Simultaneous determination of microcystin-LR and its glutathione conjugate in fish tissues by liquid chromatography-tandem mass spectrometry

A sensitive and selective liquid chromatography-tandem mass spectrometry method was developed and validated for the simultaneous quantitative determination of microcystin-LR (MC-LR) and its glutathione conjugate (MC-LR-GSH) in fish tissues. The analytes were extracted from fish liver and kidney using 0.01 M EDTA-Na-2-5% acetic acid, followed by a solid-phase extraction (SPE) on Oasis HLB and silica cartridges. High-performance liquid chromatography (HPLC) with electrospray ionization mass spectrometry, operating in selected reaction monitoring (SRM) mode, was used to quantify MC-LR and its glutathione conjugate in fish liver and kidney. Recoveries of analytes were assessed at three concentrations (0.2, 1.0, and 5 mu g g(-1) dry weight [DW]) and ranged from 91 to 103% for MC-LR, and from 65.0 to 75.7% for MC-LR-GSH. The assay was linear within the range from 0.02 to 5.0 mu g g(-1) DW, with a limit of quantification (LOQ) of 0.02 mu g g(-1) DW. The limit of detection (LOD) of the method was 0.007 mu g g(-1) DW in both fish liver and kidney. The overall precision was determined on three different days. The values for within- and between-day precision in liver and kidney were within 15%. This method was applied to the identification and quantification of MC-LR and its glutathione conjugate in liver and kidney of fish with acute exposure of MC-LR. (c) 2007 Elsevier B.V. All rights reserved.

Biochemical and ultrastructural changes of the liver and kidney of the phytoplanktivorous silver carp feeding naturally on toxic Microcystis blooms in Taihu Lake, China

Many experimental studies have documented the impact of microcystins (MC) on fish based on either intraperitoneal injection, or oral gavaging via the diet, but few experiments were conducted by MC exposure through natural food uptake in lakes. In this study, the phytoplanktivorous silver carp were stocked in a large pen set in Meiliang Bay of Taihu Lake where toxic Microcystis blooms occurred in the warm seasons. Fish samples were collected monthly and MC concentrations in liver and kidney of the fish were determined by LC-MS. The maximum MC concentrations in liver and kidney were present in July when damages in ultrastructures of the liver and kidney were revealed by electron microscope. In comparison with previous studies on common carp, silver carp showed less damage and presence of lysosome proliferation in liver and kidney. Silver carp might eliminate or lessen cell damage caused by MC through lysosome activation. Recovery in the ultrastructures of liver and kidney after Microcystis blooms was companied with a significant decrease or even disappearance of MC. Catalase and glutathione S-transferase in liver and kidney of silver carp during Microcystis blooms were significantly higher than before and after Microcystis blooms. The high glutathione pool in liver and kidney of silver carp suggests their high resistance to MC exposure. The efficient antioxidant defence may be an important mechanism of phytoplanktivorous fish like silver carp to counteract toxic Microcystis blooms. (C) 2007 Published by Elsevier Ltd.

In situ studies on the distribution patterns and dynamics of microcystins in a biomanipulation fish - bighead carp (Aristichthys nobilis)

The distribution and dynamics of microcystins in various organs of the phytoplanktivorous bighead carp were studied monthly in Lake Taihu, which is dominated by toxic cyanobacteria. There was a good agreement between LC-MS and HPLC-UV determinations. Average recoveries of spiked fish samples were 63% for MC-RR and 71% for MC-LR. The highest MC contents in intestine, liver, kidney and spleen were 85.67, 2.83, 1.70 and 1.57 mu g g(-1) DW, respectively. MCs were much higher in mid-gut walls (1.22 mu g g(-1) DW) than in hind- and fore-gut walls (0.31 and 0.18 mu g g(-1) DW, respectively), suggesting the importance of mid-gut wall as major site for MC absorption. A cysteine conjugate of MC-LR was detected frequently in kidney. Among the muscle samples analyzed, 25% were above the provisional tolerable daily intake level by WHO. Bighead is strongly resistant to microcystins and can be used as biomanipulation fish to counteract cyanotoxin contamination in eutrophic waters. (c) 2006 Elsevier Ltd. All rights reserved.

Relationships between microcystins and environmental parameters in 30 subtropical shallow lakes along the Yangtze River, China

1. A survey of 30 subtropical shallow lakes in the middle and lower reaches of the Yangtze River area in China was conducted during July-September in 2003-2004 to study how environmental and biological variables were associated with the concentration of the cyanobacterial toxin microcystin (MC). 2. Mean MC concentration in seasonally river-connected lakes (SL) was nearly 33 times that in permanently river-connected lakes (RL), and more than six times that in city lakes (NC) and non-urban lakes (NE) which were not connected to the Yangtze River. The highest MC (8.574 mu g L-1) was detected in Dianshan Lake. 3. MC-RR and MC-LR were the primary toxin variants in our data. MC-RR, MC-YR and MC-LR were significantly correlated with Ch1 a, biomass of cyanobacteria, Microcystis and Anabaena, indicating that microcystins were mainly produced by Microcystis and Anabaena sp. in these lakes. 4. Nonlinear interval maxima regression indicated that the relationships of Secchi depth, total nitrogen (TN) : total phosphorus UP) and NH4+ with MC were characterised by negative exponential curves. The relationships between MC and TN, TP, NO3- + NO2- were fitted well with a unimodal curve. 5. Multivariate analyses by principal component and classifying analysis indicated that MC was mainly affected by Microcystis among the biological factors, and was closely related with temperature among physicochernical factors.

In situ studies on the bioaccumulation of microcystins in the phytoplanktivorous silver carp (Hypophthalmichthys molitrix) stocked in Lake Taihu with dense toxic Microcystis blooms

The phytoplanktivorous silver carp is an important biomanipulation fish to control cyanobacterial blooms and is also a food fish with the greatest production in China. The accumulation of the hepatotoxic microcystins (MCs) determined by LC-MS in various organs of silver carp was studied monthly in Lake Taihu dominated by toxic Microcystis aeruginosa. Average recoveries of spiked fish samples were 78% for MC-RR and 81% for MC-LR. The highest content of MCs was found in the intestine (97.48 mu g g(-1) DW), followed by liver (6.84 mu g g(-1) DW), kidney (4.8 8 mu g g(-1) DW) and blood (1.54 mu g g(-1) DW), and the annual mean MC content was in the order of intestine > liver > kidney > blood > muscle > spleen > gallbladder > gill. Silver carp could effectively ingest toxic Microcystis cells (up to 84.4% of total phytoplankton in gut contents), but showed fast growth (from 141 g to 1759 g in I year in mean weight). Silver carp accumulated less microcystins in liver than other animals in the same site or other fish from different water bodies at similar level of toxin ingestion. There was possible inhibition of the transportation of the most toxic MC-LR across the gutwall. Muscle of silver carp in Lake Taihu should not be consumed during period of dense Microcystis blooms while viscera were risky for consumption in more months. (c) 2006 Elsevier B.V. All rights reserved.

First report of aphantoxins in China - waterblooms of toxigenic Aphanizomenon flos-aquae in Lake Dianchi

The oligohaline cyanobacterium Aphanizomenon flos-aquae (L.) Ralfs (A. flos-aquae) has been reported in several countries to produce paralytic shellfish poisons (PSPs) or protracted toxic effects. In the past years, A. flos-aquae blooms have occurred annually in the eutrophic Lake Dianchi (300 km(2) in area, located in southwestern China). Material from natural blooms dominated by A. flosaquae was collected and lyophilized. Acute toxicity testing was performed by mouse bioassay using extracts from the lyophilized material. Clear symptoms of PSPs, intoxications were observed. To confirm the production of PSPs, a strain of A. flos-aquae (DC-1) was isolated and maintained in culture. Histopathological effects were studied by examining the organ damages using transmission electron microscopy (TEM). Slight hepatocytic damage with swollen mitochondria was found. The ultrastructural pulmonary lesions were characterized by distortied nuclei and indenting of karyotheca, together with degeneration and tumefaction of mitochondria and endoplasmic reticulum. Control animals injected with acetic acid did not exhibit histopathological damage in any organ. Toxic effects of cultured algal cells on enzymatic systems in the mouse were studied using sublethal doses of extracts. Significant glutathione-S-transferase (GST) and lactate dehydrogenase (LDH) increases, together with decrease of the glutathione (GSH) level, were measured. These results indicated a potential role of PSPs intoxicating and metabolizing in the test animals. HPLC-FLD and LC/MS analysis of extracts from cultured material demonstrated the PSP toxins produced by A. flos-aquae bloom. To the best of our knowledge, this is the first study reporting chemically and toxicologically confirmed PSP toxins related to A. flosaquae in China. (c) 2005 Elsevier Inc. All rights reserved.

Seasonal dynamics of the hepatotoxic microcystins in various organs of four freshwater bivalves from the large eutrophic Lake Taihu of subtropical China and the risk to human consumption

So far, little is known on the distribution of hepatotoxic microcystin (MC) in various organs of bivalves, and there is no study on MC accumulation in bivalves from Chinese waters. Distribution pattern and seasonal dynamics of MC-LR, -YR and -RR in various organs (hepatopancreas, intestine, visceral mass, gill, foot, and rest) of four edible freshwater mussels (Anodonta woodiana, Hyriopsis cumingii, Cristaria plicata, and Lamprotula leai) were studied monthly during Oct. 2003-Sep. 2004 in Lake Taihu with toxic cyanobacterial blooms in the summer. Qualitative and quantitative determinations of MCs in the organs were done by LC-MS and HPLC. The major toxins were present in the hepatopancreas (45.5-55.4%), followed by visceral mass with substantial amount of gonad (27.6-35.5%), whereas gill and foot were the least (1.8-5.1%). The maximum MC contents in the hepatopancreas, intestine, visceral mass, gill, foot, and rest were 38.48, 20.65, 1.70, 0.64, 0.58, and 0.61 mu g/g DW, respectively. There were rather good positive correlation in MC contents between intestines and hepatopancreas of the four bivalves (r = 0.75-0.97, p < 0.05). There appeared to be positive correlations between the maximum MC content in the hepatopancreas and the delta(13)C (r = 0.919) or delta(15)N (r = 0.878) of the foot, indicating that the different MC content in the hepatopancreas might be due to different food ingestion. A glutathione (GSH) conjugate of MC-LR was also detected in the foot sample of C. plicata. Among the foot samples analyzed, 54% were above the provisional WHO tolerable daily intake (TDI) level, and the mean daily intakes from the four bivalves were 8-23.5 times the TDI value when the bivalves are eaten as a whole, suggesting the high risk of consuming bivalves in Lake Taihu. (C) 2005 Wiley Periodicals, Inc.

In vivo studies on toxin accumulation in liver and ultrastructural changes of hepatocytes of the phytoplanktivorous bighead carp i.p.-injected with extracted microcystins

Phytoplanktivorous bighead carp were injected i.p. with extracted microcystins (mainly MC-RR and -LR) at two doses, 200 and 500 MC-LReq. mu g kg(-1) bw, and the changes in extractable MCs in liver and in the ultrastructure of hepatocytes were studied at 1, 3, 12, 24 and 48 h after injection. Quantitative and qualitative determinations of MCs in the liver were conducted by HPLC and LC-MS, respectively. MC concentration in the liver reached the maxima at 12 It (2.89 mu g MCs g(-1) dry weight at the lower dose) or at 3 h (5.43 mu g MCs g(-1) dry weight at the higher dose) post-injection, followed by sharp declines afterwards, whereas the ultrastructural changes of hepatocytes in both dose groups suggest progressive increases in severity toward the directions of apoptosis and necrosis from I to 24 h, respectively. There were two new findings in fish: widening of intercellular spaces was among the early ultrastructural changes induced by MCs and ultrastructural recovery of hepatocytes was evident at 48 h post-injection in both dose groups. Both the present and previous studies suggest that with in vivo or in vitro exposure to microcystins, hepatocyte damage in fish tends to proceed toward the direction of apoptosis at lower MC concentrations but toward the direction of necrosis at high MC concentrations. The temporal dynamics of MCs in the liver suggest that bighead carp may have a mechanism to degrade or bind MC-LR actively after it enters the blood system. (c) 2005 Elsevier Ltd. All rights reserved.

Tissue distributions and seasonal dynamics of the hepatotoxic microcystins-LR and -RR in a freshwater snail (Bellamya aeruginosa) from a large shallow, eutrophic lake of the subtropical China

Tissue distributions and seasonal dynamics of the hepatotoxic microcystins-LR and -RR in a freshwater snail (Bellamya aeruginosa) were studied monthly in a large shallow, eutrophic lake of the subtropical China during June-November, 2003. Microcystins (MCs) were quantitatively determined by High-Performance Liquid Chromatography (HPLC) with a qualitative analysis by a Finnigan LC-MS system. On the average of the study period, hepatopancreas was the highest in MC contents (mean 4.14 and range 1.06-7.42 mug g(-1) DW), followed by digestive tracts (mean 1.69 and range 0.8-4.54 mug g(-1) DW) and gonad (mean 0.715 and range 0-2.62 mug g(-1) DW), whereas foot was the least (mean 0.01 and range 0-0.06 mug g(-1) DW). There was a positive correlation in MC contents between digestive tracts and hepatopancreas. A constantly higher MC content in hepatopancreas than in digestive tracts indicates a substantial bioaccumulation of MCs in the hepatopancreas of the snail. The average ratio of MC-LR/MC-RR showed a steady increase from digestive tracts (0.44) to hepatopancreas (0.63) and to gonad (0.96), suggesting that MC-LR was more resistant to degradation in the snail. Since most MCs were present in the hepatopancreas, digestive tracts and gonad with only a very small amount in the edible foot, the risk to human health may not be significant if these toxic parts are removed prior to snail consumption. However, the possible transference of toxins along food chains should not be a negligible concern. (C) 2004 Elsevier Ltd. All rights reserved.

几种植物药材的色谱－质谱联用分析

本论文由四章组成，第一、二、三章为实验论文，分别报道了中药羌活、菊花、全缘叶绿绒蒿的化学成分的高效液相色谱(HPLC)和液相色谱-质谱(LC-MS)联用分析以及挥发油的气相色谱-质谱(GC-MS)联用分析。第四章概述了重要藏药材化学成分的研究进展。 第一章首先对28批不同产地的羌活药材进行了HPLC分析，建立了羌活的指纹图谱。结果表明，不同产地羌活的化学成分基本相似，但是各组分在含量上存在较大差异。其次，对羌活的主要化学组分包括紫花前胡苷、紫花前胡素、6'-O-反式阿魏酸紫花前胡苷、茴香酸对羟基苯乙酯、羌活醇和异欧前胡素进行了定量分析。此外，针对同一产地不同采集时间的羌活挥发油进行系统分析，结果表明它们的化学成分基本相似，主要含有a－蒎烯、b－蒎烯、柠檬烯和龙脑乙酸酯等，只是各组分含量有所变化，这说明采集药材时要注意采集时间。 第二章分别报道了不同产地不同品种菊花非挥发性成分的液相色谱－二极管阵列检测－串联质谱(LC-PDA-MSn)分析和挥发性成分的气相色谱－质谱(GC-MS)联用分析比较。首先通过液相色谱－质谱－串联质谱对各色谱峰进行定性分析，通过与标准品对照，以及UV和MSn提供的结构信息，结合文献报道共鉴定了11个化学成分，包括绿原酸和10个黄酮化合物，并比较了不同品种菊花的化学成分相同之处和不同之处。另外，对七种不同品种不同产地的菊花挥发性成分通过GC-MS分析表明其主要挥发性成分为单萜类、倍半萜类化合物，共有成分樟脑、龙脑和龙脑乙酸酯等，各成分在不同挥发油中的含量变化明显。 第三章为藏药全缘叶绿绒蒿不同部位挥发油成分的气相色谱－质谱(GC-MS)联用分析，比较其挥发油化学成分及其含量变化的异同点。研究结果表明，全缘叶绿绒蒿花精油的化学成分明显多于全草部位，且两者主要成分有较大的差别。 第四章综述了青藏高原重要藏药材化学成分的研究进展。分别对藏药的资源特色和110多种常用重要藏药材的化学成分的研究情况以及藏药未来发展思路进行了阐述，以期对相关的研究提供一些信息。 This dissertation consists of four parts. The first part reports studies on the fingerprint of Notopterygium incisum and N. forbesii by HPLC-PDA-MSn, and on the constitutents of essential oil by GC-MS. The second part elaborates the chemical constitutents of Chrysanthemum L. by LC-MS and GC-MS analysis. The third part reports the chemical compositions of the essential oil from the different parts of Meconopsis integrifolia. The fourth part reviews on the progress of the studies on the chemical constitutents in Tibetan medicines. The first chapter is about HPLC analysis of a traditional Chinese herbal medicine Qiang-huo (Notopterygium incisum and N. forbesii ). Firstly, based on analyzing and contrasting the relative retention time and relative paek area in chromatographic fingerprint, the HPLC chromatographic fingerprint of Notopterygium incisum was established, which can used as a scientific basement for the quality evalution of this herb. Secondly, quantitative analysis were performed on the main chemical constitutents of Notopterygium incisum and N. forbesii including nodakenin, nodakenetin, 6’-O-trans-feruloylnodakenin, p-hydroxypenethylanisate, notopterol and isoimperatorin. The results indicated that the contents were variable related to different growth regions. Lastly, the essential oil of Notopterygium incisum collected in different harvest times is analyzed by GC-MS. The second chapter is about HPLC-MS and GC-MS analysis of several species of Chrysanthemum L. Firstly, eleven compounds including chlorogenic acid and ten flavone compounds were identified in the methanol extract of Chrysanthemum morifolium Ramat. from different regions by HPLC-MS analysis. Secondly, the essential oil of seven different species of Chrysanthemum L.were extracted by steam distillation, and its compositions were isolated and identified by GC-MS. The main active constitutents such as camphor, borneol and bornyl acetate were detected, but the relative content varied notably. The third chapter is about GC-MS analysis of the essential oil from different parts of Meconopsis integrifolia. It indicated great difference of the chemical compositions of their oil in the flowers and residual overground part. The last chapter is a review of the research progress of the Tibetan medicines, which includes their features and their main chemical constitutents.

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.

低极性化合物的电喷雾质谱检测方法及几种中藏药材的活性成分研究

本学位论文首先报道了为解决低极性化合物的电喷雾质谱（ESI-MS）分析难题而建立的一种衍生化分析方法；然后从色谱-质谱联用分析、分离纯化和结构鉴定等方面分别报道了几种中藏药材的活性成分研究。论文由下述六章组成： 第一章报道了盐酸羟胺衍生化方法在电喷雾质谱 (ESI-MS) 分析中的应用。该方法利用盐酸羟胺和羰基成肟的快速反应，建立了针对三萜酮等含酮或醛羰基低极性化合物的ESI-MS 信号增强技术。此方法不仅可应用于增强羰基化合物的ESI-MS 质谱信号，还可检测化合物中羰基的个数以及辨别涉及羰基官能团的同分异构体。此外，通过简单的氧化反应，还可将该方法拓展到三萜醇、甾醇等含羟基的低极性化合物，增强它们的ESI-MS 信号。对比已报道的相关ESI-MS 增强质谱信号的衍生化方法，此方法有经济、实用、快速和简便的显著特点。 第二章是关于野生羌活及其栽培品种化学成分的色谱-质谱联用分析。对不同产地野生羌活生长过程中活性成分的动态变化、野生羌活不同形态部位和人工栽培羌活中的活性成分含量进行了HPLC 定量分析。结果表明主要活性成分羌活醇和异欧前胡素都随生长期存在规律性变化，羌活不同形态部位中的活性成分含量也有明显不同。这些实验结果有些较好地印证了传统中医的用药理论，有些也对羌活的传统使用方法提出了新的建议。 第三章介绍了几种传统中藏药材的色谱-质谱联用及串联质谱分析。通过GC-MS 方法，从藏药材长花党参挥发油中共分离鉴定出45 个化合物；利用HPLC方法测定了该藏药材中的主要化学成分——木犀草素的含量（0.7%）；利用串联质谱技术，对西番莲和射干中的主要成分进行了快速鉴定，从西番莲中鉴定了4个黄酮碳苷；从不同产地的射干和川射干中鉴定了8 个主要异黄酮成分，其中包括一个未见报道的化合物。 第四章的内容为藏药材石莲叶点地梅的活性成分研究。从植物石莲叶点地梅(Androsace integra (Maxim.) Hand.-Mazz.) 乙醇提取物的正丁醇萃取部分共分离和鉴定了6 个化合物，利用MS 和NMR 等现代波谱学技术阐明了它们的结构：其中包括4 个三萜类化合物：分别是androsacin (1)、 ardisiacrispin A (2) 、saxifragifolin A (3) 和20(29)-lupen-3-one (4)；一个神经酰胺：4-羟基-Δ8,9(Z)-鞘氨醇-2'-羟基正二十四碳酸酰胺(5)；一个甾体类化合物：胡萝卜苷(6)。化合物1为新的13,28-epoxy-oleanane 型三萜皂苷，在其结构表征的过程中，采用LC-MS 进行糖分析，获得了值得推广的好结果。通过活性筛选发现化合物1~3 对HepG2肝癌细胞表现出不同程度的抑制活性，其中化合物2 活性最好，其IG50 为1.65μg/mL。 第五章是关于一些传统中藏药材的农药活性筛选。利用Syngenta 公司的活性筛选平台对68 种传统中藏药材醇提物进行了抗菌和除草的生物源农药活性筛选。结果表明所筛选的68 种植物提取物中，共有14 种样品表现出明显的除草/杀虫活性，其中水母雪莲花、松萝和茯神木等植物提取物还具有多种生物活性。活性成分还有待进一步追踪分离、纯化和结构鉴定。 第六章为文献综述，概述了羌活药材的研究进展。对羌活属及药用羌活植物从分类学、本草学、品质评价、人工栽培、化学成分及药理作用等方面进行了文献归纳和总结。 In this dissertation, an electrospray ionization mass spectrometry (ESI-MS) signal enhancement method, as well as the work of bioactive components study, HPLC-MS/MS application, bioassay screening, chromatograph separation and structure identification of the metabolites in several medicinal herbs have been reported. First chapter expounded a rapid, simple ESI-MS sensitivity enhancement method for detecting carbonyl groups in natural products has been developed by using hydroxylamine hydrochloride (NH2OH·HCl) as a derivatization reagent. We use the oxime formed during the derivatization reactions and its Beckmann rearrangement intermediates as a means of detecting the carbonyl groups originally present in these triterpenoids. In comparison with other derivatization methods in the literature, this method is simple, specific and can be used to detect carbonyl groups in triterpenoids which have low polarity and are poorly or non-ionizable. Moreover, it can also be used to detect hydroxyl groups by using the Dess-Martin periodinane (DMP) to convert primary and secondary hydroxyls into carbonyl groups. Chapter 2 reported an HPLC-MS method for analyzing the main bioactive compounds in both wild and cultured Notopterygium incisum. The results indicated that the main bioactive compounds varied through different seasons regularly, and in different commercial parts of this herb the content of these compounds also differed from each other. The quantitative analysis results showed that in the traditional commercial parts, the content of main chemical constitutes in Silkworm Notopterygium, Bamboo Notopterygium and Irregular-nodal Notopterygium are higher than that in Striped Notopterygium. This result is tally with the traditionally concept that the quality of Notopterygium, Bamboo Notopterygium and Irregular-nodal Notopterygium are better than that of Striped Notopterygium, which means that the quality of rhizomes is better than main roots. The chemical constituents of cultured N. incisum is reported for the first time in this dissertation and the analysis results showed some growth curves of chemical constituents in this plant, but still left some questions unanswered. Chapter 3 discussed the GC/LC-MS analysis of the traditional Chinese medicines Codonopsis thalictrifolis, Passiflora incarnate, Belamcanda chinensis and Passiflora incarnate. The main constituent, luteolin was isolated and identified from the traditional Tibet medicine of C. thalictrifolis. The quantitative analysis by HPLC has revealed that the content of luteolin in this herb is 0.7%. GC-MS was employed to analyzed chemical constituents of the essential oil from the flower of C. thalictrifolis. More than 60 peaks were detected and 45 of them were identified by comparing their spectra with that of the standards in the database and literatures. ESI-MS/MS was used to analyze the n-butanol extract of Passiflora incarnate. Based on the information of pseudo molecular ions and fragment ions of the glycosides, four major flavone-C-glycosides have been detected and identified as 7-methoxyluteolin-6-C-β-D-glucopyranoside, vitexin, swertisin and orientin. The isoflavone compounds in theextracts of three samples of B. chinensis collected in Gansu, Sichuan and Hunan, and the extract of Iris tectorum collected in Sichuan were analyzed by using TOF-HRMS and IT-MS. From the extracts of these herbs, a new isoflavone, identified as 5’,5,6,7-tetrahydroxy-3’4’-dimethoxyl isoflavon, and 7 known ones have been identified by analyzing the fragmentation patterns and their molecular formulas given by HRMS and the tandem mass spectrometry acquired by IT-MS. Chapter 4 elucidated the isolation and identification of a new triterpene saponin, androsacin (1), along with five known compounds (2-6) were isolated from the whole plants of Androsace integra (Maxim.) Hand.-Mazz., an herb used in traditional Chinese and Tibetan medicine. The chemical structure of the new compound was established as 3β-O-{β-D-glucopyranosyl-(1→4)-O-β-D-xylopyranosyl-(1→2)-O-β-D-glucopyranosyl-(1→4)-[O-β-D-glucopyranosyl-(1→2)]-α-L-arabinopyranosyl}-16α-hydroxy-13β,28-epoxy-olean-30-al by analyzing its MS, 1D- and 2D-NMR spectra. Compound 2 was cytotoxic toward HepG2 cancer cell with the GI50 value of 1.65 μg/mL. Chapter 5 described the biogenic pesticide activity screening of 68 traditional Chinese and Tibetan medicine extractions. The intention of this study is to explore bioactive natural compounds from these traditional medicinal herbs for biogenic insecticides use. Based on Syngenta’s bioassay, 14 extractions of these traditional medicines showed pesticide activities, and some of them had multi-activities on antibacterial and insecticidal. Chapter 6 is a review on the chemical and bioactivity research progress of Notopterygium incisum and N. forbesii.

高效液相色谱-质谱法测定花生中的植物螯合肽

以镉胁迫花生为原料,建立了液相色谱-电喷雾电离质谱联用仪测定花生中的植物螯合肽的LC-MS方法。以C18反相柱为分析柱,1%三氟乙酸、乙腈为梯度洗脱淋洗液,能够检测出花生中的植物螯合肽。在本实验条件下,检出限为0.2 mg/L;线性范围在0.50～20.0 mg/L(r≥0.9974);回收率大于80%。方法灵敏度高,实用性强。应用本方法对实际花生样品中的PC4进行了定量分析。初步证实了花生中存在丙氨酸类植物螯合肽。结果表明,受镉胁迫的花生中均检出不同浓度的PC4。

Determination of peptides and amino acids from wool and beer with sensitive fluorescent reagent 2-(9-carbazole)-ethyl chloroformate by reverse phase high-performance liquid chromotography and liquid chromotography mass spectrometry

A new method for the sensitive determination of amino acids and peptides using the tagging reagent 2-(9-carbazole)-ethyl chloroformate (CEOC) with fluorescence (FL) detection has been developed. Identification of derivatives was carried out by liquid chromotography mass spectrometry. The chromophore in the 2-(9-fluorenyl)-ethyl chloroformate (FMOC) reagent was replaced by carbazole, which resulted in a sensitive fluorescence lerivatizing agent CEOC. CEOC can easily and quickly label peptides and amino acids. Derivatives are stable enough to be efficiently analyzed by high-performance liquid chromatography. Studies on derivatization demonstrate excellent derivative yields over the pH range 8.8-10.0. Maximal yields close to 100% are observed with three- to fourfold molar reagent excess. Derivatives exhibit strong fluorescence and allow direct injection of the reaction mixture with no significant disturbance from the major fluorescent reagent degradation by-products, such as 2(9-carbazole)-ethanol and bis-(2-(9-carbazole)-ethyl) carbonate. In addition, the detection responses for CEOC derivatives are compared to those obtained with FMOC. The ratios AC(CEOC)/AC(FMOC) = 1.00-1.82 for fluorescence (FL) response and AC'(CEOC)/AC'(FMOC) = 1.00-1.21 for ultraviolet (UV) response are observed (here, AC and AC' are, respectively, FL and UV F response). Separation of the derivatized peptides and amino acids has been optimized on a Hypersil BDS C18 column. Excellent linear responses are observed. This method was used successfully to analyze protein hydrolysates from wool and from direct-derivatized beer. (C) 2003 Elsevier Science (USA). All rights reserved.

Analysis of Saponins from Leaves of Aralia elata by Liquid Chromatography and Multi-stage Tandem Mass Spectrometry

Four saponins were isolated from the leaves of Aralia elata, and established using NMR and other spectroscopic methods, as well as data reported in the literature. Three Aralia saponins from the leaves of Aralia elata sharing the same structures as those isolated from the root bark suggested that the leaves would be a good substitute for the root bark of Aralia elata. These four Aralia saponins were then extensively investigated using complementarily positive and negative electrospray ionization multistage tandem mass spectrometry (ESI-MSn). Two isomers of saponins with different sugar linkages were then successfully differentiated by positive ESI-MSn and verified with different retention times and the collision-induced dissociation (CID) spectra by LC-MS. A simple and effective LC-MS method was thus developed for the rapid identification and screening of these saponins in plant extracts from leaves of Aralia elata.