Analysis on Iridoid Glycosides in Crude and Processed Extracts from Cornus Officinals by Liquid Chromatography-electrospray Ionization Mass Spectrometry

The iridoid glycosides in crude and processed extracts from cornus officinals have been analyzed by high performance liquid chromatography-electrospray ionization mass spectrometry. Samples were analyzed by a reversed-phase C18 column using a binary eluent under gradient conditions. Seven iridoid glycosides could be separated and detected. The [M-H](-) ions of iridoid glycosides in the negative ion mode were observed, which reflect their molecule mass information. An in-source collision induced dissociation (in-source CID) experiment was carried out in order to identify the structures and to measure the contents of iridoid glycosides. The epimers were discovered in the experiment for the first time, namely 7 alpha-O-ethyl-morroniside and 7 beta-O-ethylmorroniside.

Pressurized electrochromatography coupled with electrospray ionization mass spectrometry for analysis of peptides and proteins

Pressurized capillary electrochromatography (pCEC) was coupled with electrospray ionization mass spectrometry (ESI-MS) using a coaxial sheath liquid interface. It was used for separation and analysis of peptides and proteins. The effects of organic modifier and applied voltage on separation were investigated, and the effects of pH value of the mobile phase and the concentration of the electrolyte on ESI-MS signal were investigated. The resolution and detection sensitivity with different separation methods (pCEC, capillary high-performance liquid chromatography) coupled on-line with mass spectrometry were compared for the separation of a peptide mixture. To evaluate the feasibility and reliability of the experimental setup of the system, tryptic digests of cytochrome c and modified protein as real samples were analyzed by using pCEC-ESI-MS.

几种中药材的化学成分及其定性定量检测方法研究

本论文由四部分组成。第一部分报道了佛手参提取物的化学成分研究，建立了活性成分含量测定的高效液相测定和指纹图谱研究，采用液质联用技术鉴定了主要色谱峰；第二部分报道了丹参及其复方制剂的特征图谱研究；第三部分探讨了两面针生物碱的电喷雾质谱裂解规律，并采用液质联用技术分离鉴定了提取物中的多种生物碱。第四部分概述了液质联用在药物代谢研究中的运用。 第一部分包括第一、第二和第三章。第一章针对佛手参（Gymnadeniaconopsea）块茎的甲醇提取物，采用大孔树脂和反相硅胶柱层析等各种分离方法，共分离鉴定出4 个化合物，通过波谱分析将它们的结构确定为dactylorhin B (1)、loroglossin (2)、dactylorhin A (3)和militarine (4)。这4 个化合物均是首次从佛手参中分离得到的琥珀酸葡萄糖苷类成分。第二章采用高效液相色谱法对西藏、四川、河北、青海和尼泊尔等不同地区产的十个佛手参样品进行腺嘌呤核苷和对羟基苯甲醇的定量分析，结果表明这2 个成份可视为佛手参的特征成分，但也注意到产地不同该2 个特征成分的含量也有所不同。第三章采用标准中药指纹图谱相似度计算软件，以10 个佛手参样品HPLC 图谱的平均值为相似性评价对照模板，对10 个样品进行了相似度评价，并经液质联用分析指认了7 个共有峰，分别为腺嘌呤核苷(1)、对羟基苯甲醇(2)、对羟基苯甲醛(3) 、dactylorhin B(4) 、loroglossin(5)、dactylorhin A(6)和militarine(7)。 第二部分包括第四、第五、第六和第七章。第四章运用电喷雾质谱检测了对照药材和五个不同产地的丹参药材中脂溶性和水溶性成分，系统地探讨了多种成分的电喷雾质谱规律，并以对照药材为标准建立了特征指纹图谱。五个产地的药II材通过与对照药材相对比，采用聚类分析的方法，得到了定性的鉴别与判断。并采用液质联用技术对丹参药材提取液中的化学成份进行分析，推测了九个特征峰，并对六样品的液相色谱图进行了聚类分析。第五章探讨了三七皂苷的电喷雾质谱电离和裂解规律，并采用电喷雾质谱法对三七标准药材，血通片中的皂苷成分进行了分析。第六章运用电喷雾质谱研究复方丹参片提取液的特征图谱，并和单味药材丹参和三七的特征图谱进行了对比研究。并运用HPLC-ESI MSn 分析鉴定了复方丹参片提取液中的化学成分，推测了12 个色谱峰。第七章总结了电喷雾质谱和液质联用技术在丹参药材，三七药材及复方丹参制剂中的运用的优势和局限性。 第三部分（第八章）研究了两面针生物碱中二氢白屈菜红碱(1)、二氢两面针碱(2)、8-酮基二氢白屈菜红碱(3)、8-丙酮基二氢两面针碱(4)、两面针碱(5)、和1,3-二(8-二氢两面针碱)丙酮(6)等六个苯并菲啶型生物碱的电喷雾质谱裂解规律，其中二氢两面针碱和二氢白屈菜红碱，8-丙酮基二氢两面针碱和8-酮基二氢白屈菜红碱是两对二个甲氧基分别在C-9 和C-10，C-10 和C-11 的同分异构体。实验结果表明，在相同的碰撞能下，这类位置异构体的ESI MS2 质谱二级碎片离子的相对峰度存在很大差异，这可以用于区分该类同分异构体，采用液-质联用可以对两面针的总生物碱提取物中的这些同分异构体加于区分。同时在本实验采用的液相色谱条件下，多种生物碱得到较好的分离，通过和对照品的保留时间，紫外吸收光谱及电喷雾质谱图对照，鉴定了11 个主要色谱峰。 第四部分（第九章）对液质联用技术在药物代谢中的运用进行了综述。 This dissertation consisted of four sections. The first two sections elaborated thephytochemical investigation of the rhizomes Gymnadenia conopsea R. Br., methoddevelopment for rapid identifying and qutifying the chemical condtituent of thistibetant medicine, and the chemical fingerprint analysis of rhizomes of G. conopsea,Salviae miltiorrhiza and P. notoginseng. The third section studied the fragmentationmechanism of six alkaloids from Zanthoxylum nitidium and method development forrapid identifying varieties of alkaloids from the extract of this herbal medicine. Thefourth section reviewed HPLC- MS method in drug metabolism studies. The first section consisted of chapters 1, 2, 3. Chapter 1 elaborated the phytochemicalinvestigation of Gymnadenia conopsea R. Br. Four succinate derivative esters wereisolated from the methanol extract of the rhizomes of G. conopsea through repeatedcolumn chromatography on normal and reversed phase silica gel, their structures weredetermined by ESI-MS, 1D and 2D NMR evidence. They were firstly discoveredfrom this species. In chapter 2, a high-performance liquid chromatography.diodearray detection (HPLC-DAD) method has been firstly developed for quantitation oftwo characteristic constituents, adenosine and 4-hydroxybenzyl alcohol, from theextract of rhizomes of G. conopsea. All 10 samples of G. conopsea contained differentamount of adenosine and 4-hydroxybenzyl alcohol. Adenosine and the4-hydroxybenzyl alcohol can be applied in identification and quality control for theroots of G. conopsea. In chapter 3, a high-performance liquid chromatography.diodearray detection.tandem mass spectrometry (HPLC-DAD-MSn) method has been firstly developed for chemical fingerprint analysis of rhizomes of G. conopsea andrapid identification of major compounds in the fingerprints. Comparing the UV andMS spectra with those of authentic compounds, seven main peaks in the fingerprintswere identified as adenosine, 4-hydroxybenzyl alcohol, 4-hydroxybenzyl aldehyde,dactylorhin B, loroglossin, dactylorhin A and militarine. The Computer AidedSimilarity Evaluation System for Chromatographic Fingerprint of TraditionalChinese Medicine (CASES) was employed to evaluate the similarities of 10 samplesof the rhizomes of G. conopsea collected from Sichuan, Qinghai and Hebei provincesand Tibet autonomous region of China, and Nepal. These samples from differentsources had similar chemical fingerprints to each other. The second section consisted of chapters 4, 5, 6 and 7. In chapter 4，both thecharacteristic spectra of liposoluble tanshinones and aqueous-soluble salvianolic acidswere established by the electrospray ionization mass spectrometry (ESI MS)technique and the differences between standard and crude rhizomes of Salviaemiltiorrhiza Bge. from 5 sources were analyzed. The law of electrospray ion trap mass(ESI ITMS) of typical tanshinones and salvianolic acids is studied.The analysis of the chemical constituent of rhizomes of Salviae miltiorrhiza Bge. byliquid chromatography coupled with mass spectrum (LC/MS) technique wasestablished，and the distances among standard herb and crude herb from 5 sourceswere calculated by clustering analysis. According the DAD spectra and MS2 data，9tanshinones could be speculated. In chapter 5, the character spectra of total saponinsin P. notoginseng extracts were established by ESI ITMS and selective ion monitoring(SIM) technology. The law of notoginsenosides by ESI MS2 was studied. In chapter 6,the characteristic spectra of Compound Danshen Tablet established and compared byESI-MS and HPLC/DAD/MS, 6 known tanshinones and 3 saponins were speculated.In chapter 7, the advantage and disadvantage of the strategy, using the ESI ITMS andLC/MS techniques for study of characteristic spetra of danshen and Compound Danshen Tablet, were summerized. The third section (chapter 8) studied the fragmentation mechanism of six alkaloids,dihydronitidine, dihydrochelerythrine, 8-acetonyl dihydronitidine,8-acetonyldrochelerythrine, nitidine and 1,3-bis (8-dihydronitidinyl)-acetone, by ESIMSn. Tandem mass spectrometry experiments indicated that different substitutionsites of the methoxyl groups at C-9 and C-10 or at C-10 and C-11 determined thedifferent abundances of the MS2 fragmentation ions using the same collision energy.According to the different abundances of MS2 product ions, positional isomericbenzo[c] phenanthridine alkaloids can be differentiated. Moreover, ten constituents inthe crude alkaloids extract from the roots of Zanthoxylum nitidium were rapidlyidentified by high-performance liquid chromatography coupled with tandem massspectrometry (HPLC-MSn), through comparing the retention times and ESI MSn spectra with the authentic standards. The fourth section (chapter 9) is a review on HPLC-MS method development in drug metabolism studies.

中国沿海典型区域织纹螺毒性及毒素成分研究

织纹螺（Nassarius spp.）味道鲜美，是中国及其它一些亚洲国家沿海地区居民习惯食用的一种水产品。但是，近几十年来，中国沿海频繁发生食用织纹螺中毒事件，严重威胁着人们的身体健康和生命安全。加之人们对织纹螺体内的毒素成分、来源及其毒性变化规律还没有清晰的认识，因此难以有效预防和控制食用织纹螺引起的中毒事件。本文根据文献报道，在中国沿海食用织纹螺中毒事件多发的典型区域，包括江苏省的连云港市和盐城市、浙江省的舟山市和宁波市、福建省的宁德市、厦门市和莆田市设立了监测点，于2006年和2007年间进行了连续采样，应用小鼠生物测试法调查了织纹螺毒性的消长情况，并利用高效液相色谱-质谱联用（Liquid Chromatography-Mass Spectrometry，LC-MS）和高效液相色谱技术（High Performance Liquid Chromatography，HPLC）对织纹螺体内的毒素成分进行了分析。 实验结果表明，2006年于江苏省盐城市射阳海域采集的织纹螺样品中，阳性样品检出率为56%，毒性在2-5 MU/g组织（湿重）之间变化，在2007年于同地采集的8个样品中，除一个样品毒性为3.14 MU/g组织（湿重）以外，其余样品均表现为阴性；而2007年采集自连云港市赣榆海域的织纹螺样品，在采样期间则呈现出极高的毒性，最高达到846.52 MU/g 组织（湿重），毒性在监测期间呈“M”状波动，在5月和7月下旬出现两个毒性高峰。2006年于浙江省宁波市象山港采集的织纹螺样品中，阳性样品检出率为25%，毒性均在2.5 MU/g组织（湿重）左右；而同年采集自舟山市定海的织纹螺样品中，阳性样品检出率为100%，最高毒性达18.40 MU/g组织（湿重），毒性在监测期间也呈“M”状波动，高峰期出现在6月初和7月底。2006年3-9月采集自福建省宁德霞浦、厦门同安和莆田涵江采集的织纹螺样品中，阳性样品检出率分别为20%、43%和14%，除7月中旬采集自宁德霞浦的一个样品毒性达到16.19 MU/g组织（湿重）之外，其余样品毒性均在2-5 MU/g组织（湿重）间波动。从阳性样品的时间分布规律来看，3月份和6、7月份是阳性样品集中出现的时期。根据以上调查结果可以看出，织纹螺的毒性消长呈现出较明显的地域性和季节性特征，不同地区的织纹螺毒性存在差异，而同一区域织纹螺毒性的消长则表现出明显的季节性集中趋势。除了2007年采集自连云港赣榆的织纹螺样品毒性与其平均个体组织重量有相似的变化趋势以外，其余地区的织纹螺样品毒性和个体大小无明显相关性。 利用LC-MS和HPLC技术对织纹螺样品中的毒素成分进行了分析，确定河豚毒素（tetrodotoxin, TTX）及其同系物（trideoxyTTX，4-epi-TTX，anhydroTTX，oxoTTX）是所采集织纹螺中的主要致毒成分，样品中没有检测到麻痹性贝毒毒素（Paralytic Shellfish Poison, PSP）。自不同地区采集的织纹螺中毒素成分基本一致，但组成存在一定差异。其中，采自江苏省连云港赣榆和浙江省舟山定海的织纹螺样品中，trideoxyTTX是主要的成分，其次是TTX；而从其它采样地点采集的织纹螺中，TTX都是主要的毒素成分，其次才是trideoxyTTX及其它同系物。对采集自江苏省连云港赣榆和浙江舟山定海的织纹螺体内毒素的解剖学分布进行了分析，结果表明肌肉、消化腺和剩余部分中的毒素组成基本一致，其中trideoxyTTX是主要的毒素成分，其次为TTX，但采自浙江舟山的织纹螺剩余部分中的TTX是主要的毒素成分。在监测期间，各组织中的毒素组成没有明显变化，但毒素含量随季节变化表现出了一定的差异。 综上所述，在中国沿海典型区域开展的织纹螺毒性调查结果表明其毒性消长具有一定的地域性和季节性特征。分析结果显示织纹螺体内的毒素成分是河豚毒素及其同系物，采自不同区域的织纹螺体内毒素成分基本一致，但毒素组成稍有差异。对织纹螺中毒素的解剖学分布研究显示，各组织中的毒素含量随季节变化而表现出一定差异，但毒素组成没有明显的季节性变化。这些结果显示中国沿海的织纹螺应具有相似的毒素来源，研究结果将为相关部门有效监测、预防和控制食用织纹螺中毒事件提供有力的科学依据。

Analysis of carbohydrates in a tibetan medicine using new labeling reagent, 1-(2-naphthyl)-3-methyl-5-pyrazolone, by HPLC with DAD detection and ESI-MS identification

A new labeling reagent, 1-(2-naphthyl)-3-methyl-5-pyrazolone (NMP), coupling with liquid chromatography (LC) with electrospray ionization mass spectrometry (ESI-MS) for the detection of carbohydrates from a famous Tibetan medicine is reported. Carbohydrates were derivatized to their bis-NMP-labeled derivatives. The method, in conjunction with a gradient elution, offered a baseline resolution of carbohydrate derivatives on a reversed phase Hypersil ODS-2 column. The carbohydrates such as mannose, galacturonic acid, glucuronic acid, rhamnose, glucose, galactose, xylose, arabinose, and fucose could be successfully detected by UV and ESI-MS. Derivatives showed intense protonated molecular ion at m/z [M+H]+ in positive ion mode. The mass to charge ratios of characteristic fragment ions at m/z 473.0 could be used for the accurately qualitative identification of carbohydrates; this characteristic fragment ion was from the cleavage of C2-C3 bond in the carbohydrate chain giving the specific fragment ions at m/z [MH-CmH2m+1Om-H2O](+) for pentose, hexose, and glyceraldehydes, and at m/z [MH-CmH2m-1Om+1-H2O](+) for alduronic acids, such as galacturonic acid and glucuronic acid (m=n-2, n is carbon atom number of carbohydrate). Compared with the traditional 1-phenyl-3-methyl-5-pyrazolone (PMP) reagent, currently synthesized NMP show the advantage of higher sensitivity to carbohydrate compounds with UV and ESI-MS detection.

Reversed-phase high-performance liquid chromatographic investigation of urinary normal and modified nucleosides of cancer patients

Post-transcriptional modifications in RNA give rise to free modified ribonucleosides circulating in the blood stream and excreted in urine. Due to their abnormal levels in conjunction with several tumor diseases, they have been suggested as possible tumor markers. The developed RP-HPLC method has been applied to analyze the urinary nucleosides in 34 urinary samples from 15 kinds of cancer patients. The statistical analyses showed the urinary nucleoside excretion, especially modified nucleoside levels, in cancer patients were significantly higher than those in normal healthy volunteers. Factor analysis was used to classify the patients with cancer and normal healthy humans. It was found that using 15 urinary nucleoside levels or only five modified nucleoside levels as data vectors the factor analysis plot displayed two almost separate clusters representing each group. (C) 1999 Elsevier Science B.V. All rights reserved.

Quantitative determination of compounds in tobacco essential oils by comprehensive two-dimensional gas chromatography coupled to time-of-flight mass spectrometry

A quantitative analysis of the individual compounds in tobacco essential oils is performed by comprehensive two-dimensional gas chromatography (GC x GC) combined with flame ionization detector (FID). A time-of-flight mass spectrometer (TOF/MS) was coupled to GC x GC for the identification of the resolved peaks. The response of a flame ionization detector to different compound classes was calibrated using multiple internal standards. In total, 172 compounds were identified with good match and 61 compounds with high probability value were reliably quantified. For comparative purposes, the essential oil sample was also quantified by one-dimensional gas chromatography-mass spectrometry (GC/MS) with multiple internal standards method. The results showed that there was close agreement between the two analysis methods when the peak purity and match quality in one-dimensional GC/MS are high enough. (c) 2005 Elsevier B.V. All rights reserved.

Characterization of complex hydrocarbons in cigarette smoke condensate by gas chromatography-mass spectrometry and comprehensive two-dimensional gas chromatography-time-of-flight mass spectrometry

Gas chromatography-mass spectrometry with electron ionization and positive-ion chemical ionization and comprehensive two-dimensional gas chromatography-time-of-flight mass spectrometry (GC x GC-TOF-MS) were applied for the characterization of the chemical composition of complex hydrocarbons in the non-polar neutral fraction of cigarette smoke condensates. Automated data processing by TOF-MS software combined with structured chromatograms and manual review of library hits were used to assign the components from GC x GC-TOF-MS analysis. The distributions of aliphatic hydrocarbons and aromatics were also investigated. Over 100 isoprenoid hydrocarbons were detected, including carotene degradation products, phytadiene isomers and carbocyclic diterpenoids. A total of 1800 hydrocarbons were tentatively identified, including aliphatic hydrocarbons, aromatics, and isoprenoid hydrocarbons. The identified hydrocarbons by GC x GC-TOF-MS were far more than those by GC-MS. (C) 2004 Elsevier B.V. All rights reserved.

Preparation and characterization of long methacrylate monolithic column for capillary liquid chromatography

Long methacrylate monolithic columns (100 cm x 320 mum i.d.) were prepared from silanized fused-silica capillaries of 320 mum i.d. by in situ copolymerization of butyl methacrylate (BMA) with ethylene dimethacrylate (EDMA) in the presence of a suitable porogen. The separation performance and selectivity of the column were evaluated and compared with a 25 cm x 320 mum i.d. column prepared in the same way by capillary high-performance liquid chromatography (mu-HPLC) The results showed that the 1 m long monolithic column can generate 33 x 10(3) plate number and exhibited good permeability, higher sample loadability, and separation capability. (C) 2004 Elsevier B.V. All rights reserved.

Synthesis of a silica-bonded bovine serum albumin s-triazine chiral stationary phase for high-performance liquid chromatographic resolution of enantiomers

A novel method of synthesizing protein chiral stationary phase (protein-CSP) is proposed with 2,4,6-trichloro-1,3,5-triazine as the activator. The bovine serum albumin (BSA) based chiral columns (150x4.6 mm I.D.) were prepared successfully within 8 h. With tryptophan as the probe solute, it was observed that the BSA immobilized by this method had a better ability to distinguish enantiomers than that activated by glutaric dialdehyde. This may be due to the well-maintained BSA conformation and the larger amount of BSA immobilized on the silica gel. The BSA-CSP prepared by this method was relatively stable under experimental conditions, and the resolution of 13 chiral compounds was achieved. The coupling reaction in this method is mild, reliable and reproducible; it is also suitable for the immobilization of various biopolymers in the preparation of bioreactor, biosensor and affinity chromatography columns. (C) 2000 Elsevier Science B.V. All rights reserved.

Rapid quantification of total microcystins in cyanobacterial samples by periodate-permanganate oxidation and reversed-phase liquid chromatography

Microcystins (MCs) comprise a family of more than 80 related cyclic hepatotoxic heptapeptides. Oxidation of MCs causes cleavage of the chemically unique C-20 beta-amino acid (2S, 3S, 8S, 9S)-3-amino-9-methoxy-2,6,8-trimethyl-10-phenyldeca-4,6-dienoic acid (Adda) amino to form 2-methyl-3-methoxy-4-phenylbutanoic acid (MMPB), which has been exploited to enable analysis of the entire family. In the present study, the reaction conditions (e.g. concentration of the reactants. temperature and pH) used in the production of MMPB by oxidation of cyanobacterial samples with permanganate-periodate were optimized through a series of well-controlled batch experiments. The oxidation product (MMPB) was then directly analyzed by high-performance liquid chromatography with diode array detection. The results of this study provided insight into the influence of reaction conditions on the yield of MMPB. Specifically, the optimal conditions, including a high dose of permanganate (>= 50 mM) in saturated periodate solution at ambient temperature under alkaline conditions (pH similar to 9) over 1-4 h were proposed, as indicated by a MMPB yield of greater than 85%. The technique developed here was applied to determine the total concentration of MCs in cyanobacterial bloom samples, and indicated that the MMPB technique was a highly sensitive and accurate method of quantifying total MCs. Additionally, these results will aid in development of a highly effective analytical method for detection of MMPB as an oxidation product for evaluation of total MCs in a wide range of environmental sample matrices, including natural waters, soils (sediments) and animal tissues. (C) 2009 Elsevier B.V. All rights reserved.