoside and wogonin, in rat plasma after oral administration of Radix Scutellariae extract. Clarithromycin was used as an internal standard (IS). Plasma samples were processed by protein precipitation with methanol. The separation was performed on an Acquity BEH C₁₈ column (100 × 2.1 mm, 1.7 μm) at a flow rate of 0.4 mL/min, using 0.1% formic acid–acetonitrile as mobile phase. The MS/MS ion transit ions monitored were 447.5 → 270.1 for baicalin, 270.1 → 168.1 for baicalein, 461.2 → 284.0 for wogonoside, 284.2 → 168.1 for wogonin and 748.5 → 158.1 for IS. Method validation was performed according to US Food and Drug Administration guidelines and the results met the acceptance criteria. The lower limit of quantification (LLOQ) achieved was 1.13 ng/mL for baicalin, 1.23 ng/mL for baicalein, 0.82 ng/mL for wogonoside and 0.36 ng/mL for wogonin. The calibration curves obtained were linear (r > 0.99) over the concentration range ~ 1–1000 ng/mL. The intra‐ and inter‐day precision was <15% and the accuracy was within ±14.7%. After validation, this method was successfully applied to a pharmacokinetic study of Radix Scutellariae extract.     

Structural characterization and identification of iridoid glycosides,saponins, phenolic acids and flavonoids in Flos Lonicerae Japonicae by a fast liquid chromatography method with diode‐array detection and time‐of‐flight mass spectrometry

A fast liquid chromatography method with diode‐array detection (DAD) and time‐of‐flight mass spectrometry (TOF‐MS) has been developed for analysis of constituents in Flos Lonicerae Japonicae (FLJ), a traditional Chinese medicine derived from the flower bud of Lonicera japonica. The chromatographic analytical time decreased to 25 min without sacrificing resolution using a column packed with 1.8‐µm porous particles (4.6 × 50 mm), three times faster than the performance of conventional 5.0‐µm columns (4.6 × 150 mm). Four major groups of compounds previously isolated from FLJ were structurally characterized by DAD‐TOF‐MS: iridoid glycosides showed maximum UV absorption at 240 nm; phenolic acids at 217, 242, and 326 nm; flavonoids at 255 and 355 nm; while saponins had no absorption. In electrospray ionization (ESI)‐TOF‐MS experiments, elimination of a glucose unit (162 Da), and successive losses of H₂O, CH₃OH and CO, were generally observed in iridoid glycosides; saponins were characterized by a series of identical aglycone ions; phenolic acids typically generated a base peak at [M–H–caffeoyl]^? by loss of a caffeic acid unit (162 Da) and several marked quinic acid moiety ions; cleavage of the glycosidic bond (loss of 162 or 308 Da), subsequent losses of H₂O, CO, RDA and C‐ring fragmentation were the most possible fragmentation pathways for flavonoids. By accurate mass measurements within 4 ppm error for each molecular ion and subsequent fragment ions, as well as the ‘full mass spectral’ information of TOF‐MS, a total of 41 compounds including 13 iridoid glycosides, 11 phenolic acids, 7 saponins, and 10 flavonoids were identified in a methanolic extract of FLJ. Copyright © 2009 John Wiley & Sons, Ltd.     

银黄口服液的质量控制及其高效液相色谱指纹图谱的研究

利用高效液相色谱方法,建立了复方银黄口服液及其原料药材黄芩和金银花的指纹图谱,测定了银黄口服液中黄芩苷 和绿原酸的含量。以Lichrospher C18色谱柱(250 mm×4.6 mm i.d.,5 μm)为分离柱,以甲醇和0.1%磷酸水溶液为流动 相进行二元梯度洗脱,流速0. 6 mL/min,在254 nm波长下检测。采用夹角余弦和相关系数的方法,计算6批银黄口服液指 纹图谱的相似度均在0.99以上,表明银黄口服液的质量未见显著差异。将银黄口服液指纹图谱中的共有峰与原料药材黄芩 和金银花的指纹图谱中的共有峰相比较,对其归属进行确认,大部分共有峰可以匹配。     

高效液相色谱-质谱联用快速筛选并鉴定黄芩甲醇提取物中抗氧化活性成分

建立了高效液相色谱-质谱联用技术快速筛选和鉴定黄芩甲醇提取物中抗氧化活性成分的方法.在黄芩甲醇提物中加入适量的二苯基三硝基苯肼( DPPH)作为实验组,避光室温反应30 min后直接经液相色谱分析,并与不加入DPPH的空白组进行比较.有抗氧化活性的成分因会与DPPH反应而使峰面积减少,而不具有抗氧化活性的成分峰面积则不变.基于色谱数据可以获得有抗氧化活性组分的相对活性强度,基于质谱数据可获得活性组分的结构信息用于结构鉴定.本方法成功地从黄芩甲醇提取物中筛选并鉴定出了黄芩苷、千层纸素A-7-O-B-D-葡萄糖醛酸苷、汉黄芩苷和千层纸素A4种较强的抗氧化活性成分.本方法利用HPLC-MS技术对复杂体系(如中药提取物)中小分子的分离和鉴定优势,可以直接筛选出黄芩甲醇提取物中的抗氧化活性成分,而不需要繁琐的前期分离和纯化,并且不需要仪器改装工作,有利于实现复杂体系中抗氧化活性成分的高通量筛选.     

Capillary high-performance liquid chromatography with mass spectrometry for simultaneous determination of major flavonoids, iridoid glucosides and saponins in Flos Lonicerae

Flos Lonicerae, a traditional herbal medicine, has been used in China to treat some inflammatory disease. Several different classes of compounds have been separated from the herb to assess their pharmacological activities. Among these classes, flavonoids, iridoid glycosides and saponins have been well studied and may be responsible for its clinical application. Therefore, quality control of Flos Lonicerae is an important issue for drug safety and validity evaluations. A quantitative method consisting of solid phase extraction followed by capillary high-performance liquid chromatography-diode array detection/electrospray ionization mass spectrometry (capillary HPLC-DAD/ESI-MS) was developed for simultaneously assay of 24 compounds in Flos Lonicerae. Under optimized capillary HPLC-DAD/ESI-MS conditions, these compounds, including nine flavonoids, eight iridoid glucosides and seven saponins, were separated with high efficiency in the selected-ion monitoring (SIM) mode. Linearity of the method was good with correlation coefficients (r(2)) in the range of 0.9935-0.9998 and detection limits were lower than 2.57 ng/mL for most of analytes. The obtained recoveries varied between 91.0 and 108.7% with the relative standard deviations (RSDs) within 8.74% (n=3). The capillary HPLC-DAD/ESI-MS method was also successfully applied to the analysis of these compounds in five species of Flos Lonicerae. It was demonstrated to be a powerful tool for comprehensive analysis of herbal medicines, owing to its exclusive selectivity and excellent sensitivity.     

Qualitative and quantitative determination of ten major saponins in Platycodi Radix by high performance liquid chromatography with evaporative light scattering detection and mass spectrometry

Saponins in Platycodi Radix (platycosides) exhibit potent biological activities in mammalian systems, including several beneficial effects such as anti-inflammatory, immunomodulatory and anti-obesity activities. In this study, we developed a new HPLC separation coupled with evaporative light scattering detector (ELSD) for the simultaneous quantitative determination of ten major saponins in Platycodi Radix. Simultaneous separation of these saponins was achieved on a C18 analytical column. The mobile phase consisted of a gradient of aqueous acetonitrile. The method was validated for linearity, precision, accuracy, limit of detection and quantification. Electrospray ionization mass spectrometry (ESI-MS) and liquid chromatography coupled with on-line mass spectrometry (LC-ESI MS/MS) were applied to identify platycosides in the purified fractions and in the crude extract. Under ESI-MS/MS conditions, the fragmentation patterns of [M-H]- ions exclusively show signals corresponding to cleavage of the glycosidic bonds, thus allowing a rapid identification of saponins in the crude extract of Platycodi Radix. The validated HPLC method provides a new basis of overall assessment on quality of Platycodi Radix, and ESI-MS/MS and LC-ESI MS/MS approaches offers analytical tools for a rapid screening of platycosides in the crude extract.     

Simultaneous determination of wogonin,oroxylin a,schisandrin, paeoniflorin and emodin in rat serum by HPLC–MS/MS and application to pharmacokinetic studies

Wogonin and oroxylin A in Scutellariae Radix, schisandrin in Chinensis Fructus, paeoniflorin in Moutan Cortex and emodin in Polygoni Cuspidate Rhizome et Radix are anti‐inflammatory active compounds. A method for simultaneous determination of the five compounds in rat was developed and validated using high‐performance liquid chromatography with tandem mass spectrometry (HPLC–MS/MS). The separation was performed on a Symmetry C₁₈ column (4.6 × 50 mm, 3.5 μm) with acetonitrile and 0.1% formic acid aqueous solution as the mobile phases. The detection was performed using multiple‐reaction monitoring with electrospray ionization source in positive–negative ion mode. The calibration curves showed good linearity (r ≥ 0.9955). The lower limit of quantification (LLOQ) was 5 ng/mL for wogonin and schisandrin, 10 ng/mL for oroxylin A and emodin, and 15 ng/mL for paeoniflorin, respectively. The relative standard deviations of intraday and interday precisions were <11.49 and 14.28%, respectively. The extraction recoveries and matrix effects were acceptable. The analytes were stable under the experiment conditions. The validated method has been successfully applied to pharmacokinetic studies of the five compounds in rats after oral administration of Hu‐gan‐kan‐kang‐yuan capsule. This paper would be a valuable reference for pharmacokinetic studies of Chinese medicine preparations containing the five compounds.     

高效液相色谱-质谱分析指导下制备黄芩中系列黄酮成分对照品

在高效液相色谱-质谱分析指导下,针对性地分离制备了黄芩药材中系列黄酮成分对照品。首先对黄芩药材乙醇提取物进行液相色谱-质谱分析,获得各色谱峰的保留时间、紫外光谱和质谱特征。经波谱数据解析结合文献对比,鉴定了黄芩药材中的19种黄酮类成分。然后根据液相色谱-质谱分析结果和文献,设计了目标成分对照品的制备流程,采用低压制备柱色谱法依次制备了黄芩苷、汉黄芩苷、黄芩素、汉黄芩素和千层纸素A共5种黄酮类成分的对照品。结果表明这5种黄酮类成分对照品的纯度均大于98%。该方法可用于针对性地快速分离制备中药中的化学成分。     

Inhibitory effect of eleven herbal extracts on advanced glycation end-products formation and aldose reductase activity

The formation of advanced glycation end-products(AGEs) and aldose reductase(AR) activity have been implicated in the development of diabetic complications. Our study sought to characterize the capacities of eleven herbal extracts against the formation of AGEs and the AR activity. An ultrahigh performance liquid chromatography and tandem mass spectrometry(UPLC–MS/MS) method was used for the detection of AR activity and the screening of AR inhibitors in this research. The amount of sorbitol from each analyte was directly detected using the multiple reaction monitoring mode and the sorbitol level could be reduced via the addition of an inhibitor. Moreover, the BSA/glucose(fructose) system was applied to investigate their inhibitory activities of AGEs formation in glycation model reactions.Compared with other screened herbs used in our study, Flos Sophorae Immaturus and Radix Scutellariae seemed to be more effective on inhibiting the formation of AGEs and AR activity. The inhibiting capacities of herbal extracts against AR activity and AGEs formation may be correlated with the bioactive components of the herbal extracts. The differences were correlated with the amount of polyphenol and flavonoid components. In the study, we have investigated the potential anti-hyperglycemic bioactivity of eleven herbal extracts in vitro, which could provide a reference for further in vivo research in the prevention and treatment of diabetic complications.     

A 2‐D‐HPLC‐CE platform coupled to ESI‐TOF‐MS to characterize the phenolic fraction in olive oil

A 2‐D‐HPLC/CE method was developed to separate and characterize more in depth the phenolic fraction of olive oil samples. The method involves the use of semi‐preparative HPLC (C18 column 250×10 mm, 5 μm) as a first dimension of separation to isolate phenolic fractions from commercial extra‐virgin olive oils and CE coupled to TOF‐MS (CE‐TOF‐MS) as a second dimension, to analyze the composition of the isolated fractions. Using this method, a large number of compounds were tentatively identified, some of them by first time, based on the information concerning high mass accuracy and the isotopic pattern provided by TOF‐MS analyzer together with the chemical knowledge and the behavior of the compounds in HPLC and CE. From these results it can be concluded that 2‐D‐HPLC‐CE‐MS provides enough resolving power to separate hundreds of compounds from highly complex samples, such as olive oil. Furthermore, in this paper, the isolated phenolic fractions have been used for two specific applications: quantification of some components of extra‐virgin olive oil samples in terms of pure fractions, and in vitro studies of its anti‐carcinogenic capacity.