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.     

Characterization and identification of multiple constituents in Yinhuang granules by high-performance liquid chromatography with diode-array and time-of-flight mass spectrometry detection

A fast high-performance liquid chromatography (HPLC) method with diode-array detection (DAD) and time-of-flight mass spectrometry (TOF/MS) has been developed for the analysis of multi-constituent in Yinhuang granules, a well-known combined herbal remedy prepared from the extract mixtures of Flos Lonicerae and Radix Scutellariae. The fast HPLC analysis was performed on an Agilent ZorBax SB-C(18) column (4.6×50 mm, 1.8 μm) and 0.2% aqueous formic acid and acetonitrile was the optimum mobile phase for gradient elution in 17 min, which is five times faster than the performance of conventional columns packed with 5.0 μm particles. With various fragmentor voltages in TOF/MS, accurate mass measurements (<5 ppm error) for molecular ions and characteristic fragment ions represented reliable identification criteria for different constituents. A total of 28 compounds, including nine phenolic acids, three iridoid glycosides and nine saponins from Flos Lonicerae and seven flavonoids from Radix Scutellariae, were identified or tentatively characterized in the extract of Yinhuang granules. The established fast HPLC-DAD-TOF/MS method turns out to be useful and efficient for quality control of this commonly used Chinese herbal preparation.     

Development of the Fingerprints for the Quality Evaluation of Scutellariae Radix by HPLC-DAD and LC-MS-MS

A high-performance liquid chromatography coupled with photodiode array detection and electrospray ionization tandem mass spectrometry (HPLC-DAD-ESI-MS²) method was firstly developed for a chemical fingerprint analysis of Scutellariae Radix and rapid identification of major compounds in the fingerprints. The experimental data for chromatography were used for hierarchical clustering analysis and similarity calculation, and those for UV and MS spectra were applied for identifying characteristic peaks. Twenty samples including S. baicalensis Georgi., S. viscidula Bge. and S. amoena C. H. Wright were classified into three groups. By comparing the UV and MS spectra data with those of the authentic standards and literature, 20 main peaks in the fingerprints were identified for the first time. The developed fingerprint assay was specific and could be readily utilized for comprehensive evaluation of Scutellariae Radix.     

格列美脲治疗的2型糖尿病大鼠的尿液代谢组学研究

采用快速高分离度液相色谱-质谱技术(RRLC-MS)检测经格列美脲治疗的2型糖尿病大鼠尿液中代谢物的变化, 对糖尿病组、 给药组和健康对照组大鼠的尿液代谢物谱进行了分析. 采用主成分分析(PCA)对3组大鼠进行分类并寻找潜在生物标记物. 结果表明, 3组大鼠的尿液代谢物谱得到了很好的区分, 发现并鉴定了2个潜在生物标记物, 分别为4-脱氧三羟基丁酸和4-胍基丁酸. 格列美脲对2型糖尿病大鼠的药物作用可能体现为对氨基酸代谢的调节作用.     

Enantioseparation and absolute configuration determination of angular-type pyranocoumarins from peucedani radix using enzymatic hydrolysis and chiral HPLC-MS/MS analysis

Angular-type pyranocoumarins from Peucedani Radix (Chinese name: Qian-hu) have exhibited potential for use on treatment of cancer and pulmonary hypertension. Due to the existence of C-3' and C-4' chiral centers, compounds belonging to this chemical type commonly exist in enantiomers and/or diastereoisomers, which may elicit distinct activities during their interactions with the human body. In the present study, a new method, which combines enzymatic hydrolysis with chiral LC-MS/MS analysis, has been developed to determine the absolute configurations of these angular-type pyranocoumarins. Pyranocoumarins isolated from Qian-hu, their enantiomers, or metabolites were individually incubated with rat liver microsomes. As the common end product from enzymatic hydrolysis of all tested pyranocoumarins, cis-khellactone was collected and its absolute configuration was determined by comparison with (+)-cis-khellactone and (-)-cis-khellactone using chiral LC-MS/MS. The absolute configurations of all tested parent pyranocoumarins were determined by combination of LC-MS/MS, NMR and polarimetric analysis. The results revealed that the metabolite cis-khellactone retained the same absolute configurations of the stereogenic carbons as the respective parent compound. This method was proven to be rapid and sensitive and also has advantages in discriminating single enantiomers and mixtures of optical isomers with different ratios.     

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.     

糖肾方对自发性Ⅱ型糖尿病鼠磷脂代谢的影响

利用正相液相色谱飞行时间质谱建立了大鼠血浆磷脂的轮廓谱。通过对磷脂轮廓谱的PLS-DA判别分析及14种化合物的定量,考察了中药糖肾方对自发性II型糖尿病鼠磷脂代谢的影响,并对比了糖肾方与西药蒙诺在糖尿病发展过程中的不同作用。结果表明：与正常对照组相比,糖尿病鼠的血浆磷脂代谢发生异常。糖肾方能够调节糖尿病鼠的磷脂代谢紊乱,蒙诺则加剧了病鼠的磷脂代谢异常。此外,8种磷脂化合物可能成为糖尿病发展过程中的潜在生物标志物。     

快速高分辨液相色谱-质谱联用技术分析2型糖尿病大鼠口服四君子汤的药代动力学

研究四君子汤在2型糖尿病大鼠体内的药代动力学。 将四君子汤水提物以18 g/kg给正常大鼠和2型糖尿病大鼠灌胃,分别在不同时间点取尿液和粪便,检测其成分及代谢产物的含量;比较正常大鼠和2型糖尿病大鼠的药代动力学参数。 采用快速高分辨液相色谱-质谱联用技术对人参皂苷Rc、甘草甜素及其脱糖代谢物进行表征。 结果表明,四君子汤中人参皂苷Rc和甘草甜素在2型糖尿病大鼠体内的累计排泄率均高于正常大鼠,大部分人参皂苷通过尿液排出体外、粪便代谢为稀有人参皂苷;大部分甘草甜素在尿液和粪便中转化为甘草次酸,2型糖尿病大鼠与正常大鼠人参皂苷Rc和甘草甜素的药代动力学参数存在明显差异。 初步总结了四君子汤在体内的药代动力学机制和代谢途径。本分析方法具有高度自动化,灵敏性和准确性,这将有助于四君子汤的临床研究的不断发展。     

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。     

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.     

UHPLC-MS-Based Serum and Urine Metabolomics Reveals the Anti-Diabetic Mechanism of Ginsenoside Re in Type 2 Diabetic Rats

Panax ginseng was employed in the treatment of “Xiao-Ke” symptom, which nowadays known as diabetes mellitus, in traditional Chinese medicine for more than a thousand years. Ginsenoside Re was the major pharmacologic ingredient found abundantly in ginseng. However, the anti-diabetic of Ginsenoside Re and its underlying mechanism in metabolic level are still unclear. Serum and urine metabolomic method was carried out to investigate the anti-diabetic pharmacological effects and the potential mechanism of Ginsenoside Re on high-fat diet combined streptozotocin-induced type 2 diabetes mellitus (T2DM) rats based on ultra-high-performance liquid chromatography coupled with quadrupole exactive orbitrap mass spectrometry (UHPLC-Q-Exactive Orbitrap/MS). Serum and urine samples were collected from the control group (CON), T2DM group, metformin (MET) treatment group, and ginsenoside Re treatment group after intervention. The biochemical parameters of serum were firstly analyzed. The endogenous metabolites in serum and urine were detected by UHPLC-MS. The potential metabolites were screened by multivariate statistical analysis and identified by accurate mass measurement, MS/MS, and metabolite databases. The anti-diabetic-related metabolites were analyzed by KEGG metabolic pathway, and its potential mechanism was discussed. The treatment of ginsenoside Re significantly reduced the blood glucose and serum lipid level improved the oxidative stress caused by T2DM. Biochemical parameters (urea nitrogen, uric acid) showed that ginsenoside Re could improve renal function in T2DM rats. Respective 2 and 6 differential metabolites were found and identified in serum and urine of ginsenoside Re compared with T2DM group and enriched in KEGG pathway. Metabolic pathways analysis indicated that the differential metabolites related to T2DM were mainly involved in arachidonic acid metabolism, Vitamin B6, steroid hormone biosynthesis, and bile secretion metabolic pathways. This study verified the anti-diabetic and anti-oxidation effects of ginsenoside Re, elaborated that ginsenoside Re has a good regulation of the metabolic disorder in T2DM rats, which could promote insulin secretion, stimulated cannabinoid type 1 receptor (CB₁), and CaMKK β to activate AMPK signaling pathway, inhibited insulin resistance, and improved blood glucose uptake and diabetic nephropathy, so as to play the role of anti-diabetic.     

The overall quality control of Radix scutellariae by capillary electrophoresis fingerprint

The qualitative and quantitative analyses are developed for the overall quality control of Radix Scutellariae, a Chinese herb, by capillary electrophoresis fingerprint (CEFP). All experiments are carried out in an uncoated fused silica capillary (75 cm x 75-microm i.d., effective length 63 cm) with a 50 mmol/L sodium borate solution (containing 5% acetonitrile, pH 9.30) under 12 kV. The detection wavelength is set at 280 nm, and naproxen is selected as the referential peak. Some parameters are used to evaluate the similarities of CEFPs. Eight co-eluting peaks are selected as the fingerprint peaks of Radix Scutellariae. The similarities between each of the ten samples and the referential CEFP of Radix Scutellariae are evaluated both qualitatively and quantitatively. Determination results indicate that the baicalin contents from different locations are varied. The methods of CEFP and quantitative analysis are rapid, simple, and accurate, with good repeatability, and can be used for the quality control of Radix Scutellariae.     

Supercritical fluid extraction of flavonoids from Scutellariae Radix

An optimal condition of supercritical fluid extraction (SFE) for flavonoids of Scutellaria baicalensis was developed. In this study, various temperatures, pressures and modifiers were studied. The conventional extraction methods were conducted in parallel for comparison. The crude extracts were qualitatively compared by TLC and GC–MS, and the contents of flavonoids were determined by HPLC. The amounts of baicalin, baicalein and wogonin in the Scutellariae Radix obtained by supercritical fluid extraction and a conventional sonic shaking method were 137.6 mg/g, 8.6 mg/g and 2.2 mg/g, 113.5 mg/g, 5.7 mg/g and 2.3 mg/g, respectively. Application of SFE for extraction of the flavonoids from Scutellariae Radix was preferable. The optimal conditions of SFE was as follows: supercritical carbon dioxide–MeOH–water (20:2.1:0.9), 50°C and 200 bar.     

A simple isocratic HPLC method for the simultaneous determination of bioactive components of Scutellariae Radix extract

Scutellariae Radix, the dried root of Scutellaria baicalensis Georgi, has been widely used in Asian countries for the treatment of dermatitis, diarrhoea, inflammatory disease and hepatic disease. A simple, sensitive and precise reversed-phase liquid chromatographic method with isocratic elution was developed to simultaneously determine four bioactive compounds in Scutellariae Radix: baicalein, baicalin, wogonin and wogonoside. Chromatographic analysis was performed on a YMC Pack Pro C(8) column (150?×?4.6?mm(2), 3?μm), with a mobile phase of 0.1% formic acid?:?acetonitrile (70?:?30, v/v) at a flow rate of 1.0?mL?min(-1), and UV detection at 280?nm. Linear behaviour was observed over the investigated concentration range (0.25-10?μg?mL(-1)) for all analytes, with a correlation coefficient of >0.997. The intra- and inter-day precisions were <8.07%, and accuracies were 92.3-102.9%. This method was successfully applied for the analysis of marker compounds for the quality control of Scutellariae Radix extract.     

Differential distribution of phytochemicals in Scutellariae Radix and Scutellariae Amoenae Radix using microscopic mass spectrometry imaging

Scutellariae Radix, the root of Scutellaria baicalensis Georgi, is widely applied in China for the treatment of fever, ulcer, bronchitis, hepatitis and inflammatory symptoms. Sctuellariae Amoenae Radix, the root of Scutellaria amoena C.H. Wright, is often prescribed as the substitute for Scutellariae Radix. Up to now, no attempt has been made to unveil and compare the localization of phytochemicals in Scutellariae Radix and its succedaneum. This investigation succeeded to look into the differential distribution of natural compounds in Scutellariae Radix and Scutellariae Amoenae Radix using microscopic mass spectrometry imaging. Compounds presenting different distribution modes in two kinds of roots were sorted out, then a quick method for the differentiation between Scutellariae Radix and Scutellariae Amoenae Radix was established. Accumulation sites of baicalein, baicalin, wogonin and wogonoside in Scutellariae Radix were also uncovered using microscopic mass spectrometry imaging. Moreover, the application of matrix assisted laser desorption ionization-quadrupole-time of flight mass spectrometry allowed the on-tissue dissociation of major flavonoids. Overall, the utilization of microscopic mass spectrometry imaging and matrix assisted laser desorption ionization-quadrupole-time of flight mass spectrometry provided a novel perspective for the discovery of natural compounds within medicinal plants.     

Comparative analysis of the main active components and hypoglycemic effects after the compatibility of Scutellariae Radix and Coptidis Rhizoma

In this study, a rapid and highly sensitive ultra high performance liquid chromatography with triple quadrupole mass spectrometry method with the mobile phase of acetonitrile and 0.1% aqueous formic acid was established and successfully applied to comparatively analyze main active components after their compatibility. Besides, the effects of Scutellariae Radix, Coptidis Rhizoma and combined extracts on type 2 diabetic rats induced by high‐fat diet along with low dose of streptozocin were investigated. Under the optimized chromatographic conditions, good separation of seven target components was achieved within 12 min. All calibration curves exhibited good linearity (R² ≥ 0.999). The relative standard deviation of precision, repeatability and stability varied from 0.69 to 2.23, 0.98 to 2.56, and 0.92 to 2.57%, respectively. The recovery ranged from 91.11 to 105.35%. The contents of seven active components were notably reduced after compatibility; however, the hypoglycemic effect of combined extracts was stronger than single drug by decreasing the activities of fructose‐1,6‐bisphosphatase, glucose 6‐phosphatase, phosphoenolpyruvate carboxykinase and increasing the activities of glucokinase, phosphofructokinase, pyruvate kinase. Accordingly, the established analytical method was accurate and sensitive enough for quantitative evaluation of seven investigated compounds. Moreover, the combined extract had definite effects on type 2 diabetes through multiple components against multiple targets.     

Simultaneous LC-MS/MS quantitation of a highly hydrophobic pharmaceutical compound and its metabolite in urine using online monolithic phase-based extraction

This article describes the use of monolithic material as extraction support in simultaneous LC-MS/MS quantitation of a highly hydrophobic pharmaceutical compound and its hydroxylated metabolite in urinary matrix. DMSO was added to urine samples to aid the solubilization of analytes during storage and transfer. Samples were then diluted and injected onto an online extraction system for high-throughput analysis in an automated fashion. A linear range of 1.03-103 ng/mL was validated for the parent compound and 7.02-1400 ng/mL for the metabolite. The accuracy (%bias) at the lower LOQ (LLOQ) for the parent compound was -2.9% and the precision (%CV) at the LLOQ was 5.4%, while the accuracy at LLOQ for the metabolite was 6.3% and the precision at LLOQ was 5.5%. The results show that quantitative LC-MS/MS analysis by online extraction using the monolithic support is reproducible, sensitive, and accurate enough to satisfy bioanalytical testing requirements in a good laboratory practice (GLP) regulated environment. Monolithic phase based online extraction provided efficient sample cleanup, which was evidenced in matrix effect testing against multiple lots of urinary matrix. The method described within can be a generic approach for quantitative analysis of analytes with poor solubility in aqueous matrix.     

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.     

基于快速高分辨液相色谱串联质谱技术的代谢组学尿液分析方法的建立

采用快速高分辨液相色谱(RRLC)分离系统与QTRAP型及QTOF型MS/MS仪联用技术,通过考察尿液样本前处理方法,优化液相色谱条件和质谱检测参数,建立了用于尿液中代谢物分析的RRLC-MS方法.采用本方法对尿液浓度下的20种代表性代谢物进行了检测,考察了方法的灵敏度和精密度,证明本方法适用于尿液代谢组学的研究.对穿...     

Design of online solid phase extraction-liquid chromatography-tandem mass spectrometry (SPE-LC-MS/MS) hyphenated systems for quantitative analysis of small organic compounds in biological matrices

Three online solid phase extraction-liquid chromatography-tandem mass spectrometry (SPE-LC-MS/MS) method examples are presented where two different types of chromatographic columns or solvent systems were coupled to meet specific analytical objectives: (i) SPE of target analytes by restricted access media from high ionic strength urine matrix was coupled with reversed phase LC-MS/MS conditions accommodating high ionization potentials of the analytes (urinary bisphenol A and other phenolic derivatives); (ii) strong cation exchange SPE of analytes of diverse polarity and pK(a) was coupled with reversed phase LC-MS/MS analysis (urinary atrazine metabolites); (iii) pre-concentration of low pg per sample analytes by weak anion exchange SPE was hyphenated with ion pair LC-MS analysis (intracellular nucleotide triphosphate analogs). With these examples we suggest a conductive generic work flow for the development of online SPE-LC-MS methods and show how advanced commercial LC devices and software allow for the design of complex yet highly versatile analytical separation systems suited to the unique physicochemical properties of the target analytes.