多肽类药物体内分析方法

多肽类药物是新药研究的新领域,也是当今新药研究最活跃和发展最迅速的领域之一。目前,建立测定生物基质中多肽类药物的方法学是世界范围存在的难题,面临着诸多挑战。酶联免疫法(ELISA)及放射同位素标记是定量多肽类药物最常用的方法,应用广泛。但液相色谱-质谱联用技术发展迅速,高专属性、高灵敏度、应用广泛、简便快速等特点势必使其成为多肽类药物体内定量分析的重要手段。本文就目前国际上几种检测方法的优劣、应用现状及发展趋势进行了综述。     

厄贝沙坦氢氯噻嗪片中氢氯噻嗪人体生物等效性研究

目的：评价两种复方厄贝沙坦片中氢氯噻嗪的生物等效性。方法：20名健康男性志愿者分别单剂量po受试制剂和参比制剂，采用高效液相色谱-质谱联用法测定血浆中氢氯噻嗪的浓度并拟合药动学参数。结果：受试制剂和参比制剂在受试者体内的药动学参数如下：血浆中氢氯噻嗪的Cmax（72．6±33．8）和（74．7±31．9）ng·ml^-1，tmax（2．0±0．5）和（1．8±0．4）h，t1／2（2．9±1．2）和（2．5±1．0）h，AUC0-48h（372．3±168．7）和（377．5±210．4）ng·h·ml^-1，AUC0-∞（398．3±191．2）和（396．5±223．5）ng·h·ml^-1。与参比制剂相比，受试制剂中氢氯噻嗪的平均相对生物利用度为（106．7±26．1）％。结论：两种制剂中氢氯噻嗪具生物等效性。     

高效液相色谱-串联质谱法测定犬血浆中盐酸二甲双胍浓度

目的：建立测定犬血浆中二甲双胍浓度的高效液相色谱．质谱．质谱联用方法，并用于二甲双胍缓释片非临床药物动力学研究。方法：血浆样品经乙腈沉淀蛋白，取上清液直接进行LC—MS／MS测定，以10mmol·L^-1甲酸铵缓冲液．乙腈（50：50）为流动相，采用ZORBAX EclipseXDB—C18柱（150mm×2．1mm，3．5μm）分离，在三级四极杆串联质谱中经电喷雾电离源（ESI）离子化，以多反应监测（MRM）方式进行检测。用于定量分析的离子反应分别为m／z 130→60（二甲双胍）和m/z304→182（可卡因，内标）。结果：LC-MS／MS法测定血浆中二甲双胍的线性范围为2．0～4117ng·ml^-1，定量下限为2．0ng．ml^-1。以3个浓度水平的质量控制样品求得各浓度水平日内、日间精密度（RSD）均小于13％。在非临床药代动力学研究中。应用此法测定了爱试Beazle犬血浆中二甲双胍的浓度．结论：该法灵敏、快速、操作简便，适用于二甲双胍的动物药物动力学研究。     

现代分析仪器在中药新药分析中的应用

随着国内外现代分析技术的发展，越来越多的新技术、新方法被应用到天然药物的研究中。在我国，对中药及其复方的分析仍然是现代仪器分析基本应用领域。此外，随着国家对药品监管力度的加强，药物毒副作用和不良反应也越来越受到人们的关注。因此，对各种检测方法、分析手段的操作简便性、分析速度快慢、应用范围等方面也提出了更高要求，其中包括分光光度法、色谱法、毛细管电泳法及其他联用分析方法等，     

进食对健康受试者口服盐酸川丁特罗片剂药代动力学的影响

目的研究在进食和空腹状态下中国健康受试者口服盐酸川丁特罗片(平喘药)药代动力学的差异。方法8名健康志愿者分别于禁食10h后空腹和统一早餐后,立即口服盐酸川丁特罗片50μg,用HPLC-MS/MS测定血浆中川丁特罗的浓度,用WinNonLin5.0计算其主要药代动力学参数。结果8名受试者在进食和空腹状态下,单次口服盐酸川丁特罗片50μg的主要药代动力学参数:Cmax分别为(17.3±4.0)、(20.8±4.1)pg.mL-1;tmax分别为(1.0±0.5)、(1.2±0.5)h;t1/2分别为(16.7±4.3)、(18.5±7.3)h;AUC0-tn分别为(80.9±23.8)、(106.8±57.1)pg.h.mL-1。结论进食状态下的Cmax和AUC0-tn分别为空腹状态时的83%和76%;而tmax和t1/2基本相近。     

First episode of shellfish contamination by palytoxin-like compounds from Ostreopsis species (Aegean Sea, Greece).

In order to investigate the toxicity of Ostreopsis species present in Greek coastal waters, cultures of Ostreopsis sp. and Ostreopsis ovata, mixed Ostreopsis field populations and shellfish collected from coastal waters of North Aegean Sea during late summer and autumn periods of 2004, 2005 and 2006 were examined by both mouse bioassay (MBA) and hemolysis neutralization assay (HNA). MBA testing was based on two different extraction protocols, while HNA also included the use of ouabain, a known palytoxin (PLT) antagonist. Results indicated the presence of a compound in both Ostreopsis cells and shellfish tissues, which was strongly toxic to mice. This compound exhibited characteristic symptomatology in mice (death, numbness, waddling gait and blindness) to that of PLT, as well as delayed hemolytic activity, which was neutralized by ouabain. HNA indicated that Ostreopsis cells contained a PLT-like compound (putative PLT, p-PLT) at concentrations ranging between 0.4 and 0.9 pg/cell, whereas concentration in shellfish tissues was estimated to range from about 33.3 to 97.0 microg p-PLT/kg tissue. To our knowledge, this is the first report of p-PLT contamination of shellfish by natural Ostreopsis species populations in European coastal waters and possibly globally, and also the first evidence on Ostreopsis cells' toxicity in the Eastern Mediterranean Sea.     

Determination of paralytic shellfish poisoning toxins in Norwegian shellfish by liquid chromatography with fluorescence and tandem mass spectrometry detection.

A novel extraction and clean-up method has been developed for the determination of paralytic shellfish poisoning (PSP) toxins in shellfish samples. Raw shellfish material was extracted with an acidic acetonitrile/water (80:20, v/v) solution, whilst being homogenised. During the homogenisation the sample extraction solution was cooled with ice water. Subsequently, the extract was frozen at -20 degrees C for at least 4h. During freezing, two layers were formed, only the lower predominantly aqueous layer was used for the determination. The final extract solution was cleaned-up using a combination of Oasis HLB and Carbograph activated carbon SPE columns. The developed extraction and clean-up methods combined with gradient elution liquid chromatography (LC)-mass spectrometry/mass spectrometry (MS/MS) has resulted in a method which can determine the analogs GTX 1-5, C1-2, DcGTX 2-3, DcSTX, Neo, STX in a single analysis with an overall detection limit of 313mug STXdiHCL-eq./kg shellfish meat. The use of the developed extraction method with post-column high performance liquid chromatography (HPLC) with fluorescence detection (FLD) method provided an overall limit of detection of 89mug STXdiHCL-eq./kg shellfish meat for the same toxins. Both post-column HPLC-FLD and LC-MS/MS was used to investigate the Norwegian PSP toxin profile. It was found that the PSP toxins could be detected in shellfish samples from the Norwegian coastline for 10 months of the year, from March till December. The toxin profile consisted mainly of the carbamate toxins, GTX 1-4, Neo and STX, in terms of both concentrations and contribution to the overall toxicity. In addition, several of the n-sulfo-carbamoyl toxins were either detected in the samples at relatively low concentrations or their presence in the samples were indicated but could not be confirmed by the post-column HPLC-FLD and LC-MS/MS analyses.     

Azaspiracid: first evidence of protein binding in shellfish.

The occurrence of azaspiracid (AZA) toxins in contaminated shellfish has been the focus of much research. The present study investigated the binding properties of these toxins in mussels of the species Mytilus edulis. The work involved extraction of proteins and AZAs from contaminated mussel hepatopancreas and examination of the extracts by isoelectric focusing (IEF), size exclusion chromatography (SEC) and sodium docecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Liquid chromatography coupled with tandem mass spectrometry analysis (LC-MS/MS) was also performed in this study to identify AZAs. Blank mussels were subjected to the same purification and analytical procedures. AZAs were found to be weakly bound to a protein with a molecular weight of 45 kDa, in samples of contaminated mussels. This protein, which was abundant in contaminated mussels, was also present in blank mussels, albeit at much lower concentrations. It was further noted that a 22 kDa protein was also present only in contaminated mussel samples.     

太子参道地药材指纹图谱的构建

目的:构建太子参道地药材的指纹图谱,为苏产太子参GAP实施和发展道地药材生产提供科学依据.方法:用HPLC、HPCE、GC-MS、LC-MS及ICP技术对道地和其它产区太子参进行分析,测定其指纹图谱.结果:初步建立了太了参道地药材的HPLC、HPCE、GC-MS、LC-MS及无机元素指纹谱.结论:方法准确可靠,重视性好,可作为太子参内在质量评价的依据.     

利用LC／MS检测中药总成分中各大类成分含量的方法学探讨

目的:利用液相-质谱(LC-Ms)技术检测总成分,并对其含量计算进行研究.方法:以感冒中药复方YL2000为例,利用LC-MS测定其中的总黄酮、总生物碱和总香豆素等主要大类成分,以HPLC测定的其中单体成分含量作参照,对LC-MS中各大类成分百分比进行含量计算.结果:以此方法计算的总黄酮、总生物碱、总香豆含量以及总含量与所用紫外分光光度法所测的含量基本一致.结论:利用LC-MS对成分结构的归属来确定各大类成分的构成比例,以所测代表成分HPLC含量结果作参照来计算各大类成分的含量具有一定的可行性.