藤黄灰链霉菌099生产麦拓莱霉素与脂肪酸酯的关系

为确定藤黄灰链霉菌099经聚酮途径合成麦拓莱霉素与初级代谢产物脂肪酸酯间的关系,利用柱层析,对发酵液中的脂肪酸酯加以分离纯化,用核磁共振谱、电喷雾电离质谱鉴定其结构,构建该菌株合理的代谢关系,并通过添加脂肪酸的发酵实验加以验证. 结果表明该菌株中不仅存在聚酮合成途径,而且存在脂肪酸酯代谢途径.     

普那霉素发酵与吸附分离耦合过程动力学

根据S.pristinaespiralis E-71普那霉素发酵过程的代谢特性提出了一个针对产物合成期的普那霉素合成动力学方程,结合液膜-孔扩散理论,建立了描述2.5L通气搅拌发酵罐中JD-1大孔吸附树脂吸附分离原位耦合发酵过程的动力学模型,并获得产物生成速率常数、产物抑制常数及液膜传质和孔内扩散系数。结果表明该发酵-吸附耦合过程动力学模型能较好地描述这一原位分离耦合发酵生产普纳霉素过程。在此基础上,考察了树脂添加量及树脂半径对原位分离耦合发酵过程的影响,结果表明添加JD-1树脂能有效移走发酵液主体相中高达90%的普那霉素,且原位分离效果随树脂粒径减小而增强,在一定范围内随树脂添加量的增加而增强,当添加70g.L-1半径为0.195mm的树脂时,普纳霉素产量达到1.01g.L-1,其中被树脂吸附的高于95%。     

二维气相色谱-质谱在香精香料分析中的应用

香精香料成分复杂,常规的气相色谱-质谱联用仪难以满足检测要求。研究采用中心切割二维气相色谱分析烟用香精香料中的香味成分。通过单独切割与多段同时切割实验,以烟酸甲酯和十七烷为内标物质,对其中的香味成分进行定性及半定量分析,实验结果显示,多段同时切割与单独切割实验结果基本一致。相对于单独切割,多段同时切割具有分析时间短、定性定量准确、实验重复性好等优势,在香精香料成分分析方面具有广阔的应用前景。     

超高效液相色谱-串联质谱检测蔬果中多杀霉素的残留

[目的]建立超高效液相色谱-串联质谱(UPLC-MS/MS)检测蔬果中残留分析方法。[方法]采用乙腈提取多杀霉素A、D,UPLC-MS/MS进行定性和定量。[结果]多杀霉素A、D在0.005~0.5 mg/L范围内线性关系良好,相关系数均在0.999 5以上;在0.02~0.5 mg/L添加质量浓度范围内,回收率为72.3%~116.3%,相对标准偏差为1.10%~4.92%;最低检出质量浓度为0.5μg/L。[结论]方法简便、快速、定量准确、灵敏度和精密度好,可实现蔬菜水果中多杀霉素农残分析的要求。     

气相色谱-质谱联用法对3-氯亚氨基二苄中杂质的研究

应用气相色谱-质谱联用方法研究3-氯亚氨基二苄中的杂质。在30.0m×0.25mm,0.5μm的ZB-5MS毛细管柱上,柱温100°C保持3min,后以20°C/min的速率程序升温至250°C,气化室温度270°C,载气氦气流量0.8mL/min的条件下,3-氯亚氨基二苄及其中杂质获得很好分离。通过对各组分质谱图分析,并结合反应过程确定了3-氯亚氨基二苄中的四种杂质分别为亚氨基二苄,甲基亚氨基二苄酮,10-氯-5H-二苯并[b,f]氮杂和3,7-二氯-10,11-二氢-二苯并[b,f]氮杂。     

染料中痕量多氯联苯的气相色谱-质谱联用分析

本文介绍了染料产品中痕量的多氯联苯(PCBs)的定量测定方法。采用正己烷作提取剂,用DB-5MS毛细管色谱柱进行分离,气相色谱-质谱联用仪进行定性分析,外标法定量。由于采用了灵敏度较高的选择离子检测(SIM)方式定量,本方法对目标化合物的检测限可达到0.01mg/kg,回收率为80%～100%,相对标准偏差≤4.0%。     

高效液相色谱-串联质谱同时测定蜂胶中氯霉素、甲砜霉素和氟甲砜霉素残留量

建立了高效液相色谱-串联质谱同时测定蜂胶中氯霉素、甲砜霉素和氟甲砜霉素残留的方法。结果表明:在1~30μg/L范围内氯霉素、甲砜霉素和氟甲砜霉素3种目标物的线性关系良好,相关系数R~2均大于0.997;氯霉素的检出限为0.01μg/kg,甲砜霉素和氟甲砜霉素的检出限均为0.05μg/kg;3种目标物的加标回收率为67.3%~121%、变异系数为0.82%~12.2%。该方法准确可靠、灵敏简便,能够满足对蜂胶中氯霉素类药物的残留确证。     

气相色谱-嗅辨仪/质谱联用法对龙井茶中差异标志物的高通量筛查

龙井茶为中国十大名茶之首,其特征成分决定了其品质差异。因此,研究龙井茶成分群并寻找差异标志物对其质控起着重要的作用。利用气相色谱-嗅辨仪(GC-O)和气相色谱-质谱仪(GC-MS)结合移动窗口偏最小二乘判别分析(MWPLSDA)方法,确定了可以区分不同产地和等级龙井茶的5种差异标志物。通过比较不同色谱变量范围获得的偏最小二乘模式识别(PLSDA)结果,各差异标志物的代表性排序为戊醛>(Z)-3-己烯-1-醇和3-烯烃>苯甲醇>2-乙基-1-己醇。此外,当选择戊醛对应色谱变量区间时,可实现对6种龙井的100%区分。     

紫茉莉根中油脂化合物的气相色谱-质谱分析

为鉴定紫茉莉根的油脂化合物化学成分,采用气相色谱-质谱联用仪分析了新鲜紫茉莉根中和干燥紫茉莉根中的油脂类化学成分,采用锋面积归一化法测定其相对含量。结果表明,从紫茉莉新鲜根中分离得到9种化合物,主要为脂肪酸及其酯、烷烃等化合物。从紫茉莉干燥根的乙醇提取物中分离得到7种化合物,主要为醛类、脂肪酸及其酯等化合物。     

基于液相色谱-质谱联用技术分析鸡胚发育期间血清代谢物的变化

目的应用液相色谱-质谱联用技术(liquid chromatograph-mass spectrometer,LC-MS)分析肉鸡胚胎发育期间血清代谢物的变化。方法将120枚罗氏308肉鸡种蛋随机分为2组,分别于孵化的第14天(E14)和出壳1日龄(H1)收集血清样品。应用LC-MS和高分辨串联质谱SYNAPT G2-XS QTOF,结合主成分分析(principal component analysis,PCA)、偏最小二乘判别分析(partial least squares-discriminate analysis,PLS-DA)、变量投影重要性指标(variable important for the projection,VIP)确定差异代谢产物,并采用代谢物富集分析(metabolite set enrichment analysis,MESA)法分析差异代谢产物涉及的主要代谢途径。结果与E14比较,38个血清代谢物于H1发生显著变化,其中1个显著升高,37个显著下降。差异代谢物主要富集于22个代谢途径中,其中显著富集于蛋白质合成途径的差异代谢产物主要有L-苏氨酸、L-亮氨...     

Detection of differential levels of proteins in the urine of patients with endometrial cancer: analysis using two-dimensional gel electrophoresis and o-glycan binding lectin

Cancers can cause some proteins to be aberrantly excreted or released in the urine, which can be used as biomarkers. To screen for potential biomarkers for endometrial cancer (ECa), the urinary proteins from patients who were newly diagnosed with early stage ECa and untreated controls were separated using two-dimensional gel electrophoresis (2-DE) and followed by image analysis. The altered levels of zinc alpha-2 glycoprotein, alpha 1-acid glycoprotein, and CD59 were detected in the patients compared to the controls. In addition, the urine of the ECa patients was also found to contain relatively lower levels of a fragment of nebulin when the 2-DE separated urinary proteins were probed using champedak galactose binding (CGB) lectin. The different levels of the nebulin fragment were further validated by subjecting the urinary protein samples to CGB lectin affinity chromatography and analysis of the bound fractions by LC-MS/MS. Our data is suggestive of the potential use of the differentially expressed urinary proteins as biomarkers for ECa although this requires further extensive validation on clinically representative populations.     

Differential Proteomic Analysis of Anthers between Cytoplasmic Male Sterile and Maintainer Lines in Capsicum annuum L

Cytoplasmic male sterility (CMS), widely used in the production of hybrid seeds, is a maternally inherited trait resulting in a failure to produce functional pollen. In order to identify some specific proteins associated with CMS in pepper, two-dimensional gel electrophoresis (2-DE) was applied to proteomic analysis of anthers/buds between a CMS line (designated NA3) and its maintainer (designated NB3) in Capsicum annuum L. Thirty-three spots showed more than 1.5-fold in either CMS or its maintainer. Based on mass spectrometry, 27 spots representing 23 distinct proteins in these 33 spots were identified. Proteins down-regulated in CMS anthers/buds includes ATP synthase D chain, formate dehydrogenase, alpha-mannosidas, RuBisCO large subunit-binding protein subunit beta, chloroplast manganese stabilizing protein-II, glutathione S-transferase, adenosine kinase isoform 1T-like protein, putative DNA repair protein RAD23-4, putative caffeoyl-CoA 3-O-methyltransferase, glutamine synthetase (GS), annexin Cap32, glutelin, allene oxide cyclase, etc. In CMS anthers/buds, polyphenol oxidase, ATP synthase subunit beta, and actin are up-regulated. It was predicted that male sterility in NA3 might be related to energy metabolism turbulence, excessive ethylene synthesis, and suffocation of starch synthesis. The present study lays a foundation for future investigations of gene functions associated with pollen development and cytoplasmic male sterility, and explores the molecular mechanism of CMS in pepper.     

Revealing the Hidden Diagnostic Clues of Male Infertility from Human Seminal Plasma by Dispersive Solid Phase Extraction and MALDI-TOF MS

Seminal plasma (SP) mirrors the local pathophysiology of the male reproductive system and represents a non-invasive fluid for the study of infertility. Matrix-Assisted Laser Desorption/Ionization-Time-of-Flight Mass Spectrometry (MALDI-TOF-MS) provides a high-throughput platform to rapidly extrapolate the diagnostic profiles of information-rich patterns. In this study, dispersive solid phase extraction (d-SPE) combined with MALDI-TOF-MS was applied for the first time to the human SP, with the aim of revealing a diagnostic signature for male infertility. Commercially available octadecyl (C₁₈)-, octyl (C₈)-bonded silica sorbents and hexagonal mesoporous silica (HMS) were tested and the robustness of MALDI-TOF peptide profiling was evaluated. Best performances were obtained for C₁₈-bonded silica with the highest detection of peaks and the lowest variation of spectral features. To assess the diagnostic potential of the method, C₁₈-bonded silica d-SPE and MALDI-TOF-MS were used to generate enriched endogenous peptide profiles of SP from 15 fertile and 15 non-fertile donors. Principal component analysis (PCA) successfully separated fertile from non-fertile men into two different clusters. An array of seven semenogelin-derived peptides was found to distinguish the two groups, with high statistical significance. These findings, while providing a rapid and convenient route to selectively enrich native components of SP peptidome, strongly reinforce the prominent role of semenogelins in male infertility.     

3-氨基喹啉与常用基体在MALDI-TOFMS测定天然提取植物多糖的比较

采用3-氨基喹啉(3-AQ)、二元基体3-AQ/2,5-二羟基苯甲酸(DHB)及常用于糖类物质测定的DHB和二元基体DHB/1-羟基异喹啉(1-HIQ)对一天然提取植物糖样进行了基体辅助激光解吸/电离飞行时间质谱(MALDI-TOF MS)分析。实验结果表明:此植物糖样由线形和环形聚己糖组成,其最大分子量至少达3000 Da;3-AQ与常用基体DHB及DHB/HIQ相比较,能提供更准确、更全面的样品组成及分子结构信息,且具有优越于常用基体解吸电离环状糖分子的能力。     

Characterization of paraffin oils and petrolatum using LDI‐TOF MS and principle component analysis

Laser desorption/ionization time‐of‐flight mass spectrometry (LDI‐TOF MS) followed by evaluation of the mass spectra with principal component analysis (PCA) was used for the in‐depth characterization of paraffin oils (mineral oils) and petrolatum (paraffin jelly) samples. These raw materials are liquid and semisolid mixtures of hydrocarbons obtained from petroleum. Mass spectrometric analysis was done using a solvent‐free sample preparation with silver trifluoroacetate. The analysis was carried out on a commercially available matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometer. Mass spectra were evaluated using parameters calculated from the areas of different alkane and cycloalkane species and by PCA. The ratios between specific peak areas were chosen as PCA‐input data instead of the less reproducible absolute peak areas. The principal components enable comparison of a large number of samples and can also be used for visualization of data. In this work, it is clearly demonstrated that combining LDI‐TOF MS and PCA provides a fast and efficient tool for the characterization of paraffin oils and petrolatum. Petrolatum and four different kinds of paraffin oil were analyzed and the results compared with other analytical methods.     

Analysis of platinum adducts with DNA nucleotides and nucleosides by capillary electrophoresis coupled to ESI-MS: indications of guanosine 5'-monophosphate O6-N7 chelation

DNA is the ultimate target of platinum-based anticancer therapy. Since the N7 of guanine is known to be the major binding site of cisplatin and its analogues, adduct formation with model nucleotides, especially 2'-deoxyguanosine 5'-monophosphate (dGMP), has been studied in detail. During the last few years a coupled capillary eletrophoresis/electrospray-ionization mass spectrometry (CE/ESI-MS) method has been advantageously used in order to separate and identify platinum adducts with nucleotides in submillimolar concentrations in aqueous solutions. Beside the bisadduct, [Pt(NH(3))(2)(dNMP)(2)](2-) (NMP=2'-deoxynucleoside 5'-monophosphate), and the well-known monochloro and monohydroxo adducts, [Pt(NH(3))(2)Cl(dNMP)](-) and [Pt(NH(3))(2)(dNMP)OH](-), respectively, a third kind of monoadduct species with a composition of [Pt(NH(3))(2)(dNMP)](-) can be separated by CE and detected through the m/z values measured with ESI-MS. Different experimental setups indicate the existence of an O(6)-N7 chelate, whereas the formation of N7-alphaPO(4) macrochelates or dinuclear species is unlikely. Additionally, offline MS experiments with 2'-deoxyguanosine (dG) and stabilization of the controversially discussed O(6)-N7 chelate by oxidation with hydrogen peroxide support the assumption of the existence of O(6)-N7 chelation.     

Solution- and crystal-phase covalent modification of lysozyme by a purpose-designed organoruthenium complex. A MALDI-TOF MS study of its metal binding sites

Study of the reaction between the transition organometallic complex 4-ruthenocenyl 2,6-dimethylpyrylium tetrafluoroborate and the enzyme hen egg white lysozyme (HEWL) in solution and by diffusion in crystals was performed by use of a combination of spectroscopic and chromatographic methods. Conjugation involving the lysine residues of lysozyme appeared to occur readily, yielding very stable ruthenocenyl pyridinium adducts with average degrees of incorporation ranging from 0.2 to 1.8 metal complexes per protein molecule, depending on reaction conditions. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) revealed that the protein conjugates were in fact mixtures of unmodified, mono-, di- and sometimes tripyridinium adducts. In combination with reversed-phased HPLC, we were able to show that six different monoruthenocenyl pyridinium adducts were formed in solution. This result was confirmed by trypsin digestion of a ruthenocenyl pyridinium conjugate and MALDI-TOF MS analysis of the peptide mixture, which showed that lysines 1, 13, 33, 96, 97 and 116 were involved in the reaction with the pyrylium complex, lysines 13, 33 and 116 being the major binding sites. In the tetragonal crystal state, no binding of the ruthenium complex was shown to occur at lysine 116, owing to steric hindrance at this particular position.     

Sensitivity of positive ion mode electrospray ionization mass spectrometry (ESI MS) in the analysis of purine bases in ESI MS and on-line electrochemistry ESI MS (EC/ESI MS)

A cone-shaped MS inlet and on-line electrochemistry (EC) were used to enhance the ionization efficiency in electrospray ionization mass spectrometry (ESI MS) of purine bases. A pathway of positive ion mode ESI may involve oxidation of purine bases, guanine, adenine, xanthine and hypoxanthine, by 1e⁻, 1H⁺ processes. The electrospray process generates dimers of purine bases that are detected in ESI MS as protonated ions, except for xanthine, for which a protonated radical dimer is detected. Thus electrochemical oxidation of purine bases during ESI may generate reactive radicals that can subsequently dimerize. Dimer formation is facilitated in ESI MS when the carrier solution pH is high. The positive ion mode ESI MS ionization is consistent with the reactivity of the bases toward oxidation. Furthermore, the formation of the protonated ions, and Na⁺ and K⁺ adducts of the bases, expected in positive ion ESI MS, are observed. In addition, unusual H-bonding of purine bases guanine and xanthine is confirmed by ESI MS. Application of low EC voltage to the on-line EC cell in EC/ESI MS improves the sensitivity and correlates with the decrease of the intensity of the dimers, possibly as a result of their further oxidation.     

Description of osmotic dehydration of apple using two-dimensional diffusion models considering shrinkage and variations in process parameters

The main purpose of this paper is to study osmotic dehydration of apple in water and sucrose solutions. The kinetics of water quantity and solid gain are described by two mathematical models using numerical solutions of the two-dimensional diffusion equation in Cartesian coordinates with boundary condition of third kind. For the first model, both the process parameters and the product dimensions have been considered constant. For the second model these quantities have been taken as variable. The numerical solutions have been obtained via the finite volume method with fully implicit formulation. Process parameter estimation, using experimental data, was obtained by an optimizer based on the inverse method. The third-kind boundary condition was shown to be appropriate in view of the fact that the resistances to mass flows on the product surface are not at all negligible. The temperature and concentration of the osmotic solution used in the experiments influenced significantly the kinetics of solid uptake and water quantity as well as the values of estimated parameters. Mathematical model which considers the variations in the process parameters and the product shrinkage presents good physical consistency and well describes the mass migrations.     

Determination of Branched-Chain Amino Acids in Food Supplements and Human Plasma by a CE-MS/MS Method with Enhanced Resolution

In the presented study, a capillary electrophoresis-mass spectrometry method combining high separation efficiency and sensitive detection has been developed and validated, for the first time, to quantify branched chain amino acids (valine, isoleucine, leucine) in commercial food and sport supplement samples and human plasma samples. The separations were performed in a bare fused silica capillary. The background electrolyte was composed of 500 mM formic acid with pH 2.0. The plasma sample pretreatment was realized by simple protein precipitation with acetonitrile. Injection of a short zone of highly basic electrolyte before the sample injection and application of the negative pressure on the separation were accompanied by enhanced resolution of the isobaric amino acids—isoleucine and leucine. The developed method was characterized by favorable validation parameters, such as linearity (r² > 0.99), accuracy and precision, the limit of detection, lower limit of quantification, or robustness. These parameters were more than sufficient for the quantification of branched chain amino acids in various samples. The determined concentrations of branched chain amino acids in food and sports supplements were in very good agreement with the content declared by the manufacturer. The investigated concentrations of branched chain amino acids were in the range 294.68–359.24 µM for valine, 91.76–95.67 µM for isoleucine, and 196.78–251.24 µM for leucine. These concentrations fall within the physiological limits. The developed CE-MS/MS method represents a suitable alternative to traditional approaches used in branched chain amino acid quality control and bioanalysis.