An UPLC-MS/MS application to investigate the chemical composition of the ethanol extract from Anoectochilus chapaensis and its hypoglycemic activity in insulin-resistant HepG2 cells

Anoectochilus chapaensis Gagnep. (Orchidaceae) was named as the “king of medicine” because of its excellent efficacy for the treatment of diabetes. However, the bioactive constituents are unknown. An ethanol extract from A. chapaensis showed significant stimulating effect on glucose consumption in HepG2 cells. The chemical composition was investigated by UPLC-MS/MS in negative electrospray ionization (ESI) mode, and 63 compounds including flavonoids, triterpenoids, and aliphatic acids were tentatively identified by accurate mass and characteristic fragments. Moreover, the method of hypoglycemic screening with insulinresistant HepG2 cells and UPLC-MS/MS might be potentially useful in rapid and efficient characterization and primary prediction of natural products prior to traditional isolation.     

Analysis of the constituents in the rat plasma after oral administration of Yin Chen Hao Tang by UPLC/Q-TOF-MS/MS

A UPLC/Q-TOF-MS/MS method for analyzing the constituents in rat plasma after oral administration of Yin Chen Hao Tang (YCHT), a traditional Chinese medical formula, has been established. The UPLC/MS fingerprints of the samples were established first in vitro and in vivo, with 45 compounds in YCHT and 21 compounds in rat plasma after oral administration of YCHT were detected. Of the 45 detected compounds in vitro, 30 were identified, and all of the 21 compounds detected in rat plasma were identified either by comparing the retention time and mass spectrometry data with that of reference compounds or by mass spectrometry analysis and retrieving the reference literatures. Of the identified 21 compounds in rat plasma, 19 were the original form of compounds absorbed from the 45 detected compounds in vitro, 2 were the metabolites of the compounds existed in YCHT. It is concluded that a rapid and validated method has been developed based on UPLC-MS/MS, which shows high sensitivity and resolution that is more suitable for identifying the bioactive constituents in plasma after oral administration of Chinese herbal medicines, and provides helpful chemical information for further pharmacology and active mechanism research on the Chinese medical formula.     

UPLC-MS/MS在中药材农残分析中的应用

超高效液相色谱-串联质谱技术（UPLC-MS/MS）是一项应用范围广泛的新型技术,本文根据国内外学者使用超高效液相色谱-串联质谱技术进行中药材中农残检测的研究工作,就目前该技术的应用进行了系统综述,对基质效应的应对方法进行了总结与讨论,为UPLC-MS/MS在中药农残分析领域的后续研究及应用提供思路和借鉴,并对应用前景进行了展望。     

Metabolite fingerprinting of Camptotheca acuminata and the HPLC–ESI-MS/MS analysis of camptothecin and related alkaloids

The major phytochemical constituents, namely, alkaloids, flavonoids and ellagic acid derivatives, of leaves of Camptotheca acuminata were identified using high performance liquid chromatography (HPLC) coupled with electrospray mass spectrometry (ESI-MS) in extracts of plants cultivated in Italy and collected at different growth stages. Alkaloids related to camptothecin were identified and quantified by HPLC coupled with ESI-tandem mass spectrometry (MS/MS) employing, respectively, an ion trap and a triple quadrupole mass analyser. The fragmentation patterns of alkaloids related to camptothecin were analysed and a specific Multiple Reaction Monitoring HPLC–MS/MS method was developed for the quantitative determination of these constituents. The described method provides high sensitivity and specificity for the characterisation and quantitative determination of the alkaloids in C. acuminata.     

Development and validation of a highly sensitive UPLC-MS/MS method for simultaneous determination of aconitine, mesaconitine, hypaconitine, and five of their metabolites in rat blood and its application to a pharmacokinetics study of aconitine, mesaconitine, and hypaconitine

A rapid, specific and sensitive method was developed for the simultaneous determination of eight Aconitum alkaloids: aconitine (AC), mesaconitine (MA), hypaconitine (HA), benzoylaconine (BAC), benzoylmesaconine (BMA), benzoylhypaconine (BHA), aconine and mesaconine in rat blood by ultra-performance liquid chromatography coupled tandem mass spectrometry (UPLC-MS/MS). The UPLC-MS/MS system coupled with an electrospray ionization (ESI) source was operated in a positive mode via multiple-reaction monitoring (MRM). Samples were treated with methanol to remove protein prior to analysis by UPLC-MS/MS. The analytes were separated with a Waters C18 column (1.7 μm, 50?×?2.1?mm) and a gradient elution using acetonitrile and 0.1% formic acid-water as the mobile phases. The linear response range was from 0.125 to 1000 nmol/L for these eight alkaloids and the correlation coefficients (r(2) values) were all higher than 0.997. The method was validated with respect to precision, accuracy, recovery, matrix effect, carryover effect and sample stability, and found to be within the acceptable limits. The developed and validated method was successfully applied to simultaneously determine the eight Aconitum alkaloids in rats blood after intravenous administration of a mixture of AC, MA and HA.     

高效液相色谱串联质谱法分析菊花水提取液的化学成分

目的采用高效液相色谱串联质谱法分析菊花水提取液的化学成分。方法采用水为溶剂加热回流提取法提取菊花药材,提取液采用高效液相色谱串联质谱法对其主要的化学成分进行分离和鉴定。结果通过高效液相色谱可将20个主要的化学成分较好的分离,其中13个通过串联质谱进行了结构鉴定,8个通过对照品得到了确证。结论高效液相色谱串联质谱法快速、准确,可用于菊花药材的化学成分鉴定。     

UPLC-MS/MS法测定中成药制剂中添加的11种降压药物

目的 建立一种UPLC-MS/MS条件下同时对中成药制剂中非法添加的11种降压类药物进行快速、准确、灵敏洲定的方法.方法 采用超高效液相色谱-串联点喷雾四极杆质谱,在多反应监测(MRM)模式下测定中成药胶囊荆、片荆及丸剂中11种降压药物的含量.结果 在本实验条件下11种降压药物在0.025μg/m～5.000μg·mL...     

UHPLC‐MS/MS and UHPLC‐HRMS identification of zolpidem and zopiclone main urinary metabolites and method development for their toxicological determination

Zolpidem and zopiclone (Z‐compounds) are non‐benzodiazepine hypnotics of new generation that can be used in drug‐facilitated sexual assault (DFSA). Their determination in biological fluids, mainly urine, is of primary importance; nevertheless, although they are excreted almost entirely as metabolites, available methods deal mainly with the determination of the unmetabolized drug. This paper describes a method for the determination in urine of Z‐compounds and their metabolites by ultra‐high‐pressure liquid chromatography/tandem mass spectrometry (UHPLC‐MS/MS) and UHPLC coupled with high resolution/high accuracy Orbitrap® mass spectrometry (UHPLC‐HRMS). The metabolic profile was studied on real samples collected from subjects in therapy with zolpidem or zopiclone; the main urinary metabolites were identified and their MS behaviour studied by MS/MS and HRMS. Two carboxy‐ and three hydroxy‐ metabolites, that could be also detected by gas chromatography/mass spectrometry (GC‐MS) as trimethylsylyl derivatives, have been identified for zolpidem. Also, at least one dihydroxilated metabolite was detected. As for zopiclone, the two main metabolites detected were N‐demethyl and N‐oxide zopiclone. For both substances, the unmetabolized compounds were excreted in low amounts in urine. In consideration of these data, a UHPLC‐MS/MS method for the determination of Z‐compounds and their main metabolites after isotopic dilution with deuterated analogues of zolpidem and zopiclone and direct injection of urine samples was set up. The proposed UHPLC‐MS/MS method appears to be practically applicable for the analysis of urine samples in analytical and forensic toxicology cases, as well as in cases of suspected DFSA. Copyright © 2013 John Wiley & Sons, Ltd.     

Polyphenolic extract isolated from Korean Lonicera japonica Thunb. induce G2/M cell cycle arrest and apoptosis in HepG2 Cells: Involvements of PI3K/Akt and MAPKs

Lonicera japonica Thunb. (L. japonica T.) has been used in Korean traditional medicine for long time because of its anti-cancer and hepatic protective effect. In this study, we investigated polyphenolic extract in L. japonica T. using high-performance liquid chromatography coupled with tandem mass spectrometry (HPLC–MS/MS) and its anti-cancer effect on hepatocarcinoma cells. Human HepG2 cell line was treated with various concentrations of polyphenolic extract. Apoptosis was detective by cell morphology, cell cycle analysis and immunoblot analysis. Polyphenolic extract inhibited cell proliferation at 48 h in a dose-dependent manner. Polyphenolic extract affected HepG2 cell viability by inhibiting cell cycle progression at the G2/M transition and inducing apoptosis. Polyphenolic extract also decreased the expression of CDK1, CDC25C, cyclin B1, pro-caspases-3 and -9 and poly ADP ribose polymerase, and affected the levels of mitochondrial apoptotic-related proteins. The phosphorylation of extracellular signal-related kinase ½ (ERK 1/2), c-Jun N-terminal kinase (JNK), and p-38 mitogen-activated protein kinases (MAPKs) were increased in HepG2 cells treated with polyphenolic extract, whereas Akt was dephosphorylated. These results indicate that inhibition of PI3K/Akt and activation of MAPKs are pivotal in G2/M cell cycle arrest and apoptosis of human hepatocarcinoma cells mediated by polyphenolic extract.     

Analysis of the bioactive constituents of ChanSu in rat plasma by high performance liquid chromatography with mass spectrometric detection

A plasma pharmacochemistry analysis of the bioactive constituents in rat plasma after oral administration of ChanSu was performed. High performance liquid chromatography coupled with the electrospray ionization mass spectrometry techniques of HPLC/ESI-IT-MS/MS and RRLC/ESI-Q-TOF-MS were used for the chemical profiling of samples of dosed plasma, control plasma and ChanSu extract. Comparative analysis of the resulting chemical profiles revealed 20 prototype compounds and 4 metabolites derived from ChanSu as potential bioactive components. By MS/MS analysis and accurate molecular weight assessments, the majority of these bioactive components (19 prototype compounds and 2 metabolites) were structurally identified. Moreover, seven were confirmed by comparing their retention behaviors using MS and MS/MS analysis. This study proposes a series of potential bioactive components of ChanSu, which we hope will lead to new drug discovery based on ChanSu and other traditional Chinese medicines.     

超高压液相/电喷雾-LTQ-Orbitrap质谱联用技术分析三七根中皂苷类成分

建立三七药材的UPLC-ESI-HRMS定性分析方法。采用Kinetics色谱柱,梯度洗脱,经DAD检测器后采用ESI-LTQ-Orbitrap质谱负离子模式采集,三七成分的一级全扫描离子在离子阱内进行CID多级质谱,FT检测高分辨质谱数据进行分析。结果表明三七皂苷成分分离良好,根据多级质谱碎片信息和精确相对分子质量信息,结合文献,从三七中鉴定分析了43个成分,并首次从三七中检测到新型的三七皂苷母核和乙酰取代皂苷成分。本方法分离度良好、灵敏度高,可用于三七药材的定性分析,并有助于对三七进行进一步的活性成分研究和质量控制。     

Identification and determination of the major constituents in Deng's herbal tea granules by rapid resolution liquid chromatography coupled with mass spectrometry

Deng's herbal tea (DHT), a famous traditional Chinese herbal tea consisting of six traditional Chinese medicines (Honeysuckle, Chrysanthemum, Rhizoma imperatae, Folium mori, dandelion and liquorice), is widely used in China for its health benefits. In this paper, a rapid resolution liquid chromatography coupled with mass spectrometry (RRLC-MS) method was developed for the identification and determination of the major constituents in DHT granules. A good RRLC separation was achieved using an Agilent Poroshell 120 SB-C₁₈ column and gradient elution (0.5% formic acid in water/acetonitrile) within 30 min. Twenty-eight compounds were identified or tentatively characterized based on their exact molecular weights and fragmentation patterns. Fifteen major bioactive constituents of those 28 compounds were chosen as the benchmark substances. Their quantitative analyses were performed by a triple quadrupole tandem mass spectrometer (MS/MS) operating in multiple-reaction monitoring mode, and a full quantitative analysis of the 15 major constituents was performed by our developed RRLC-MS/MS method in only 10 min. Of the 16 DHT granule samples tested, the quality of the results was stable, which confirms that the developed method was efficient and robust for the quality control of DHT granules.     

UPLC-MS/MS法测定氯霉素滴眼液中氯霉素的含量

目的建立超高效液相色谱-串联质谱法(UPLC-MS/MS)测定氯霉素滴眼液中氯霉素含量。方法采用UPLC BEH C18色谱柱,以乙腈-水为流动相,经梯度洗脱后,利用质谱仪的多反应监测(MRM)模式测定氯霉素含量。结果氯霉素质量浓度在1.0~20.0 ng/ml范围内与质谱相应值呈良好的线性关系,检出限为0.15 ng/ml,定量限为0.5 ng/ml,RSD为0.83%,加标回收率平均值在94.0%~100.3%。结论UPLC-MS/MS法准确度高、灵敏度高、重现性好。     

Phytochemical analysis of an antiviral fraction of Radix astragali using HPLC–DAD–ESI–MS/MS

Radix astragali (Huangqi in Chinese) is a well-known traditional Chinese medicinal herb that has been used clinically in China for centuries to cure various diseases. To profile the antiviral constituents of this herb, a high-performance liquid chromatography–diode array detector–electrospray ionization–tandem mass spectrometry (HPLC–DAD–ESI–MS/MS) analytical method was developed to separate and determinate the active part of the extract of Radix astragali, which showed potent inhibition of several viruses. By comparing their retention time and MS data with those obtained from the authentic compounds and the published data, a total of 18 compounds, comprising 11 flavonoids and 7 saponins, were identified. This study provides an approach to rapidly characterize bioactive constituents in traditional Chinese medicines (TCMs).     

五加生化胶囊UPLC-MS指纹图谱研究

目的:建立五加生化胶囊UPLC-MS的指纹图谱,为科学客观地评价其质量提供可靠的方法。方法:色谱柱:ACQUITYUPLC BEH(2.1mm×50 mm,1.7μm);流动相:乙腈(A)-0.1%甲酸和10 mmol.L-1醋酸铵(B)梯度洗脱[5.0%A(0 min),5.0%A→50%A(0～10 min),50%A～80%A(10～16 min)];柱温:40℃;流速:0.4 mL.min-1;进样量:8μL;电喷雾接口,正离子模式,毛细管电压3.50 kV,锥孔电压35.00 V,离子源温度120℃,脱溶剂温度380℃,脱溶剂气N2:500 L.h-1,锥孔气N2:50 L.h-1。结果:11批五加生化胶囊UPLC-MS指纹图谱中共标定了37个共有色谱峰,通过与对照品的保留时间及质谱信息的比对,确定4号、8号、9号、10号和30号峰分别为紫丁香苷、阿魏酸、甘草苷、异嗪皮啶和藁本内酯。结论:所建立的五加生化胶囊UPLC-MS指纹图谱满足于方法学的要求,所得的指纹图谱特征性及专属性强,可用于五加生化胶囊质量的控制。     

Metabolic profiling of roots of liquorice (Glycyrrhiza glabra) from different geographical areas by ESI/MS/MS and determination of major metabolites by LC-ESI/MS and LC-ESI/MS/MS

Liquid chromatography electrospray mass spectrometry (LC-ESI/MS) has been applied to the full characterization of saponins and phenolics in hydroalcoholic extracts of roots of liquorice (Glycyrrhiza glabra). Relative quantitative analyses of the samples with respect to the phenolic constituents and to a group of saponins related to glycyrrhizic acid were performed using LC-ESI/MS. For the saponin constituents, full scan LC-MS/MS fragmentation of the protonated (positive ion mode) or deprotonated (negative ion mode) molecular species generated diagnostic fragment ions that provided information concerning the triterpene skeleton and the number and nature of the substituents. On the basis of the specific fragmentation of glycyrrhizic acid, an LC-MS/MS method was developed in order to quantify the analyte in the liquorice root samples. Chinese G. glabra roots contained the highest levels of glycyrrhizic acid, followed by those from Italy (Calabria).     

Qualitative and quantitative characterization of chemical constituents in Xin-Ke-Shu preparations by liquid chromatography coupled with a LTQ Orbitrap mass spectrometer

Xin-Ke-Shu (XKS), a traditional Chinese medicine (TCM) preparation containing five herbal medicines, has been commonly used for the treatment of coronary heart disease in China. However, the chemical constituents in XKS have not been clarified yet. In order to quickly define the chemical profiles and control the quality of XKS preparations, liquid chromatography coupled with electrospray ionization hybrid linear trap quadrupole orbitrap (LC-LTQ-Orbitrap) mass spectrometry was applied for simultaneous identification and quantification of multi-constituent. A total of 51 compounds, including phenolic acids, isoflavone-C-glycosides, isoflavone-O-glycosides, flavonoids, and triterpenoid saponins, were identified or tentatively deduced on the base of their retention behaviors, MS and MS(n) data, or by comparing with reference substances and literatures. In addition, an optimized LC-ESI-MS method was established for quantitative determination of 15 marker compounds in XKS preparations from 7 independent pharmaceutical companies. The validation of the method, including spike recoveries, linearity, sensitivity (LOD and LOQ), precision, and repeatability, was carried out and demonstrated to be satisfied the requirements of quantitative analysis. This is the first report on the comprehensive determination of chemical constituents in XKS preparations by LC-LTQ-Orbitrap mass spectrometry. The results suggested that the established methods would be a powerful and reliable analytical tool for the characterization of multi-constituent in complex chemical system and quality control of TCM preparations.     

Development and validation of a highly sensitive UPLC-MS/MS method for simultaneous determination of aconitine,mesaconitine, hypaconitine,and five of their metabolites in rat blood and its application to a pharmacokinetics study of aconitine,mesaconitine, and hypaconitine

1. A rapid, specific and sensitive method was developed for the simultaneous determination of eight Aconitum alkaloids: aconitine (AC), mesaconitine (MA), hypaconitine (HA), benzoylaconine (BAC), benzoylmesaconine (BMA), benzoylhypaconine (BHA), aconine and mesaconine in rat blood by ultra-performance liquid chromatography coupled tandem mass spectrometry (UPLC-MS/MS).

2. The UPLC-MS/MS system coupled with an electrospray ionization (ESI) source was operated in a positive mode via multiple-reaction monitoring (MRM). Samples were treated with methanol to remove protein prior to analysis by UPLC-MS/MS. The analytes were separated with a Waters C18 column (1.7 µm, 50?×?2.1?mm) and a gradient elution using acetonitrile and 0.1% formic acid-water as the mobile phases.

3. The linear response range was from 0.125 to 1000 nmol/L for these eight alkaloids and the correlation coefficients (r² values) were all higher than 0.997. The method was validated with respect to precision, accuracy, recovery, matrix effect, carryover effect and sample stability, and found to be within the acceptable limits.

4. The developed and validated method was successfully applied to simultaneously determine the eight Aconitum alkaloids in rats blood after intravenous administration of a mixture of AC, MA and HA.

     

Effects of 2-amino-9H-pyrido[2,3-b]indole (AαC) metabolic bio-activation on oxidative DNA damage in human hepatoma G2 (HepG2) cells

2-Amino-9H-pyrido[2,3-b]indole (AαC), which is a hazardous compound present in cigarette smoke, has been listed as probable human carcinogens (Group 2B). The carcinogenicity and genotoxicity of AαC were activated by the process of metabolic bio-activation. Whereas, few studies about genotoxicity induced by AαC have been reported. In this study, we took HepG2 cells as the model to investigate the relationship between oxidative DNA damage induced by AαC and metabolic bio-activation of AαC, which is of importance to unveil the mechanism of AαC genotoxicity. Firstly, the HepG2 cells were treated with 10 and 20?μg/mL AαC, respectively. Then different concentrations of protein ranging from 0 to 1?mg/mL in S9 mixture solution were utilized to make cells have different capacities for metabolic activation. Intracellular AαC hydroxylated metabolites and 8-OHdG were estimated by using ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). The results showed that, at the same concentration of AαC, with the increment of concentration of protein in S9 mixture solution, the levels of hydroxylated metabolites and 8-OHdG/10⁶dG increased. And at the same concentration of protein in S9 mixture solution, with the increment of concentration of AαC, the levels of hydroxylated metabolites and 8-OHdG/10⁶dG increased. The hydroxylated metabolites and 8-OHdG were positively related by correlation analysis. In addition, the correlation coefficients of N-OH-AαC and 8-OHdG were maximum (R²?=?0.73 and 0.66). Taken together, these results indicated that the metabolic bio-activation of AαC might result in oxidative DNA damage.