Capillary Electrophoresis-mass Spectrometry Method Research Articles

Native Capillary Electrophoresis-Mass Spectrometry of Near 1 MDa Non-Covalent GroEL/GroES/Substrate Protein Complexes.

Protein complexes are essential for proteins' folding and biological function. Currently, native analysis of large multimeric protein complexes remains challenging. Structural biology techniques are time-consuming and often cannot monitor the proteins' dynamics in solution. Here, a capillary electrophoresis-mass spectrometry (CE-MS) method is reported to characterize, under near-physiological conditions, the conformational rearrangements of ∽1 MDa GroEL upon complexation with binding partners involved in a protein folding cycle. The developed CE-MS method is fast (30min per run), highly sensitive (low-amol level), and requires ∽10 000-fold fewer samples compared to biochemical/biophysical techniques. The method successfully separates GroEL14 (∽800 kDa), GroEL7 (∽400 kDa), GroES7 (∽73kDa), and NanA4 (∽130 kDa) oligomers. The non-covalent binding of natural substrate proteins with GroEL14 can be detected and quantified. The technique allows monitoring of GroEL14 conformational changes upon complexation with (ATPγS)4-14 and GroES7 (∽876 kDa). Native CE-pseudo-MS3 analyses of wild-type (WT) GroEL and two GroEL mutants result in up to 60% sequence coverage and highlight subtle structural differences between WT and mutated GroEL. The presented results demonstrate the superior CE-MS performance for multimeric complexes' characterization versus direct infusion ESI-MS. This study shows the CE-MS potential to provide information on binding stoichiometry and kinetics for various protein complexes.

Development of a rapid and highly accurate method for 13C tracer-based metabolomics and its application on a hydrogenotrophic methanogen.

Microfluidic capillary electrophoresis-mass spectrometry (CE-MS) is a rapid and highly accurate method to determine isotopomer patterns in isotopically labeled compounds. Here, we developed a novel method for tracer-based metabolomics using CE-MS for underivatized proteinogenic amino acids. The method consisting of a ZipChip CE system and a high-resolution Orbitrap Fusion Tribrid mass spectrometer allows us to obtain highly accurate data from 1μl of 100nmol/l amino acids comparable to a mere 1 [Formula: see text] 104-105 prokaryotic cells. To validate the capability of the CE-MS method, we analyzed 16 protein-derived amino acids from a methanogenic archaeon Methanothermobacter thermautotrophicus as a model organism, and the mass spectra showed sharp peaks with low mass errors and background noise. Tracer-based metabolome analysis was then performed to identify the central carbon metabolism in M. thermautotrophicus using 13C-labeled substrates. The mass isotopomer distributions of serine, aspartate, and glutamate revealed the occurrence of both the Wood-Ljungdahl pathway and an incomplete reductive tricarboxylic acid cycle for carbon fixation. In addition, biosynthesis pathways of 15 amino acids were constructed based on the mass isotopomer distributions of the detected protein-derived amino acids, genomic information, and public databases. Among them, the presence of alternative enzymes of alanine dehydrogenase, ornithine cyclodeaminase, and homoserine kinase was suggested in the biosynthesis pathways of alanine, proline, and threonine, respectively. To our knowledge, the novel 13C tracer-based metabolomics using CE-MS can be considered the most efficient method to identify central carbon metabolism and amino acid biosynthesis pathways and is applicable to any kind of isolated microbe.

A simple and green capillary electrophoresis-mass spectrometry method for therapeutic drug monitoring of colistin in clinical plasma samples

Colistin and other polymyxin antibiotics have become increasingly used in clinical settings as a result of treating multidrug-resistant infections in critically ill patients. The highly variable pharmacokinetics of colistin in these patients is accompanied by a high risk of toxicity or underdosing. An effective tool that allows rational optimization of the drug dosage regimen is therapeutic drug monitoring. Therefore, there is a need to dispose with appropriate, sensitive, and accurate analytical methods. Here, a simple, specific, and accurate on-line capillary electrophoresis – tandem mass spectrometry method was developed and applied for the first time to determine colistin in human plasma. Protein precipitation using acidified acetonitrile was the solitary procedure used to achieve sample pretreatment. A bare fused silica capillary was employed for the separation process, and the background electrolyte used was 50 mM formic acid (pH 2.54). The FDA's bioanalytical method validation guidelines were followed in the validation of the proposed method. For colistin A and colistin B, favorable performance and validation parameters were obtained (such as linearity, limit of detection, lower limit of quantitation, intra-day and inter-day precision, accuracy, and stability).The validated method was then effectively used to analyze real clinical samples taken from patients who were in critical condition. Our newly developed method is comparable with previously published liquid chromatography approaches and has the potential to be applied in the therapeutic monitoring of colistin in routine clinical laboratories. Moreover, according to the greenness assessment, the developed capillary electrophoresis – mass spectrometry method represents a very interesting green and sustainable tool in the field of bioanalysis.

Analysis of naphthenic acids in produced water samples by capillary electrophoresis-mass spectrometry.

A capillary electrophoresis-mass spectrometry method was used to analyze naphthenic acids in produced water samples. It was possible to detect cyclopentanecarboxylic, benzoic, cyclohexanebutyric, 1-naphthoic, decanoic, 3,5-dimethyladamantane-1-carboxylic, 9-anthracenecarboxylic, and pentadecanoic acids within ca. 13min using a buffer composed of 40mmol/L ammonium hydroxide, 32mmol/L acetic acid and 20% v/v isopropyl alcohol, pH 8.6. The proposed method showed good repeatability, with relative standard deviation (RSD) values of 6.6% for the sum of the peak areas and less than 2% for the analysis time. In the interday analysis, the RSD values for the sum of the peak areas and migration time were 10.3% and 10%, respectively. The developed method demonstrated linear behavior in the concentration range between 5 and 50mg/L for benzoic, decanoic, 3,5-dimethyladamantane-1-carboxylic and 9-anthracenecarboxylic acids, and between 10 and 50mg/L for cyclopentanecarboxylic, cyclohexanebutyric, 1- naphthoic, and pentadecanoic acids. The detection limits values ranged from 0.31 to 1.64mg/L. Six produced water samples were analyzed and it was possible to identify and quantify cyclopentanecarboxylic, benzoic, cyclohexanebutyric, and decanoic acids. The concentrations varied between 4.8 and 98.9mg/L, proving effective in the application of complex samples.

Advances in chiral separation and analysis by capillary electrophoresis-mass spectrometry

目前使用的绝大多数药物为手性化合物,它们具有相似的物理和化学性质,但药理活性不同,且常以外消旋混合物的形式存在,因此对手性化合物的分离在生物、环境、食品和医药等领域一直备受关注。与广泛使用的液相色谱-质谱(LC-MS)相比,毛细管电泳-质谱(CE-MS)作为一种新型分离分析技术,具有分离效率高、样品和试剂消耗量低、选择性高和分离模式多样化等诸多优势,已经发展成为手性分析领域中有广阔应用前景的分析方法之一。CE-MS结合了CE的高分离效率和低样品消耗以及MS的高灵敏度和强结构解析能力,在蛋白质组学和代谢组学等领域发挥了重要作用。CE杰出的手性拆分能力与MS优势的结合,亦使CE-MS成为实现手性化合物高效分离分析的完美组合。在过去的十几年里,基于不同CE-MS分离模式的高性能手性分析体系层出不穷,如电动色谱-质谱(EKC-MS)、胶束电动色谱-质谱(MEKC-MS)和毛细管电色谱-质谱(CEC-MS)等,并成功应用于医药、生物、食品和环境科学等领域的手性化合物分析。该文主要综述了2011~2021年,CE-MS在手性化合物分析领域的技术、手性选择剂(如改性环糊精和聚合物表面活性剂等)的使用以及在医药等领域应用方面的研究进展,并讨论了不同手性分析模式的局限性,为未来的CE-MS手性分离分析技术发展及应用提供借鉴。

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.

Spray-Capillary-Based Capillary Electrophoresis Mass Spectrometry for Metabolite Analysis in Single Cells.

Single-cell capillary electrophoresis mass spectrometry (CE-MS) is a promising platform to analyze cellular contents and probe cell heterogeneity. However, current single-cell CE-MS methods often rely on offline microsampling processes and may demonstrate low sampling precision and accuracy. We have recently developed an electrospray-assisted device, spray-capillary, for low-volume sample extraction. With the spray-capillary, low-volume samples (pL-nL) are drawn into the sampling end of the device, which can be used directly for CE separation and online MS detection. Here, we redesigned the spray-capillary by utilizing a capillary with a <15 μm tapered tip so that it can be directly inserted into single cells for sample collection and on-capillary CE-MS analysis. We evaluated the performance of the modified spray-capillary by performing single-cell microsampling on single onion cells with varying sample injection times and direct MS analysis or online CE-MS analysis. We have demonstrated, for the first time, online sample collection and CE-MS for the analysis of single cells. This application of the modified spray-capillary device facilitates the characterization and relative quantification of hundreds of metabolites in single cells.

Separation, identification and quantification of associated impurities in cobratide using sheathless CE-MS and CE-UV.

Cobratide is a peptide drug extracted from the venom of Chinese cobra, and has been widely used in the clinical treatment of chronic, intractable and persistent pain. In a recent study, it was reported that it has the potential to treat COVID-19. In order to control the quality of commercial cobratide drugs, a protocol was established for the separation, identification and quantification of cobratide and its associated impurities, in which sheathless capillary electrophoresis-mass spectrometry (CE-MS) was used for identification and a rapid capillary electrophoresis-ultraviolet-visible detector (CE-UV) method was developed for accurate quantification. Separation conditions that affect the resolution and MS intensities of cobratide and its associated impurities were investigated, including pH value, concentration of background electrolyte (BGE), ratio of organic additive and sample solution. The optimized CE conditions (BGE: 50 mM NH4Ac, pH 4.0; sample solution: deionized water) were used for both sheathless CE-MS and CE-UV methods. Three associated impurities were separated and identified for the first time by sheathless CE-MS. Then, a rapid CE-UV method was validated and used for accurate quantification of cobratide and its associated impurities. The CE-UV method showed good linearity between concentration and corrected peak area of cobratide in the concentration range of 5.36-536.30 μg mL-1. The limit of quantification of the CE-UV method was 4.16 μg mL-1. The relative standard deviations of migration time were less than 1% for both intra-day and inter-day experiments, and those of corrected peak area were less than 5%. Finally, different cobratide drugs were analyzed to evaluate the batch-to-batch consistency. This established protocol combining sheathless CE-MS and CE-UV methods would provide useful information for both quality control and process analysis of peptide drugs.

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.

Enantioselective analysis in complex matrices using capillary electrophoresis-mass spectrometry: A case study of the botanical drug Corydalis Rhizoma

Enantioselective analysis is critically important in the pharmaceutical and agricultural industries. However, most of the methods reported were developed for the analysis of pure racemates acquired from chemical synthesis or purification. Direct analysis of chiral enantiomers in complex matrices has rarely been reported. This work demonstrated capillary electrophoresis-mass spectrometry (CE-MS) for the enantioselective analysis of botanical drugs for the first time, using a widely used botanical drug, Corydalis Rhizoma, as an example. The method was used for the simultaneous enantioselective analysis of dl-tetrahydropalmatine and (RS)-tetrahydroberberine (canadine) in Corydalis Rhizoma extract. Using (2-hydroxypropyl)-β-cyclodextrin as the chiral selector, a partial filling technique was used to avoid signal suppression and contamination of the MS detector. Post column organic modifier was used to assist with ionization in the flow through microvial CE-MS interface, therefore, organic solvents was not used in the background electrolyte. The completely aqueous background electrolyte contributed to better chiral separations. The CE-MS method established here can directly determine the analytes in their complex matrix without any pre-purification steps, while also offering high sensitivity and low operational costs (including sample, chiral selector and solvent). In the method validation process, good linearity (r > 0.993), sensitivity and accuracy (recoveries within 89.1–110.0%) were demonstrated. The CE-MS technique was shown to be able to provide good selectivity for the simultaneous chiral separation of multiple pairs of enantiomers in complex matrices.

Direct profiling of endogenous metabolites in rat brain microdialysis samples by capillary electrophoresis-mass spectrometry with on-line preconcentration

Metabolic profiling of body fluids from small animal models is often used in (translational) biological studies in order to obtain insight into the underlying molecular mechanisms of (complex) diseases. An example is the use of brain microdialysis samples from rats to study neurological disorders by means of a metabolomics approach. From an analytical point of view, the profiling of (endogenous) metabolites in rat brain microdialysates is challenging because of the limited sample volume for both sample preparation and injection, notably in longitudinal studies. In this work, we have assessed the analytical performance of capillary electrophoresis-mass spectrometry (CE-MS) for the direct profiling of endogenous metabolites in rat brain microdialysates, i.e. without using any sample preparation or derivatization. An on-line preconcentration procedure with sample stacking, which was fully compatible with the high-salt concentration in microdialysates, was used to significantly improve the detection sensitivity of the CE-MS method for metabolic profiling. A response surface methodology, applying a Box-Behnken design, was considered to determine the optimal conditions for preconcentration. A linear response (R2>0.99) for selected metabolites in the concentration range from 0.05 to 10 µM was obtained in perfusate samples. Interday RSD values for peak area and migration time were 2.6-19% and below 3.8%, respectively. Limits of detection ranged from 11 to 284 nM when employing an injection volume of about 291 nL, corresponding to 17% of the total capillary volume. The utility of the CE-MS approach was demonstrated by the direct profiling of endogenous metabolites in rat brain microdialysates. At least 48 compounds could be analyzed of which 25 were provisionally identified and quantified.

Online capillary electrophoresis - mass spectrometry analysis of histatin-5 and its degradation products.

Histatin-5 (Hst-5) is a human salivary peptide with antibacterial and antifungal activities. Thorough characterization and reliable quantification of Hst-5 and its degradation products are essential for understanding the Hst-5 degradation pathway. Due to the highly basic and strong cationic nature of the Hst-5 peptide, the quantitative analysis of Hst-5 and its degradation forms by online mass spectrometry remains challenging. Here, we adopt a recently developed electrokinetically pumped sheath liquid capillary electrophoresis - mass spectrometry (CE-MS) coupling technology, and successfully apply it for the analysis of Hst-5 and its degradation products. Our CE-MS method is demonstrated to be robust and quantitative. This novel analytical platform is reproducible and free of sample carryover. The efficacy of this method is demonstrated with a kinetic study of Hst-5 degradation by Sap9, a secreted aspartic peptidase. Our work demonstrates the potential of online CE-MS as a powerful approach for characterizing highly basic peptides.

On-line Aptamer Affinity Solid-Phase Extraction Capillary Electrophoresis-Mass Spectrometry for the Analysis of Blood α-Synuclein.

In this paper, an on-line aptamer affinity solid-phase extraction capillary electrophoresis-mass spectrometry method is described for the purification, preconcentration, separation, and characterization of α-synuclein (α-syn) in blood at the intact protein level. A single-stranded DNA aptamer is used to bind with high affinity and selectivity α-syn, which is a major component of Lewy bodies, the typical aggregated protein deposits found in Parkinson's disease (PD). Under the conditions optimized with recombinant α-syn, repeatability (2.1 and 5.4% percent relative standard deviation for migration times and peak areas, respectively) and microcartridge lifetime (around 20 analyses/microcartridge) were good, the method was linear between 0.5 and 10 μg·mL-1, and limit of detection was 0.2 μg·mL-1 (100 times lower than by CE-MS, 20 μg·mL-1). The method was subsequently applied to the analysis of endogenous α-syn from red blood cells lysate of healthy controls and PD patients.

Capillary electrophoresis-mass spectrometry using robust poly(ether ether ketone) capillary for tolerance to high content of organic solvents

Nonaqueous capillary electrophoresis (CE) and organic-aqueous CE using high content of organic solvents are common techniques in capillary electrophoresis-mass spectrometry (CE-MS) due to various advantages. However, the long-term stability of commonly used polyimide coated fused silica capillary remains a major problem that polyimide coating is prone to swelling in organic solvents and aminolysis in alkaline buffer, which cause serious problems like irregular electrospray, current drop or clogging event. In this work, robust PEEK capillary was used as separation capillary for tolerance to high content of organic solvents in CE-MS system. It is interesting that the high mechanical and chemical stability of PEEK capillary ensured stable CE separation and electrospray when used in CE-MS. All quantitative experiments were accomplished by single PEEK capillary. In addition, the using of organic solvents as running buffer reduced adsorption of hydrophobic compounds on PEEK capillary wall and improved the symmetry of peak shape. The PEEK-based CE-MS method showed good separation performance in analysis of several groups of active alkaloids in traditional Chinese medicines and was applied for quantitative analysis of peimine and peiminine in Bulbus Fritillariae thunbergii herb with high sensitivity. Based on the advantages like simple pretreatment, easy to cut, stable and good separation performance of PEEK column in CE-MS, this study provides an effective way for improving the stability and employment of high content of organic solvents in CE-MS analysis.

Front Cover: Nonaqueous capillary electrophoresis mass spectrometry method for determining highly hydrophobic peptides

Front Cover: Nonaqueous capillary electrophoresis mass spectrometry method for determining highly hydrophobic peptides

Reliability of plasma polar metabolite concentrations in a large-scale cohort study using capillary electrophoresis-mass spectrometry.

BackgroundCohort studies with metabolomics data are becoming more widespread, however, large-scale studies involving 10,000s of participants are still limited, especially in Asian populations. Therefore, we started the Tsuruoka Metabolomics Cohort Study enrolling 11,002 community-dwelling adults in Japan, and using capillary electrophoresis-mass spectrometry (CE-MS) and liquid chromatography–mass spectrometry. The CE-MS method is highly amenable to absolute quantification of polar metabolites, however, its reliability for large-scale measurement is unclear. The aim of this study is to examine reproducibility and validity of large-scale CE-MS measurements. In addition, the study presents absolute concentrations of polar metabolites in human plasma, which can be used in future as reference ranges in a Japanese population.MethodsMetabolomic profiling of 8,413 fasting plasma samples were completed using CE-MS, and 94 polar metabolites were structurally identified and quantified. Quality control (QC) samples were injected every ten samples and assessed throughout the analysis. Inter- and intra-batch coefficients of variation of QC and participant samples, and technical intraclass correlation coefficients were estimated. Passing-Bablok regression of plasma concentrations by CE-MS on serum concentrations by standard clinical chemistry assays was conducted for creatinine and uric acid.Results and conclusionsIn QC samples, coefficient of variation was less than 20% for 64 metabolites, and less than 30% for 80 metabolites out of the 94 metabolites. Inter-batch coefficient of variation was less than 20% for 81 metabolites. Estimated technical intraclass correlation coefficient was above 0.75 for 67 metabolites. The slope of Passing-Bablok regression was estimated as 0.97 (95% confidence interval: 0.95, 0.98) for creatinine and 0.95 (0.92, 0.96) for uric acid. Compared to published data from other large cohort measurement platforms, reproducibility of metabolites common to the platforms was similar to or better than in the other studies. These results show that our CE-MS platform is suitable for conducting large-scale epidemiological studies.

Nonaqueous capillary electrophoresis mass spectrometry method for determining highly hydrophobic peptides.

A nonaqueous capillary electrophoresis mass spectrometry (NACE-MS) method was developed to separate and determine highly hydrophobic temporin peptides. The nonaqueous background electrolyte solution was a mixture of 20% acetonitrile, 78% methanol and 2% formic acid, with 20 mM ammonium formate. The separation of six peptides was completed within 12 min. The CE system was connected to a triple quadrupole mass spectrometer operating in MRM mode using a chemical modifier solution of 2 mM ammonium formate in ethanol with the flow through microvial interface. The mass spectrometer offered a second dimension of separation for peptides having identical migration times but different structures. The new method represents the first system capable of reliably determining hydrophobic peptides without using reversed phase liquid chromatography mass spectrometry.

Microfluidic Capillary Electrophoresis-Mass Spectrometry for Analysis of Monosaccharides, Oligosaccharides, and Glycopeptides.

Glycomics and glycoproteomics analyses by mass spectrometry require efficient front-end separation methods to enable deep characterization of heterogeneous glycoform populations. Chromatography methods are generally limited in their ability to resolve glycoforms using mobile phases that are compatible with online liquid chromatography-mass spectrometry (LC-MS). The adoption of capillary electrophoresis-mass spectrometry methods (CE-MS) for glycomics and glycoproteomics is limited by the lack of convenient interfaces for coupling the CE devices to mass spectrometers. Here, we describe the application of a microfluidics-based CE-MS system for analysis of released glycans, glycopeptides and monosaccharides. We demonstrate a single CE method for three different modalities, thus contributing to comprehensive glycoproteomics analyses. In addition, we explored compatible sample derivatization methods. We used glycan TMT-labeling to improve electrophoretic migration and enable multiplexed quantitation by tandem MS. We used sialic acid linkage-specific derivatization methods to improve separation and the level of information obtained from a single analytical step. Capillary electrophoresis greatly improved glycoform separation for both released glycans and glycopeptides over that reported for chromatography modes more frequently employed for such analyses. Overall, the CE-MS method described here enables rapid setup and analysis of glycans and glycopeptides using mass spectrometry.

Quantitative Nonaqueous Capillary Electrophoresis-Mass Spectrometry Method for Determining Active Ingredients in Plant Extracts.

Nonaqueous capillary electrophoresis (NACE) is very well suited for online coupling with mass spectrometry due to the relatively high volatility and low surface tension of most organic solvents. Here we present a quantitative NACE-ESI-MS/MS method for separating and determining physcion, chrysophanol, and aloe-emodin in rhubarb. Dantron was used as an internal standard to ensure accuracy and reproducibility in quantitative analyses. Parameters including the pH, background electrolyte (BGE) composition, flow-through microvial chemical modifier solution composition, and modifier solution flow rate were carefully optimized. The developed method was validated by assessing its precision, LODs, and linear range. The contents of physcion, chrysophanol, and aloe-emodin in rhubarb were determined to be 0.22%, 1.0%, and 0.17%, respectively.

Metabolic Profiling of Total Physical Activity and Sedentary Behavior in Community-Dwelling Men.

ObjectivePhysical activity is known to be preventive against various non-communicable diseases. We investigated the relationship between daily physical activity level and plasma metabolites using a targeted metabolomics approach in a population-based study.MethodsA total of 1,193 participants (male, aged 35 to 74 years) with fasting blood samples were selected from the baseline survey of a cohort study. Information on daily total physical activity, classified into four levels by quartile of metabolic equivalent scores, and sedentary behavior, defined as hours of sitting per day, was collected through a self-administered questionnaire. Plasma metabolite concentrations were quantified by capillary electrophoresis mass spectrometry method. We performed linear regression analysis models with multivariable adjustment and corrected p-values for multiple testing in the original population (n = 808). The robustness of the results was confirmed by replication analysis in a separate population (n = 385) created by random allocation.ResultsHigher levels of total physical activity were associated with various metabolite concentrations, including lower concentrations of amino acids and their derivatives, and higher concentrations of pipecolate (FDR p <0.05 in original population). The findings persisted after adjustment for age, body mass index, smoking, alcohol intake, and energy intake. Isoleucine, leucine, valine, 4-methyl-2-oxoisopentanoate, 2-oxoisopentanoate, alanine, and proline concentrations were lower with a shorter sitting time.ConclusionsPhysical activity is related to various plasma metabolites, including known biomarkers for future insulin resistance or type 2 diabetes. These metabolites might potentially play a key role in the protective effects of higher physical activity and/or less sedentary behavior on non-communicable diseases.