Liquid chromatography/mass spectrometry for metabonomics investigation of the biochemical effects induced by aristolochic acid in rats: the use of information-dependent acquisition for biomarker identification

The toxic effects of oral administrations of nephrotoxic and carcinogenic aristolochic acid (AA) to male Sprague-Dawley rats were investigated by using high-performance liquid chromatography coupled with a quadrupole time-of-flight mass spectrometer. Analysis of the urine and plasma samples revealed distinct changes in the biochemical patterns in the AA-dosed rats. After peak finding and alignment, principal component analysis (PCA) and partial least-squares discriminant analysis (PLS-DA) were used for multivariate data analysis. Potential biomarkers were studied by high-resolution mass spectrometry (MS) and tandem mass spectrometry (MS/MS) analyses. The MS/MS spectra of all endogenous metabolites satisfying the pre-defined criteria were acquired in a single information-dependent acquisition (IDA) analysis, demonstrating that IDA was an efficient approach for structural elucidation in metabonomic studies. Citric acid and a glucuronide-containing metabolite were observed as potential biomarkers in rat urine. A significant increase in plasma creatinine concentration was also observed in the AA-dosed rats, which indicated that AA induced an adverse effect on the renal clearance function.     

Determination of 12 potential nephrotoxicity biomarkers in rat serum and urine by liquid chromatography with mass spectrometry and its application to renal failure induced by Semen Strychni

In previous nephrotoxicity metabonomic studies, several potential biomarkers were found and evaluated. To investigate the relationship between the nephrotoxicity biomarkers and the therapeutic role of Radix Glycyrrhizae extract on Semen Strychni‐induced renal failure, 12 typical biomarkers are selected and a simple LC–MS method has been developed and validated. Citric acid, guanidinosuccinic acid, taurine, guanidinoacetic acid, uric acid, creatinine, hippuric acid, xanthurenic acid, kynurenic acid, 3‐indoxyl sulfate, indole‐3‐acetic acid, and phenaceturic acid were separated by a Phenomenex Luna C₁₈ column and a methanol/water (5 mM ammonium acetate) gradient program with a runtime of 20 min. The prepared calibration curves showed good linearity with regression coefficients all above 0.9913. The absolute recoveries of analytes from serum and urine were all more than 70.4%. With the developed method, analytes were successfully determined in serum and urine samples within 52 days. Results showed that guanidinosuccinic acid, guanidinoacetic acid, 3‐indoxyl sulfate, and indole‐3‐acetic acid (only in urine) were more sensitive than the conventional renal function markers in evaluating the therapeutic role of Radix Glycyrrhizae extract on Semen Strychni‐induced renal failure. The method could be further used in predicting and monitoring renal failure cause by other reasons in the following researches.     

Metabolic characterization of the early stage of hepatic fibrosis in rat using GC‐TOF/MS and multivariate data analyses

The aim of this study was to explore the changes in the urine metabolic spectrum in rats with the early stage of liver fibrosis using gas chromatography–time of flight/mass spectrometry (GC‐TOF/MS), try to search for potential biomarkers and elucidate the probably metabonomic pathogenesis. The early stage of liver fibrosis was established with a single subcutaneous injection of carbon tetrachloride twice each week for 4 weeks continuously. At the end of the experiment, GC‐TOF/MS technology with multivariate statistical approaches such as principal component analysis, partial least squares‐discriminant analysis and orthogonal partial least squares‐discriminant analysis was used to analyze the changes in the metabolic spectrum trajectory and identify potential biomarkers. Twelve potential biomarkers in the model group, such as succinic acid, threonine and lactose, were selected, which indicate that the metabonomic pathogenesis of the early stage of liver fibrosis may be related to disorders of energy metabolism, amino acid metabolism and fatty acid metabolism.     

Development and validation of a gas chromatography/mass spectrometry metabonomic platform for the global profiling of urinary metabolites

This paper presents a simple and reliable gas chromatography/mass spectrometry (GC/MS) method for the metabonomic analysis of human urine samples. The sample preparation involved the depletion of excess urea via treatment with urease and subsequent protein precipitation using ice-cold ethanol. An aliquot of the mixture was separated, dried, trimethylsilyl (TMS)-derivatized and 1.0 microL of the derivatized extract was injected into the GC/MS system via split injection (1:10). Approximately 150 putative metabolites belonging to different chemical classes were identified from the pooled human urine samples. All the identified metabolites were selected to evaluate precision and stability of the GC/MS assay. More than 95% of the metabolites demonstrated good reproducibility, with intra-day and inter-day precision values below 15%. Metabolic profiling of 53 healthy male and female urine samples in combination with pattern recognition techniques was performed to further validate the GC/MS metabolite profiling assay. Principal component analysis (PCA) followed by orthogonal partial least squares analysis (OPLS) revealed differences between urinary metabolite profiles of healthy male and female subjects. This validated GC/MS metabolic profiling method may be further applied to the metabonomic screening of urinary biomarkers in clinical studies. Copyright (c) 2008 John Wiley & Sons, Ltd.     

Lysophosphatidylcholine Biomarkers of Lung Cancer Detected by Ultra-performance Liquid Chromatography Coupled with Quadrupole Time-of-flight Mass Spectrometry

Centrifugal ultrafiltration after methanol extraction of whole plasma was used as an optimal condition for the preparation of blood plasma before metabonomic studies. The plasma samples from 102 lung cancer patients and 34 healthy volunteers were prepared with this approach. With ultra-performance liquid chromatography(UPLC) coupled with quadrupole time-of-flight mass spectrometry(Q-TOF MS) analysis, the samples were investigated in order to find potential disease biomarkers. After data acquisition, orthogonal signal correction partial least squares models were built to differentiate the healthy volunteers from lung cancer patients and to identify metabolites that showed significantly different expression between the two groups. Several metabolite ions were identified as potential biomarkers according to the variable importance in the project(VIP) value in both ion modes. Five lysophosphatidylcholines were further identified as specifically lysoPC 16:0, isomer of lysoPC 16:0, lysoPC 18:0, lysoPC 18:1 and lysoPC 18:2. These results suggest that UPLC coupled with Q-TOF MS is an effective technique for the analysis of plasma metabolites in metabonomic studies.     

Nontargeted urine metabolomics analysis of the protective and therapeutic effects of Citri Reticulatae Chachiensis Pericarpium on high-fat feed-induced hyperlipidemia in rats

In this study, we focused on studying the changes in urine metabolites in hyperlipidemic rats using ultra-performance liquid chromatography coupled with quadrupole time-of-fight mass spectrometry (UPLC–Q-TOF/MS) and metabolomics, as well as the effect of Citri Reticulatae Chachiensis Pericarpium (CRCP) on hyperlipidemia. These urine samples were examined by UPLC–Q-TOF/MS to obtain MS data. The MS data were analyzed by principal component analysis and partial least squares-discriminant analysis to identify the differential metabolites. CRCP reduced the body weight and levels of triglycerides, total cholesterol and low-density lipoprotein cholesterol and abnormally decreased high-density lipoprotein cholesterol in hyperlipidemic rats, which were significantly raised by a high-fat diet. Twenty-seven potential biomarkers were identified within the complex sample matrix of urine. Fourteen biomarkers increased in the hyperlipidemia rats compared with normal rats. Meanwhile, 13 biomarkers decreased. CRCP reversed abnormal changes in biomarkers, including 5-l -glutamyl-taurine, 5-aminopentanoic acid, cis-4-octenedioic acid and 2-octenedioic acid. These biomarkers show that hyperlipidemia is related to the metabolic pathways of taurine and hypotaurine metabolism, fatty acid biosynthesis , and arginine and proline metabolism . CRCP mainly prevents hyperlipidemia by intervening in these metabolic pathways.     

Metabonomic study of biochemical changes in urinary of type 2 diabetes mellitus patients after the treatment of sulfonylurea antidiabetic drugs based on ultra‐performance liquid chromatography/mass spectrometry

A metabonomic study on biochemical changes in the urine of type 2 diabetes mellitus (T2DM) patients after the treatment of sulfonylurea (SU) antidiabetic drugs was performed. An ultra‐performance liquid chromatography/mass spectrometry (UPLC/MS) method was used to generate metabolic fingerprints for the metabonomic analysis of urinary samples obtained from 20 T2DM patients without any drug treatment and 20 T2DM patients treated with SU antidiabetic drugs and 20 normal glucose tolerance subjects. The resulting data were subjected to chemometric analysis (principal component analysis and partial least squares discriminant analysis) to investigate the effect of SU antidiabetic drugs on urinary metabolite profiles of T2DM patients. Biomarkers such as xanthine, phenylalanine, tryptophan, hippurate, phenylacetylglutamine, carnitine C8:1, carnitine C10:3, uric acid and citrate were found to be responsible for the separation of T2DM and SU‐treated groups, which indicates a potential effect of SU on energy metabolism, Tricarboxylic acid (TCA) cycle, gut microflora metabolism and oxidative stress. The study may be helpful to the understanding of the action of mechanism of SU antidiabetic drugs. Copyright © 2014 John Wiley & Sons, Ltd.     

Therapeutic Effects of Selaginella tamariscina on the Model of Acute Gout with Hyperuricemia in Rats Based on Metabolomics Analysis

Gout is a disease of purine metabolic disorders which results from long‐term hyperuricemia and the sodium urate deposition in and around the joints. Selaginella tamariscina (ST ) is an important traditional Chinese herbal medicine and is used for the treatment of gout and hyperuricemia. In this study, the rat model of acute gout with hyperuricemia was established by intraperitoneal injection of xanthine and oxonic acid potassium salt and articular injection monosodium urate (MSU ). The effect of ST in the treatment of gout was investigated by measuring joint swelling, the expression of IL ‐1β in serum and histological changes of joint by haematoxylin eosin (H&E) staining. Subsequently, urine metabolomics analysis for biomarkers discovery in acute gout with hyperuricemia rats was performed by the ultra‐performance liquid chromatography‐electrospray ionization quadruple time‐of‐flight mass spectrometry (UPLC‐ESI‐QTOF /MS ) combined with chemometric approach. Principal component analysis (PCA ) and orthogonal partial least squares‐discriminant analysis (OPLS‐DA ) were used to detect potential biomarkers. A total of 18 potential biomarkers were identified mainly including tryptophan metabolism; tyrosine metabolism; lysine methylation; pyrimidine metabolism; purine metabolism; TCA cycle and fatty acid metabolisms. This study indicates that ST could efficiently ameliorate the disease of acute gout with hyperuricemia in rats. The related metabolic biomarkers could provide useful information and the metabolic mechanism could be used for further study about the model of acute gout with hyperuricemia in rats.     

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。     

A rapid screening approach to metabonomics using UPLC and oa-TOF mass spectrometry: application to age, gender and diurnal variation in normal/Zucker obese rats and black, white and nude mice

The use of Ultra Performance Liquid Chromatography (UPLC), with a rapid 1.5 minute reversed-phase gradient separation on a 1.7 microm reversed-phase packing material to provide rapid "high throughput" support for metabonomic screening is demonstrated. The peak capacity and the number of marker ions detected using these fast UPLC separations and oa-TOF MS was found to be similar to that generated by conventional HPLC-MS methods with a 10 minute separation. The UPLC-MS methodology was applied to the analysis of urine samples from rodents, including normal and Zucker obese rats and three strains of mice (of both sexes), and was found to provide rapid discrimination between age, strain, gender and diurnal variation.     

Targeted metabolomic analysis of 33 amino acids and biogenic amines in human urine by ion‐pairing HPLC‐MS/MS: Biomarkers for tacrolimus nephrotoxicity after renal transplantation

Calcineurin inhibitor nephrotoxicity, especially for the widely used tacrolimus, has become a major concern in post‐transplant immunosuppression. Multiparametric amino acid metabolomics is useful for biomarker identification of tacrolimus nephrotoxicity, for which specific quantitative methods are highlighted as a premise. This article presents a targeted metabolomic assay to quantify 33 amino acids and biogenic amines in human urine by high‐performance liquid chromatography coupled with tandem mass spectrometry. Chromatographic separation was carried out on an Agilent Zorbax SB‐C₁₈ column (3.0 × 150 mm, 5 μm) with addition of an ion‐pairing agent in the mobile phase, and MS/MS detection was achieved in both the positive and negative multiple reaction monitoring modes. Good correlation coefficients (r² > 0.98) were obtained for most analytes. Intra‐ and inter‐day precision, stability, carryover and incurred sample reanalysis met with the acceptance criteria of the guidance of the US Food and Drug Administration. Analysis on urine from healthy volunteers and renal transplantation patients with tacrolimus nephrotoxicity confirmed symmetric dimethylarginine and serine as biomarkers for kidney injury, with AUC values of 0.95 and 0.81 in receiver operating characteristic analysis, respectively. Additionally, symmetric dimethylarginine exhibited a tight correlation with serum creatinine, and was therefore indicative of renal function. The targeted metabolomic assay was time and cost prohibitive for amino acid analysis in human urine, facilitating the biomarker identification of tacrolimus nephrotoxicity.     

核磁共振技术测试不同性别大鼠尿液代谢物的研究

采用核磁共振波谱技术测试不同性别大鼠尿液代谢物,分析性别因素对大鼠尿液代谢成分的影响.大鼠尿液核磁共振氢谱（1HNMR谱）结果采用主成分分析（principal component analysis,PCA）和正交偏最小二乘判别分析（orthogonal to partial least squares discriminant analysis,OPLS-DA）方法分析,得到不同性别大鼠尿液中的差异性代谢物.PCA分析结果显示2组尿液代谢成分有明显的差异,进一步进行OPLS-DA分析可以判别出2组尿液中具有差异性的代谢物.结果显示,雌性大鼠尿液中的丙氨酸、缬氨酸、鸟氨酸等氨基酸类以及乙酸、硫胺、氨基马尿酸、苯乙胺、氧氨嘧啶等代谢物含量高于雄性大鼠,差异有统计学意义（p〈0.05）.雄性大鼠尿液中的甲胺、二甲胺、三甲胺、肌酸酐、尿囊素、延胡索酸、甲酸等代谢物则明显高于雌性大鼠,差异有统计学意义（p〈0.05）.性别因素对大鼠尿液中的代谢成分有一定的影响.     

Urinary metabolite profiling provides potential differentiation to explore the mechanisms of adjuvant‐induced arthritis in rats

To explore the pathogenesis of rheumatoid arthritis (RA) from the perspective of metabolomics, gas chromatography time‐of‐flight mass spectrometry (GC‐TOF/MS) technology was used to observe changes in the metabolic profiles of urine output from rats with adjuvant‐induced arthritis (AA). Sprague–Dawley rats were randomly divided into a control group and an experimental group, with eight in each. Rats in the experimental group were induced by intracutaneous innoculation of 0.1 mL Freund's complete adjuvant to right paws. On day 20 after immunization, the metabolic profiles between rat control and experimental groups were compared by combining GC‐TOF/MS technology with multivariate statistical approaches, including principal component analysis, partial least squares discriminant analysis and orthogonal projections to latent structures–discriminant analysis. Nine potential biomarkers were identified, including 2,2‐dimethylsuccinic acid, tartronic acid, dehydroshikimic acid, hippuric acid, adenine, phenaceturic acid, l ‐dopa, 1,4‐dihydroxy‐2‐naphthoic acid and melibiose. The findings indicate that the rats with AA are disturbed in metabolism of purine, amino acid, fat and energy. This study also demonstrates that the dysfunction in a range of biosynthetic and catabolic pathways, which leads to increased oxygen free radicals and inflammation, could cause underlying pathogenesis of RA. Copyright © 2016 John Wiley & Sons, Ltd.     

Liquid chromatography–mass spectrometry methods for urinary biomarker detection in metabonomic studies with application to nutritional studies

The effects of sample preparation and chromatographic method differences on the classification and recovery of metabolic biomarkers from UPLC‐MS measurements on urine samples of humans exposed to different dietary interventions have been investigated. Eight volunteers consumed three high‐fat meals (rich in saturated, monounsaturated and polyunsaturated fatty acids, respectively) in randomized order with a washout period in between. For each participant, urine samples were obtained prior to and at three timed intervals after each meal. Samples were processed either by dilution (1 : 4) or by liquid–liquid extraction and then run under two different gradient conditions. For each analysis method, a total of 96 observations (eight participants, four time points, three diets) were measured. The total ion count chromatograms were analyzed using partial‐least‐squares discriminant analysis. All three dietary classes could be discriminated irrespective of sample preparation and chromatographic method. However, the main discriminating metabolites varied according to sample preparation, indicating that sample treatment and chromatographic conditions influence the ability to extract biomolecular information. Diluted samples showed higher m/z compounds (ca 400 u) while liquid–liquid extraction samples showed low m/z at the same retention time span. Optimized methods for metabolite identification (e.g. organic acids) were statistically inferior to global screening for mixed compound identification, confirming that multiple compound class‐based metabolic profiles are likely to give superior metabonomic (diagnostic) classification, although great care has to be taken in the interpretation in relation to matrix effects. Copyright © 2009 John Wiley & Sons, Ltd.     

Lysophosphatidylcholine—biomarker of Metformin action: studied using UPLC/MS/MS

An UPLC/MS/MS based metabonomic method was developed and applied to the elucidation of biomarker of metformin action. The plasma metabolite profiling in healthy volunteers before and after per os metformin was determined with UPLC/MS/MS and analyzed by using multivariate statistics. Significant difference in endogenous metabolite profiles was revealed before and after administration of metformin. Four biomarkers found were lysophosphatidylcholines (LPCs), and their structures were tentatively identified to be 16:0 LPC, 18:0 LPC, 18:1 LPC and 18:2 LPC according to the molecular ions information and corresponding fragments of product ion scan. Lysophosphatidylcholine in blood may be involved in metformin treatment. Copyright © 2009 John Wiley & Sons, Ltd.     

Method for rapidly discovering active components in Yupingfeng granules by UPLC‐ESI‐Q‐TOF‐MS

Yupingfeng granules (YPFG) were isolated from a traditional Chinese medicine (TCM) formulation composed of three herbs (Astragali Radix, Atractylodis Macrocephalae Rhizoma, and Saposhnikoviae Radix). This formulation is used in TCM to tonify qi, and it can help strengthen exterior and reduce sweating. Nevertheless, the active components of YPFG remain unclear. In this study, the chemical constituents of YPFG were systematically characterized by ultra‐performance liquid chromatography coupled with electrospray ionization/ quadrupole time‐of‐flight mass spectrometry (UPLC‐ESI‐Q‐TOF‐MS). Fifty‐eight compounds, namely, 20 flavonoids, 19 saponins, nine organic acids, four volatile coumarins, three lactones, one alkaloid, and two other components, were identified. In addition, the constituents of YPFG with the potential for in vivo bioactivities following oral administration were investigated in Sprague–Dawley rats. Thirteen compounds, namely, 11 flavonoid‐related and 2 saponin‐related components, were detected in rat plasma. After enriching flavonoids and saponins in YPFG by extraction, the extracts and YPFG were administrated to immunosuppressed rats, respectively. Plasma samples were analyzed by UPLC‐ESI‐Q‐TOF‐MS, and principal component analysis (PCA) confirmed that the extracts had similar effects to YPFG. This method could discover active ingredients in YPFG quickly and provide a scientific basis for quality control and mechanism research.     

Metabonomics study of atherosclerosis rats by ultra fast liquid chromatography coupled with ion trap-time of flight mass spectrometry

An ultra fast liquid chromatography coupled with IT-TOF mass spectrometry (UFLC/MS-IT-TOF) metabonomic approach was employed to study the plasma and urine metabolic profiling of atherosclerosis rats. Acquired data were subjected to principal component analysis (PCA) for differentiating the atherosclerosis and the control groups. Potential biomarkers were screened by using S-plot and were identified by the accurate mass and MSⁿ fragments information obtained from UFLC/MS-IT-TOF analysis. 12 metabolites in rat plasma and 8 metabolites in urine were identified as potential biomarkers. Concentrations of leucine, phenylalanine, tryptophan, acetylcarnitine, butyrylcarnitine, propionylcarnitine and spermine in plasma and 3-O-methyl-dopa, ethyl N2-acetyl-l-argininate, leucylproline, glucuronate, t6A N(6)-(N-threonylcarbonyl)-adenosine and methyl-hippuric acid in urine decreased in atherosclerosis rats. Ursodeoxycholic acid, chenodeoxycholic acid, LPC (C16:0), LPC (C18:0) and LPC (C18:1) in plasma and hippuric acid in urine were in higher levels in atherosclerosis rats. The alterated metabolites demonstrated abnormal metabolism of phenylalanine, tryptophan, bile acids and amino acids. This research proved that metabonomics is a promising tool for disease research.     

基于超高效液相色谱-高分辨质谱和渗透压校正样本浓度变异性的尿液代谢组学分析

尿液是代谢组学研究中主要关注的体液样本之一。尿液样本中的代谢物浓度受饮食、疾病等因素影响变异较大,这极大阻碍了高质量组学数据的获取和可靠生物标志物的鉴定。研究为克服尿液样本的浓度变异性,在原始数据采集前,根据样本渗透压的大小,针对性地调整进样量或者稀释样本,从而确保代谢组学分析样本的渗透压与进样量的乘积相当,再经超高效液相色谱-高分辨质谱技术(UPLC-HRMS)分析,采用总离子丰度或总有用峰面积(MSTUS)对数据集进行归一化处理。研究利用临床样本及其梯度稀释的溶液,对该方法与现有研究普遍使用的方法进行了比较,随后通过先天性肾积水患者及健康志愿者的尿液样本做了进一步的方法学验证。数据集经校正后,峰面积RSD<30%的提取峰数量增加,主成分分析结果较校正前有更高的组内聚集和组间分群效应,正交偏最小二乘判别分析的统计模型更不易过拟合。与肌酐比较,渗透压值与质谱信号间呈现了更好的线性关系。以上结果表明,数据采集前通过样本渗透压进行校正,能有效消除因样本本身代谢物浓度变化引起的组内差异,提高方法的重复性和统计模型的可靠度。以渗透压为基准的校正策略,比肌酐校正法适用范围更广,结果也更准确。研究可对后续各类来源的尿液代谢组学研究提供数据归一化的指导和参考。