•LC–MS is now the major analytical platform for metabolomics and metabonomics.•Metabolomics represents an upcoming field and a major application area for LC–MS technology.•A critical review of ...challenges, limitations of current practice and prospects of metabolomics is provided.•Selected paradigms show the potential of metabolomics in the life sciences and biomarker discovery.
Based on publication and citation numbers liquid chromatography (LC–MS) has become the major analytical technology in the field of global metabolite profiling. This dominance reflects significant investments from both the research community and instrument manufacturers. Here an overview of the approaches taken for LC–MS-based metabolomics research is given, describing critical steps in the realisation of such studies: study design and its needs, specific technological problems to be addressed and major obstacles in data treatment and biomarker identification. The current state of the art for LC–MS-based analysis in metabonomics/metabolomics is described including recent developments in liquid chromatography, mass spectrometry and data treatment as these are applied in metabolomics underlining the challenges, limitations and prospects for metabolomics research. Examples of the application of metabolite profiling in the life sciences focusing on disease biomarker discovery are highlighted. In addition, new developments and future prospects are described.
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► LC–MS is increasingly used in global metabolite profiling despite stability issues. ► Data extraction and marker identification remain at the centre of interest. ► Pre-analytical ...issues and sample preparation are also important factors. ► Method validation and protocol harmonisation are needed to obtain quality data. ► Large scale efforts and their outcome (e.g. HMDB) provide breakthrough paradigms.
Untargeted, global metabolite profiling (often described as metabonomics or metabolomics) represents an expanding research topic and is, potentially, a major pillar for systems biology studies. To obtain holistic metabolic profiles from complex samples, such as biological fluids or tissue extracts, requires powerful, high resolution and information-rich analytical methods and for this spectroscopic technologies are generally used. Mass spectrometry, coupled to liquid chromatography (LC–MS), is increasingly being used for such investigations as a result of the significant advances in both technologies over the past decade. Here we try to critically review the topic of LC–MS-based global metabolic profiling and describe and compare the results offered by different analytical strategies and technologies. This review highlights the current challenges, limitations and opportunities of the current methodology.
•Accurate and fast determination method for amino acids and derivatives.•Surrogate matrix approach provides accurate determination of endogenous metabolites in urine.•Wide range of applications in ...nutritional and metabolomics studies
Aminoacids and their derivatives are key biologically important metabolites and reliable, rapid and accurate, quantification for these analytes in urine remains an important analytical challenge. Here a fast and reliable HILIC-tandem MS method is presented for application in clinical or nutritional studies. The developed method was validated according to existing guidelines adapted for endogenous analytes. The validation strategy provided evidence of linearity, LOD and LOQ, accuracy, precision, matrix effect and recovery. The surrogate matrix approach was applied for calibration proving satisfactory accuracy and precision based on standard criteria over the working concentration ranges. Intra and inter day accuracy was found to range between 0.8 and 20% for the LQC (low QC) and between 0.05 and 15 % for MQC (medium QC) and HQC (high QC). Inter and intraday precision were found to be between 3 and 20 % for the LQC and between 1 and 15% for the MQC and HQC. The stability of the analytes, in both surrogate and pooled urine QC samples, was found to be within 15% over a short period at 4 °C or after a up to 3 freeze-thaw cycles. The uncertainty of the method was also assessed to provide increased confidence for the acquired measurements. The method was successfully applied to a subset of human urine samples involved in a study of amino acids dietary uptake. This method may provide a valuable tool for many applications or studies where amino acid metabolic signatures in the excreted urine are under investigation.
•Untargeted metabolomics often lack standardisation and quality control assessment.•Data quality assessment should be thorough employing different statistical tools.•A roadmap is proposed for the QC ...of LC-HRMS untargeted metabolomics data.•The current protocol allows monitoring the analytical processes.•Set up of analytical sequence, data collection, analysis and processing are studied.
The process of untargeted metabolic profiling/phenotyping of complex biological matrices, i.e., biological fluids such as blood plasma/serum, saliva, bile, and tissue extracts, provides the analyst with a wide range of challenges. Not the least of these challenges is demonstrating that the acquired data are of “good” quality and provide the basis for more detailed multivariate, and other, statistical analysis necessary to detect, and identify, potential biomarkers that might provide insight into the process under study. Here straightforward and pragmatic “quality control (QC)” procedures are described that allow investigators to monitor the analytical processes employed for global, untargeted, metabolic profiling. The use of this methodology is illustrated with an example from the analysis of human urine where an excel spreadsheet of the preprocessed LC–MS output is provided with embedded macros, calculations and visualization plots that can be used to explore the data. Whilst the use of these procedures is exemplified on human urine samples, this protocol is generally applicable to metabonomic/metabolomic profiling of biofluids, tissue and cell extracts from many sources.
•A study of derivatization prior to GC-MS analysis of 10 organic acids including markers of Organic Acidurias.•The stability of standard solutions and analyte derivatives is evaluated.•Findings show ...that method's performance should be examined for each analyte before application in real samples.
In the current paper the analytical conditions for the determination of ten free organic acids by GC-MS are studied with the aim to establish a method for organic acid profiling in human urine to be used as a tool for the detection of metabolic or other health disorders. Studies included the GC-MS method development, the derivatization (trimethylsilylation) reaction conditions, the stability of the standard solutions during storage in the freezer, and the stability of the formed trimethylsilyl derivatives. Best results were obtained at a derivatization temperature of 50°C, and a reaction time of 30min. Standard solutions were stable for 22 days, derivatized samples were stable at least for 30h when stored at −24°C. GC-MS analysis achieved sensitive determination of the organic acids under study with limits of detection ranging from 0.03mmol/mol creatinine for glutaric acid, to 0.34mmol/mol creatinine for glycolic acid. Within-day and day-to-day assay imprecision was found satisfactory with relative standard deviations being below 10%. The developed method was successfully applied to the quantitative analysis of free organic acids in urine samples obtained from hospitalized children. Creatinine-corrected excretion rates of all analyzed organic acids were within reference intervals.
•Detection and quantification method of nine widely used insecticides and fungicides in blood and urine.•Extraction of the analytes by three different sample preparation procedures, protein ...precipitation, SPE, and d-SPE.•Method validation in the two substrates for accurate determination to evaluate exposure.•Application to postmortem blood and urine samples from cases of acute poisoning.
Pesticide poisoning is a common occurrence due to their widespread use, easy access and high toxicity even in small concentrations. The most common poisoning fatalities have been observed due to exposure to organophosphates, carbamates and neonicotinoids, thus development of a method for the rapid determination of these compounds in blood and urine is of great importance for clinical and toxicology laboratories. A simple, fast and reliable method was developed for the determination of 9 pesticides in blood and urine using HPLC-MS/MS instrumentation. In order to find the most suitable sample pretreatment technique, three different sample preparation procedures: SPE, protein precipitation and QuEChERS were compared. The final optimized analytical method was fully validated with the values of parameters such as calibration linearity, accuracy, precision, recovery, matrix effect and stability being acceptable. The method proved reliable, accurate, robust and sensitive and was successfully applied for the quantitation of pesticides in three postmortem cases of pesticides poisoning.
Liquid chromatography (LC) hyphenated to mass spectrometry is currently the most widely used means of determining metabolic phenotypes via both untargeted and targeted analysis. At present a range of ...analytical separations, including reversed-phase, hydrophilic interaction and ion-pair LC are employed to maximise metabolome coverage with ultra (high) performance liquid chromatography (UHPLC) increasingly displacing conventional high performance liquid chromatography because of the need for short analysis times and high peak capacity in such applications. However, it is widely recognized that these methodologies do not entirely solve the problems facing researchers trying to perform comprehensive metabolic phenotyping and in addition to these “routine” approaches there are continuing investigations of alternative separation methods including 2-dimensional/multi column approaches. These involve either new stationary phases or multidimensional combinations of the more conventional materials currently used, as well as application of miniaturization or “new” approaches such as supercritical HP and UHP- chromatographic separations. There is also a considerable amount of interest in the combination of chromatographic and ion mobility separations, with the latter providing both an increase in resolution and the potential to provide additional structural information via the determination of molecular collision cross section data. However, key problems remain to be solved including ensuring quality, comparability across different laboratories and the ever present difficulty of identifying unknowns.
•Separation approaches for untargeted metabolic phenotyping by LC-MS are described.•The development and application of multi-column/multi-dimensional separations are illustrated.•Emerging techniques including SFC, miniaturization and IMS are discussed.•Limitations in metabolic phenotyping using LC-MS in biomedicine and the life sciences are highlighted.•Aspects of standardization and metabolite identification are described.
•A review of the advantages, constrains and issues in MS-based metabolomics.•Perspective for future research seen in six fields.•Design of experiment, analytical developments for quality ...control-validation, improvement of metabolome coverage, fusion of data, metabolite identification, and data visualisation.
The present review aims to critically discuss some of the major problems and limitations of LC–MS based metabolomics as experienced from an analytical chemistry standpoint. Metabolomics offers distinct advantages to a variety of life sciences. Continuous development of the field has been realised due to intensive efforts from a great many scientists from widely divergent backgrounds and research interests as demonstrated by the contents of this special issue. The aim of this commentary is to describe current hindrances to field's progress, (some unique to metabolomics, some common with other omics fields or with conventional targeted bioanalysis) to propose some potential solutions to overcome these constraints and to provide a future perspective for likely developments in the field.
The paper reports the development of a multianalyte method and its application in metabolic profiling of biological fluids. The initial aim of the method was the quantification of metabolites ...existing in cell culture medium used in in‐vitro fertilization (IVF) and in other biological fluids related to embryo growth. Since most of these analytes are polar primary metabolites a hydrophilic interaction liquid chromatography system was selected. The analytical system comprised Ultra‐HPLC with detection on a triple quadrupole mass spectrometer operating in both positive and negative modes. Mobile phase and gradient elution conditions were studied with the aim to achieve the highest coverage of metabolic space in a single injection namely the largest number of analytes that could be detected and quantified. The developed method provides absolute quantitation of ca. 100 metabolites belonging to key metabolite classes such as sugars, aminoacids, nucleotides, organic acids, and amines. Following validation, the method was applied for the metabolic profiling of hundreds of samples of spent culture medium originating from human IVF procedures and several hundreds of biological samples such as amniotic fluid, human urine and blood serum from pregnant women. The bioanalytical end‐point was to provide assistance in the process of embryo transfer and improving IVF success rates but also to provide insight in complications related to the subsequent embryo growth during pregnancy.
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