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.

High-performance liquid chromatographic determination of 17 beta-estradiol and 17 beta-estradiol-3-acetate solubilities and diffusion coefficients in silicone elastomeric intravaginal rings

A rapid, sensitive reversed-phase high-performance liquid chromatographic method has been developed for the determination of in vitro release of 17 beta-estradiol and its ester prodrug, 17 beta-estradiol-3-acetate, from silicone intravaginal rings. Partial hydrolysis of the acetate under the aqueous conditions provided by the 1% benzalkonium chloride release medium necessitates its conversion to 17 beta-estradiol prior to HPLC analysis. Both steroid peaks have been fully resolved from the benzalkonium chloride peaks by the reported chromatographic method,which employs a C-18 bonded reversed-phase column, an acetonitrile-water (50:50, v/v) mobile phase and a UV detection wavelength of 281 nm. The peak area versus 17 beta-estradiol concentration was found to be linear over the range of 0.0137-1347 mu g ml(-1) The HPLC method has also been used to determine the silicone solubilities and diffusion coefficients of the two related steroids. The almost 100-fold increase in 17 beta-estradiol-3-acetate release from the silicone core-type intravaginal rings compared to 17 beta-estradiol is shown to be due to a 60-fold increase in silicone solubility and a one and a half-fold increase in diffusitivity. The results demonstrate that an effective estrogen replacement therapy dose of 17 beta-estradiol may be administered from a silicone intravaginal reservoir device containing the labile 17 beta-estradiol-3-acetate prodrug. (C) 2000 Elsevier Science B.V. All rights reserved.

Indirect hydrogen analysis by gas chromatography coupled to mass spectrometry (GC-MS).

Gas chromatography (GC) is an analytical tool very useful to investigate the composition of gaseous mixtures. The different gases are separated by specific columns but, if hydrogen (H2 ) is present in the sample, its detection can be performed by a thermal conductivity detector or a helium ionization detector. Indeed, coupled to GC, no other detector can perform this detection except the expensive atomic emission detector. Based on the detection and analysis of H2 isotopes by low-pressure chemical ionization mass spectrometry (MS), a new method for H2 detection by GC coupled to MS with an electron ionization ion source and a quadrupole analyser is presented. The presence of H2 in a gaseous mixture could easily be put in evidence by the monitoring of the molecular ion of the protonated carrier gas. Copyright © 2013 John Wiley & Sons, Ltd.

Validation of an analytical method for nitrous oxide (N2O) laughing gas by headspace gas chromatography coupled to mass spectrometry (HS-GC-MS): Forensic application to a lethal intoxication.

Drug abuse is a widespread problem affecting both teenagers and adults. Nitrous oxide is becoming increasingly popular as an inhalation drug, causing harmful neurological and hematological effects. Some gas chromatography-mass spectrometry (GC-MS) methods for nitrous oxide measurement have been previously described. The main drawbacks of these methods include a lack of sensitivity for forensic applications; including an inability to quantitatively determine the concentration of gas present. The following study provides a validated method using HS-GC-MS which incorporates hydrogen sulfide as a suitable internal standard allowing the quantification of nitrous oxide. Upon analysis, sample and internal standard have similar retention times and are eluted quickly from the molecular sieve 5Å PLOT capillary column and the Porabond Q column therefore providing rapid data collection whilst preserving well defined peaks. After validation, the method has been applied to a real case of N2O intoxication indicating concentrations in a mono-intoxication.

Determination of volatile organic compounds in recycled polyethylene terephthalate and high-density polyethylene by headspace solid phase microextraction gas chromatography mass spectrometry to evaluate the efficiency of recycling processes

A method for the determination of volatile organic compounds (VOCs) in recycled polyethylene terephthalate and high-density polyethylene using headspace sampling by solid-phase microextraction and gas chromatography coupled to mass spectrometry detection is presented. This method was used to evaluate the efficiency of cleaning processes for VOC removal from recycled PET. In addition, the method was also employed to evaluate the level of VOC contamination in multilayer packaging material containing recycled HDPE material. The optimisation of the extraction procedure for volatile compounds was performed and the best extraction conditions were found using a 75 mu m carboxen-polydimethylsiloxane (CAR-PDMS) fibre for 20 min at 60 degrees C. The validation parameters for the established method were linear range, linearity, sensitivity, precision (repeatability), accuracy (recovery) and detection and quantification limits. The results indicated that the method could easily be used in quality control for the production of recycled PET and HDPE. (C) 2011 Elsevier B.V. All rights reserved.

Cholesterol Hydroperoxides Generate Singlet Molecular Oxygen [O(2) ((1)Delta(g))]: Near-IR Emission, (18)O-Labeled Hydroperoxides, and Mass Spectrometry

In mammalian membranes, cholesterol is concentrated in lipid rafts. The generation of cholesterol hydroperoxides (ChOOHs) and their decomposition products induces various types of cell damage. The decomposition of some organic hydroperoxides into peroxyl radicals is known to be a potential source of singlet molecular oxygen [O(2) ((1)Delta(g))] in biological systems. We report herein on evidence of the generation of O(2) ((1)Delta(g)) from ChOOH isomers in solution or in liposomes containing ChOOHs, which involves a cyclic mechanism from a linear tetraoxide intermediate originally proposed by Russell. Characteristic light emission at 1270 nm, corresponding to O(2) ((1)Delta(g)) monomolecular decay, was observed for each ChOOH isomer or in liposomes containing ChOOHs. Moreover, the presence of O(2) ((1)Delta(g)) was unequivocally demonstrated using the direct spectral characterization of near-infrared light emission. Using (18)O-labeled cholesterol hydroperoxide (Ch(18)O(18)OH), we observed the formation of (18)O-labeled O(2) ((1)Delta(g)) [(18)O(2) ((1)Delta(g))] by the chemical trapping of (18)O(2) ((1)Delta(g)) with 9,10-diphenylanthracene (DPA) and detected the corresponding (18)O-labeled DPA endoperoxide (DPA(18)O(18)O) and the (18)O-labeled products of the Russell mechanism using high-performance liquid chromatography coupled to tandem mass spectrometry. Photoemission properties and chemical trapping clearly demonstrate that the decomposition of Ch(18)O(18)OH generates (18)O(2) ((1)Delta(g)), which is consistent with the Russell mechanism and points to the involvement of O(2) ((1)Delta(g)) in cholesterol hydroperoxide-mediated cytotoxicity.

Reactivity of Beer Bitter Acids Toward the 1-Hydroxyethyl Radical as Probed by Spin-Trapping Electron Paramagnetic Resonance (EPR) and Electrospray Ionization-Tandem Mass Spectrometry (ESI-MS/MS)

The iso-alpha-acids or isohumulones are the major contributors to the bitter taste of beer, and it is well-recognized that they are degraded during beer aging. In particular, the trans-isohumulones seem to be less stable than the cis-isohumulones. The major radical identified in beer is the 1-hydroxyethyl radical; however, the reactivity between this radical and the isohumulones has not been reported until now. Therefore, we studied the reactivity of isohumulones toward the 1-hydroxyethyl radical through a competitive kinetic approach. It was observed that both cis- and trans-isohumulones and dihydroisohumulones are decomposed in the presence of 1-hydroxyethyl radicals, while the reactivities are comparable. On the other hand, the tetrahydroisohumulones did not react with 1-hydroxyethyl radicals. The apparent second-order rate constants for the reactions between the 1-hydroxyethyl radical and these compounds were determined by electron paramagnetic resonance (EPR) spectroscopy and electrospray ionization-tandem mass spectrometry [ESI(+)-MS/MS]. It follows that degradation of beer bitter acids is highly influenced by the presence of 1-hydroxyethyl radicals. The reaction products were detected by liquid chromatography electrospray ionization-ion trap-tandem mass spectrometry (LC-ESI-IT-MS/MS), and the formation of oxidized derivatives of the isohumulones was confirmed. These data help to understand the mechanism of beer degradation upon aging.

New method for determination of (E)resveratrol in wine based on microextraction using packed sorbent and ultra-performance liquid chromatography

An ultra-fast and improved analytical methodology based on microextraction by packed sorbent (MEPS) combined with ultra-performance LC (UPLC) was developed and validated for determination of (E)-resveratrol in wines. Important factors affecting the performance of MEPS such as the type of sorbent material (C2, C8, C18, SIL, and M1), number of extraction cycles, and sample volume were studied. The optimal conditions of MEPS extraction were obtained using C8 sorbent and small sample volumes (50–250mL) in one extraction cycle (extract–discard) and in a short time period (about 3 min for the entire sample preparation step). (E)-Resveratrol was eluted by 1 250mL of the mixture containing 95% methanol and 5% water, and the separation was carried out on a highstrength silica HSS T3 analytical column (100 mm 2.1 mm, 1.8mm particle size) using a binary mobile phase composed of aqueous 0.1% formic acid (eluent A) and methanol (eluent B) in the gradient elution mode (10 min of total analysis). The method was fully validated in terms of linearity, detection (LOD) and quantification (LOQ) limits, extraction yield, accuracy, and inter/intra-day precision, using a Madeira wine sample (ET) spiked with (E)-resveratrol at concentration levels ranging from 5 to 60mg/mL. Validation experiments revealed very good recovery rate of 9575.8% RSD, good linearity with r2 values 40.999 within the established concentration range, excellent repeatability (0.52%), and reproducibility (1.67%) values (expressed as RSD), thus demonstrating the robustness and accuracy of the MEPSC8/UPLC-photodiode array (PDA) method. The LOD of the method was 0.21mg/mL, whereas the LOQ was 0.68mg/mL. The validated methodology was applied to 30 commercial wines (24 red wines and six white wines) from different grape varieties, vintages, and regions. On the basis of the analytical validation, the MEPSC8/UPLC-PDA methodology shows to be an improved, sensitive, and ultra-fast approach for determination of (E)-resveratrol in wines with high resolving power within 6 min.

Investigation of urinary volatile organic metabolites as potential cancer biomarkers by solid-phase microextraction in combination with gas chromatography-mass spectrometry

BACKGROUND: Non-invasive diagnostic strategies aimed at identifying biomarkers of cancer are of great interest for early cancer detection. Urine is potentially a rich source of volatile organic metabolites (VOMs) that can be used as potential cancer biomarkers. Our aim was to develop a generally reliable, rapid, sensitive, and robust analytical method for screening large numbers of urine samples, resulting in a broad spectrum of native VOMs, as a tool to evaluate the potential of these metabolites in the early diagnosis of cancer. METHODS: To investigate urinary volatile metabolites as potential cancer biomarkers, urine samples from 33 cancer patients (oncological group: 14 leukaemia, 12 colorectal and 7 lymphoma) and 21 healthy (control group, cancer-free) individuals were qualitatively and quantitatively analysed. Dynamic solid-phase microextraction in headspace mode (dHS-SPME) using a carboxenpolydimethylsiloxane (CAR/PDMS) sorbent in combination with GC-qMS-based metabolomics was applied to isolate and identify the volatile metabolites. This method provides a potential non-invasive method for early cancer diagnosis as a first approach. To fulfil this objective, three important dHS-SPME experimental parameters that influence extraction efficiency (fibre coating, extraction time and temperature of sampling) were optimised using a univariate optimisation design. The highest extraction efficiency was obtained when sampling was performed at 501C for 60min using samples with high ionic strengths (17% sodium chloride, wv 1) and under agitation. RESULTS: A total of 82 volatile metabolites belonging to distinct chemical classes were identified in the control and oncological groups. Benzene derivatives, terpenoids and phenols were the most common classes for the oncological group, whereas ketones and sulphur compounds were the main classes that were isolated from the urine headspace of healthy subjects. The results demonstrate that compound concentrations were dramatically different between cancer patients and healthy volunteers. The positive rates of 16 patients among the 82 identified were found to be statistically different (Po0.05). A significant increase in the peak area of 2-methyl3-phenyl-2-propenal, p-cymene, anisole, 4-methyl-phenol and 1,2-dihydro-1,1,6-trimethyl-naphthalene in cancer patients was observed. On average, statistically significant lower abundances of dimethyl disulphide were found in cancer patients. CONCLUSIONS: Gas chromatographic peak areas were submitted to multivariate analysis (principal component analysis and supervised linear discriminant analysis) to visualise clusters within cases and to detect the volatile metabolites that are able to differentiate cancer patients from healthy individuals. Very good discrimination within cancer groups and between cancer and control groups was achieved.

A sensitive microextraction by packed sorbent-based methodology combined with ultra-high pressure liquid chromatography as a powerful technique for analysis of biologically active flavonols in wines

A new approach based on microextraction by packed sorbent (MEPS) and reversed-phase high-throughput ultra high pressure liquid chromatography (UHPLC) method that uses a gradient elution and diode array detection to quantitate three biologically active flavonols in wines, myricetin, quercetin, and kaempferol, is described. In addition to performing routine experiments to establish the validity of the assay to internationally accepted criteria (selectivity, linearity, sensitivity, precision, accuracy), experiments are included to assess the effect of the important experimental parameters such as the type of sorbent material (C2, C8, C18, SIL, and C8/SCX), number of extraction cycles (extract-discard), elution volume, sample volume, and ethanol content, on the MEPS performance. The optimal conditions of MEPS extraction were obtained using C8 sorbent and small sample volumes (250 μL) in five extraction cycle and in a short time period (about 5 min for the entire sample preparation step). Under optimized conditions, excellent linearity View the MathML source(Rvalues2>0.9963), limits of detection of 0.006 μg mL−1 (quercetin) to 0.013 μg mL−1 (myricetin) and precision within 0.5–3.1% were observed for the target flavonols. The average recoveries of myricetin, quercetin and kaempferol for real samples were 83.0–97.7% with relative standard deviation (RSD, %) lower than 1.6%. The results obtained showed that the most abundant flavonol in the analyzed samples was myricetin (5.8 ± 3.7 μg mL−1). Quercetin (0.97 ± 0.41 μg mL−1) and kaempferol (0.66 ± 0.24 μg mL−1) were found in a lower concentration. The optimized MEPSC8 method was compared with a reverse-phase solid-phase extraction (SPE) procedure using as sorbent a macroporous copolymer made from a balanced ratio of two monomers, the lipophilic divinylbenzene and the hydrophilic N-vinylpyrrolidone (Oasis HLB) were used as reference. MEPSC8 approach offers an attractive alternative for analysis of flavonols in wines, providing a number of advantages including highest extraction efficiency (from 85.9 ± 0.9% to 92.1 ± 0.5%) in the shortest extraction time with low solvent consumption, fast sample throughput, more environmentally friendly and easy to perform.

Development of a new high-performance liquid chromatographic method for the determination of ceftazidime

A rapid, accurate, and sensitive high-performance liquid chromatographic (HPLC) method was developed and validated for the determination of ceftazidime in pharmaceuticals. The method validation parameters yielded good results and included range, linearity, precision, accuracy, specificity, and recovery. The excipients in the commercial powder for injection did not interfere with the assay. Reversed-phase chromatography was used for the HPLC separation on a Waters C18 (WAT 054275; Milford, MA) column with methanol-water (70 + 30, v/v) as the mobile phase pumped isocratically at a flow rate of 1.0 mL/min. The effluent was monitored at 245 nm. The calibration graph for ceftazidime was linear from 50.0 to 300.0 mu g/mL. The values for interday and intraday precision (relative standard deviation) were < 1 %. The results obtained by the HPLC method were calculated statistically by analysis of variance. We concluded that the HPLC method is satisfactory for the determination of ceftazidime in the raw material and pharmaceuticals.