超高效液相色谱/飞行时间质谱法分析尿液中的代谢物用于区分人类性别的研究

尿中的代谢产物可以反映生命个体的生理状态。为了考察在非严格控制条件下(即对志愿者的饮食、生活方式以及样品采集时间等诸多条件不加以控制)基于尿中代谢物的指纹图谱对男女性别进行区分的可行性,采用超高效液相色谱/飞行时间质谱(UPLC/TOF-MS)联用技术分析了31个随机尿样,并用主成分分析法（PCA）和偏最小二乘法判别分析（PLS-DA）两种数据处理方法对数据进行处理,与PCA法比较,PLS-DA法能提高分类效果,并筛选出4种可能的与性别相关的生物标记物。研究结果表明,UPLC/MS联用技术通量高,数据量丰富;模式识别数据处理方法适合于从大量数据中提取信息,两者的结合有利于代谢组学的研究。     

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。     

超高效液相色谱-四极杆-静电场轨道阱质谱联用法用于宫颈癌患者尿液代谢组学研究

利用超高效液相色谱-四极杆-静电场轨道阱质谱联用(UHPLC-Q-Orbitrap MS)方法对宫颈癌(Cervical cancer,CC)患者和健康人(Healthy control,HC)的尿液进行分析,研究宫颈癌患者尿液中的潜在标志物,为其发病机制和诊断提供科学依据。筛选11例宫颈癌患者(Age(45.7±5.6)years)及11例健康人(Age(45.9±3.2)years)尿液样本,采用液相色谱-质谱联用技术对尿液进行测定,通过主成分分析(PCA)和偏最小二乘法-判别分析(OPLS-DA)处理数据,结果表明,两组人群代谢轮廓有显著差别,发现并鉴定了12种潜在的生物标志物,提示特定的肿瘤代谢途径中潜在的代谢标志物可能在宫颈癌发生发展中发挥重要作用。     

血清代谢轮廓分析在评估乙肝肝硬化病程中的应用

采用高效液相色谱-质谱联用(HPLC-MS)作为代谢组学研究平台,分析不同Child-Pugh分级肝硬化病人和健康人群的血清标本,获取代谢轮廓.对数据进行主成分分析(PCA)和正交偏最小二乘判别分析(OPLS-DA),用各组病例的80%作为训练数据构建疾病的OPLS-DA区分模型,以剩余的20%作为检测数据,观察模型对...     

基于液相色谱-质谱联用技术的代谢组学方法研究薄荷烟对大鼠代谢的影响

将基于液相色谱-质谱联用(LC-MS)技术的代谢组学分析平台用于薄荷烟对大鼠代谢影响的研究。分析了3组大鼠的尿样,包括对照大鼠、吸食普通烟大鼠和吸食薄荷烟大鼠,并采用主成分分析(PCA)方法对数据进行模式识别。PCA得分图表明吸食薄荷烟大鼠与对照组大鼠尿样的代谢差异要小于吸食普通烟大鼠。从PCA载荷图中找到并鉴定了犬尿喹啉酸等8种重要代谢物。通过考察代谢物在对照大鼠、吸食薄荷烟大鼠和吸食普通烟大鼠尿样中的相对含量变化,进一步说明了烟草中添加薄荷醇可减少烟草对大鼠代谢的影响。     

阿卡波糖对Ⅱ型糖尿病大鼠尿液代谢轮廓的影响

采用超高效液相色谱-质谱联用(UPLC-MS/MS)方法研究了阿卡波糖对Ⅱ型糖尿病大鼠代谢轮廓的影响, 分析了健康组、 糖尿病模型组和糖尿病给予阿卡波糖组的大鼠尿样, 采用主成分分析法(PCA)和偏最小二乘法-判别分析(PLS-DA)对数据进行分析. PCA得分图表明, 健康组、 糖尿病组和阿卡波糖组的代谢轮廓有显著差别, 根据PLS-DA载荷图筛选, 将对各组分离贡献大的化合物的串联质谱分析数据经Human Metabolome Database(HMDB)和Mass Bank.jp等数据库检索, 进行质谱信息匹配, 鉴定出苯乙酰甘氨酸、 肌酐及葡萄糖酸等8种内源性代谢物为潜在生物标记物.     

基于液相色谱-质谱技术的代谢组学分析方法新进展

液相色谱-质谱联用技术是代谢组学研究领域的主要技术平台之一,近年来基于液相色谱-质谱联用技术的代谢组学分析方法获得了巨大发展。本文结合本研究组在代谢组学方面的研究成果,综述了近年来液相色谱-质谱联用技术在代谢组学分析方法方面的新进展,并对其发展前景进行了展望。综述引用文献41篇。     

小儿肺炎患者尿液的代谢组学初步研究

利用液相色谱-质谱联用法对小儿肺炎( Childhood pneumonia, CP)患者和健康儿童( Healthy control)的尿液进行分析,发现小儿肺炎患者尿液中的潜在标记物,为其发病机制及早期筛查提供科学依据。筛选10例小儿肺炎患者(age 47.72±2.35 months)及10例健康儿童(age 46.65±1.97 months)尿液样本,采用快速高分辨液相色谱四极杆-飞行时间质谱联用( RRLC-Q TOF/MS)技术对其尿液代谢物进行分析,通过主成分分析方法( PCA)对两组代谢物进行分类,并发现潜在生物标记物。 RRLC-Q TOF/MS检测表明,CP组和Healthy Control组尿液代谢物图谱能得到很好的区分,并鉴定了5种生物标记物,提示嘌呤代谢、氨基酸代谢可能在小儿肺炎发生发展中有重要作用。     

基于UPLC-oaTOF-MS的糖尿病及糖尿病肾病的代谢组学研究

以糖尿病患者、糖尿病肾病患者和正常人的血清为研究对象, 采用超高效液相色谱-飞行时间质谱建立其代谢指纹图谱, 并结合主成分分析进行模式识别, 实现患者和正常人的区分, 并试图发现潜在的标志物.     

UHPLC/Q-Orbitrap-MS结合化学计量学方法研究酸枣仁入血成分

建立了一种基于超高效液相色谱-质谱联用技术结合化学计量学快速分析酸枣仁入血成分的方法。以腹腔注射对氯苯丙氨酸(PCPA)建立失眠大鼠模型,给药组连续灌胃给予酸枣仁水提物(30 g/kg)5 d后,分别收集模型组和给药组的血清样品。采用Oasis PRIME HLB 96孔板对血清样品预处理,以超高效液相色谱-静电场轨道离子阱质谱(UHPLC/Q-Orbitrap-MS)进行数据采集,最后采用主成分分析(PCA)、正交偏最小二乘判别分析(OPLS-DA)及变量投影重要性分析(VIP)筛选差异性的原型化合物及代谢产物。共鉴定和推断了20个入血成分,包括11个原型成分及9个代谢产物。研究结果为进一步深入探讨酸枣仁改善失眠的效应物质提供了基础。     

Profiling of grape monoterpene glycosides (aroma precursors) by ultra‐high performance‐liquid chromatography‐high resolution mass spectrometry (UHPLC/QTOF)

A ‘suspect screening analysis’ method for grape metabolomics by ultra‐high performance‐liquid chromatography (UHPLC) and high‐resolution quadrupole‐time of flight (QTOF) mass spectrometry was recently developed. This method was applied to study grape monoterpene glycosides, the main grape aroma precursors. Since standard compounds were not available, they were tentatively identified by overlapping various analytical approaches, in agreement with the indications recommended in mass spectrometry (MS)‐based metabolomics. Accurate mass and isotopic pattern, MS/MS fragmentation, correlation between fragments observed and putative structures and between liquid chromatography coupled with mass spectrometry (LC/MS) and gas chromatography/mass spectrometry signals were studied. Seventeen monoterpene glycosides were identified without performing the hydrolytic artifacts commonly used to study these compounds which may affect sample profile. This is the first time that a detailed study of these aroma precursors has been carried out by direct LC/MS analysis. Copyright © 2014 John Wiley & Sons, Ltd.     

The use of online heart‐cutting high‐performance liquid chromatography coupled with linear ion trap mass spectrometry in the identification of impurities in vidarabine monophosphate

It is difficult to identify unknown impurities in nucleotide analogues by mass spectrometry because mass‐spectrometry‐incompatible mobile phases need to be used to separate the major ingredient from impurities. In this study, vidarabine monophosphate was selected, and unknown impurities were identified by online heart‐cutting two‐dimensional high‐performance liquid chromatography and linear ion trap mass spectrometry. The one‐dimensional reversed‐phase column was filled with a mobile phase containing nonvolatile salt. In two‐dimensional high‐performance liquid chromatography, we used an Acclaim Q1 column with volatile salt, and the detection wavelength was 260 nm. The mass spectrum was scanned in positive‐ and negative‐ion mode. The online heart‐cutting and online demineralization technique ensured that the mobile phase was compatible with mass spectrometry; seven impurities were identified by MS² and MS³ fragments. The mass fragmentation patterns of these impurities were investigated. The two isomers were semiprepared and complemented by nuclear magnetic resonance. The results were further compared with those of normal‐phase high‐performance liquid chromatography with mass spectrometry. The online heart‐cutting two‐dimensional high‐performance liquid chromatography with mass spectrometry was superior in identifying more impurities. The method solves the problem of incompatibility between the mobile phase and mass spectrometry, so it is suitable for identifying unknown impurities. This method may also be used for investigating impurities in other nucleotide analogues.     

Development of a method to screen and isolate bioactive constituents from Stellera chamaejasme by ultrafiltration and liquid chromatography combined with semi‐preparative high‐performance liquid chromatography and high‐speed counter current chromatography

A simple and efficient method based on ultrafiltration with liquid chromatography and mass spectrometry was used for the rapid screening and identification of ligands in the extracts of Stellera chamaejasme. The bound ligands, i.e. daphnoretin, isopimpinellin, chamaechromone, neochamaejasmin A, and chamaejasmine (purity of 96.8, 90.75, 91.41, 93.98, and 98.91%, respectively), were separated by semi‐preparative high‐performance liquid chromatography combined with high‐speed counter‐current chromatography. To the best of our knowledge, this is the first study to report the detection of potent lipoxidase and lactate dehydrogenase inhibitors in Stellera chamaejasme extracts. The results demonstrate that our method of ultrafiltration with liquid chromatography and mass spectrometry combined with mixed chromatography can be used to screen and confirm the bioactivity of all isolated compounds. This method also eliminates the need for separation of inactive compounds, thereby improving efficiency when studying bioactive substances. For some complex mixtures, neither semi‐preparative high‐performance liquid chromatography nor high‐speed counter‐current chromatography can purify all the target active compounds with high purity in a one‐step separation. The combination of the two methods allow for efficient purification of target bioactive compounds with different polarities and physicochemical properties based on their complementary properties.     

Enhancing the selectivity of liquid chromatography–mass spectrometry by using trilinear decomposition on LC‐MS data: An application to three‐way calibration of coeluting analytes in human plasma

The high selectivities of liquid chromatography and mass spectrometry make liquid chromatography–mass spectrometry one of the most popular tools for quantitative analysis in complex chemical, biological, and environmental systems, while the potential mathematical selectivity of liquid chromatography–mass spectrometry is rarely investigated. This work discussed the mathematical selectivity of liquid chromatography–mass spectrometry by three‐way calibration based on the trilinear model, with an application to quantitative analysis of coeluting aromatic amino acids in human plasma. By the trilinear decomposition of the constructed liquid chromatography–mass spectrometry‐sample trilinear model and individual regression of the decomposed relative intensity versus concentration, the proposed three‐way calibration method successfully achieved quantitative analysis of coeluting aromatic amino acids in human plasma, even in the presence of uncalibrated interferent(s) and a varying background. This analytical method can ease the requirements for sample preparation and complete chromatographic separation of components, reduce the use of organic solvents, decrease the time of chromatographic separation, and increase the peak capacity of liquid chromatography–mass spectrometry. As a “green analytical method”, the liquid chromatography–mass spectrometry three‐way calibration method can provide a promising tool for direct and fast quantitative analysis in complex systems containing uncalibrated spectral interferents, especially for the situation where the coelution problem is difficult to overcome.     

Separation and purification of two minor typical diarrhetic shellfish poisoning toxins from harmful marine microalgae via combined liquid chromatography with mass spectrometric detection

A novel method was developed for the purification of two typical diarrhetic shellfish poisoning toxins from toxin‐producing marine microalgae using macroporous resin, high‐speed countercurrent chromatography–mass spectrometry, and semipreparative high‐performance liquid chromatography–mass spectrometry. Analytical high‐performance liquid chromatography–mass spectrometry was used for identification and purity analysis of okadaic acid and dinophysistoxin‐1 because they exhibit no visible or ultraviolet absorption. First, four kinds of macroporous resins were investigated, and HP‐20 macroporous resin was selected for the preenrichment and cleanup of the two target toxins. Second, the resin‐purified sample was further purified using high‐speed countercurrent chromatography coupled with a mass spectrometer. The purities of the obtained okadaic acid and dinophysistoxin‐1 were 89.0 and 83.0%, respectively, as determined through analytical high‐performance liquid chromatography–mass spectrometry. Finally, further purification was carried out using semipreparative high‐performance liquid chromatography with mass spectrometry, and the purities of the final okadaic acid and dinophysistoxin‐1 products were both over 98.0% based on the analytical high‐performance liquid chromatography–mass spectrometry chromatograms and fraction spectra. This work demonstrates that the proposed purification process is a powerful method for the preparation of high‐purity okadaic acid and dinophysistoxin‐1 from toxin‐producing marine microalgae. Moreover, it is particularly important for the purification and preparation of minor toxins that exhibit no visible or ultraviolet absorption from harmful marine algae.     

Rapid and accurate liquid chromatography and tandem mass spectrometry method for the simultaneous quantification of ten metabolic reactions catalyzed by hepatic cytochrome P450 enzymes

The hepatic cytochrome P450 enzymes play a central role in the biotransformation of endogenous and exogenous substances. A sensitive high‐throughput liquid chromatography with tandem mass spectrometry assay was developed and validated for the simultaneous quantification of the products of ten metabolic reactions catalyzed by hepatic cytochrome P450 enzymes. After the substrates were incubated separately, the samples were pooled and analyzed by liquid chromatography with tandem mass spectrometry using an electrospray ionization source in the positive and negative ion modes. The method exhibited linearity over a broad concentration range, insensitivity to matrix effects, and high accuracy, precision, and stability. The novel method was successfully applied to study the kinetics of phenacetin‐O deethylation, coumarin‐7 hydroxylation, bupropion hydroxylation, taxol‐6 hydroxylation, omeprazole‐5 hydroxylation, dextromethorphan‐O demethylation, tolbutamide‐4 hydroxylation, chlorzoxazone‐6 hydroxylation, testosterone‐6β hydroxylation, and midazolam‐1 hydroxylation in rat liver microsomes.     

Rapid screening,identification, and purification of neuraminidase inhibitors from Lithospermum erythrorhizon Sieb.et Zucc. by ultrafiltration with HPLC–ESI‐TOF‐MS combined with semipreparative HPLC

We put forward an efficient strategy based on bioassay guidance for the rapid screening, identification, and purification of the neuraminidase inhibitors from traditional Chinese medicines, and apply to the discovery of anti‐influenza components from Lithospermiun erythrorhizon Sieb.et Zucc. Ultrafiltration with high‐performance liquid chromatography and electrospray ionization time‐of‐flight mass spectrometry was employed for the rapid screening and preliminarily identification of anti‐influenza components from Zicao. Semipreparative high‐performance liquid chromatography was used for the rapid separation and purification of the target compounds. NMR spectroscopy, mass spectrometry, and UV spectroscopy were used for further structural identification, and the activity of the compounds was verified by in vitro assay. Five compounds were found to have neuraminidase inhibitory activity by this method. Subsequently, the five compounds were separated by semipreparative high‐performance liquid chromatography with the purity over 98% for all of them by high‐performance liquid chromatography test. Combined with the NMR spectroscopy, mass spectrometry, and UV spectroscopy data, they were identified as alkannin, acetylalkannin, isobutyrylalkannin, β,β‐dimethylacryloylalkannin and isovalerylalkannin. The in vitro assay showed that all five compounds had good neuraminidase inhibitory activities. These results suggested that the method is highly efficient, and it can provide platform and methodology supports for the rapid discovery of anti‐influenza active ingredients from complex Chinese herbal medicines.     

Fast and sensitive analysis of beta blockers by ultra‐high‐performance liquid chromatography coupled with ultra‐high‐resolution TOF mass spectrometry

This paper presents a method for the determination of acebutolol, betaxolol, bisoprolol, metoprolol, nebivolol and sotalol in human serum by liquid–liquid extraction and ultra‐high‐performance liquid chromatography coupled with ultra‐high‐resolution TOF mass spectrometry. After liquid–liquid extraction, beta blockers were separated on a reverse‐phase analytical column (Acclaim RS 120; 100 × 2.1 mm, 2.2 μm). The total run time was 6 min for each sample. Linearity, limit of detection, limit of quantification, matrix effects, specificity, precision, accuracy, recovery and sample stability were evaluated. The method was successfully applied to the therapeutic drug monitoring of 108 patients with hypertension. This method was also used for determination of beta blockers in 33 intoxicated patients.     

Metabolite profiling and quantification of phytochemicals of Tianma–Gouteng granule in human and rat urine using ultra high performance liquid chromatography coupled with tandem mass spectrometry

Tianma–Gouteng granule has been used for the treatment of hypertension, headache, and stroke in China. However, the metabolism of Tianma–Gouteng granule has not been clear. In the present study, an ultra high performance liquid chromatography coupled with quadrupole time‐of‐flight mass spectrometry method has been developed for rapid identification of 35 prototypes and 43 metabolites in human and rat urine after single oral administration of Tianma–Gouteng granule. The results showed that glucuronidation and sulfation were the main metabolic pathways for flavonoids, alkaloids, iridoidic glycosides, anthraquinones, phenols, and stilbenes that were found in Tianma–Gouteng granule. Moreover, a validated ultra high performance liquid chromatography coupled with tandem mass spectrometry method was applied for the quantification of 14 compounds in rat urine after an oral administration of Tianma–Gouteng granule (2.5 g/kg). During 0–48 h after dosing, the cumulative excretion rates of nine prototype components were 53% for gastrodin, 0.07～1.6% for geniposide, baicalin and baicalein, wogonoside, rhynchophylline and isorhynchophylline, leonurine, and emodin, indicating that urinary excretion is the major way for gastrodin to eliminate from the body. This study provides a comprehensive understanding of metabolism and excretive kinetics of Tianma–Gouteng granule in human and/or rat, and helpful information for screening of its active components in vivo and clinical application.     

Study on chemical constituents and fingerprints of Phellodendron amurense Rupr. based on ultra-performance liquid chromatography–quadrupole–time-of-flight–mass spectrometry

The chemical constituents from Phellodendron amurense Rupr. were characterized systematically by ultra-performance liquid chromatography—quadrupole–time-of-flight–mass spectrometry method for collecting mass spectrometry data, and the fingerprints method was established, providing reference for its quality control. The chromatographic column was ACQUITY UPLC BEH-C₁₈ (100 mm×2.1 mm, 1.7 μm). The mobile phase was acetonitrile-0.1% formic acid aqueous solution and the compounds from P. amurense Rupr. were identified by Qualitative Analysis 10.0 software, reference substance, retention time, mass spectrometry fragmentation pattern and database retrieval. Meanwhile, liquid chromatography–mass spectrometry fingerprint methods of P. amurense Rupr. and Phellodendron chinense Schneid. were established by using the similarity evaluation system of chromatographic fingerprint of traditional Chinese medicine (2012 edition), and the differences were analyzed by multivariate statistical analysis methods. A total of 105 compounds were identified, including 102 alkaloids, two phenolic acids, and one lactone compound. Liquid chromatography–mass spectrometry fingerprint method was established with ideal precision, stability and repeatability, and 12 quality differential markers were recognized between the above two herbs. Liquid chromatography–mass spectrometry method can be used for qualitative analysis of the constituents of Phellodendron amurense Rupr., providing reference for clarifying the material basis and promoting the clinical precision medication and quality evaluation of P. amurense Rupr.