Georgios Theodoridis

Liquid chromatography–mass spectrometry based global metabolite profiling: A review

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.

Solid-phase microextraction for the analysis of biological samples

Solid-phase microextraction (SPME) has been introduced for the extraction of organic compounds from environmental samples. This relatively new extraction technique has now also gained a lot of interest in a broad field of analysis including food, biological and pharmaceutical samples. SPME has a number of advantages such as simplicity, low cost, compatibility with analytical systems, automation and the solvent-free extraction. The last few years, SPME has been combined with liquid chromatography and capillary electrophoresis, besides the generally used coupling to gas chromatography, and has been applied to various biological samples such as, e.g., urine, plasma and hair. The objective of the present paper is a survey of the application of SPME for the analysis of biological samples. Papers about the analysis of biologically active compounds are categorised and reviewed. The impact of SPME on various analytical fields (toxicological, forensic, clinical, biochemical, pharmaceutical, and natural products) is illustrated. The main features of SPME and its modes are briefly described and important aspects about its application for the determination of pharmaceuticals, drugs of abuse and compounds of clinical and toxicological interest are discussed. SPME is compared with other sample pretreatment techniques. The potential of SPME and its main advantages are demonstrated. Special attention is paid to new trends in applications of SPME in bioanalysis.

Current practice of liquid chromatography-mass spectrometry in metabolomics and metabonomics.

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.

Evaluation of the repeatability of ultra-performance liquid chromatography–TOF-MS for global metabolic profiling of human urine samples ☆

The application of reversed-phase ultra-performance liquid chromatography, based on the use of sub 2 microm particles, combined with time-of-flight mass spectrometry has been investigated for the production of global metabolite profiles from human urine. The stability and repeatability of the methodology, which employed gradient elution, was determined by the repeat analysis of a pooled quality control (QC) sample. As seen in previous studies conducted with conventional LC-MS an element of system conditioning was required to obtain reproducible data, as the initial injections were unrepresentative. However, once the system had equilibrated excellent repeatability in terms of retention time, signal intensity and mass accuracy was seen providing confidence that for this matrix, the within-day repeatability of UPLC-TOF-MS was sufficient to assure data quality in global metabolic profiling applications.

Hydrophilic interaction chromatography coupled to MS for metabonomic/metabolomic studies

Hydrophilic interaction chromatography (HILIC) is a relatively recently introduced mode of liquid-phase separations. Recently, HILIC has been used for coupling to MS in metabonomic/metabolomic studies to provide a complementary tool to the widely used reversed-phase (RP) chromatographic separations. The combination of HILIC with MS detection covers a number of polar metabolites that are typically nonretained in RPLC-mode separations and thus enlarging the number of detected analytes. This way of metabolite profiling thus provides more comprehensive metabolome coverage than using RP chromatography alone. This review describes the applications and the utility of HILIC-MS in metabolomic/metabonomic studies and highlights certain characteristic examples in the life and plant-food sciences.

Site and Strain-Specific Variation in Gut Microbiota Profiles and Metabolism in Experimental Mice

Background The gastrointestinal tract microbiota (GTM) of mammals is a complex microbial consortium, the composition and activities of which influences mucosal development, immunity, nutrition and drug metabolism. It remains unclear whether the composition of the dominant GTM is conserved within animals of the same strain and whether stable GTMs are selected for by host-specific factors or dictated by environmental variables. Methodology/Principal Findings The GTM composition of six highly inbred, genetically distinct strains of mouse (C3H, C57, GFEC, CD1, CBA nu/nu and SCID) was profiled using eubacterial –specific PCR-DGGE and quantitative PCR of feces. Animals exhibited strain-specific fecal eubacterial profiles that were highly stable (c. >95% concordance over 26 months for C57). Analyses of mice that had been relocated before and after maturity indicated marked, reproducible changes in fecal consortia and that occurred only in young animals. Implantation of a female BDF1 mouse with genetically distinct (C57 and Agoutie) embryos produced highly similar GTM profiles (c. 95% concordance) between mother and offspring, regardless of offspring strain, which was also reflected in urinary metabolite profiles. Marked institution-specific GTM profiles were apparent in C3H mice raised in two different research institutions. Conclusion/Significance Strain-specific data were suggestive of genetic determination of the composition and activities of intestinal symbiotic consortia. However, relocation studies and uterine implantation demonstrated the dominance of environmental influences on the GTM. This was manifested in large variations between isogenic adult mice reared in different research institutions.

Selective solid-phase extraction sorbent for caffeine made by molecular imprinting

A molecularly imprinted polymer (MIP) was prepared with caffeine as the template molecule. Thermal polymerisation (60 degrees C) was optimised, varying ratios of monomer, cross linker and template. The polymer was used as a solid-phase extraction (SPE) sorbent, for selective trapping and pre-concentration of caffeine. Caffeine was loaded on the MIP-SPE cartridge using different loading conditions (solvents, pH value). Washing and elution of the caffeine bound to the MIP was studied utilising different protocols. The extraction protocol was successfully applied to the direct extraction of caffeine from beverages and spiked human plasma.

HILIC-UPLC-MS for Exploratory Urinary Metabolic Profiling in Toxicological Studies

Hydrophilic interaction ultra performance liquid chromatography (HILIC-UPLC) permits the analysis of highly polar metabolites, providing complementary information to reversed-phase (RP) chromatography. HILIC-UPLC-TOF-MS was investigated for the global metabolic profiling of rat urine samples generated in an experimental hepatotoxicity study of galactosamine (galN) and the concomitant investigation of the protective effect of glycine. Within-run repeatability and stability over a large sample batch (>200 samples, 60 h run-time) was assessed through the repeat analysis of a quality control sample. Following system equilibration, excellent repeatability was observed in terms of retention time (CV < 1.7%), signal intensity (CV < 14%), and mass variability (<0.005 amu), providing a good measure of reproducibility. Classification of urinary metabolic profiles according to treatment was observed, with significant changes in specific metabolites after galN exposure, including increased urocanic acid, N-acetylglucosamine, and decreased 2-oxoglutarate. A novel finding from this HILIC-UPLC-MS approach was elevated urinary tyramine in galN-treated rats, reflecting disturbed amino acid metabolism. These results show HILIC-UPLC-MS to be a promising method for global metabolic profiling, demonstrating high within-run repeatability, even over an extended run time. Retention of polar endogenous analytes and xenobiotic metabolites was improved compared with RP studies, including galN, N-acetylglucosamine, oxoglutarate, and urocanic acid, enhancing metabolome coverage and potentially improving biomarker discovery.

Hydrophilic interaction and reversed-phase ultra-performance liquid chromatography TOF-MS for metabonomic analysis of Zucker rat urine.

Hydrophilic interaction chromatography (HILIC) provides a complementary technique to RP methods for the retention of polar analytes for LC-MS-based metabonomic studies. Combining the advantages of both RP and HILIC separations with the efficient and rapid separations obtained using sub-2 mum particles via the recently introduced ultra-performance LC (UPLC) enables increased coverage of the metabolites present in biological samples to be achieved. Here an HILIC-UPLC-MS method was developed to provide metabolite profiles for urine samples obtained from male Zucker rats. The resulting data were compared with results obtained for the same samples by RP-UPLC-MS and demonstrated the complementary nature of the two separations with both methods enabling discrimination between the different sample types. Interestingly sample type differentiation was based on different markers.

Preparation of a molecularly imprinted polymer for the solid-phase extraction of scopolamine with hyoscyamine as a dummy template molecule

Molecularly imprinted polymers (MIPs) selective for scopolamine were produced using hyoscyamine (a close structural analogue) as template molecule. The produced polymers were used as media for solid-phase extraction, exhibiting selective binding properties for the analyte from biological samples. Human and calf urine and serum were processed on the MIP under various extraction protocols. The best performance was observed after loading the analyte in aqueous environment facilitating retention on the MIP by non-selective hydrophobic interactions. The MIPs were subsequently washed using an optimised solvent system to enable selective desorption of the analyte. Other related and non-related compounds were accessed to evaluate molecular recognition properties. Recoveries of up to 79% were achieved for the analyte of interest from biological samples.