Pressurized CEC coupled with QTOF‐MS for urinary metabolomics

Pressurized CEC (pCEC) coupled with ESI‐QTOF‐MS using a sheathless interface was applied for metabolomics to develop an alternative analytical method for metabolic profiling of complex biofluid samples such as urine. The hyphenated system was investigated with mixed standards and pooled urine samples to evaluate its precision, repeatability, linearity, sensitivity, and selectivity. The applied voltage, mobile phase, and gradient elution were optimized and applied for the analysis of urinary metabolites. Multivariate data analysis was subsequently performed and used to distinguish lung cancer patients from healthy controls successfully. High separation efficiency has been achieved in pCEC due to the EOF. For metabolite identification, the pCEC‐MS separation mechnism was helpful for discriminating the fragment ions of glutamine conjugates from co‐eluted metabolites. Three glutamine conjugates, including phenylacetylglutamine, acylglutamine C8:1, and acylglutamine C6:1 were identified among 16 differential urinary metabolites of lung cancer. Receiver‐operating‐characteristic analysis of acylglutamine C8:1 resulted in an area‐under‐curve value of 0.882. Overall, this work suggests that this pCEC‐ESI‐QTOF‐MS method may provide a novel and useful platform for metabolomic studies due to its superior separation and identification.     

Profiling nucleotides in low numbers of mammalian cells by sheathless CE–MS in positive ion mode: Circumventing corona discharge

Negative ion mode nano-ESI–MS is often considered for the analysis of acidic compounds, including nucleotides. However, under high aqueous separation conditions, corona discharge is frequently observed at emitter tips, which may result in low ion abundances and reduced nano-ESI needle emitter lifetimes. In this work, we introduce a sheathless CE-MS method for the highly efficient and sensitive analysis of nucleotides employing ESI in positive ion mode, thereby fully circumventing corona discharge. By using a background electrolyte of 16 mM ammonium acetate (pH 9.7) a mixture of 12 nucleotides, composed of mono-, di-, and tri-phosphates, could be efficiently analyzed with plate numbers per meter above 220 000 and with LODs in the range from 0.06 to 1.3 nM, corresponding to 0.4 to 8.6 attomole, when using an injection volume of about 6.5 nL only. The utility of the method was demonstrated for the profiling of nucleotides in low numbers of mammalian cells using HepG2 cells as a model system. Endogenous nucleotides could be efficiently analyzed in extracts from 50 000 down to 500 HepG2 cells only. Moreover, apart from nucleotides, also some nicotinamide-adenine dinucleotides and amino acids could be analyzed under these conditions, thereby clearly illustrating the utility of this approach for metabolic profiling of low amounts of biological material.     

CE–MS for anionic metabolic profiling: An overview of methodological developments

The efficient profiling of highly polar and charged metabolites in biological samples remains a huge analytical challenge in metabolomics. Over the last decade, new analytical techniques have been developed for the selective and sensitive analysis of polar ionogenic compounds in various matrices. Still, the analysis of such compounds, notably for acidic ionogenic metabolites, remains a challenging endeavor, even more when the available sample size becomes an issue for the total analytical workflow. In this paper, we give an overview of the possibilities of capillary electrophoresis‐mass spectrometry (CE–MS) for anionic metabolic profiling by focusing on main methodological developments. Attention is paid to the development of improved separation conditions and new interfacing designs in CE–MS for anionic metabolic profiling. A complete overview of all CE–MS‐based methods developed for this purpose is provided in table format (Table 1) which includes information on sample type, separation conditions, mass analyzer and limits of detection (LODs). Selected applications are discussed to show the utility of CE–MS for anionic metabolic profiling, especially for small‐volume biological samples. On the basis of the examination of the reported literature in this specific field, we conclude that there is still room for the design of a highly sensitive and reliable CE–MS method for anionic metabolic profiling. A rigorous validation and the availability of standard operating procedures would be highly favorable in order to make CE–MS an alternative, viable analytical technique for metabolomics.     

CE-MS for metabolomics: Developments and applications in the period 2018–2020

Capillary electrophoresis-mass spectrometry (CE-MS) is now a mature analytical technique in metabolomics, notably for the efficient profiling of polar and charged metabolites. Over the past few years, (further) progress has been made in the design of improved interfacing techniques for coupling CE to MS; also, in the development of CE-MS approaches for profiling metabolites in volume-restricted samples, and in strategies that further enhance the metabolic coverage. In this article, which is a follow-up of a previous review article covering the years 2016–2018 (Electrophoresis 2019, 40, 165–179), the main (technological) developments in CE-MS methods and strategies for metabolomics are discussed covering the literature from July 2018 to June 2020. Representative examples highlight the utility of CE-MS in the fields of biomedical, clinical, microbial, plant and food metabolomics. A complete overview of recent CE-MS-based metabolomics studies is given in a table, which provides information on sample type and pretreatment, capillary coatings, and MS detection mode. Finally, some general conclusions and perspectives are given.     

Gas chromatography/flame ionisation detection mass spectrometry for the detection of endogenous urine metabolites for metabonomic studies and its use as a complementary tool to nuclear magnetic resonance spectroscopy

Metabonomics is a relatively new field of research in which the total pool of metabolites in body fluids or tissues from different patient groups is subjected to comparative analysis. Nuclear magnetic resonance (NMR) spectroscopy is the technology that is currently most widely used for the analysis of these highly complex metabolite mixtures, and hundreds of metabolites can be detected without any upfront separation. We have investigated in this study whether gas chromatography (GC) separation in combination with flame ionisation detection (FID) and mass spectrometry (MS) detection can be used for metabolite profiling from urine. We show that although GC sample preparation is much more involved than for NMR, hundreds of metabolites can reproducibly be detected and analysed by GC. We show that the data quality is sufficiently high--particularly if appropriate baseline correction and time-warping methods are applied--to allow for data comparison by chemometrics methods. A sample set of urines from eleven healthy human volunteers was analysed independently by GC and NMR, and subsequent chemometrics analysis of the two datasets showed some similar features. As judged by NIST database searches of the GC/MS data some of the major metabolites that are detected by NMR are also visible by GC/MS. Since in contrast to NMR every peak in GC corresponds to a single metabolite, the electron ionisation spectra can be used to quickly identify metabolites of interest if their reference spectra are present in a searchable database. In summary, we show that GC is a method that can be used as a complementary tool to NMR for metabolite profiling of urine samples.     

Characterization of chemical constituents and rats metabolites of Shuanghua Baihe tablets by HPLC‐Q‐TOF‐MS/MS

A high‐performance liquid chromatography/quadrupole time‐of‐flight mass spectrometry method was established to detect as many constituents in rat biological fluids as possible after oral administration of Shuanghua Baihe tablets (SBT). An Agilent Poroshell 120 EC‐C₁₈ column was adopted to separate the samples, and mass spectra were acquired in positive and negative modes. First, the fingerprints of SBT were established, resulting in 32 components being detected within 40 min. Among these compounds, 12 were tentatively identified by comparing the retention times and mass spectral data with those of reference standards and the reference literature; the other 20 components were tentatively assigned solely based on the MS data. Furthermore, metabolites in rat plasma and urine after oral administration of SBT were also analyzed. A total of 19 compounds were identified, including 13 prototypes and six metabolites through metabolic pathways of demethylation and glucuronide conjugation. Glucuronidated alkaloids were the main constituents in the plasma, and were then excreted from urine. This is the first systematic study on the metabolic profiling of SBT. Copyright © 2014 John Wiley & Sons, Ltd.     

Biological Sample Injection for Open Tubular Liquid Chromatography

Abstract

Two injection procedures for open tubular liquid chromatography have been evaluated for their ability to directly sample biological components in minute volumes of body fluids. The techniques provided R.S.D.s in peak heights of less than 12% for injections of low nL volumes of human serum spiked with a test compound. A dynamically modified open tubular column was employed to separate the anti-tumor drug doxorubicin hydrochloride and two of its metabolites.     

Metabonomics investigation of human urine after ingestion of green tea with gas chromatography/mass spectrometry, liquid chromatography/mass spectrometry and (1)H NMR spectroscopy

A method using gas chromatography/mass spectrometry (GC/MS), liquid chromatography/mass spectrometry (LC/MS) and (1)H NMR with pattern recognition tools such as principle components analysis (PCA) was used to study the human urinary metabolic profiles after the intake of green tea. From the normalized peak areas obtained from GC/MS and LC/MS and peak heights from (1)H NMR, statistical analyses were used in the identification of potential biomarkers. Metabolic profiling by GC/MS provided a different set of quantitative signatures of metabolites that can be used to characterize the molecular changes in human urine samples. A comparison of normalized metabonomics data for selected metabolites in human urine samples in the presence of potential overlapping peaks after tea ingestion from LC/MS and (1)H NMR showed the reliability of the current approach and method of normalization. The close agreements of LC/MS with (1)H NMR data showed that the effects of ion suppression in LC/MS for early eluting metabolites were not significant. Concurrently, the specificity of detecting the stated metabolites by (1)H NMR and LC/MS was demonstrated. Our data showed that a number of metabolites involved in glucose metabolism, citric acid cycle and amino acid metabolism were affected immediately after the intake of green tea. The proposed approach provided a more comprehensive picture of the metabolic changes after intake of green tea in human urine. The multiple analytical approach together with pattern recognition tools is a useful platform to study metabolic profiles after ingestion of botanicals and medicinal plants.     

Development of a sheathless CE‐ESI‐MS interface

A sheath‐flow interface is the most common ionization technique in CE‐ESI‐MS. However, this interface dilutes the analytes with the sheath liquid and decreases the sensitivity. In this study, we developed a sheathless CE‐MS interface to improve sensitivity. The interface was fabricated by making a small crack approximately 2 cm from the end of a capillary column fixed on a plastic plate, and then covering the crack with a dialysis membrane to prevent metabolite loss during separation. A voltage for CE separation was applied between the capillary inlet and the buffer reservoir. Under optimum conditions, 52 cationic metabolite standards were separated and selectively detected using MS. With a pressure injection of 5 kPa for 15 s (ca. 1.4 nL), the detection limits for the tested compounds were between 0.06 and 1.7 μmol/L (S/N = 3). The method was applied to analysis of cationic metabolites extracted from a small number (12 000) of cancer cells, and the number of peaks detected was about 2.5 times higher than when using conventional sheath‐flow CE‐MS. Because the interface is easy to construct, it is cost‐effective and can be adapted to any commercially available capillaries. This method is a powerful new tool for highly sensitive CE‐MS‐based metabolomic analysis.     

Ganglioside analysis in body fluids by liquid-phase separation techniques hyphenated to mass spectrometry

The expression of gangliosides in central nervous system is a few times higher than in the extraneural tissue, a characteristic highlighting their major role at this level. Although in very low amounts, gangliosides are ubiquitously distributed in body fluids too, where, depending on many factors, including pathological states, their composition fluctuates, thus having diagnostic value. Ganglioside investigation in biological fluids, which, except for cerebrospinal fluid (CSF), may be sampled noninvasively, was for years impeded by the limited sensitivity of the analytical instrumentation available in glycomics. However, because the last decade has witnessed significant developments in biological mass spectrometry (MS) and the hyphenated separation techniques, marked by a major increase in sensitivity, reproducibility, and data reliability, ganglioside research started to be focused on biofluid analysis by separation techniques coupled to MS. In this context, our review presents the achievements in this emerging field of gangliosidomics, with a particular emphasis on modern liquid chromatography (LC), thin-layer chromatography, hydrophilic interaction LC, and ion mobility separation coupled to high-performance MS, as well as the results generated by these systems and allied experimental procedures in profiling and structural analysis of gangliosides in healthy or diseased body fluids, such as CSF, plasma/serum, and milk.     

A metabolomic strategy to screen the prototype components and metabolites of Qingkailing injection in rat urine by high‐performance liquid chromatography with tandem mass spectrometry

Xenobiotic metabolome identification of Chinese herbal formula in biological systems is a very challenging task. Qingkailing injection is a typical Chinese herbal injection, which is wildly used clinically in China. However, the holistic metabolic fate of the ingredient from Qingkailing injection remains unclear. In this work, a metabolomic strategy for comprehensively elucidating Qingkailing injection derived prototype components and metabolites in rat urine conducted by hybrid linear ion trap high‐resolution mass spectrometry was developed. High‐performance liquid chromatography coupled with hybrid linear ion trap high‐resolution mass spectrometry was developed to obtain the urine profiling between the control group and Qingkailing injection treated group. Orthogonal partial least squares discriminate analysis was applied to distinguish the exogenous and the endogenous. In the S‐plot, 37 xenobiotics derived from Qingkailing injection were found in urine, including 18 prototype compounds and 19 metabolites. The characterization of the prototype components and metabolites in rat's urine provided essential data for further pharmacological studies of Qingkailing injection. Our results indicated that the metabolomic approach was an effective tool to discover, screen, and analyze the multiple prototype components and their metabolites from complicated traditional Chinese preparations in vivo.     

Negative mode sheathless capillary electrophoresis electrospray ionization-mass spectrometry for metabolite analysis of prokaryotes

Capillary electrophoresis (CE) was coupled to negative mode electrospray ionisation-mass spectrometry (MS) for separation and detection of phosphorylated and acidic metabolites in extracts of prokaryotes. Unlike previous CE-MS systems for metabolite analysis, a sheathless interface was used to improve sensitivity. To accomplish this, the separation capillary was modified by creating a porous junction near the outlet where the electrospray voltage and cathodic voltage for CE were applied. The outlet of the capillary was pulled to a 5 microm inner diameter to form an electrospray emitter and had a frit fabricated near the exit to prevent clogging. During analysis pressure was applied at the inlet of the separation column to create sufficient flow towards the detector. Limits of detection for 19 metabolites in full scan mode ranged from 20 nM for ADP ribose to 2.5 microM for alpha-ketoglutarate for 40 nL injections. Extracts of Escherichia coli, strain DH5-alpha, were analyzed using this system. In full scan mode, 118 different metabolites were detected. Tandem mass spectrometry was also employed to attempt identification. Reproducible fragmentation of 19 parent peaks was found and 10 of these produced spectra that were consistent with identification obtained from matching to compounds in the MetaCyc database. These results demonstrate the utility of a sensitive CE-MS system for large scale metabolite detection in biological samples.     

阳虚体质者血清和尿液的核磁共振代谢组学

采用基于核磁共振(NMR)的代谢组学方法研究了中医阳虚体质及平和体质(正常对照)的血清和尿液, 分析了阳虚体质的特征代谢物. 实验收集阳虚体质及平和体质各30人的血清及尿液样品, 采用多元统计分析方法研究了阳虚质组和对照组血清和尿液中的内源性代谢差异. 结果表明, 阳虚质血清中乳酸、 极低密度脂蛋白/低密度脂蛋白、N-乙酰糖蛋白、脂肪酸及不饱和脂肪酸的含量降低, 谷氨酰胺、葡萄糖、磷脂酰胆碱及高密度脂蛋白的含量增多; 尿液中肌酐的含量降低, 乳酸、二甲胺、柠檬酸及马尿酸的含量增多. 阳虚体质的潜在生物标志物的发现从代谢组学角度为个体差异提供了新的依据. 阳虚体质与平和体质存在能量代谢、脂代谢及糖代谢的差异以及相关脏腑功能的改变.     

Isotopic labeling-assisted metabolomics using LC–MS

Metabolomics has emerged as the latest of the so-called “omics” disciplines and has great potential to provide deeper understanding of fundamental biochemical processes at the biological system level. Among recent technological developments, LC–HRMS enables determination of hundreds to thousands of metabolites over a wide range of concentrations and has developed into one of the most powerful techniques in non-targeted metabolomics. The analysis of mixtures of in-vivo-stable isotopic-labeled samples or reference substances with un-labeled samples leads to specific LC–MS data patterns which can be systematically exploited in practically all data-processing steps. This includes recognition of true metabolite-derived analytical features in highly complex LC–MS data and characterization of the global biochemical composition of biological samples. In addition, stable-isotopic labeling can be used for more accurate quantification (via internal standardization) and identification of compounds in different organisms.     

Strategy of using microsome-based metabolite production to facilitate the identification of endogenous metabolites by liquid chromatography mass spectrometry

One of the challenges in metabolomic profiling of complex biological samples is to identify new and unknown compounds. Typically, standards are used to help identify metabolites, yet standards cannot be purchased or readily synthesized for many unknowns. In this work we present a strategy of using human liver microsomes (HLM) to metabolize known endogenous human metabolites (substrates), producing potentially new metabolites that have yet to be documented. The metabolites produced by HLM can be tentatively identified based on the associated substrate structure, known metabolic processes, tandem mass spectrometry (MS/MS) fragmentation patterns and, if necessary, accurate mass measurements. Once identified, these metabolites can be used as references for identification of the same compounds in complex biological samples. As a proof of principle, a total of 9 metabolites have been identified from individual HLM incubations using 5 different substrates. Each metabolite was used as a standard. In the analysis of human urine sample by liquid chromatography MS/MS, four spectral matches were found from the 9 microsome-produced metabolite standards. Two of them have previously been documented as endogenous human metabolites, the third is an isomer of a microsome-metabolite and the fourth compound has not been previously reported and is also an isomer of a microsome-metabolite. This work illustrates the feasibility of using microsome-based metabolism to produce metabolites of endogenous human metabolites that can be used to facilitate the identification of unknowns in biological samples. Future work on improving the performance of this strategy is also discussed.     

Qualitative Metabolome Analysis of Human Cerebrospinal Fluid by <Superscript>13</Superscript>C-/<Superscript>12</Superscript>C-Isotope Dansylation Labeling Combined with Liquid Chromatography Fourier Transform Ion Cyclotron Resonance Mass Spectrometry

Metabolome analysis of human cerebrospinal fluid (CSF) is challenging because of low abundance of metabolites present in a small volume of sample. We describe and apply a sensitive isotope labeling LC-MS technique for qualitative analysis of the CSF metabolome. After a CSF sample is divided into two aliquots, they are labeled by ¹³C-dansyl and ¹²C-dansyl chloride, respectively. The differentially labeled aliquots are then mixed and subjected to LC-MS using Fourier-transform ion cyclotron resonance mass spectrometry (FTICR MS). Dansylation offers significant improvement in the performance of chromatography separation and detection sensitivity. Moreover, peaks detected in the mass spectra can be readily analyzed for ion pair recognition and database search based on accurate mass and/or retention time information. It is shown that about 14,000 features can be detected in a 25-min LC-FTICR MS run of a dansyl-labeled CSF sample, from which about 500 metabolites can be profiled. Results from four CSF samples are compared to gauge the detectability of metabolites by this method. About 261 metabolites are commonly detected in replicate runs of four samples. In total, 1132 unique metabolite ion pairs are detected and 347 pairs (31%) matched with at least one metabolite in the Human Metabolome Database. We also report a dansylation library of 220 standard compounds and, using this library, about 85 metabolites can be positively identified. Among them, 21 metabolites have never been reported to be associated with CSF. These results illustrate that the dansylation LC-FTICR MS method can be used to analyze the CSF metabolome in a more comprehensive manner.     

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.     

Surface sampling capillary electrophoresis–mass spectrometry for a direct chemical characterization of tissue and blood samples

Capillary electrophoresis (CE) is a powerful separation tool for non-targeted analysis of chemically complex samples, such as blood, urine, and tissue. However, traditionally CE requires samples in solution for analysis, which limits information on analyte distribution and heterogeneity in tissue. The recent development of surface sampling CE–mass spectrometry (SS-CE–MS) brings these advantages of CE to solid samples and enables chemical mapping directly from the tissue surface without laborious sample preparation. Here, we describe developments of SS-CE–MS to increase reproducibility and stability for metabolite, lipid, and protein extraction from tissue sections and dried blood spots. Additionally, we report the first electrokinetic sequential sample injection for high throughput analysis. We foresee that the wide molecular coverage from a distinct tissue region in combination with higher throughput will provide novel information on biological function and dysfunction.     

Interface solution isoelectric focusing with in situ MALDI‐TOF mass spectrometry

This paper describes a simple and reusable microfluidic device combining solution IEF (sIEF) with MALDI‐TOF MS for rapid proteomic and metabolic analysis of microliter samples. The device contains two glass plates with nanoliter microwell arrays, which can be assembled to form a fluidic path for sIEF separation, and reconfigured for dividing separated bands. One microliter samples can be loaded and separated by sIEF into static bands in 10～30 min. After a slipping operation, the static IEF bands can be divided into nanoliter droplets in microwells without mobilization, and the device can be opened for in situ MALDI‐TOF MS detection without loss of separation resolution. The performance of the device is characterized by separating and identifying intact proteins. The applicability in metabolic analysis is demonstrated by preliminary experiments on profiling small molecular metabolites in cerebrospinal fluid microdialysates from rat brain.     

超高效液相色谱-四极杆-飞行时间质谱检测和鉴定猪尿中氯丙那林的主要代谢产物

采用超高效液相色谱-四极杆-飞行时间质谱(UPLC/Q-TOF MS)检测和鉴定了猪尿中氯丙那林的主要代谢产物,并讨论了氯丙那林在猪体内的主要代谢途径。按10 mg/kg(b. w.)的剂量口服灌食氯丙那林,分别采集给药前及给药后的猪尿液样品。采用UPLC/Q-TOF MS对样品进行分析,并应用质量亏损过滤和离子色谱峰提取等数据处理技术,在给药后24 h内的猪尿中检测和鉴定了9种氯丙那林的代谢产物,其中,Ⅰ相代谢产物2种,Ⅱ相代谢产物7种。然后,根据氯丙那林原形和代谢产物的碎片离子特征,对代谢产物的结构进行鉴定。最后,根据所鉴定的代谢产物,推测氯丙那林在猪体内的代谢途径包括苯环羟基化、β -羟基和仲氨基的葡萄糖醛酸轭合、羟基化后的葡萄糖醛酸和硫酸轭合等。研究结果表明,羟基化氯丙那林及其轭合产物的相对含量大于60%,明显高于氯丙那林原形及其轭合产物,是尿液中的主要代谢产物。本研究将为确定氯丙那林在动物体内的残留标示物及加强对氯丙那林非法使用的监控提供科学依据。