基于超高效液相色谱-四极杆飞行时间质谱联用技术的血瘀模型大鼠血浆代谢组学分析

采用盐酸肾上腺素加冰水浴建立急性血瘀大鼠模型，使用超高效液相色谱-四极杆飞行时间质谱（UPLC-Q-TOF/MS）检测空白对照组与血瘀模型组中血浆代谢物，用主成分分析（PCA）、有监督偏最小二乘法判别分析（PLS-DA）及正交偏最小二乘法判别分析（OPLS-DA）对代谢组学数据进行多维统计分析，筛选潜在生物标志物。与对照组相比，在血瘀模型组大鼠血浆中检测出46个差异代谢物，血瘀模型组中乙酰胆碱、N6，N6，N6-三甲基-L-赖氨酸、胞嘧啶、乙酰肉碱等21个代谢物显著上调，吲哚丙酸、LysoPC（14：0）等25个代谢物显著下调，可能与脂质代谢、半乳糖代谢、亚油酸代谢、不饱和脂肪酸生物合成、糖酵解、花生四烯酸代谢等通路有关。代谢产物可作为血瘀证研究中的重要标记物，该研究结果有助于揭示血瘀证的发病机制，可为临床血瘀疾病的诊断及选用药物治疗提供思路，为后续治疗手段提供参考依据。     

基于拟靶向液相色谱-质谱联用的胃癌患者血清代谢组分析

胃癌是一种高发的恶性肿瘤,是癌症相关死亡的第二大病因。早期筛查是提高患者生存率的有效手段,但目前临床上尚缺乏实现胃癌无创筛检的可靠标志物。本研究采用了基于液相色谱-质谱联用的拟靶向代谢组学方法分析了20例胃癌患者及40例正常人血清代谢组,以期发现新的潜在代谢标志物。代谢组数据的主成分分析和偏最小二乘法数据分析结果显示,胃癌患者与健康人群的血清代谢组存在明显的差异,结合非参数检验进一步筛选并定性出57个差异代谢物。其中二氢胆固醇经验证组样本验证,具有成为胃癌代谢标志物的潜力。本研究在发现胃癌的潜在代谢标志物的同时,也为胃癌患者代谢分型提供了重要的科学依据。     

基于超高效液相色谱-质谱的胶质瘤患者血浆代谢组学研究

采用超高效液相色谱-四极杆-静电场轨道阱质谱(UHPLC-Q-OrbitrapHRMS)技术对胶质瘤患者和正常对照人群的血浆进行代谢轮廓分析,筛选胶质瘤代谢标志物,为其发病机制阐明和临床早期诊断提供科学依据。通过对UHPLC-Q-OrbitrapHRMS采集得到的谱图进行峰识别、峰匹配和去噪等处理后,应用主成分和正交偏最小二乘-判别分析法对代谢组学数据进行统计分析,筛选VIP1.0及P0.05的差异代谢物,并进一步对其诊断能力进行评价。结果显示,胶质瘤患者的血浆代谢轮廓发生明显变化,发现并鉴定得到10个差异代谢物,其中亮氨酸、缬氨酸、色氨酸、胆碱和牛磺酸在胶质瘤患者血浆中含量降低,组氨酸、柠檬酸、乳酸、肌酸和丙酮酸含量升高,与正常对照组比较具有显著性差异(P0.05),提示氨基酸和能量等代谢异常可能对胶质瘤的发生发展具有重要影响。此外,各差异性代谢物对胶质瘤均显示出较好的诊断能力(AUC0.8),可作为潜在诊断标志物。     

铜催化2-（2,2-二溴乙烯基）苯酚化合物与苯酚衍生物的串联醚化反应

首次报道铜催化2-（2,2-二溴乙烯基）苯酚类化合物与苯酚衍生物的串联醚化反应. 以2-（2,2-二溴乙烯基）苯酚衍生物与苯酚类化合物为起始原料,醋酸铜为催化剂、碳酸铯作为碱,在N,N-二甲基甲酰胺中通过一锅法合成了24个未见文献报道的2-芳氧基苯并呋喃类化合物. 此合成方法具有原料易得、操作简便、分离纯化简单和无需昂贵配体等优点.     

基于液相色谱-串联质谱的结肠癌血清氧固醇标志物筛选

结肠癌(CC)是全球常见恶性肿瘤之一,发病率呈逐年上升趋势,目前没有有效的标志物用于疾病早期诊断和干预跟踪。胆固醇及其氧化衍生物氧固醇在众多恶性肿瘤发生发展中发挥关键作用。该研究采用液相色谱-串联质谱(LC-MS/MS)技术,对CC临床血清样本中胆固醇及相关10种氧固醇代谢物进行了定性定量分析,并采用偏最小二乘判别分析(PLS-DA)和正交偏最小二乘判别分析(OPLS-DA)进行多元统计分析,发现上述目标代谢物能够较好地区分CC组与健康对照组。为防止数据过拟合,该研究在PLS-DA模型各代谢物变量投影重要性(VIP)基础上,结合最优组分数及K-均值聚类结果,筛选得到3种代谢标志物。通过受试者操作特征曲线(ROC)的曲线下面积(AUC)分析,发现筛选得到的3种潜在标志物联合预测CC达到0.998,说明模型性能优良。GO(基因本体论)富集分析显示3种潜在标志物主要分布在内质网和包被囊泡上,参与胆固醇代谢、运输、低密度脂蛋白重塑等生物进程,发挥胆固醇运输活性和低密度脂蛋白颗粒受体结合的分子功能。KEGG(京都基因与基因组百科全书)通路分析显示3种潜在标志物富集于类固醇生物合成、PPAR(过氧化物酶体增殖物激活受体)信号通路及ABC(ATP结合盒)转运等通路上。该研究为寻找CC标志物及进一步阐明胆固醇及氧固醇在CC发病过程中的作用奠定了一定的基础。     

基于气相色谱-质谱联用和图模型分析的脂代谢异常患者血浆代谢组学研究

利用气相色谱-质谱联用技术(GC-MS)和图模型分析方法,寻找脂代谢异常患者可能的血浆代谢标志物群。采用GC-MS技术对37例脂代谢异常患者和10例健康人的血浆样品进行分析,得到血浆代谢物的表达谱。偏最小二乘-判别分析(Partial least squares-discriminant analysis,PLS-DA)得分图可区分脂代谢异常患者与健康人,运用PLS-DA载荷图及t检验发现有9个代谢物在两组间存在显著性差异。经NIST谱库检索,它们分别为缬氨酸、甘氨酸、丙氨酸、焦谷氨酸、葡萄糖醛酸、半乳糖、甘露糖、亚油酸和甘油。在脂代谢异常患者血浆中,除甘油浓度显著高于健康人外,其余8种代谢物浓度均明显低于健康人。图模型分析结果发现这些代谢物与脂代谢异常临床常用诊断指标之间具有很好的相关性。它们可能是脂代谢异常疾病早期诊断和预后新的特异性代谢标志物群。     

基于核磁共振技术的临床尿毒症的代谢组学研究

采用核磁共振技术与统计学分析方法,结合尿液的生化指标,进行临床尿毒症尿样的代谢组学研究。结果表明,尿毒症患者和健康对照人群尿样的代谢轮廓存在明显差异,通过核磁共振氢谱鉴定出70种代谢物,其中20种具有显著差异。与健康人群相比,尿毒症患者尿液中2-羟基异丁酸、3-羟基丁酸、丙酮、丁酸、谷氨酸、肌氨酸、肌酐、赖氨酸、N,N-二甲基组氨酸、柠檬酸、天冬酰胺、乙醇和乙醇胺的含量明显偏低,而支链氨基酸(包括亮氨酸、缬氨酸、异亮氨酸)、牛磺酸、乳酸、α-葡萄糖和β-葡萄糖的含量明显偏高。这些代谢物涉及氨基酸代谢、能量代谢和脂质代谢中的多条代谢途径,可作为尿毒症代谢影响的潜在的生物标志物,有助于理解尿毒症发病的生化机制。     

基于液相色谱/电喷雾飞行时间质谱技术的结石症尿液代谢组学探讨

探索了一种简便的尿液样品前处理流程,通过反相液相色谱(RPLC)与亲水相互作用色谱(HILIC)相结合的高效液相色谱/电喷雾飞行时间质谱的联用技术(HPLC/ESI-TOFMS),建立了泌尿系结石症的尿液代谢组学模型,研究结石症患者尿液样品中的代谢物浓度水平与健康人对照组间的差异情况。采用主成分分析(PCA)及偏最小二乘法-判别分析(PLS-DA)等多变量数据分析方法,寻找结石症患者组和对照组之间的差异性代谢谱。模型结果显示:结石症患者尿液代谢物水平与健康对照组具有明显差异,寻找其中潜在的差异性标志物群组,有望将该模型用于泌尿系结石症的快速预测和诊断。     

pH响应性含氟三嵌段共聚物的制备及性质研究

以苄醇(BzOH)与氢化钾(KH)反应形成的氧阴离子作为引发剂, 依次引发甲基丙烯酸-2-(N,N-二甲氨基)乙酯(DMAEMA, 简称DMA)、甲基丙烯酸-2-(N,N-二乙氨基)乙酯(DEAEMA, 简称DEA)和甲基丙烯酸-(2,2,3,3,4,4,5,5-八氟)戊酯(OFPMA)进行氧阴离子聚合, 获得含氟三嵌段共聚物PDMA-b-PDEA-b-POFPMA和PDEA-b-PDMA-b-POFPMA. 共聚物的化学结构可以通过不同单体的加料顺序和各种单体的投料量加以控制. 通过¹H NMR, ¹⁹F NMR和GPC测试, 研究聚合物的结构、分子量及分子量分布. 利用表面张力、荧光探针法、Zeta电位和透射电镜等测试方法, 研究共聚物在不同pH值的水溶液中的聚集行为.     

基于UPLC-Q/TOF MS技术的痛风患者血清代谢组学研究

采用超高效液相色谱-飞行时间质谱联用技术（UPLC-Q/TOF MS）对恩施土家族苗族自治州60例健康志愿者（对照组）和65例痛风患者（痛风组）的血清样本建立代谢图谱,基于主成分分析及正交偏最小二乘判别分析对所得数据进行模式识别,并结合变量权重投影分析及火山图筛选出痛风患者的血清代谢标志物。通过数据分析和数据库检索,共筛选出63种差异代谢物,其中27种代谢物显著上调（P <0.05）,36种代谢物显著下调（P <0.05）,主要包括甘油磷脂类、氨基酸类及胆碱等成分。首先,对以上差异代谢物进行受试者工作特征曲线（ROC）分析,其中曲线下面积（AUC）大于0.8的14种代谢物是诊断效能较好的代谢物;然后对筛选的63种差异代谢物进行代谢通路富集分析,以Impact> 0.1且P <0.05为标准,得到影响最大的代谢通路主要有甘油磷脂代谢、醚性脂质代谢、亚油酸代谢、半胱氨酸和蛋氨酸代谢、花生四烯酸代谢及戊糖和葡萄醛酸的互相转化等。综上,痛风患者和健康对照人群的血清代谢水平有明显差异,差异代谢物的鉴定为痛风的发病机制和早期筛查提供了实验依据。     

Metabolite profiling analysis of hepatitis B virus–induced liver cirrhosis patients with minimal hepatic encephalopathy using gas chromatography-time-of-flight mass spectrometry and ultra-performance liquid chromatography-quadrupole-time-of-flight mass spectrometry

This study used gas chromatography-time-of-flight mass spectrometry (GC-TOFMS) and ultra-performance liquid chromatography-quadrupole TOFMS (UPLC-QTOFMS) metabonomic analytical techniques in combination with bioinformatics and pattern recognition analysis methods to analyze the serum metabolite profiling of hepatitis B virus (HBV)–induced liver cirrhosis patients with minimal hepatic encephalopathy (MHE), to find the specific biomarkers of MHE, to reveal the pathogenesis of MHE, and to determine a promising approach for early diagnosis of MHE. Serum samples of 100 normal controls (NC group), 29 HBV-induced liver cirrhosis patients with MHE (MHE group), and 24 HBV-induced liver cirrhosis patients without MHE [comprising 12 cases of compensated cirrhosis (CS group) and 12 cases of decompensated cirrhosis (DS group)] were collected and employed into GC-TOFMS and UPLC-QTOFMS platforms for serum metabolite detection; the outcome data were then analyzed using principal component analysis and orthogonal partial least squares-discriminant analysis (OPLS-DA). There were no significant differential metabolites between the NC group and the CS group. A series of key differential metabolites were detected. According to the variable influence in projection values and P-values, 60 small-molecule metabolites were considered to be dysregulated in the MHE group (compared to the NC group); 27 of these 60 dysregulated differential metabolites were considered to be the potential biomarkers (see Table 4, marked in bold); 66 small-molecule metabolites were considered to be dysregulated in the DS group (compared to the NC group); 34 of these 66 dysregulated differential metabolites were considered to be the potential biomarkers (see Table 5, marked in bold). According to the fold-change values, 9 of these 27 metabolites, namely valine, oxalic acid, erythro-sphingosine, 4,7,10,13,16,19-docosahexaenoic acid, isoleucine, allo-isoleucine, thyroxine, rac-octanoyl carnitine, and tocopherol (vitamin E), were downregulated in the MHE group (compared to the NC group); the other 18, namely adenine, glycochenodeoxycholic acid, fucose, allothreonine, glycohyocholic acid, glycoursodeoxycholic acid, tyrosine, taurocheno-deoxycholate, phenylalanine, 2-hydroxy-3-methyl-butanoic acid, hydroxyacetic acid, taurocholate, sorbitol, rhamnose, tauroursodeoxycholate, tolbutamide, pyroglutamic acid, and malic acid, were upregulated; 6 of these 34 metabolites were downregulated in the DS group (compared to the NC group), and the other 28 were upregulated, as shown in Table 5. (a) GC-TOFMS and UPLC-QTOFMS metabonomic analytical platforms can detect a range of metabolites in the serum; this might be of great help to study the pathogenesis of MHE and may provide a new approach for the early diagnosis of MHE. (b) Metabonomics analysis in combination with pattern recognition analysis might have great potential to distinguish the HBV-induced liver cirrhosis patients who have MHE from the normal healthy population and HBV-induced liver cirrhosis patients without MHE.     

Comprehensive two-dimensional gas chromatography/time-of-flight mass spectrometry for metabonomics: Biomarker discovery for diabetes mellitus

Comprehensive two-dimensional gas chromatography/time-of-flight mass spectrometry (GC × GC-TOFMS) coupled with pattern recognition methods was applied to analyze plasma from diabetic patients and healthy controls. After sample preparation and GC × GC-TOFMS analysis, collected data were transformed, the peak alignment between different chromatograms was performed to generate the metabolites’ peak table, then orthogonal signal correction filtered partial least-squares discriminant analysis (OSC-PLSDA) was carried out to model the data and discover metabolites with a significant concentration change in diabetic patients. With the method above, diabetic patients and healthy controls could be correctly distinguished based on the metabolic abnormity in plasma. Five potential biomarkers including glucose, 2-hydroxyisobutyric acid, linoleic acid, palmitic acid and phosphate were identified. It was found that elevated free fatty acids were essential pathophysiological factors in diabetes mellitus which reflected either the hyperglycemia or the deregulation of fatty acids metabolism. These potential biomarkers in plasma, e.g. palmitic acid, linoleic acid and 2-hydroxybutyric acid might be helpful in the diagnosis or further study of diabetes mellitus. This study shows the practicability and advantage of GC × GC-TOFMS coupled with data analysis and mining for metabonomics in biomarker discovery.     

Serum metabolomics as a novel diagnostic approach for gastrointestinal cancer

Conventional tumor markers are unsuitable for detecting carcinoma at an early stage and lack clinical efficacy and utility. In this study, we attempted to investigate the differences in serum metabolite profiles of gastrointestinal cancers and healthy volunteers using a metabolomic approach and searched for sensitive and specific metabolomic biomarker candidates. Human serum samples were obtained esophageal (n = 15), gastric (n = 11), and colorectal (n = 12) cancer patients and healthy volunteers (n = 12). A model for evaluating metabolomic biomarker candidates was constructed using multiple classification analysis, and the results were assessed with receiver operating characteristic curves. Among the 58 metabolites, the levels of nine, five and 12 metabolites were significantly changed in the esophageal, gastric and colorectal cancer patients, respectively, compared with the healthy volunteers. Multiple classification analysis revealed that the variations in the levels of malonic acid and l ‐serine largely contributed to the separation of esophageal cancer; gastric cancer was characterized by changes in the levels of 3‐hydroxypropionic acid and pyruvic acid; and l ‐alanine, glucuronoic lactone and l ‐glutamine contributed to the separation of colorectal cancer. Our approach revealed that some metabolites are more sensitive for detecting gastrointestinal cancer than conventional biomarkers. Our study supports the potential of metabolomics as an early diagnostic tool for cancer. Copyright © 2011 John Wiley & Sons, Ltd.     

气相色谱-质谱法同时快速测定血清中5种剧毒灭鼠剂

建立了同时快速测定血清样品中毒鼠强、氟乙酰胺、氟乙酸钠、甘氟Ⅰ与甘氟Ⅱ 5种灭鼠剂的气相色谱-质谱联用法。在pH 2.0条件下,以N,N-二乙基对苯二胺为衍生剂,N,N'-二环己基碳二亚胺为催化剂,氟乙酸钠在室温下振荡衍生5 min,衍生物与毒鼠强、氟乙酰胺、甘氟Ⅰ、甘氟Ⅱ一并被乙酸乙酯萃取,经50 ℃下氮吹浓缩后用气相色谱-质谱同时测定,采用选择离子监测(SIM)模式,基质标准外标法定量。方法选用SLB-IL59离子液体毛细管柱(30 m×0.25 mm×0.20 μm,最高温度:300 ℃),流速1.0 mL/min,经程序升温在15 min内成功地分离了5种灭鼠剂。结果显示,血清中氟乙酰胺的线性范围为0.02~2.0 mg/L,毒鼠强的线性范围为0.02~10 mg/L,其他目标物的线性范围为0.01~1.0 mg/L;检出限为0.001~0.002 mg/L (S/N=3),相关系数R²>0.995。在3个加标水平下,方法加标回收率介于84.0%和110.0%之间,相对标准偏差(n=6)介于2.9%和7.5%之间。方法操作简便、准确,灵敏度高,适于中毒病人的快速诊断检测。     

液相色谱-质谱联用系统在肝硬化不同阶段代谢轮廓研究中的应用

采用高效液相色谱-轨道离子阱质谱联用(HPLC-LTQ Orbitrap XL MS)代谢组学研究平台分析不同阶段肝硬化病人和健康人群的血清标本,获取代谢轮廓。采用模式识别方法结合非参数检验对数据进行分析。研究发现,由肝硬化A级组、B级组、C级组和健康对照组的代谢轮廓构建的正交偏最小二乘判别分析(OPLS-DA)模型(R2（Y）=90.1%, Q2=66.7%),对检测组数据的预测准确率达到93.8%,具有很好的判别能力。从代谢轮廓中可以鉴别出用于区分不同疾病阶段的特异性代谢标志物,如溶血磷脂酰胆碱、甘氨鹅去氧胆酸、半胱氨酸、甘氨酸、氨基己二酸、哌可酸等。研究结果表明: 利用代谢组学方法获得的血清代谢轮廓可以用来构建区分模型和寻找代谢标志物,为乙肝肝硬化的诊断和监测提供支持和依据。     

液相色谱-串联质谱法检测干血点中的同型半胱氨酸

建立了一种高通量液相色谱-串联质谱技术检测干血点(DBS)中同型半胱氨酸(homocycteine, Hcy)的方法。以DBS为样本,homocystine-D8为同位素内标,二硫苏糖醇(DTT)为蛋白结合态Hcy的还原剂,使用含0.1%(v/v)甲酸、0.05%(v/v)三氟乙酸的乙腈溶液萃取。整个前处理过程使用自动移液平台及96孔板实现高通量自动化操作。处理后的样本经过Phenomenex CN柱分离,使用多反应监测模式进行LC-MS/MS分析。结果表明:Hcy的检出限为0.12 μ mol/L(S/N=3),定量限为0.46 μ mol/L(S/N=10)。Hcy在1.16~148.00 μ mol/L范围内线性关系良好,R²=0.994。Hcy的平均回收率为(103.0±4.97)%~(112.0±2.13)%,日内相对标准偏差(RSD)为1.9%~4.6%,日间RSD为1.5%~7.1%。DBS样本在不同温度(-4、-20、22和37℃)下储存不同时间(0、1、2、3、4、5、6、14天)后的稳定性试验显示样本总体RSD<15%,经前处理后的样本在48 h内的稳定性试验显示样本总体RSD<5%。该方法与传统生化分析方法的相关性好(R²=0.9818, n=47)。     

LC–MS analysis of low molecular weight organic acids derived from root exudation

A sensitive method for quantification of citric, fumaric, malic, malonic, oxalic, trans aconitic, and succinic acid in soil- and root-related samples is presented. The method is based on a novel, fast, and simple esterification procedure and subsequent analysis via liquid chromatography–mass spectrometry. Derivatization comprises in situ generation of HCl, which catalyzes the Fischer esterification with benzyl alcohol. As a key advance, the esterification with the aromate allows reversed-phase separation and improves electrospray ionization efficiency. The method provided procedural detection limits of 1 nM for citric, 47 nM for fumaric, 10 nM for malic, 10 nM for malonic, 16 nM for oxalic, 15 nM for succinic, and 2 nM for aconitic acid utilizing 500 μL of liquid sample. The working range was 3 nM to 10 μM for citric acid, 158 nM to 10 μM for fumaric acid, 34 nM to 10 μM for malic acid, 33 nM to 10 μM for malonic acid, 53 nM to 10 μM for oxalic acid, 48 nM to 10 μM for succinic acid, and 6 nM to 10 μM for aconitic acid. Quantification of the analytes in soil-related samples was performed via external calibration of the entire procedure utilizing ¹³C-labeled oxalic and citric acid as internal standards. The robustness of the method was tested with soil extracts and samples from hydroponic experiments. The latter concerned the regulation of phosphorus solubilization via plant root exudation of citric, malic, and oxalic acid.     

基于超高效液相色谱-四极杆飞行时间质谱的缺血性脑卒中的代谢组学研究

采用基于超高效液相色谱-四极杆飞行时间质谱的代谢组学方法,研究缺血性脑卒中患者和健康人群的血浆,分析了缺血性脑卒中的生物标志物.实验收集30例缺血性脑卒中患者和17例健康志愿者的血浆样品,采用主成分分析和正交偏最小二乘判别分析,研究了缺血性脑卒中患者组和健康对照组血浆中的代谢物差异,并进行了代谢通路分析.实验结果表明,患者组血浆中的二氢神经鞘氨醇、植物鞘氨醇等物质含量升高,谷氨酰胺、焦谷氨酸和2-酮丁酸等物质含量降低.结果表明,脑卒中不仅影响了鞘脂类和氨基酸的代谢,还对能量代谢产生了显著的影响.     

新型镍离子亲和色谱介质的制备及其性能考察

以氯甲基化聚苯乙烯交联微球(氯球)为载体基质、N,N-二甲基甲酰胺(DMF)为溶剂、N,N-二异丙基乙胺(DIEA)催化,将合成的Lys(Boc)-OEt、氯乙酸乙酯连接到微球上。经水解使羧基裸露,与Ni²⁺螯合,制得新型镍离子亲和色谱介质。将其应用于His-Lys-Tyr三肽及Phe-His-Thr三肽纯化研究。以不含组氨酸的六肽作为杂质模型,用所制备的镍离子亲和介质进行分离。结果表明,所制备的镍离子亲和介质对两个含组氨酸的三肽具有很好的亲和作用,并实现了与不含组氨酸六肽的分离。     

膀胱癌血清及尿液代谢组学研究

应用代谢组学研究方法,对与膀胱癌(Bladder cancer,BC)发病相关的生物标志物进行筛选,采用液相色谱-电喷雾质谱(LC-ESI/MS)联用技术对20名膀胱癌患者与24名正常人的血清和尿液进行研究.多变量统计分析结果表明,膀胱癌患者和正常人聚类明显,血清和尿液中分别发现13个潜在标志物.其中,(2E,6E,8E)-二十二碳三烯-1-醇、7-((1S,2S)-2-(庚胺)环己基)庚酸和(11E,14E,17E)-三烯-二十碳-1-醇首次在血清中发现,有潜力成为膀胱癌诊断标志物.液相色谱-质谱联用结合多变量分析的代谢组学研究技术在膀胱癌诊断中展现出巨大潜力.