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。     

HPLC-ESI-MS快速筛选并鉴定补骨脂甲醇提取物中的抗氧化活性成分

采用HPLC-ESI-MS法建立了一种快速筛选并鉴定补骨脂甲醇提取物中抗氧化活性成分的方法,即利用二苯基三硝基苯肼(DPPH·)与补骨脂甲醇提取物中抗氧化活性成分反应前后色谱图中峰面积的变化来筛选出抗氧化活性物质,同时利用电喷雾质谱对这些活性组分进行结构鉴定.利用该方法从补骨脂甲醇提取物中筛选出了8个抗氧化活性组分,分别为新补骨脂异黄酮,补骨脂二氢黄酮,补骨脂异黄酮,补骨脂定,异补骨脂查尔酮,补骨脂二氢黄酮甲醚,corylifol和补骨脂酚.这种方法较传统的筛选方法省时省力,既不容易造成微量组分丢失,又不需要仪器改装工作,易于实现复杂体系抗氧化活性物质的高通量筛选.     

红车轴草总异黄酮成分DNA结合剂的超滤质谱筛选

采用离心超滤和液相色谱-质谱联用的方法, 从红车轴草异黄酮提取物中筛选DNA结合剂. 结果表明, 红车轴草中10种异黄酮成分与DNA具有不同的结合能力. 采用液相色谱-串联质谱联用技术对其结构进行了鉴定, DNA结合能力较强的5种化合物分别是德鸢尾素-4'-O-β-D-葡萄糖苷、 德鸢尾素、 黄豆黄葡萄糖苷、 红车轴草素和鹰嘴豆牙素A. 为从中药提取物等复杂体系筛选并鉴定DNA结合剂建立了快速的超滤质谱平台.     

2,2-二苯基-1-苦肼基-高效液相色谱法快速筛选亚麻籽抗氧化活性成分

为了更加迅速地筛选到亚麻籽复杂提取物中的抗氧化活性物质及其活性强弱,以开环异落松树脂酚二葡萄糖苷(SDG)、开环异落叶松树脂酚(SECO)和肠二醇(ED)3种木酚素为研究对象,建立了抗氧化活性能力筛选的2,2-二苯基-1-苦肼基-高效液相色谱法(DPPH-HPLC法)。液相色谱条件为:流动相为乙腈和水,经反相色谱柱Waters XBridge C18分离,检测波长为280 nm。DPPH-HPLC法的步骤是:将单一或者混合抗氧化剂分别与DPPH混合,放置20 min,作为待测液,将同样量的相应抗氧化剂作为对照液,分别进行液相色谱分析获得其含量,计算出相应的抗氧化剂清除率(SRA),活性顺序为:SDG>SECO>ED。结合超高效液相色谱-飞行时间质谱联用法,鉴定出亚麻籽中5种抗氧化物,筛选其抗氧化能力分别为:SDG异构体(5)>SDG(4)>7α-[(β-D-glupyranosyl)oxy]-1-methoxyisolariciresinol(1)>(6R,7R,8S)-1-Methoxyisolariciresinol(2)>蜀葵苷元二葡萄糖苷(3)。结果表明,建立的DPPH-HPLC法能够有效完成复杂亚麻籽提取物的抗氧化活性物质筛选。     

用高表达EGFR细胞膜色谱-HPLC/MS联用快速筛选独活中抗EGFR活性成分

报道了用高表达表皮生长因子受体细胞膜色谱与高效液相色谱/质谱在线联用方法(EGFR/CMC-online-HPLC/MS)快速筛选发现中药独活中的活性成分.实验中,采用高表达EGFR的细胞膜制备色谱固定相,建立EGFR/细胞膜色谱(EGFR/CMC)模型,利用柱切换和固相萃取技术,将EGFR/CMC模型与高效液相色谱/质谱(HPLC/MS)在线联用,构成一种新的可同时"识别-鉴定"目标成分的二维色谱系统,并应用于快速筛选独活中具有抗EGFR活性的目标成分.结果发现独活中的蛇床子素具有与对照药物达沙替尼类似的色谱保留特性,能够作用于EGFR;同时MTT及Elisa分析实验证实蛇床子素对HEK293EGFR细胞增殖及EGFR表达均有抑制作用.本文建立的EGFR/CMC-online-HPLC/MS二维色谱方法,可以选择性地从中药复杂体系中快速"识别-鉴定"目标组分,且筛选结果与特定生物效应显著相关.     

青翘清热解毒活性组分及成分筛选

探讨青翘清热解毒活性组分。以C_(18)为分离材料将青翘60%乙醇提取物分离为6个化学成分群F_1～F_6;以清除DPPH·的能力、抑菌效果和改善LPS诱导的小鼠巨噬细胞RAW264.7产生炎症反应的程度为评价指标,评价不同化学成分群的抗氧化、抑菌和抗炎活性。F_2化学成分群在抗氧化、抑菌和抗炎活性等方面均具有较强的活性;进一步化学成分分析表明青翘清热解毒活性组分F_2主要是由苯乙醇苷类成分组成,主要成分为连翘酯苷A和连翘酯苷I。青翘清热解毒活性活性成分主要为苯乙醇苷类成分。     

超高效液相色谱法测定大黄与铜离子络合能力

中药肝豆汤治疗铜代谢障碍疾病具有疗效显著、副作用小的优势,但由于缺乏评价铜离子(Cu²⁺)络合能力的技术手段,限制了肝豆汤配位活性成分的筛查和发现。为评价复方肝豆汤中主药大黄对铜离子的络合能力,该文通过优化大黄活性成分与铜离子配位反应条件,建立了超高效液相色谱法(UHPLC)测定中药大黄与铜离子络合能力的方法。样品经Agilent Eclipse Plus C18色谱柱(50 mm×2.1 mm, 1.8μm)分离,以甲醇-0.1%(v/v)磷酸水溶液为流动相进行梯度洗脱,柱温30℃,流速为0.3 mL/min,检测波长为254 nm,进样体积为5μL。研究通过UHPLC分析比较大黄与铜离子配位反应前后色谱峰变化,超高效液相色谱四极杆-飞行时间质谱(UPLC-Q-TOF-MS)鉴定大黄提取物中配位活性成分,计算大黄提取物与铜离子配位反应前后其色谱峰面积变化率来评价大黄中活性成分与Cu²⁺的络合能力。结果显示,大黄提取物与Cu²⁺在pH为9的体系中配位反应12 h达到平衡;鉴定出大黄提取物中20种主要成分,根据Cu     

超高效液相色谱-串联质谱法同时测定黄芩提取物中4种黄酮类成分

黄芩提取物样品中加入水,经甲醇超声提取2次,离心后,合并上清液,用水定容至50.0mL,过0.22μm微孔滤膜,采用超高效液相色谱-串联质谱法同时测定滤液中黄芩素、黄芩苷、汉黄芩苷、野黄芩苷等4种黄酮类成分的含量。以ACQUITY UPLC HSS T3色谱柱为固定相,以不同体积比的0.01%(质量分数)氨水和乙腈的混合液为流动相进行梯度洗脱,串联质谱分析中采用电喷雾离子源负离子模式(ESI-)和多反应监测模式。4种黄酮类成分的质量浓度在5.0～500.0μg·L~(-1)内与其对应的质谱响应值呈线性关系,检出限(3S/N)为0.3～1.5μg·kg~(-1),测定下限(10S/N)为1.0～5.0μg·kg~(-1)。以空白黄芩提取物样品为基体进行加标回收试验,所得回收率为91.1%～99.1%,测定值的相对标准偏差(n=6)为2.0%～4.1%。     

二维液相色谱-质谱联用分离中药复方葛根芩连汤中的有效成分

建立了二维液相色谱-质谱联用方法分离中药复方葛根芩连汤的成分。以CN柱作第一维色谱柱,水和甲醇梯度洗脱分离;以ODS柱作第二维色谱柱,20 mmol/L乙酸铵缓冲液和乙腈梯度洗脱分离;质谱检测采用电喷雾电离/大气压化学电离（ESI/APCI）复合离子源,正负离子扫描。实验结果表明搭建的二维液相色谱的峰容量显著高于一维色谱,分离效率得到了明显的提高。以第一维色谱的第3个流分为例,对其二维分离进行仔细分析,发现质谱比紫外光谱检测到的组分多,质谱中采用负离子模式比正离子模式检测到的组分多。表明搭建的二维液相色谱-质谱分离平台分离效果好,提高了液相色谱的峰容量和分离效率。该方法操作简便,可作为中药等复杂体系分离分析的有效手段。     

HPLC-DAD-ESI/MS~n-DPPH在线筛选与鉴别丹参和康定鼠尾草中抗氧化活性成分

本研究建立了在线高效液相色谱-质谱-二苯基三硝基苯肼(HPLC-DAD-ESI/MSn-DPPH)快速筛选和鉴别丹参和康定鼠尾草中抗氧化活性成分和含量的方法。经液相色谱、质谱和文献报道综合分析鉴定出丹参和康定鼠尾草中的3种抗氧化活性化合物,分别为咖啡酸、异迷迭香酸苷和迷迭香酸。比较了热回流、超声辅助提取和快速溶剂萃取的提取效果,并对色谱条件进行优化。在优化条件下,这3种化合物均可有效分离,并在1.7~35.3μg/m L范围内线性关系良好,相关系数为0.999 5~0.999 8,检出限为0.05~1.85μg/m L,定量下限为0.18~6.16μg/m L,平均回收率为96.6%~97.2%,相对标准偏差为1.1%~1.3%。运用该方法测定丹参和鼠尾草样品中这3个化合物的含量分别为:咖啡酸0.303,0.254 mg/g;异迷迭香酸苷1.019,1.401 mg/g;迷迭香酸17.279,8.104 mg/g。本方法简便、快速、准确、重现性好,适用于从复杂天然产物中快速筛选与鉴别抗氧化活性成分。     

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.     

Rapid and simple method for screening of natural antioxidants from Chinese herb Flos Lonicerae Japonicae by DPPH-HPLC-DAD-TOF/MS

A rapid and simple method has been developed for the screening and identification of natural antioxidants of Flos Lonicerae Japonicae (FLJ), derived from the flower buds of Lonicera japonica. The hypothesis is that upon reaction with 1,1-diphenyl-2-picrylhydrazyl (DPPH), the peak areas (PAs) of compounds with potential antioxidant effects in the HPLC chromatograms will be significantly reduced or disappeared, and the identity confirmation could be achieved by HPLC-DAD-TOF/MS hyphenated technique. Using the proposed approach, about 14 compounds in the FLJ extract were found to possess a potential antioxidant activity. They were identified as chlorogenic acid (1), 1-O-caffeoylquinic acid (1-O-CQA, 2), caffeic acid (4), 4-O-CQA (5), rutin (7), isoquercitrin (8), luteolin-7-O-glucoside (9), lonicerin (10), 4,5-O-dicaffeoylquinic acid (4,5-O-diCQA, 11), 3,5-O-diCQA (12), 1,3-O-diCQA (13), 3,4-O-diCQA (14), 1,4-O-diCQA (16), and luteolin (17). In addition, the free radical scavenging capacities of the available identified compounds were also investigated by HPLC assay. The results indicated that the compounds with PAs significantly decreasing were natural antioxidants, whereas those with PAs not changing presented no activities, which accordingly indicated that this newly proposed method could be widely applied for rapid screening and identification of natural antioxidants from complex matrices including Chinese herbal medicines.     

Comparative evaluation of three methods based on high-performance liquid chromatography analysis combined with a 2,2'-diphenyl-1-picrylhydrazyl assay for the rapid screening of antioxidants from Pueraria lobata flowers

Traditional activity-guided fractionation of natural products is a time-consuming, labor intensive, and expensive strategy, which cannot compete with high-throughput and rapid screening of natural products. Therefore, more efficient approaches are necessary for searching active compounds from natural products. Three main methods based on high-performance liquid chromatography (HPLC) analysis combined with 2,2′-diphenyl-1-picrylhydrazyl (DPPH) assay, DPPH spiking HPLC analysis, on-line post-column HPLC-DPPH analysis, and HPLC-based DPPH activity profiling, were then developed for the rapid screening of antioxidants from complex mixtures. In the present study, a comparative study of these three methods has been conducted to identify antioxidants from an ethyl acetate fraction of Pueraria lobata flowers. The parameters in HPLC analysis and DPPH assay were optimized. The results indicated that all three methods could achieve similar information with regard to antioxidants, without the need for preparative isolation techniques. However, there were differences in instrumental set-up, sensitivity, and efficiency. DPPH spiking HPLC analysis seemed to be more sensitive and effective with simpler instrumental set-up and easier operation, which could also detect the total antioxidant capacity of color complexes. Eighteen antioxidants were tentatively screened and identified from P. lobata flowers by DPPH spiking HPLC-MS/MS. Among them, ten compounds including one new compound were first isolated from P. lobata flowers, and the DPPH radical scavenging activity of the new compound was reported for the first time.     

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.     

Quantitative analysis of urinary endogenous markers for the treatment effect of Radix Scutellariae on type 2 diabetes rats

The effect of Radix Scutellariae treated on type 2 diabetic rats has been investigated by a liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) based urinary quantitative approach. In this research, multiple reactions monitoring mode of MS/MS in LC-MS/MS analysis was used to quantitatively analyze the concentrations of 7 endogenous compounds in urine of normal control group, type 2 diabetic model group and Radix Scutellariae-treated group, and multivariate statistical analysis was utilized for MS data processing. The above-mentioned three groups can be distinguished via pattern recognition. The obtained results indicated that Radix Scutellariae affect the urinary metabolic profiling of type 2 diabetic rats on the polyol pathway, protein glycation reaction and amino acids metabolism pathway. According to these results, Radix Scutellariae should have the pharmacological effect on preventing or delaying the onset and progression of diabetes and its complications.     

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.     

大黄中鞣质成分的分离与液相色谱/质谱联用分析

研究了大黄中鞣质类成分的提取、分离与分析方法。优化了大黄原药材中鞣质类物质的提取方法;建立了大黄鞣质类成分的梯度洗脱反相高效液相色谱(HPLC)分析方法,使鞣质类成分得到了良好的分离;采用液相色谱-质谱(LC-MS)对大黄中主要的鞣质类化合物进行了结构分析,并总结了一部分鞣质类化合物在高效液相色谱-电喷雾质谱(HPLC-ESI-MS)谱图上的裂解规律。     

高效液相色谱-质谱法快速鉴定复方毛冬青冲剂中三萜皂苷活性成分

建立了高效液相色谱-质谱法(HPLC-MS)快速鉴定复方毛冬青冲剂中三萜皂苷活性成分的方法.以甲醇为萃取剂超声萃取复方毛冬青冲剂30 min.采用高效液相色谱-离子阱质谱(HPLC-IT-MS)和高效液相色谱-飞行时间质谱(HPLC-TOF-MS)对萃取液进行分析,选用Agilent Zorbax Eclipse XDB C18色谱柱(250 mm×4.6 mm, 5 μm),以水(含0.1 %甲酸)-乙腈为流动相进行梯度洗脱,柱后流出液采用电喷雾离子阱质谱(ESI-IT-MS)和电喷雾飞行时间质谱(ESI-TOF-MS)的正、负离子模式进行检测.检测结果经离子阱一级质谱(IT-MS1)、离子阱二级质谱(IT-MS2)和分析时间质谱(TOF-MS)信息分析,并与相关文献报道进行比较,鉴定出1种三萜酸和8种三萜皂苷成分,并推测了其它3种可能的三萜皂苷化学成分,通过对照品对比分析,三萜酸确证为Ilexgenin A,其中一种三萜皂苷确证为Ilexsaponin A1.本方法无需对照品即可快速有效地鉴定出复方毛冬青冲剂中的三萜皂苷活性成分,为建立冲剂的质量标准提供了依据.     

高效液相色谱-串联质谱法检测花生中的黄曲霉毒素B_1

应用高效液相色谱-电喷雾串联四极杆质谱联用系统(HPLC-MS/MS),在多反应离子检测方式(MRM)下,对花生中的黄曲霉毒素B1进行检测.对花生中黄曲霉毒素B1的提取、净化、液相分离及串联质谱等相关检测参数进行了优化研究.用V(甲醇):V(水)=6:4提取,OASIS HLB SPE小柱净化,定容过滤.采用V(甲醇):V(水)(含体积分数0.1%甲酸)=7:3为流动相,前级离子313.0,二级离子241.1、269.1,ESI正离子方式检测,在3.3 min出峰.结果表明,在ESI正离子模式下,黄曲霉毒素B1在其线性定量范围0.1～50μg/kg内,相关系数达到0.9999,检出限为0.03μg/kg,最低定量限为0.1μg/kg.低、中、高浓度添加回收率范围为93%～105%.