重组人粒细胞巨噬细胞集落刺激因子的生物质谱和毛细管电泳法

目的：为准确分析重组人粒细胞巨噬细胞集落刺激因子(rHuGM-CSF)半成品及其制剂的纯度和表达的正确性。方法：用毛细管区带电泳测定了2批rHuGM-CSF半成品和3批rHuGM-CSF注射液的纯度。用基体辅助激光解吸飞行时间质谱和电喷雾质谱测定了2批rHuGM-CSF半成品的分子量。用毛细管等电聚集法测定了rHuGM-CSF半成品的等电点。结果：2批半成品中1批含有少量二聚体,且含有4个不同等电点的组分,另1批表达不正确;2批成品均存在大量杂质。结论：发现有些rHuGM-CSF样品有生物学活性,但表达不正确,纯度也不高;各分析方法是互补的,不能相互替代。     

基因工程药物的现代仪器分析法

随着分子生物学的发展,出现了许多基于重组ＤＮＡ技术的特效药物,如治疗肾性贫血的基因工程人红细胞生成素（ｒＨｕＥＰＯ）,治疗白细胞缺乏的基因工程人粒细胞集落刺激因子（ｒＨｕＧ－ＣＳＦ）和粒细胞巨噬细胞集落刺激因子（ｒＨｕＧＭ－ＣＳＦ）,这些药物与传统...     

Pharmacokinetics of recombinant human granulocyte macrophage colony-stimulating factor in Macaca mulata

目的：研究重组人粒细胞巨噬细胞集落刺激因子（ｒｈＧＭＣＳＦ）在恒河猴体内的药物动力学．方法：用酶连接免疫吸附测定法检测血浆中ｒｈＧＭＣＳＦ的含量．结果：ｉｖｒｈＧＭＣＳＦ后血药浓度时间曲线符合三房室模型．第１，２和３相的Ｔ１２分别为００５－００７ｈ，０１４－０５８ｈ和１４－４１ｈ．ＡＵＣ随剂量成比例增加．ｉｖ高剂量和低剂量的Ｃｌ和Ｋ１０都相似．ｓｃｒｈＧＭＣＳＦ后血药浓度的峰值为０９３±０１６μｇ·Ｌ－１，达峰时间为２６５±０１４ｈ，生物利用度为０６１．结论：恒河猴ｒｈＧＭＣＳＦ药物动力学数据为临床试验提供有用参考．     

中国姥鲨软骨新生血管抑制因子的分离纯化及理化性质检测

实验用盐酸胍抽提，超滤，层析等方法，从中国姥鲨软骨中分离纯化到一种新生血管抑制因子，得率为１／６００００经毛细管电泳检测，其纯度为９０．６７％，ＳＤＳ－ＰＡＧＥ电泳及等电聚集电泳结果表明：ＳＣＤＩ的分子量为１６１００Ｄ、等电点为８．１５；氨基酸组成分析表明ＳＣＤＩ含有２０种氨基酸，其中以ｌＥＩ，Ｉｌｅ和Ｇｌｙ２含量最为丰富。     

rhGM-CSF生物活性的测定方法及工作标准品的标定

目的：研究用ＴＦ－１细胞测定ｒｈＧＭ－ＣＳＦ（重组人粒／巨噬细胞集落刺激因子）生物学活性的方法并用ＷＨＯ ｒｈＧＭ－ＣＳＦ国际标准品进行工作标准品和对照品（生白能先灵产品）的联合标定。方法：采用 ＭＴＴ染色法。结果：首次将（４,４）法引入ｒｈＧＭ－ＣＳＦ生物活性的测定,使所标定的工作标准品生物效价为每瓶１．３７×１０６ ＩＵ,所标定的对照品生物效价为每瓶１．７９×１０６ＩＵ,效价的平均可信限率分别为 ４． ９０４％和 ４． ３３３％。结论：（４, ４）法可用于ｒｈＧＭ－ＣＳＦ生物活性的测定,结果便于统计分析。所标定的工作标准品可作为ｒｈＧＭ－ＣＳＦ生物活性测定的工作标准。     

重组人红细胞生成素单克隆抗体的制备和鉴定

以重组人红细胞生成素免疫ＢＡＬＢ／ｃ小鼠，通过淋巴细胞杂交瘤技术将ＢＡＬＢ／ｃ小鼠脾细胞和Ｓｐ２／０骨髓瘤细胞融合，经ＥＬＩＳＡ法筛及有限稀释法克隆化，制备了２株能稳定分泌抗ｒＨｕＥＰＯ单克隆抗体的小鼠杂交瘤细胞系Ｇ５０和Ａ９４。用鼠阊我隆抗体分型试剂盒鉴定，其分泌的ＭｃＡｂ亚类均是ＩｇＧ１。经ＥＬＩＳＡ鉴定，两组ＭｃＡｂ只与ｒＨｕＥＰＯ特异性结合，与ＩＬ－１，ＧＭ－ＣＳＦ，ＩＦＮ－α和ＩＦＮ－ｒ     

人粒细胞—巨噬细胞集落刺激因子转移表达及对慢性乙型肝炎细胞免…

为了探讨人ＧＭ－ＣＳＦ基因转移表达对慢性乙型肝炎患者细胞免疫功能的影响，运用携带人ＧＭ－ＣＳＦ基因的重组腺病毒转染慢性乙型肝炎患者ＰＢＭＣｓ，表明以重组腺病毒为载体的人ＧＭ－ＣＳＦ基因在转染细胞中，可持续表达１．２６±０．０６５￣２．０９±０．１１ｎｇｓ·ｍｌ＾－１·２４ｈ＾－１水平２６天左右。通过观察慢性乙型肝炎患者ＰＢＭＣｓ在ＧＭ－ＣＳＦ基因转染前后及分泌ＧＭ－ＣＳＦ细胞和慢性乙型肝炎患者ＰＢ     

GM-CSF基因增强HCV非结构蛋白DNA疫苗诱导的细胞免疫应答

作者分别构建了编码丙型肝炎病毒（ＨＣＶ）非结构蛋白ＮＳ３、ＮＳ４和ＮＳ５的质粒ｐＴＶ－ＮＳ３４５、编码粒细胞－巨噬细胞－集落刺激因子（ＧＭ－ＣＳＦ）的质粒ｐＴＶ－ＧＭＣＳＦ以及编码ＮＳ３４５和ＧＭ－ＣＳＦ的双顺反子质粒ｐＴＶ－ＮＳ３４５／ＧＭＣＳＦ，分别将这三种质粒转染ＣＯＳ－７细胞，用免疫沉淀法检测ＨＣＶＮＳ３４５蛋白的表达，用ＥＬＩＳＡ检测培养上清中的ＧＭ－ＣＳＦ。将Ｂｕｆｆａｌｏ大鼠分为４组，第１、２、４组分别肌注ｐＴＶ－ＮＳ３４５、ｐＴＶ－ＮＳ３４５／ＧＭＣＳＦ和对照载体ｇＴＶ ４００μ…     

1-(2,6-二甲基苯氧基)-2-(3,4-二甲氧基苯乙胺基)丙烷盐酸盐对自发性高血压大鼠血管平滑肌细胞增殖及对PDGF-B,bFGF,c-sis,c-myc的影响

用氚胸腺嘧啶核苷（３ＨＴｄＲ）掺入法，电镜，免疫组化，原位杂交方法，在自发性高血压大鼠（ＳＨＲ）观察了１（２，６二甲基苯氧基）２（３，４二甲氧基苯乙胺基）丙烷盐酸盐（ＤＤＰＨ）对血管平滑肌细胞（ＶＳＭＣ）增殖的作用及对生长因子ＰＤＧＦＢ，ｂＦＧＦ及其相关癌基因ｃｓｉｓ与ｃｍｙｃ表达的影响。结果发现：ＤＤＰＨ在降低ＳＨＲ血压同时，能减少肾动脉ＶＳＭＣ的线粒体，粗面内质网和３ＨＴｄＲ掺入量，并能逆转ＶＳＭＣ增殖时ＰＤＧＦＢ，ｂＦＧＦ抗原及ｃｓｉｓ与ｃｍｙｃｍＲＮＡ的表达增强。提示：ＤＤＰＨ能抑制ＳＨＲ的ＶＳＭＣ增殖，与生长因子及癌基因调控的分子生物学机制有关。     

Anionic subsite of active center of Torpedo acetylcholinesterase constructs a part of its conformati

目的：研究电鳐乙酰胆碱酯酶（ＡＣｈＥ）结构功能关系，探讨ＡＣｈＥ负矩部位是否是其构象决定簇的一个组成部分．方法：用ＥＬＩＳＡ及酶抑制实验观察ＡＣｈＥ负矩部位探针２ＰＡＭ对ＡＣｈＥ与其ＭｃＡｂ３Ｆ３之间的免疫反应性的影响．结果：ＭｃＡｂ３Ｆ３不能与２ＰＡＭ及ＡＣｈＥ的复合物反应；２ＰＡＭ浓度依赖性地降低ＭｃＡｂ３Ｆ３对ＡＣｈＥ的抑制率；但不能解离ＡＣｈＥ与３Ｆ３构成的抗原抗体复合物．结论：电鳐ＡＣｈＥ活性中心负矩部位构成其活性中心构象抗原决定簇的一部分．     

Simultaneous separation and confirmation of amphetamine and related drugs in equine plasma by non-aqueous capillary-electrophoresis-tandem mass spectrometry

A non-aqueous capillary electrophoresis-mass spectrometry (NACE-MS) method was developed for simultaneous separation and identification of 12 amphetamine and related compounds in equine plasma. Analytes were recovered from plasma by liquid-liquid extraction using methyl tertiary butyl ether (MTBE). A bare fused-silica capillary was used for separation of the analytes. Addition of sheath liquid to the capillary effluent allowed the detection of the analytes by positive electrospray ionization mass spectrometry using full scan data acquisition. The limit of detection (LOD) for the target analytes was 10-200 ng/mL and that of confirmation (LOC) was 50-1000 ng/mL in equine plasma. Capillary electrophoresis (CE) and mass spectrometry (MS) parameters were optimized for full CE separation and MS detection of the analytes. Separation buffer comprised 25 mM ammonium formate in acetonitrile/methanol (20: 80, v/v) plus 1 M formic acid. Sheath liquid was isopropanol-water-formic acid (50:50:0.5, v/v/v). Samples were hydrodynamically injected and separated at 25 kV. Analytes were electrokinetically separated and mass spectrometrically identified and confirmed. This simple, fast, inexpensive and reproducible method was successfully applied to post race equine plasma and research samples in screening for amphetamine and related drugs.     

Application of surface ionization methods for highly sensitive and selective analysis of benzodiazepine derivatives

In this work, the results of studying the possibilities of surface ionization mass spectrometry (SI/MS) and atmosphere pressure thermodesorption surface ionization (APTDSI) spectroscopy for high-sensitivity and selective detection and identification of psychotropic preparations of benzodiazepine derivatives – medazepam, diazepam and chlordiazepoxide – are presented. It has been established that the SI mass spectra of benzodiazepines have a small number of lines and are significantly different from those obtained by electron ionization (EI) and that the molecules of benzodiazepines can be ionized by surface ionization with high efficiency (≥100 times and more than by EI) and the current density increases from diazepam to medazepam.

It has been found that the APTDSI spectra of benzodiazepine derivatives have two characteristic maxima connected with energy of sublimation and desorption.

The ionization efficiency is several C/g (Coulomb per gram), the linear range of the concentration dependence is 3–4 orders of a magnitude. The results obtained by the SI/MS and APTDSI methods have been compared with those obtained by the conventional TLC and GC/MS with electron ionization.     

Direct and rapid characterization of illicit drugs in adulterated samples using thermal desorption electrospray ionization mass spectrometry

Foods and drinks have been adulterated with illicit drugs to facilitate criminal activities. Unfortunately, conventional analytical methods are incapable of rapidly characterizing these drugs in samples, as serious interferences from sample matrices must be removed through tedious and time-consuming pretreatment. Ambient ionization mass spectrometry (AMS) generally does not require sample pretreatment and is thus a suitable tool for directly and rapidly detecting illicit drugs in samples in different physical states. In this study, thermal desorption electrospray ionization mass spectrometry (TD-ESI/MS), an AMS technique, was utilized to efficiently characterize illicit drugs spiked in samples including drinks, powders, and jelly candies. To perform sensitive analysis, the mass analyzer was operated in multiple reaction monitoring mode to monitor the molecular and fragment ions of the target analytes. The time required to complete a typical TD-ESI/MS analysis was less than 30 s. The limits of detection (LODs) for illicit drugs were found to be 100 ppb in drinks, 100–1000 ppb in instant powders, and 1.3–6.5 ng/mm² on stamp surfaces. FM2 and nitrazepam laced in the inner layer of a jelly candy were detected by TD-ESI/MS, showcasing the advantage of the technique for direct and rapid analysis as opposed to conventional methods.     

Characterization of recombinant human granulocyte colony stimulating factor (rHuG-CSF) by capillary zone electrophoresis, capillary isoelectric focusing electrophoresis and electrospray ionization mass spectrometry

Recombinant human granulocyte colony-stimulating factor (rHuG-CSF) is a hematopietic cytokine that stimulates and regulates the proliferation and differentiation of neutrophils. Glycosylated and non-glycosylated forms of rHuG-CSF cannot be distinguished by traditional biological assays. In addition, it is very difficult to characterize impurities of the same molecular weight in biologicals. In this study, non-glycosylated rHuG-CSF, two glycosylated rHuG-CSF isoforms and their commercial dosages were successfully separated by capillary zone electrophoresis (CZE) using 50mM Tricine containing 20mM NaCl and 2.5mM 1,4-diaminobutane (DAB) at pH 8.0, which could be employed for the qualitative discrimination assay of rHuG-CSF related products. CZE, capillary isoelectric focusing electrophoresis (CIEF), and mass spectrometry (MS) were used to effectively characterize non-glycosylated rHuG-CSF. It was found that proteins in the samples with different pIs in the CIEF profile could not be detected by CZE, while no difference was observed between these proteins and rHuG-CSF. Further analysis by electrospray ionization mass spectrometry with the resolution of 2000 showed that the components with different pIs in the non-glycosylated rHuG-CSF bulk sample are nearly equal in molecular weight. Therefore, it is necessary to combine several modern analytical techniques for quality control to get well-characterized biologicals.     

Qualitative analysis of seized cocaine samples using desorption electrospray ionization‐ mass spectrometry (DESI‐MS)

Desorption electrospray ionization ‐ mass spectrometry (DESI‐MS) is a useful technique for the qualitative analysis of compounds found in seized drug material. In this study, DESI‐MS was utilized in the screening analysis of illicit cocaine samples. The technique was also applied to the geographical origin determination of these samples. The limit of detection was determined to be 24.3 µg (or 3.47 µg/mm²) and the analysis time was less than 1 minute per sample. The intra‐day and inter‐day precision for the detection of cocaine was 11 % and 42 %, respectively; therefore the quantitative data provided by DESI‐MS was limited in its use for accurate determination of cocaine concentration in a sample. Using the quadrupole time‐of‐flight (QTOF) mass spectrometer, the presence of cocaine and impurities detected were confirmed by accurate tandem MS data. The qualitative chemical profiles obtained using DESI‐MS were compared to two popular analysis techniques, GC‐MS and LC‐MS. The effects of a range of adulterants including caffeine, procaine, levamisole, lignocaine, paracetamol, and atropine on the detectability of cocaine were also investigated. It was found that the addition of these adulterants in a cocaine sample did not prevent the detection of the analyte itself (there was slight enhancement in some samples), which was useful in drug detection. The detection of truxillines in the seized samples by DESI‐MS aided in the preliminary determination of geographical origin, i.e., Bolivian, Peruvian or Colombian leaf origin. The application of DESI‐MS to the qualitative analysis and screening of seized cocaine samples demonstrates the potential and applicability of the technique to the fast chemical profiling of illicit samples. Copyright © 2014 John Wiley & Sons, Ltd.     

Limitation standard of toxic aconitines in Aconitum proprietary Chinese medicines using on-line extraction electrospray ionization mass spectrometry

Development of rapid analytical methods and establishment of toxic component limitation standards are of great importance in quality control of traditional Chinese medicine. Herein, an on-line extraction electrospray ionization mass spectrometry (oEESI-MS) coupled with a novel whole process integral quantification strategy was developed and applied to direct determination of nine key aconitine-type alkaloids in 20 Aconitum proprietary Chinese medicines (APCMs). Multi-type dosage forms (e.g., tablets, capsules, pills, granules, and liquid preparation) of APCM could be determined directly with excellent versatility. The strategy has the characteristics of high throughput, good tolerance of matrix interference, small amount of sample (∼0.5 mg) and reagent (∼240 μL) consumption, and short analysis time for single sample (<15 min). The results were proved to be credible by high performance liquid chromatography−mass spectrometry (LC–MS) and electrospray ionization mass spectrometry, respectively. Moreover, the limitation standard for the toxic aconitines in 20 APCMs was established based on the holistic weight toxicity (HWT) evaluation and the Chinese Pharmacopoeia severally, and turned out that HWT-based toxicity evaluation results were closer to the real clinical applications. Hence, a more accurate and reliable APCM toxicity limitation was established and expected to play an important guiding role in clinics. The current study extended the power of ambient MS as a method for the direct quantification of molecules in complex samples, which is commonly required in pharmaceutical analysis, food safety control, public security, and many other disciplines.     

Analysis of (dichloromethylene) bisphosphonate in urine by capillary gas chromatography—mass spectrometry

An anion exchange extraction method of bisphosphonates from urine is described. More than 90% of the (dichloromethylene) bisphosphonate (Cl₂MBP, clodronate) was recovered from urine. The extracted bisphosphonates were trimethyl-silylated and analysed with capillary gas chromatography—mass spectrometry (GC/MS). The mass spectrometric techniques used were electron ionization (EI), ammonia chemical ionization (CI), ammonia CI tandem mass spectrometry and methane negative chemical ionization (NCI). The limit of detection of Cl₂MBP was 25 pg/injection in the NCI/selective ion recording (SIR)-mode. At 100 ng ml⁻¹ of Cl₂MBP the precision of the whole assay method was 17.9% (N = 6). The NCI/SIR technique offers a sensitive and highly selective method for the quantitation of Cl₂MBP in urine.     

电喷雾离子阱质谱分析17-烯丙胺基-17-脱甲基格尔德霉素及其代谢物

目的 旨在确立17-烯丙胺基-17-脱甲基格尔德霉素（17AAG）及其代谢物的质谱分析条件.方法 采用电喷雾离子阱质谱,同时用液质联用方法,对17AAG及其主要活性代谢产物17-氨基-17-脱甲基格尔德霉素（17AG）进行了探讨,同时分离并考察了其在大鼠胆汁中的代谢物及其一般裂解规律.结果 在电喷雾负离子模式下,所产生的准分子离子峰均为[M-H]-峰,在MS2质谱中均产生失去43 u的碎片峰,在MS3质谱中均产生失去98 u,18 u的碎片峰.结论 这些裂解特征可用于该类化合物的结构鉴定及代谢物的分析.     

Profiling impurities and degradants of butorphanol tartrate using liquid chromatography/mass spectrometry and liquid chromatography/tandem mass spectrometry substructural techniques

A rapid and systemic strategy based on liquid chromatography/mass spectrometry (LC/MS) profiling and liquid chromatography/tandem mass spectrometry (LC/MS/MS) substructural techniques was utilized to elucidate the degradation products of butorphanol, the active ingredient in stadol® NS. This strategy integrates, in a single instrumental approach, analytical HPLC, UV detection, full-scan electrospray mass spectrometry, and tandem mass spectrometry to rapidly and accurately elucidate structues of impurities and degradants. In these studies, several low-level degradation products were observed in long-term storage stability samples of bulk butorphanol. The resulting analytical profile includes information on five degradants including molecular structures, chromatographic behavior, molecular weight, UV data, and MS/MS substructural information. The degradation products formed during long-term storage of butorphanol tartrate included oxidative products proposed as 9-hydroxy- and 9-keto-butorphanol, norbutorphanol, a ring-contraction degradant, and Δ1, 10-butorphanol. These methodologies are applicable at any stage of the drug product cycle from discovery through to development. This library of butorphanol degradants provides a foundation for future development work regarding product monitoring, as well as a useful diagnosite tool for new degradation products.