支原体对抗生素的敏感性

近年来出现了多种新型抗生素。它们比目前常用的抗生素优点更多,而且很多种除抗细菌外,还能抑制和杀灭支原体,这对于细胞培养工作者无疑是一大“福音”。但目前,这些抗生素大都还没有在国内广泛使用,而且有关它们的文献分散在各种医学和药学杂志上,查阅不便。为了有针对性地设计杀灭支原体的配方,有必要将国际上发表的有关文献加以整理,以便应用。     

4株乳酸菌对14种抗生素的敏感性试验

实验模拟饲料中益生菌与抗生素接触环境,对益生菌的存活比率进行量化表示,对用于饲料添加剂的4株乳酸菌进行抗生素敏感性试验。结果表明除极个别乳酸菌和抗生素的组合外,乳酸菌均在此种实验方法下对抗生素有很好的耐受性,实验结果对微生物饲料添加剂的研制及其在畜禽生产中的应用具有指导意义。     

一种快速有效分析烟草花冠中花青素苷的方法

花青素苷（anthocyanins）成分和含量的分析是花色研究的重要内容之一。该文利用高效液相色谱-电喷雾离子化-质谱技术（HPLC-ESI-MS）, 建立了一种快速有效地分析烟草（Nicotiana tabacum）花冠中花青素苷成分及含量的方法。在保证良好分离效果的基础上, 将分析时间缩短至15分钟, 大大提高了分析速度, 降低了成本, 并成功应用于转基因烟草花冠中花青素苷成分的分析。为不具备高效分析仪器（如超高效液相色谱-串联质谱（UPLC-MS/MS））的一般实验室研究花青素苷合成相关基因在烟草中的异源表达提供了一种有效且实用的分析方法。     

LC-MS/MS检测癫痫患者神经递质类氨基酸

目的：建立液相色谱串联质谱同位素内标法检测神经递质类氨基酸并用于癫痫患者临床评价。方法：选用AAA-C18柱色谱柱,以乙腈水（含有0.01%七氟丁酸、0.1%甲酸）为流动相,采用梯度洗脱进行分离,血浆样品用iTRAQ-115衍生化试剂处理后,加入iTRAQ-114衍生化的氨基酸内标并进样,选用3200QTRAP型质谱仪的多重反应监测（MRM）扫描方式进行检测。疾病组与健康组的统计采用t检验和主成份分析。结果：疾病组和健康组氨基酸测定结果显示：Trp、GABA两组间没有显著性差异（P〉0.05）,Arg、Gly、Ser、Tau、Asp、Glu、EtN、两组间有显著性差异（P〈0.05）,通过PCA分析显示,疾病组与健康组之间差异明显,Asp、Glu、Ser等是引起差异的主要氨基酸。结论：试验方法灵敏、专属性强,并初步的用于癫痫患者体内氨基酸评价。     

LC-MS/MS 检测癫痫患者神经递质类氨基酸

目的:建立液相色谱串联质谱同位素内标法检测神经递质类氨基酸并用于癫痫患者临床评价.方法:选用AAA-C18柱色谱柱,以乙腈水(含有0.01%七氟丁酸、0.1%甲酸)为流动相,采用梯度洗脱进行分离,血浆样品用iTRAQ-115衍生化试剂处理后,加入iTRAQ-114衍生化的氨基酸内标并进样,选用3200QTRAP型质谱仪的多重反应监测(MRM)扫描方式进行检测.疾病组与健康组的统计采用t检验和主成份分析.结果:疾病组和健康组氨基酸测定结果显示:Trp、GABA两组间没有显著性差异(P＞0.05),Arg、Glv、Ser、Tau、Asp、Glu、EtN、两组间有显著性差异(P＜0.05),通过PCA分析显示,疾病组与健康组之间差异明显,Asp、Glu、Ser等是引起差异的主要氨基酸.结论:试验方法灵敏、专属性强,并初步的用于癫痫患者体内氨基酸评价.     

4株沙漠小球藻对几种常用抗生素的敏感性研究

目的:对分离自新疆沙漠的4株小球藻GTD8A1、GTD4A-1、GTD7C-2和TLD6B进行几种常用基因工程抗生素的敏感性研究,以期筛选出沙漠小球藻基因工程中选择标记的抗生素。方法:运用藻株在液体培养过程中藻体颜色变化和血球板细胞计数的方法,系统性研究沙漠小球藻对几种常用基因工程抗生素的敏感性。结果:4株沙漠小球藻对卡那霉素和链霉素都非常敏感,敏感浓度(细胞致死浓度,下同)均为25μg/m L;对氯霉素也很敏感,GTD8A1、GTD7C-2和TLD6B的敏感浓度也均为25μg/m L,GTD4A-1的敏感浓度为100μg/m L;4株沙漠小球藻对氨苄西林和头孢霉素的敏感性都不是很明显,在一定的浓度范围,氨苄西林和头孢霉素对藻细胞的生长还具有促进作用,当氨苄西林和头孢霉素的浓度很高时才表现出一定的抑制作用。结论:卡那霉素和链霉素可作为沙漠小球藻基因工程选择标记中的2种抗生素,为今后建立其遗传转化系统奠定了基础。     

三株螺原体对抗生素敏感性的体外测定

对我国新分离的两个螺原体和典型的柑桔僵化病螺原体,在体外条件下对10种抗生素的敏感性进行了测定;其敏感程度用最小生长抑制浓度(MIC)和最小致死浓度(MBC)表示。结果表明,红霉素、四环素、土霉素等有较强的生长抑制和致死作用;其次是氯霉素、夹竹桃霉素、庆大霉素;作用较弱的有链霉素、新霉素、卡那霉素;青霉素在试验浓度范围内(0.01—2000μg/ml)无作用。三株供试菌中,以柑桔僵化病螺原体(Sc189)对10种抗生素最敏感,新分离的两个螺原体CH-1和CB-2的敏感性相似。同时,不同培养时间对MIC测定     

三株螺原体对抗生素敏感性的体外测定

对我国新分离的两个螺原体和典型的柑桔僵化病螺原体,在体外条件下对10种抗生素的敏感性进行了测定;其敏感程度用最小生长抑制浓度(MIC)和最小致死浓度(MBC)表示。结果表明,红霉素、四环素、土霉素等有较强的生长抑制和致死作用;其次是氯霉素、夹竹桃霉素、庆大霉素;作用较弱的有链霉素,新霉素、卡那霉素;青霉素在试验浓度范围内(0.01—2000μg/ml)无作用。三株供试菌中,以柑桔僵化病螺原体(Sc189)对10种抗生素最敏感,新分离的两个螺原体CH-1和CB-2的敏感性相似。同时,不同培养时间对MIC测定     

不同时期分离的淋病奈瑟菌对5种抗生素的敏感性研究

目的：研究杭州市不同时期分离的林病奈瑟菌对5种抗生素的敏感性。方法：用琼脂烯释法对门诊1998年7月～2001年10月分离的285株淋病奈瑟菌进行青霉素、四环素、壮观霉素、氧氟沙星及头孢曲松的最小抑菌浓度（MIC）测定,并就PPNG株和non-PPNG株菌的MIC值进行了比较。结果：青霉素、四环毒等5种抗生素MIC值2001年～1998年两者之间比较,青霉素、四环素等5种抗生素MIC值2001年与1998年两者之间比较,除壮观霉素没有变化外,其余都有显著变化,而氧氟沙星变化最大,PP-NG菌株与非PPNG株菌MIC值除氧氟沙星外均存在差异。结论：表明了杭州市淋病奈瑟菌5种抗生素耐药性变迁,以便为临床选择用药提供依据。     

Multidimensional proteomic analysis of photosynthetic membrane proteins by liquid extraction-ultracentrifugation-liquid chromatography-mass spectrometry

The membrane protein components of photosystem I (PSI) and II (PSII) from different species were prefractionated by liquid extraction and sucrose gradient ultracentrifugation and subsequently analyzed by reversed-phase high-performance liquid chromatography-electrospray ionization-mass spectrometry (RP-HPLC-ESI-MS) using poly-(styrene-divinylbenzene)-based monolithic capillary columns. The analytical method was shown to be very flexible and enabled the identification of antenna proteins as well as most of the proteins of the reaction center from PSI and PSII in various plant species with few RP-HPLC-ESI-MS analyses necessitating only minor adaptations in the gradients of acetonitrile in 0.05% aqueous trifluoroacetic acid. The membrane proteins, ranging in molecular mass (Mr) from 4196 (I protein) to more than 80,000 (PSI A/B) as well as isoforms were identified on the basis of their intact Mr and comparison with Mr deduced from known DNA or protein sequences. High quality mass spectra enabled the identification and quantitation of the nonphosphorylated and phosphorylated reaction center subunits D1, D2, and CP43 of PSII, containing five to seven membrane-spanning alpha-helices. Because of its high flexibility and suitability for proteins having a very wide range of Mr and hydrophobicities, the method is generally applicable to the analysis of complex mixtures of membrane proteins.     

On-line characterization of monoclonal antibody variants by liquid chromatography-mass spectrometry operating in a two-dimensional format

Recombinant monoclonal antibodies (MAbs) have become one of the most rapidly growing classes of biotherapeutics in the treatment of human disease. MAbs are highly heterogeneous proteins, thereby requiring a battery of analytical technologies for their characterization. However, incompatibility between separation and subsequent detection is often encountered. Here we demonstrate the utility of a generic on-line liquid chromatography–mass spectrometry (LC-MS) method operated in a two-dimensional format toward the rapid characterization of MAb charge and size variants. Using a single chromatographic system capable of running two independent gradients, up to six fractions of interest from an ion exchange (IEC) or size exclusion (SEC) separation can be identified by trapping and desalting the fractions onto a series of reversed phase trap cartridges with subsequent on-line analysis by mass spectrometry. Analysis of poorly resolved and low-level peaks in the IEC or SEC profile was facilitated by preconcentrating fractions on the traps using multiple injections. An on-line disulfide reduction step was successfully incorporated into the workflow, allowing more detailed characterization of modified MAbs by providing chain-specific information. The system is fully automated, thereby enabling high-throughput analysis with minimal sample handling. This technology provides rapid data turnaround time, a much needed feature during product characterization and development of multiple biotherapeutic proteins.     

5-Methyltetrahydrofolic acid and folic acid measured in plasma with liquid chromatography tandem mass spectrometry: applications to folate absorption and metabolism

We describe a liquid chromatography (LC) tandem mass spectrometry (MS-MS) method for the determination of 5-methyltetrahydrofolic acid (5-methylTHF) and folic acid concentrations and enrichments in human plasma. It was used to study absorption and initial metabolism in five volunteers with two simultaneously administered oral test doses ([(13)C(6)]folic acid in capsules and [(2)H(2)]folic acid in a drink). [(13)C(5)]5-methylTHF and [(2)H(4)]folic acid were used as internal standards. Plasma samples (2 ml) were purified using folate binding protein affinity columns, followed by a concentration step. After LC separation, folates were detected using positive electrospray ionization MS-MS under multiple reaction monitoring conditions. Calibrations were linear for 5-methylTHF over the range 1.2 x 10(-11) (=limit of detection) to 3.2 x 10(-7)mol/L and for folic acid over the range 5 x 10(-10) (=limit of detection) to 4.5 x 10(-8)mol/L. For 5-methylTHF concentration in plasma, intraassay coefficient of variation was within 8.6% (and for unlabeled 5-methylTHF it was within 2.8%) and interassay coefficient of variation was within 9.0%. For folic acid concentrations these coefficient of variations were within 7.5% and within 6.5%, respectively. The [(13)C(6)] and [(2)H(2)] isotopomers of folic acid and 5-methylTHF were measured in the plasma of each volunteer for 8h. After accounting for the time delay due to capsule opening, the modeling results showed no significant differences in absorption time, first pass effect, and elimination rate in the folic acid test doses in capsule or drink. We conclude that LC-MS-MS offers increased sensitivity for quantification of plasma concentrations and enrichments of 5-methylTHF and folic acid and is applicable to stable-isotope studies in humans.     

A sensitive liquid chromatography-tandem mass spectrometry method for the quantification of mometasone furoate in human plasma

A robust, rapid, selective and sensitive liquid chromatography-negative atmospheric pressure chemical ionization (LC-(APCI(-))-MS-MS) method has been developed for the quantification of mometasone furoate (MF) in human plasma utilizing a solid-phase extraction clean-up step and 13C-fluticasone propionate as internal standard. The intra- and inter-day coefficients of variation were < or = 15% and the lower limit of quantification (LLOQ) was 15 pg/ml. This method is ideally suited for pharmacokinetic investigations of low MF levels following inhalation of MF.     

High-performance liquid chromatography-mass selective detection assay for adenine released from a synthetic RNA substrate by ricin A chain

High-performance liquid chromatography (HPLC) and selected ion monitoring mass spectrometry (MS) were used to develop a quantitative assay for adenine released from a synthetic RNA substrate by ricin A chain, which contains the toxin's N-glycosidase activity. Because ricin and ricin A chain have potential applications as biotherapeutics and bioweapons, assays are needed to evaluate potency and potential inhibitors of activity. The detection limit for adenine was 0.02 microM (2.4 ng/ml), and the standard curve was linear up to 27.3 microM. The lower limit of quantitation was 0.27 microM and was reproducible throughout this range. Reaction characterization showed that most adenine was released by 5h and that the reaction could not be fully stopped with formic acid concentrations up to 0.75 mM (the maximum typically used for HPLC-MS). Injections were made at 2-min intervals, 10 injections could be performed before the column was backflushed, and no ricin A chain was observed in the column effluent. This assay would also be useful for ricin since ricin A chain did not pass through the HPLC column. With minor modifications to this system, the assay should provide rapid, sensitive, selective, and quantitative assessment of the activity of most ribosome-inactivating proteins. In addition, further chromatographic and mass spectrometric improvements could reduce sample requirements and analysis times.     

The simple and rapid quantification method for L-3,4-dihydroxyphenylalanine (L-DOPA) from plant sprout using liquid chromatography-mass spectrometry

L-3,4-dihydroxyphenylalanine (L-DOPA) is one of the important secondary metabolites of plants and has been used for various purposes, such as in clinical treatment for Parkinson’s disease and dopamine-responsive dystonia. In plants, L-DOPA is a precursor of many alkaloids, catecholamines, and melanin; the L-DOPA synthesis pathway is similar to that in mammals. L-DOPA acts as an allelochemical, has an important role in several biological processes, such as stress response and metabolism, in plants. L-DOPA is widely used in the clinical treatment as well as a dietary supplement or psychotropic drug, understanding of biosynthesis of L-DOPA in plant could lead to a stable supply of L-DOPA. This paper describes an improved method for simple and rapid quantification of L-DOPA content using liquid chromatography-tandem mass spectrometry. The standard quantitative methods for L-DOPA require multiple purification steps or relatively large amounts of plant material. In our improved method, quantification of L-DOPA was possible with extract of one–two pieces of cotyledon without any partitioning or column for purification. The endogenous L-DOPA (approximately 4,000 µg g⁻¹ FW (fresh weight)) could be detected from the one pieces of cotyledon of the faba bean sprout using this method. This method was also effective for samples with low endogenous amounts of L-DOPA such as broccoli, Japanese white radish, pea, and red cabbage sprouts. Therefore, this improved method will allow to measurement of L-DOPA content easily and accurately from a small amount of plant tissue and contribute to understanding biosynthesis, catabolism, and transport of L-DOPA.     

Quantitative analysis of adenosine using liquid chromatography/atmospheric pressure chemical ionization-tandem mass spectrometry (LC/APCI-MS/MS)

Adenosine-secreting cellular brain implants constitute a promising therapeutic approach for the treatment of epilepsy. To engineer neural stem cells for therapeutic adenosine delivery, a reliable and fast analytical method is necessary to quantify cell-based adenosine release. Here we describe the development, optimization and validation of adenosine measurement using liquid chromatography–atmospheric pressure chemical ionization-tandem mass spectrometry (LC–APCI-MS/MS). LC–MS/MS in positive ion mode used selected reaction monitoring at m/z of 268.2/136.1 and 302.2/170.0 for adenosine and the internal standard, respectively. The bias was within 15% of the nominal value and evaluation of precision showed a relative standard deviation lower than 15% for all measured concentrations. The lower limit of quantification of adenosine was 15.6 ng/ml. Freeze and thaw stability and processed sample stability also fulfilled the acceptance criteria. Evaluation of the matrix effect showed that the method is not affected by relative matrix effects. The major advantages of this method are the absence of an extraction phase and the combination of the high selectivity and sensitivity characteristic for the LC–MS/MS technique, with a short run time of 4.5 min. These results demonstrate that this method is a useful tool to measure adenosine concentrations in culture medium released from stem cells in vitro.     

Identification of benzophenanthridine alkaloids from Bocconia arborea by gas chromatography-mass spectrometry

A methanol extract of the bark of Bocconia arborea was fractionated on silica gel and the fractions analysed using gas chromatography coupled with mass spectrometry (GC-MS). Several benzophenanthridine alkaloids were identified including dihydrosanguinarine, oxysanguinarine, 11-acetonyldihydrochelerythrine, dihydrochelerythrine, chelerythrine, chelerythridimerine and angoline as the principal constituents. The results show that the direct GC-MS analysis of these alkaloids is possible with a clear distinction between the compounds. The technique is shown to be a valuable tool and an alternative technique to classical phytochemical procedures permitting the fast analysis of alkaloids mixtures.     

Analysis of benidipine enantiomers in human plasma by liquid chromatography-mass spectrometry using a macrocyclic antibiotic (Vancomycin) chiral stationary phase column

We used a novel chromatographic method to rapidly and simply characterize the pharmacokinetics of benidipine enantiomers in human plasma. The stereoisomers of benidipine were extracted from plasma using diethylether under alkaline conditions. After evaporating the organic layer, the residue was reconstituted in the mobile phase (methanol:acetic acid:triethylamine, 100:0.01:0.0001, v/v/v). The enantiomers in the extract were separated on a macrocyclic antibiotic (Vancomycin) chiral stationary phase column. The mobile phase was eluted at 1 ml/min and was split by an interface. One-fifth of the eluent was used to quantify both isomers in a tandem mass spectrometer in multiple reaction-monitoring mode. The coefficient of variation of the precision of the assay was less than 8%, the assay accuracy was between 93.4 and 113.3%, and the limit of detection was 0.05 ng/ml for 1 ml of plasma. The method described above was used to measure the concentration of both benidipine enantiomers in plasma from healthy subjects who received a single oral dose of a racemate of 8 mg benidipine. The C(max) and AUC(inf) values of (+)-alpha benidipine were higher than those of (-)-alpha benidipine by 1.96- and 1.85-fold, respectively (p<0.001), whereas, the T(max) and t(1/2) for each of the benidipine stereoisomers were not significantly different.     

Protein identification in cerebrospinal fluid using packed capillary liquid chromatography Fourier transform ion cyclotron resonance mass spectrometry

The identification and characterization of proteins in complex biological samples such as body fluids, require powerful and reliable tools. Mass spectrometry is today one of the most important methods in such research. This paper reports on the results from the first experiment where a tryptic digest of cerebrospinal fluid was analyzed applying reversed phase liquid chromatography coupled on-line to a 9.4 T Fourier transform ion cyclotron resonance mass spectrometer. In total, 70 204 peaks were detected, which originated from 16 296 isotopic clusters corresponding to 6551 unique peptide masses. From these masses, 39 proteins were identified in the sample. The amount of sample required for one experiment corresponds to 32 microL of cerebrospinal fluid.