Andrew J Chetwynd

Arthur David
email: arthur.david.univ@gmail.com

A review of nanoscale LC-ESI for metabolomics and its potential to enhance the metabolome coverage

Keywords: nanoflow, nanoESI, metabolomics, exposomics, ion suppression, sample preparation

Liquid chromatography-electrospray ionisation-mass spectrometry (LC-ESI-MS) platforms are widely used to perform high throughput untargeted profiling of biological samples for metabolomics-based approaches. However, these LC-ESI platforms usually favour the detection of metabolites present at relatively high concentrations because of analytical limitations such as ion suppression, thus reducing overall sensitivity. To counter this issue of sensitivity, the latest in terms of analytical platforms can be adopted to enable a greater portion of the metabolome to be analysed in a single analytical run. Here, nanoflow liquid chromatography-nanoelectrospray ionisation (nLC-nESI), which has previously been utilised successfully in proteomics, is explored for use in metabolomic and exposomic research. As a discovery based field, the markedly increased sensitivity of these nLC-nESI platforms offer the potential to uncover the roles played by low abundant signalling metabolites (e.g. steroids, eicosanoids) in health and disease studies, and would also enable an improvement in the detection of xenobiotics present at trace levels in biological matrices to better characterise the chemical exposome. This review aims to give an insight into the advantages associated with nLC-nESI for metabolomics-based approaches. Initially we detail the source of improved sensitivity prior to reviewing the available approaches to achieving nanoflow rates and nanospray ionisation for metabolomics. The robustness of nLC-nESI platforms was then assessed using the literature available from a metabolomic viewpoint. We also discuss the challenging point of sample preparation which needs to be addressed to fully enjoy the benefits of these nLC-nESI platforms. Finally, we assess metabolomic analysis utilising nano scale platforms and look ahead to the future of metabolomics using these new highly sensitive platforms.

Introduction

The aim of untargeted metabolomics is the comprehensive analysis of all known and unknow metabolites in a biological sample such as cells, biofluids or tissues at any one time [START_REF] Dunn | Systems level studies of mammalian metabolomes: the roles of mass spectrometry and nuclear magnetic resonance spectroscopy[END_REF].

Quantitative strategy can also be used where dozens to hundreds of metabolites are targeted and this is referred to as targeted metabolomics [START_REF] Lu | Analytical strategies for LC-MS-based targeted metabolomics[END_REF]. The metabolites in question are typically <1000 Da [START_REF] Nicholson | Global systems biology, personalized medicine and molecular epidemiology[END_REF][START_REF] Guy | Global metabolic profiling analysis on human urine by UPLC-TOFMS: Issues and method validation in nutritional metabolomics[END_REF], and are reactants, intermediates or by-products of enzymatic activity [START_REF] Dunn | Systems level studies of mammalian metabolomes: the roles of mass spectrometry and nuclear magnetic resonance spectroscopy[END_REF]. In some cases, these metabolites may be exogenous in source (e.g., xenobiotics), such as food additives from the diet [START_REF] Scalbert | The food metabolome: a window over dietary exposure[END_REF], pharmaceutical intake (e.g. anti-inflammatory drugs and mild analgesics, antidepressants) [START_REF] Johnson | Xenobiotic Metabolomics: Major Impact on the Metabolome[END_REF][START_REF] Kristensen | Analgesic use -prevalence, biomonitoring and endocrine and reproductive effects[END_REF] or use of cosmetics (e.g., parabens, UV filters) [START_REF] Jiménez-Díaz | Analytical methods for the determination of personal care products in human samples: An overview[END_REF]. The combination of the metabolome (including microbial metabolism) and xenobiotics as well as their products from phase I and/or II metabolism in biological samples is known as the (xeno)metabolome [START_REF] Johnson | Xenobiotic Metabolomics: Major Impact on the Metabolome[END_REF][START_REF] Al-Salhi | The Xenometabolome and Novel Contaminant Markers in Fish Exposed to a Wastewater Treatment Works Effluent[END_REF][START_REF] Holmes | Detection of urinary drug metabolite (Xenometabolome) signatures in molecular epidemiology studies via statistical total correlation (NMR) spectroscopy[END_REF][START_REF] David | A new approach for plasma (xeno)metabolomics based on solid-phase extraction and nanoflow liquid chromatographynanoelectrospray ionisation mass spectrometry[END_REF][START_REF] Vineis | The exposome in practice: Design of the EXPOsOMICS project[END_REF]. Profiling biological samples from different populations such as diseased and healthy subjects or exposed and non-exposed subjects using an untargeted metabolomics-based approach combined with chemometric pattern analysis allows for the potential to simultaneously uncover biomarkers of effect (i.e. disruption of endogenous metabolite profiles) as well as biomarkers of exposure (i.e., xenobiotic mixtures).

Metabolomics-based approaches has thus wide applicability in medicine [START_REF] Balog | Intraoperative Tissue Identification Using Rapid Evaporative Ionization Mass Spectrometry[END_REF][START_REF] Medina | Metabolomics and the Diagnosis of Human Diseases -A Guide to the Markers and Pathophysiological Pathways Affected[END_REF], toxicology [START_REF] Johnson | Xenobiotic Metabolomics: Major Impact on the Metabolome[END_REF][START_REF] Bonvallot | Potential Input From Metabolomics for Exploring and Understanding the Links Between Environment and Health[END_REF][START_REF] Bouhifd | Review: Toxicometabolomics[END_REF], food sciences [START_REF] Scalbert | The food metabolome: a window over dietary exposure[END_REF][START_REF] Ibanez | Global Foodomics strategy to investigate the health benefits of dietary constituents[END_REF], exposomics and human health studies [START_REF] Vineis | The exposome in practice: Design of the EXPOsOMICS project[END_REF][START_REF] Jamin | Untargeted profiling of pesticide metabolites by LC-HRMS: an exposomics tool for human exposure evaluation[END_REF][START_REF] Bonvallot | Metabolomics as a powerful tool to decipher the biological effects of environmental contaminants in humans[END_REF], plant sciences [START_REF] Fiehn | Metabolomics -the link between genotypes and phenotypes[END_REF][START_REF] Sumner | Plant metabolomics: large-scale phytochemistry in the functional genomics era[END_REF], and environmental sciences [START_REF] Al-Salhi | The Xenometabolome and Novel Contaminant Markers in Fish Exposed to a Wastewater Treatment Works Effluent[END_REF][START_REF] Viant | Mass spectrometry based environmental metabolomics: a primer and review[END_REF][START_REF] David | Disruption of the Prostaglandin Metabolome and Characterization of the Pharmaceutical Exposome in Fish Exposed to Wastewater Treatment Works Effluent As Revealed by Nanoflow-Nanospray Mass Spectrometry-Based Metabolomics[END_REF].

To undertake untargeted analysis, analytical platforms capable of analysing thousands of metabolites simultaneously are required. To date, the most widely used analytical platform is liquid chromatography-mass spectrometry (LC-MS), typically utilising an electrospray ionisation source (ESI) [START_REF] Theodoridis | Liquid chromatography-mass spectrometry based global metabolite profiling: A review[END_REF][START_REF] Gika | Current practice of liquid chromatographymass spectrometry in metabolomics and metabonomics[END_REF]. These LC-ESI platforms can offer highly reproducible and very high throughput methods to perform untargeted profiling of biological samples for metabolomics-based approaches.

Furthermore, the soft ionisation process of LC-ESI platforms allows structural elucidation and the analysis of both volatile and non-volatile metabolites. However, these LC-ESI platforms usually favour the detection of metabolites present at relatively high concentrations because of analytical issues such as poor ionisation efficiency and ion suppression [START_REF] Antignac | The ion suppression phenomenon in liquid chromatography-mass spectrometry and its consequences in the field of residue analysis[END_REF][START_REF] Metz | High Resolution Separations and Improved Ion Production and Transmission in Metabolomics[END_REF]. This is a major limitation since the idea behind the use of a metabolomics-based approach is to discriminate new biomarkers of effect or exposure that have so far remained unidentified. It is therefore essential for these untargeted methods to be sensitive enough to detect signalling metabolites or xenobiotics present at trace levels and that could be important in health effect outcomes. There is therefore a need for innovative strategies which allow to overcome sensitivity issues caused by ion suppression for metabolomics-based approaches.

As a result of these deficiencies, LC and ESI technologies have slowly been miniaturised and the latest developments in LC-MS have taken miniaturisation a step further with the development of nanoLC-nanoESI (nLC-nESI) [START_REF] Nazario | Evolution in miniaturized column liquid chromatography instrumentation and applications: An overview[END_REF][START_REF] Blue | Recent advances in capillary ultrahigh pressure liquid chromatography[END_REF]. As the name suggests, flow rates are on the nL/min scale and has been defined as LC platforms that deliver flow rates of 10-1000 nL/min with ESI emitters internal diameters (ID) of between 10 and 50 µm [START_REF] Wickremsinhe | A review of nanoelectrospray ionization applications for drug metabolism and pharmacokinetics[END_REF][START_REF] Chervet | Instrumental requirements for nanoscale liquid chromatography[END_REF]. The increased sensitivity of the nLC-nESI can be attributed to factors derived from both the nLC and nESI source, the latter being the main source of the increased sensitivity. The use of nESI emitters means that the droplets formed in the ESI plume are 100-1000 fold smaller than the typical droplets emitted from conventional ESI emitters [START_REF] Wilm | Analytical properties of the nanoelectrospray ion source[END_REF]. The process of ESI is an evaporative one, and therefore the generation of significantly smaller plume droplets with lower volumes considerably increases the rate of desolvation, resulting in up to 500 times more ions being formed and entering the mass analyser [START_REF] Wilm | Analytical properties of the nanoelectrospray ion source[END_REF][START_REF] Marginean | Picoelectrospray ionization mass spectrometry using narrow-bore chemically etched emitters[END_REF][START_REF] Karas | Nano-electrospray ionization mass spectrometry: addressing analytical problems beyond routine[END_REF][START_REF] Marginean | Analytical characterization of the electrospray ion source in the nanoflow regime[END_REF]. Another advantage of nESI lies in the fact that the emitter is closer to the MS inlet compared to conventional ESI, allowing a more efficient introduction of the ions formed into the inlet [START_REF] Smith | Ultrasensitive and Quantitative Analyses from Combined Separations-Mass Spectrometry for the Characterization of Proteomes[END_REF]. In addition, due to the much lower flow rate, chromatographic dilution is significantly decreased allowing more concentrated peaks to elute from the analytical column [START_REF] Gama | Nano-Liquid Chromatography in Pharmaceutical and Biomedical Research[END_REF][START_REF] Fanali | Separation of basic compounds of pharmaceutical interest by using nano-liquid chromatography coupled with mass spectrometry[END_REF][START_REF] Hilhorst | Sense and nonsense of miniaturized LC-MS/MS for bioanalysis[END_REF]. Further advantages of the nLC-nESI include reduced desolvation temperature [START_REF] Page | Ionization and Transmission Efficiency in an Electrospray Ionization-Mass Spectrometry Interface[END_REF] and the decrease of the consumption of both mobile and stationary phases [START_REF] Nazario | Evolution in miniaturized column liquid chromatography instrumentation and applications: An overview[END_REF].

Hence, these nano scale platforms offer a significant improvement in terms of sensitivity due to increased ionisation efficiency and reduced chromatographic dilution. These characteristics offer great potential to metabolomic researchers looking to encompass as much of the metabolome as possible. This review aims to give an insight to the advantages associated with nLC-nESI for metabolomics. To do this, we first describe the different techniques that have been used to achieve nLC and nESI for metabolomics. Here, we will focus on nanoscale instrumentation that have been successfully applied to metabolomic applications since comprehensive reviews on miniaturised liquid chromatography instrumentation can be found elsewhere, e.g., [START_REF] Nazario | Evolution in miniaturized column liquid chromatography instrumentation and applications: An overview[END_REF]. We then discuss the robustness of these platforms in targeted analysis and global metabolomic studies. We also discuss a challenging point, the sample preparation, which needs to be addressed to get the most out of the platforms sensitivity while not adversely impacting upon the metabolomic analysis. Subsequently, we look ahead to the potential nLC-nESI-MS has in metabolomics for elucidating the roles played by very low abundance metabolites in health, disease and the potential nLC-nESI-MS has for the identification of mixtures of xenobiotics in exposure assessment.

Overview of nLC-nESI platforms and advantages for metabolomics

The aim of this section is to present instrumentations which can be utilised for metabolomics to generate nanoflow rates, the different nanocolumns and nESI emitters and to discuss their advantages and drawbacks. An overview of instrumentation, nanocolumns and methods used for metabolomics can be seen in Table 1.

Nanoflow generation

The method by which nanoflow rates are produced can have a dramatic effect on the quality of the chromatography and repeatability of the analysis, either in terms of peak shape or retention time. Here the generation of nanoflow rates for direct infusion metabolomics will be discussed in addition to the formation of nanoflow rate by splitting higher flow rates and direct from pump generation for nLC-MS. These methods are known as self-fed/direct infusion, split flow, and direct flow, respectively.

Self-fed/ direct infusion nanoESI

While not a nLC-nESI method strictly speaking, direct infusion nESI-MS has proven popular for a number of metabolomic studies to analyse liver extracts [START_REF] Shi | Metabolomic Analysis of the Effects of Polychlorinated Biphenyls in Non-alcoholic Fatty Liver Disease[END_REF][START_REF] Southam | Dynamic range and mass accuracy of wide-scan direct infusion nanoelectrospray Fourier transform ion cyclotron resonance mass spectrometry-based metabolomics increased by the spectral stitching method[END_REF][START_REF] Wu | High-throughput tissue extraction protocol for NMRand MS-based metabolomics[END_REF], plasma [START_REF] Boernsen | Controlled protein precipitation in combination with chipbased nanospray infusion mass spectrometry. An approach for metabolomics profiling of plasma[END_REF], urine [START_REF] Chekmeneva | Optimization and Application of Direct Infusion Nanoelectrospray HRMS Method for Large-Scale Urinary Metabolic Phenotyping in Molecular Epidemiology[END_REF], zebrafish embryos [START_REF] Raterink | Rapid metabolic screening of early zebrafish embryogenesis based on direct infusion-nanoESI-FTMS[END_REF], Daphnia magna [START_REF] Taylor | A new approach to toxicity testing in Daphnia magna: application of high throughput FT-ICR mass spectrometry metabolomics[END_REF]. These studies benefitted from the generation of nanoflow rates within a nESI emitter to achieve sensitive and high throughput analysis [START_REF] Wickremsinhe | A review of nanoelectrospray ionization applications for drug metabolism and pharmacokinetics[END_REF][START_REF] Southam | Dynamic range and mass accuracy of wide-scan direct infusion nanoelectrospray Fourier transform ion cyclotron resonance mass spectrometry-based metabolomics increased by the spectral stitching method[END_REF].

Furthermore, direct infusion nESI reduces the volume of solvent used by 1000 fold compared to conventional LC-MS [START_REF] Dethy | Demonstration of Direct Bioanalysis of Drugs in Plasma Using Nanoelectrospray Infusion from a Silicon Chip Coupled with Tandem Mass Spectrometry[END_REF]. Self-fed systems load sample directly into an emitter to which a voltage is applied initiating a nanospray via capillary action and electrostatic force [START_REF] Marginean | Picoelectrospray ionization mass spectrometry using narrow-bore chemically etched emitters[END_REF][START_REF] Rahman | Realizing nano electrospray ionization using disposable pipette tips under super atmospheric pressure[END_REF][START_REF] Kanu | Evaluation of micro-versus nano-electrospray ionization for ambient pressure ion mobility spectrometry[END_REF]. These systems were initially utilised in proteomics, however, they were known to offer poor reproducibility [START_REF] Zhang | Chip-based nanoelectrospray mass spectrometry for protein characterization[END_REF][START_REF] Zhang | Automated chip-based nanoelectrospray-mass spectrometry for rapid identification of proteins separated by two-dimensional gel electrophoresis[END_REF][START_REF] Van Pelt | Characterization of a fully automated nanoelectrospray system with mass spectrometric detection for proteomic analyses[END_REF].

In addition, samples must be manually loaded individually into the tips and be installed for each run, making automated high-throughput analysis difficult [START_REF] Zhang | Chip-based nanoelectrospray mass spectrometry for protein characterization[END_REF][START_REF] Zhang | Automated chip-based nanoelectrospray-mass spectrometry for rapid identification of proteins separated by two-dimensional gel electrophoresis[END_REF][START_REF] Van Pelt | Characterization of a fully automated nanoelectrospray system with mass spectrometric detection for proteomic analyses[END_REF].

New chip based methods automatically aspirate sample into a disposable tip which interfaces with a nESI emitter [START_REF] Wickremsinhe | A review of nanoelectrospray ionization applications for drug metabolism and pharmacokinetics[END_REF][START_REF] Zhang | Chip-based nanoelectrospray mass spectrometry for protein characterization[END_REF][START_REF] Zhang | Automated chip-based nanoelectrospray-mass spectrometry for rapid identification of proteins separated by two-dimensional gel electrophoresis[END_REF][START_REF] Van Pelt | Characterization of a fully automated nanoelectrospray system with mass spectrometric detection for proteomic analyses[END_REF][START_REF] Van Pelt | A fully automated nanoelectrospray tandem mass spectrometric method for analysis of Caco-2 samples[END_REF]. Each tip and emitter is single use, eliminating carryover, reduces the impact of emitter blockage and significantly increases sample throughput [START_REF] Dethy | Demonstration of Direct Bioanalysis of Drugs in Plasma Using Nanoelectrospray Infusion from a Silicon Chip Coupled with Tandem Mass Spectrometry[END_REF][START_REF] Zhang | Automated chip-based nanoelectrospray-mass spectrometry for rapid identification of proteins separated by two-dimensional gel electrophoresis[END_REF][START_REF] Van Pelt | Characterization of a fully automated nanoelectrospray system with mass spectrometric detection for proteomic analyses[END_REF][START_REF] Hop | Uniformity of ionization response of structurally diverse analytes using a chip-based nanoelectrospray ionization source[END_REF][START_REF] Chekmeneva | Development of nanoelectrospray high resolution isotope dilution mass spectrometry for targeted quantitative analysis of urinary metabolites: application to population profiling and clinical studies[END_REF]. A novel method known as SIM stitch, collects direct infusion Orbitrap MS data using a series of overlapping SIM windows. Once collected SIM windows are "stitched" together to create a full MS spectra covering the entire m/z range. This technique alleviates the effect of space charge effects in the Orbitrap mass analyser and reduces the impact of high abundance metabolites being preferentially analysed [START_REF] Southam | Dynamic range and mass accuracy of wide-scan direct infusion nanoelectrospray Fourier transform ion cyclotron resonance mass spectrometry-based metabolomics increased by the spectral stitching method[END_REF][START_REF] Southam | A complete workflow for highresolution spectral-stitching nanoelectrospray direct-infusion mass-spectrometry-based metabolomics and lipidomics[END_REF].

The use of direct infusion nESI for high-throughput analyses seems to fit well with the application of metabolomics in studies which requires consecutive analysis of very large numbers of samples (e.g., epidemiological studies) by reducing dramatically the time and the cost of analysis.

However, the lack of chromatographic separation in direct infusion methods means that the analysis still suffers from ion suppression [START_REF] Dethy | Demonstration of Direct Bioanalysis of Drugs in Plasma Using Nanoelectrospray Infusion from a Silicon Chip Coupled with Tandem Mass Spectrometry[END_REF][START_REF] Hop | Uniformity of ionization response of structurally diverse analytes using a chip-based nanoelectrospray ionization source[END_REF]. In addition, these methods are unable to differentiate between isobaric/isomeric molecules or provide definitive metabolite identification without the need for subsequent fragmentation and/or chromatographic experiments, such as nLC-nESI-MS [START_REF] Chekmeneva | Development of nanoelectrospray high resolution isotope dilution mass spectrometry for targeted quantitative analysis of urinary metabolites: application to population profiling and clinical studies[END_REF].

Split flow

When nLC was first introduced commercial pumps capable of delivering nanoflow rates were not available [START_REF] Chervet | Instrumental requirements for nanoscale liquid chromatography[END_REF]. To counter this, HPLC pumps provided high flow rates which were subsequently split to provide nano scale flow rates with surplus mobile phase going to waste [START_REF] Metz | High Resolution Separations and Improved Ion Production and Transmission in Metabolomics[END_REF]. Depending upon where the flow is split, up to 99% of the sample can be lost, thus, eliminating the benefit of low volume or precious samples [START_REF] Smith | Ultrasensitive and Quantitative Analyses from Combined Separations-Mass Spectrometry for the Characterization of Proteomes[END_REF]. In addition, the green chemistry benefits are lost as most of the solvent goes to waste in the splitting process [START_REF] Chervet | Instrumental requirements for nanoscale liquid chromatography[END_REF][START_REF] Hernandez-Borges | Recent applications in nanoliquid chromatography[END_REF]. A further complication is evident when trying to perform a gradient separation. Due to differing mobile phase viscosities, variation in back pressure and surface tension is observed, making it more difficult to achieve stable retention characteristics when using non-commercial flow splitters [START_REF] Hernandez-Borges | Recent applications in nanoliquid chromatography[END_REF][START_REF] Valaskovic | Automated orthogonal control system for electrospray ionization[END_REF]. One large advantage to the use of split flow systems is that they have a dual use as a conventional LC and a nanoflow system effectively giving researchers two instruments in one. The lack of split flow use in metabolomics potentially reflects upon the aforementioned disadvantages.

Direct flow

In recent years, technological advances have enabled commercially available nano reciprocal or syringe pumps to become widely available [START_REF] Šesták | Instrument platforms for nano liquid chromatography[END_REF]. These platforms have been called direct nanoflow, due to the nanoflow rates being provided directly from the LC pumps. Unlike most of their split flow counterparts, direct flow platforms make use of micro fluidic flow controllers which control flow rates much more precisely than flow split systems [START_REF] Noga | A practical guide to nano-LC troubleshooting[END_REF]. Furthermore, there are fewer connections in the system thus reducing the risk of leakages, zero dead volumes and blockages [START_REF] Noga | A practical guide to nano-LC troubleshooting[END_REF]. The use of direct nanoflow is of particular importance in analysis utilising a gradient system. This is due to its ability to allow for variation in mobile phase viscosities using sophisticated flow controllers [START_REF] Hernandez-Borges | Recent applications in nanoliquid chromatography[END_REF][START_REF] Valaskovic | Automated orthogonal control system for electrospray ionization[END_REF]. It is known that providing nanoflow rates directly from the pump improves retention and spray characteristics of the nano platform [START_REF] Gama | Nano-Liquid Chromatography in Pharmaceutical and Biomedical Research[END_REF][START_REF] Hernandez-Borges | Recent applications in nanoliquid chromatography[END_REF]. In addition, the cost and environmental benefits of reduced solvent usage are also realised using direct nanoflow pumps [START_REF] Hernandez-Borges | Recent applications in nanoliquid chromatography[END_REF].

To date direct nLC has been implemented in a small number of metabolomic analysis of urine [START_REF] Chetwynd | Solid phase extraction and splitless nanoflow liquid chromatography -nanoelectrospray ionisation mass spectrometry for improved global urine metabolomics[END_REF][START_REF] Chetwynd | Use of a pre-analysis osmolality normalisation method to correct for variable urine concentrations and for improved metabolomic analyses[END_REF][START_REF] Chetwynd | Nanoflow-Nanospray Mass Spectrometry Metabolomics Reveals Disruption of the Urinary Metabolite Profiles of HIV-Positive Patients on Combination Antiretroviral Therapy[END_REF][START_REF] Medina | Non-targeted metabolomic approach reveals urinary metabolites linked to steroid biosynthesis pathway after ingestion of citrus juice[END_REF], plasma [START_REF] David | A new approach for plasma (xeno)metabolomics based on solid-phase extraction and nanoflow liquid chromatographynanoelectrospray ionisation mass spectrometry[END_REF], tissue [START_REF] David | Disruption of the Prostaglandin Metabolome and Characterization of the Pharmaceutical Exposome in Fish Exposed to Wastewater Treatment Works Effluent As Revealed by Nanoflow-Nanospray Mass Spectrometry-Based Metabolomics[END_REF], faeces [START_REF] Stoll | Fecal metabolomics in pediatric spondyloarthritis implicate decreased metabolic diversity and altered tryptophan metabolism as pathogenic factors[END_REF], exhaled breath condensates [START_REF] Kononikhin | Exhaled breath condensate analysis from intubated newborns by nano-HPLC coupled to high resolution MS[END_REF], sweat [START_REF] Li | Nanoflow LC-MS for High-Performance Chemical Isotope Labeling Quantitative Metabolomics[END_REF], cerebrospinal fluids [START_REF] Myint | Quantitative profiling of polar cationic metabolites in human cerebrospinal fluid by reversed-phase nanoliquid chromatography/mass spectrometry[END_REF] and cell extracts [START_REF] Jones | A Nano Ultra-Performance Liquid Chromatography-High Resolution Mass Spectrometry Approach for Global Metabolomic Profiling and Case Study on Drug-Resistant Multiple Myeloma[END_REF].

Nanocolumns

According to the classification of Saito et al. [START_REF] Saito | Capillary columns in liquid chromatography: between conventional columns and microchips[END_REF], nanocolumns range from 0.01 to 0.1 mm internal diameter (ID). In this review, we have included metabolomic studies using nanocolumns with ID up to 0.2 mm but with flow rates within the nanoflow rate ranges (i.e., up to 1000 nL/min) (Table 1). The main advantages of columns with lower ID compared with conventional HPLC columns are reduced chromatographic dilution, lower consumption of mobile phases and lower consumption of sample mainly due to the lower flow rates [START_REF] Nazario | Evolution in miniaturized column liquid chromatography instrumentation and applications: An overview[END_REF][START_REF] Collins | Porous layer open tubular columns in capillary liquid chromatography[END_REF].

Due to the broad range of physico-chemical properties of small molecules (e.g., polarity, size) analysed in metabolomics, it is important that as many as possible can be retained on an analytical column with sufficient separation between each other. Nanocolumns used in metabolomic studies so far include BEH C18, HSS T3, Acclaim PepMax C18 and ZIC-HILIC for a wide range of matrices (Table 1). It must be noted that the majority of nanocolumns available utilise conventional reversed phased column chemistries. However, normal phase such as HILIC are now available from Merck (ZIC-HILIC 75-100 µm i.d), Sciex cHiPLC (75 µm x 15cm HALO HILIC), Tosoh (TSK-GEL Amide-80 HILIC, 50 µm i.d.) and NewObjective (Unison UK-Amino 75 µm -10 mm) which could then be used to improve the retention of very polar and polar metabolites for nanoscale platforms as seen in the sole nanoHILIC study to date [START_REF] Myint | Quantitative profiling of polar cationic metabolites in human cerebrospinal fluid by reversed-phase nanoliquid chromatography/mass spectrometry[END_REF].

A comparative study of three reversed phase nanocolumns with two column chemistries and three different pore sizes (BEH C18 300 and 130 Å, and HSS T3 100 Å) was performed in view of metabolomic applications for urine and plasma [START_REF] Chetwynd | Evaluation of analytical performance and reliability of direct nanoLC-nanoESI-high resolution mass spectrometry for profiling the (xeno)metabolome[END_REF] (see Fig. 1). Increased retention, separation and increased peak area were observed for most of the metabolites tested with the low porosity columns (100 and 130 Å), due to a combination of decreased chromatographic dilution, increased stationary phase surface area and increased mass transfer rate [START_REF] Hilhorst | Sense and nonsense of miniaturized LC-MS/MS for bioanalysis[END_REF][START_REF] Chetwynd | Evaluation of analytical performance and reliability of direct nanoLC-nanoESI-high resolution mass spectrometry for profiling the (xeno)metabolome[END_REF][START_REF] Motokawa | Monolithic silica columns with various skeleton sizes and through-pore sizes for capillary liquid chromatography[END_REF]. Furthermore, the retention of many analytes was favoured by the HSS phase compared with the BEH, suggesting that the use of HSS T3 phase with porosity smaller pore size could increase the performance of the system by reducing co-elution and ion suppression for metabolomic applications.

Similar phases to these used for metabolomics with conventional HPLC columns are already available for nanocolumns and allow to achieve a comprehensive coverage of the metabolome of urine [START_REF] Chetwynd | Nanoflow-Nanospray Mass Spectrometry Metabolomics Reveals Disruption of the Urinary Metabolite Profiles of HIV-Positive Patients on Combination Antiretroviral Therapy[END_REF], plasma [START_REF] Holmes | Detection of urinary drug metabolite (Xenometabolome) signatures in molecular epidemiology studies via statistical total correlation (NMR) spectroscopy[END_REF] and tissues samples [START_REF] David | Disruption of the Prostaglandin Metabolome and Characterization of the Pharmaceutical Exposome in Fish Exposed to Wastewater Treatment Works Effluent As Revealed by Nanoflow-Nanospray Mass Spectrometry-Based Metabolomics[END_REF]. However, more nanocolumns adapted in terms of column chemistry and pore sizes (i.e. lower pore size than for proteomics) would be welcome to gain optimal sensitivity and chromatographic separation of polar, mid-polar and apolar metabolites for metabolomic analysis.

Nano ESI sources

A nESI source is characterised by the narrow bore nanospray emitter. The sensitivity and reproducibility of nESI analysis is directly related to the quality of the emitter [START_REF] Lopes | Quantification of intact human insulinlike growth factor-I in serum by nano-ultrahigh-performance liquid chromatography/tandem mass spectrometry[END_REF][START_REF] Gibson | Nanoelectrospray emitters: Trends and perspective[END_REF]. Several factors such as the emitter material, tip geometry and the internal diameter influence the quality of nESI emitters [START_REF] Rahman | Realizing nano electrospray ionization using disposable pipette tips under super atmospheric pressure[END_REF][START_REF] Lopes | Quantification of intact human insulinlike growth factor-I in serum by nano-ultrahigh-performance liquid chromatography/tandem mass spectrometry[END_REF][START_REF] Kelly | Chemically etched open tubular and monolithic emitters for nanoelectrospray ionization mass spectrometry[END_REF]. For metabolomic applications, metal emitter may provide the most robust qualities and can be cleaned thus reducing costs associated with replacing damaged or blocked emitters [START_REF] Lopes | Quantification of intact human insulinlike growth factor-I in serum by nano-ultrahigh-performance liquid chromatography/tandem mass spectrometry[END_REF][START_REF] Shui | Micro-electrospray with stainless steel emitters[END_REF]. It is worth noting that homemade emitters can be constructed and these are generally less expensive that commercial ones. The geometry of the emitter is an important consideration for small molecule analysis as it impacts upon spray stability and emitter longevity.

The use of tapered emitters has been demonstrated to have a shorter usable lifetime than nontapered tips, mainly as a result of clogging [START_REF] Kelly | Chemically etched open tubular and monolithic emitters for nanoelectrospray ionization mass spectrometry[END_REF]. In other studies, tapered emitters have been found to produce a more stable nanospray, meaning that more reproducible mass spectra can be produced [START_REF] Lopes | Quantification of intact human insulinlike growth factor-I in serum by nano-ultrahigh-performance liquid chromatography/tandem mass spectrometry[END_REF][START_REF] Gibson | Nanoelectrospray emitters: Trends and perspective[END_REF]. The internal diameter of the emitter is a further source of increased ionisation efficiency of the nESI source. A study by Lopes et al. [START_REF] Lopes | Quantification of intact human insulinlike growth factor-I in serum by nano-ultrahigh-performance liquid chromatography/tandem mass spectrometry[END_REF] investigated 4 internal diameters; 5, 10, 20 and 30 µm and found that all emitters had similar sensitivity in terms of spectral features and total ion current.

However, it was determined that the 30 µm was preferred as clogging is less frequent due to the larger orifice [START_REF] Lopes | Quantification of intact human insulinlike growth factor-I in serum by nano-ultrahigh-performance liquid chromatography/tandem mass spectrometry[END_REF]. In other studies, the smaller the internal diameter, the greater the observed ionisation efficiency, however, emitter clogging became problematic at internal diameters below 10 µm [START_REF] Xiong | Separation and Sequencing of Isomeric Oligonucleotide Adducts Using Monolithic Columns by Ion-Pair Reversed-Phase Nano-HPLC Coupled to Ion Trap Mass Spectrometry[END_REF].

With these factors considered, the use of non-tapered emitters with an internal diameter in the range of 10-30 µm offers an acceptable compromise between sensitivity, system longevity and potential loss of sample.

Robustness of nanoflow platforms for metabolomics

In metabolomics, the ability to generate highly repeatable and reliable data is a key factor to ensure high quality studies can be carried out [START_REF] Benton | Intra-and Interlaboratory Reproducibility of Ultra Performance Liquid Chromatography-Time-of-Flight Mass Spectrometry for Urinary Metabolic Profiling[END_REF][START_REF] Gika | Within-day reproducibility of an HPLC-MS-Based method for metabonomic analysis: Application to human urine[END_REF]. Two major sources of variation that limit the quality of metabolomic data sets are retention time drift and poor peak intensity reproducibility [START_REF] Benton | Intra-and Interlaboratory Reproducibility of Ultra Performance Liquid Chromatography-Time-of-Flight Mass Spectrometry for Urinary Metabolic Profiling[END_REF][START_REF] Gika | A QC approach to the determination of day-today reproducibility and robustness of LC-MS methods for global metabolite profiling in metabonomics/metabolomics[END_REF]. The stability of retention time in particular is of great significance during the peak picking process prior to multivariate statistical analysis. To date, many studies have reported on retention time and mean peak area repeatability while analysing a broad range of compounds and matrices. While many of these have been in targeted approaches they provide a good indication as to the suitability for untargeted small molecule analysis.

Retention time stability

For metabolomic analysis, it has been suggested that the % coefficient of variation (%CV) for retention time should not exceed 2% [START_REF] Theodoridis | Liquid chromatography-mass spectrometry based global metabolite profiling: A review[END_REF]. The retention time variation reported in studies using both direct and split nanoflow systems for small molecule analysis in targeted and untargeted studies are detailed in Table 2. The range of CVs reported was lower with direct nanoflow systems (<0.20% to 2.2%) than for split flow ones (0.50-3.4%). Furthermore, all reported CVs were below the 2% threshold with direct nanoflow systems (with the exception of the 2D nLC system) and usually lower than 1% (in 12 out of 19 studies). On the other hand, 3 out 13 reported CVs were higher than the 2% threshold for the split flow and these systems typically have CVs greater than 1% (in 9 out of 13 studies). Overall, these studies reported highly reproducible results for retention times but seems to highlight the importance of pump fed direct nanoflow for retention time stability [START_REF] Theodoridis | Liquid chromatography-mass spectrometry based global metabolite profiling: A review[END_REF].

The number of metabolomic studies reporting retention time stability for nanoflow platforms is very limited. To date, only two have reported retention time stability of metabolites [START_REF] David | A new approach for plasma (xeno)metabolomics based on solid-phase extraction and nanoflow liquid chromatographynanoelectrospray ionisation mass spectrometry[END_REF][START_REF] Chetwynd | Solid phase extraction and splitless nanoflow liquid chromatography -nanoelectrospray ionisation mass spectrometry for improved global urine metabolomics[END_REF]. In both studies, the CV was below 1.9%. A further metabolomics study has reported the retention time stability of 6 internal standards, and CVs below 0.2% were reported for these internal standards spiked in plasma and urine [START_REF] Chetwynd | Evaluation of analytical performance and reliability of direct nanoLC-nanoESI-high resolution mass spectrometry for profiling the (xeno)metabolome[END_REF].

Comparing retention time repeatability reported in different studies is quite difficult because of differences in chromatographic methods (e.g. solvents, additives such as formic acid) and matrices used for the experiment. Nevertheless, the reported CVs for the split and direct nanoflow platforms suggest that reproducible retention characteristics are achievable on both nanoflow systems even if direct nanoflow provides slightly more retention time reproducibility.

NanoESI MS response stability

It has been suggested that a CV of up to 30% is acceptable for variation of mean peak area in metabolomics analysis [START_REF] Want | Global metabolic profiling procedures for urine using UPLC-MS[END_REF]. The mean peak CV has been reported in a number of studies using nanoflow systems, covering a wide range of analytes and samples matrices (Table 3).

Overall, these targeted and untargeted studies indicate that a high degree of peak area reproducibility can be achieved since CV of peak area calculated for a wide range of metabolites were lower than 20% in all but one study. There appears to be little difference between emitter geometry and internal diameters for those studies, showing that no single diameter or geometry appeared preferential in terms of reproducibility for these studies. The low reproducibility reported by Kiefer et al. [START_REF] Kiefer | Nanoscale Ion-Pair Reversed-Phase HPLC-MS for Sensitive Metabolome Analysis[END_REF] may be due to the fact that ion pairing solvents were in use which can be less stable in the ESI source. Moreover, this was reported for only 1 compound (all others being below 14%). While these studies investigated only a limited number of compounds, they demonstrate that nLC-nESI is reproducible in terms of peak area.

As with retention time stability, the mean peak area reproducibility in metabolomic analysis has only been investigated in a small number of cases. In these cases the variation in mean peak areas for the whole metabolome were investigated using either the method or a modification of the method proposed by Want et al. [START_REF] Want | Global metabolic profiling procedures for urine using UPLC-MS[END_REF]. In all these studies, >70% peaks of quality control samples returned CV of <30% for negative and positive nESI respectively [START_REF] David | A new approach for plasma (xeno)metabolomics based on solid-phase extraction and nanoflow liquid chromatographynanoelectrospray ionisation mass spectrometry[END_REF][START_REF] Chetwynd | Solid phase extraction and splitless nanoflow liquid chromatography -nanoelectrospray ionisation mass spectrometry for improved global urine metabolomics[END_REF][START_REF] Chetwynd | Use of a pre-analysis osmolality normalisation method to correct for variable urine concentrations and for improved metabolomic analyses[END_REF][START_REF] Chetwynd | Nanoflow-Nanospray Mass Spectrometry Metabolomics Reveals Disruption of the Urinary Metabolite Profiles of HIV-Positive Patients on Combination Antiretroviral Therapy[END_REF]. Furthermore, Jones et al. reported that >50% of all peaks measure in the metabolome of yeast cell extracts had a CV of < 14% [START_REF] Jones | A Nano Ultra-Performance Liquid Chromatography-High Resolution Mass Spectrometry Approach for Global Metabolomic Profiling and Case Study on Drug-Resistant Multiple Myeloma[END_REF]. Each of these studies indicates that the reproducibility of the nESI source is capable of supplying reliable results for metabolomics.

Sample preparation strategies for metabolomic studies using nLC-nESI

Sample preparation is an important aspect to consider for the use of nanoflow platforms for metabolomics [START_REF] David | A new approach for plasma (xeno)metabolomics based on solid-phase extraction and nanoflow liquid chromatographynanoelectrospray ionisation mass spectrometry[END_REF][START_REF] Chetwynd | Evaluation of analytical performance and reliability of direct nanoLC-nanoESI-high resolution mass spectrometry for profiling the (xeno)metabolome[END_REF][START_REF] Garcia-Villalba | Nano and rapid resolution liquid chromatography-electrospray ionization-time of flight mass spectrometry to identify and quantify phenolic compounds in olive oil[END_REF]. The narrow bore columns, emitters and connections are prone to blockages and consequently, efficient sample clean-up are recommended to remove particulates, salts and proteins [START_REF] David | A new approach for plasma (xeno)metabolomics based on solid-phase extraction and nanoflow liquid chromatographynanoelectrospray ionisation mass spectrometry[END_REF]. In the field of untargeted metabolomics, it has often been argued that selective sample preparation such as solid phase extraction (SPE) should be avoided in order to have the whole picture of the sample. However, several studies have shown that sample preparation based on SPE can increase the coverage of the metabolome compared with conventional protein precipitation (PPT) as well as the repeatability of the method [START_REF] David | A new approach for plasma (xeno)metabolomics based on solid-phase extraction and nanoflow liquid chromatographynanoelectrospray ionisation mass spectrometry[END_REF][START_REF] Yang | New sample preparation approach for mass spectrometry-based profiling of plasma results in improved coverage of metabolome[END_REF][START_REF] Tulipani | Comparative analysis of sample preparation methods to handle the complexity of the blood fluid metabolome: when less is more[END_REF][START_REF] Michopoulos | UPLC-MS-based analysis of human plasma for metabonomics using solvent precipitation or solid phase extraction[END_REF].

Sample preparation for metabolomic purposes that allow for the efficient removal of interfering matrix components other than PPT include, for instance, offline or online SPE, solid-phase microextraction (SPME), ultrafiltration, delipidation using commercially available lipid depletion plate and Quick, Easy, Cheap, Effective, Rugged and Safe (QuEChERS) methods [START_REF] David | A new approach for plasma (xeno)metabolomics based on solid-phase extraction and nanoflow liquid chromatographynanoelectrospray ionisation mass spectrometry[END_REF][START_REF] Gika | Current practice of liquid chromatographymass spectrometry in metabolomics and metabonomics[END_REF][START_REF] Tulipani | Comparative analysis of sample preparation methods to handle the complexity of the blood fluid metabolome: when less is more[END_REF][START_REF] Vuckovic | Current trends and challenges in sample preparation for global metabolomics using liquid chromatography-mass spectrometry[END_REF][START_REF] Raterink | Recent developments in sample-pretreatment techniques for mass spectrometry-based metabolomics[END_REF][START_REF] Yin | Current state-of-the-art of nontargeted metabolomics based on liquid chromatography-mass spectrometry with special emphasis in clinical applications[END_REF][START_REF] Berlioz-Barbier | Non-targeted investigation of benthic invertebrates (Chironomus riparius) exposed to wastewater treatment plant effluents using nanoliquid chromatography coupled to high-resolution mass spectrometry[END_REF].

These techniques can be used on their own or in combination with other techniques such as LLE, with or without fractionation [START_REF] Yang | New sample preparation approach for mass spectrometry-based profiling of plasma results in improved coverage of metabolome[END_REF]. Among these different techniques, delipidation (mainly lyso-and phospholipids) of the sample has gained increased interest because it can reduce ion-suppression effects and can increase column lifetime [START_REF] David | A new approach for plasma (xeno)metabolomics based on solid-phase extraction and nanoflow liquid chromatographynanoelectrospray ionisation mass spectrometry[END_REF][START_REF] Raterink | Recent developments in sample-pretreatment techniques for mass spectrometry-based metabolomics[END_REF]. These recent innovations are effective for removing phospholipids and they have been shown, in combination to solvent deproteinisation, to enhance analyte detection of non-lipid species in comparison to extractions with organic solvents and a membrane-based solvent free technique [START_REF] Tulipani | Comparative analysis of sample preparation methods to handle the complexity of the blood fluid metabolome: when less is more[END_REF]. It is also worth noting that miniaturised sample preparation techniques such as SPME [START_REF] Gionfriddo | New Generation of Solid-Phase Microextraction Coatings for Complementary Separation Approaches: A Step toward Comprehensive Metabolomics and Multiresidue Analyses in Complex Matrices[END_REF], stir bar sorptive extraction (SBSE) [START_REF] David | Stir bar sorptive extraction for trace analysis[END_REF]or microextraction by packed sorbent (MEPS) [START_REF] Abdel-Rehim | Recent advances in microextraction by packed sorbent for bioanalysis[END_REF] may be applied to nanoscale LC, and applications in metabolomic studies are numerous in the case of SPME [START_REF] Gionfriddo | New Generation of Solid-Phase Microextraction Coatings for Complementary Separation Approaches: A Step toward Comprehensive Metabolomics and Multiresidue Analyses in Complex Matrices[END_REF][START_REF] Bojko | Solid-phase microextraction in metabolomics[END_REF].

Examples of applications of these sample preparation techniques in metabolomics-based approaches using a nLC-nESI system include a study where plasma samples were extracted using phospholipid filtration plates in combination with polymeric or mixed mode exchange SPE [START_REF] David | A new approach for plasma (xeno)metabolomics based on solid-phase extraction and nanoflow liquid chromatographynanoelectrospray ionisation mass spectrometry[END_REF].

Another study described a SPE methodology to enable a high urine equivalence to be injected on a nLC-nESI-MS platform [START_REF] Chetwynd | Solid phase extraction and splitless nanoflow liquid chromatography -nanoelectrospray ionisation mass spectrometry for improved global urine metabolomics[END_REF]. Additionally, lyophilisation and SPE has been used to prepare yeast samples for metabolomic analysis using a nLC-nESI-MS platform [START_REF] Jones | A Nano Ultra-Performance Liquid Chromatography-High Resolution Mass Spectrometry Approach for Global Metabolomic Profiling and Case Study on Drug-Resistant Multiple Myeloma[END_REF]. These studies showed that injections of more concentrated extracts onto nLC-nESI-MS platforms using more selective sample preparation can result in a wider coverage of the metabolome and can also extend the column life time.

In addition, column trapping has also been used in metabolomics-based approaches using a nLC-nESI system to load a larger volume of sample. While the metabolites are "trapped", mobile phase continues to wash through the column thus removing any salts and any other unretained contaminants [START_REF] Li | Nanoflow LC-MS for High-Performance Chemical Isotope Labeling Quantitative Metabolomics[END_REF]. This method can be used to replace offline SPE and automate the entire sample preparation and analysis process [START_REF] Luo | Metabolomics of Small Numbers of Cells: Metabolomic Profiling of 100, 1000 and 10000 Human Breast Cancer Cells[END_REF].

Current and potential use of nLC-nESI in metabolomics

Although still limited, several papers have already used nLC-nESI-MS platforms in metabolomics. These studies have highlighted that these systems may be up to 2000 fold more sensitive than conventional LC-ESI-MS [START_REF] Chetwynd | Evaluation of analytical performance and reliability of direct nanoLC-nanoESI-high resolution mass spectrometry for profiling the (xeno)metabolome[END_REF] and with LOD and LOQs up to 300 fold lower using nLC-nESI-MS [START_REF] Garcia-Villalba | Nano and rapid resolution liquid chromatography-electrospray ionization-time of flight mass spectrometry to identify and quantify phenolic compounds in olive oil[END_REF]. Two studies have also shown how nLC-nESI-MS can be combined with chemical isotope labelling to further increase the coverage of the metabolome and enhance the relative quantification of metabolites [START_REF] Li | Nanoflow LC-MS for High-Performance Chemical Isotope Labeling Quantitative Metabolomics[END_REF][START_REF] Luo | Metabolomics of Small Numbers of Cells: Metabolomic Profiling of 100, 1000 and 10000 Human Breast Cancer Cells[END_REF]. In this section, we review the different studies which have used nLC-nESI-MS systems in metabolomics according to their applications.

Health and disease metabolomics

The prospect of very highly sensitive analysis of low abundance or difficult to ionise metabolite species provides an exciting prospect for metabolomic studies of health and disease. Metabolic pathways of particular interest are, for instance, conjugated and unconjugated sex steroids such as the estrogens and androgens which are implicated in several cancers, infertility and other related endocrine disruption problems [START_REF] Gouveia | Mass spectrometry techniques in the survey of steroid metabolites as potential disease biomarkers: A review[END_REF]. These metabolites are typically found at low concentrations and usually difficult to ionise using conventional ESI-MS. Also of interest and found at low concentrations are metabolites involved in inflammation and several disease pathways such as eicosanoids [START_REF] Singh | Cysteinyl Leukotrienes and Their Receptors: Molecular and Functional Characteristics[END_REF].

A couple of early metabolomics-based studies investigated the effect of dietary compounds on health and disease using nLC-nESI-MS. In one study, the potential for phenol rich olive oil was investigated for its cell proliferation inhibition capacity in colon cancer [START_REF] Fernández-Arroyo | Application of nanoLC-ESI-TOF-MS for the metabolomic analysis of phenolic compounds from extra-virgin olive oil in treated colon-cancer cells[END_REF]. The other nutraceutical investigation using nLC-nESI-MS was implemented to investigate the health benefits associated with the consumption of citrus juice [START_REF] Medina | Non-targeted metabolomic approach reveals urinary metabolites linked to steroid biosynthesis pathway after ingestion of citrus juice[END_REF]. In this study, significant changes in steroidogenesis pathways were detected following citrus juice consumption. Some of the detected steroids, in particular 17hydroxyprogesterone, are typically found at low levels in urine, thus requiring a highly sensitive analysis to detect these changes.

In human health and disease, four studies using nLC-nESI-MS have been carried out to date, one on HIV [START_REF] Chetwynd | Nanoflow-Nanospray Mass Spectrometry Metabolomics Reveals Disruption of the Urinary Metabolite Profiles of HIV-Positive Patients on Combination Antiretroviral Therapy[END_REF], one on drug resistant multiple myeloma [START_REF] Jones | A Nano Ultra-Performance Liquid Chromatography-High Resolution Mass Spectrometry Approach for Global Metabolomic Profiling and Case Study on Drug-Resistant Multiple Myeloma[END_REF], a third on paediatric spondylarthrosis [START_REF] Stoll | Fecal metabolomics in pediatric spondyloarthritis implicate decreased metabolic diversity and altered tryptophan metabolism as pathogenic factors[END_REF] and the most recent investigating lung disease in new-born neonates [START_REF] Kononikhin | Exhaled breath condensate analysis from intubated newborns by nano-HPLC coupled to high resolution MS[END_REF]. The metabolomic analysis of anti-retroviral toxicity in the urine of HIV positive patients detected for the first time all the anti-retroviral drugs and a wide range of their metabolites in addition to several low abundance endogenous metabolites in an untargeted analysis (see Fig. 2 for example of chromatograms) [START_REF] Chetwynd | Nanoflow-Nanospray Mass Spectrometry Metabolomics Reveals Disruption of the Urinary Metabolite Profiles of HIV-Positive Patients on Combination Antiretroviral Therapy[END_REF].

The most recent study investigated exhaled breath condensates from two lung morphologies in intubated neonates (<20 days old). The ability to analyse samples such as exhaled breath condensates requires a highly sensitive technique due to the low volumes available. Concentrations of a number eicosanoids were found to differentiate between the control group and the diseased lung groups highlighting an inflammatory aspect of the conditions [START_REF] Kononikhin | Exhaled breath condensate analysis from intubated newborns by nano-HPLC coupled to high resolution MS[END_REF]. Furthermore, eicosanoids were detected in 60% of all samples underlying the sensitivity of the nLC-nESI-MS approach used as previous analysis has shown these compounds are present at a pg/mL level in exhaled breath condensates [START_REF] Kononikhin | Exhaled breath condensate analysis from intubated newborns by nano-HPLC coupled to high resolution MS[END_REF][START_REF] Ciebiada | Eicosanoids in exhaled breath condensate and bronchoalveolar lavage fluid of patients with primary lung cancer[END_REF].

An example of application using targeted metabolomics includes a study where a sensitive nLC-nESI-MS method was developed to monitor 184 phosphorus-related metabolic changes in small volumes of cancer cells treated with metabolic enzyme inhibitors [START_REF] Uehara | Quantitative Phosphorus Metabolomics Using Nanoflow Liquid Chromatography-Tandem Mass Spectrometry and Culture-Derived Comprehensive Global Internal Standards[END_REF].

These studies indicate that nLC-nESI-MS platforms are well suited for metabolomic analysis in health and disease studies. Using nLC-nESI-MS to uncover low abundance signalling metabolites, it may become possible to characterise unique metabolites and metabolic pathways affected in different cancers and diseases.

Exposure assessment and biomonitoring studies

With regards to the very high diversity of xenobiotics present in the environment, metabolomics-based approaches using highly sensitive nLC-nESI platforms offer exciting perspectives to detect xenobiotic mixtures (i.e., the chemical exposome), usually present at trace levels, in environmental and biological samples and simultaneously study their potential associated health effects [START_REF] Rappaport | The blood exposome and its role in discovering causes of disease[END_REF][START_REF] Wild | The exposome: from concept to utility[END_REF].

To date, the use of nLC-nESI-MS platforms in untargeted analysis for exposure assessment has been very limited. It includes a study in which a metabolomics-based approach using a nUPLC-nESI-TOFMS platform was used to investigate the chemical mixtures accumulating in fish exposed to a treated wastewater effluent and the associated changes in the tissues metabolome [START_REF] David | Disruption of the Prostaglandin Metabolome and Characterization of the Pharmaceutical Exposome in Fish Exposed to Wastewater Treatment Works Effluent As Revealed by Nanoflow-Nanospray Mass Spectrometry-Based Metabolomics[END_REF]. A wide range of contaminants including 31 pharmaceuticals from 11 different classes, endocrine disrupters, personal care products, pesticides, antibacterials and human dietary products were detected, sometimes at very low concentrations, in blood plasma and tissues of effluent-exposed fish (see Fig. 3 for example of chromatograms). Concurrently, metabolite profiling revealed for the first time a widespread reduction (between 50% and 90%) in prostaglandin (E2 and F2α) profiles in effluentexposed fish tissues/plasma [START_REF] David | Disruption of the Prostaglandin Metabolome and Characterization of the Pharmaceutical Exposome in Fish Exposed to Wastewater Treatment Works Effluent As Revealed by Nanoflow-Nanospray Mass Spectrometry-Based Metabolomics[END_REF]. Another example of application using nLC platform for exposure assessment includes a study where an untargeted approach was used to investigate changes in a small benthic invertebrates exposed to a wastewater treatment plant effluent. Significant changes in lipid metabolism were observed and several xenobiotics were detected (e.g., ibuprofen and propranolol) in invertebrates exposed to the wastewater treatment plant effluent using very small invertebrate samples (i.e., 12 mg) [START_REF] Berlioz-Barbier | Non-targeted investigation of benthic invertebrates (Chironomus riparius) exposed to wastewater treatment plant effluents using nanoliquid chromatography coupled to high-resolution mass spectrometry[END_REF].

These studies show that highly sensitive untargeted techniques based on nanoscale could be extremely useful to identify xenobiotics that need to be prioritised for future toxicological investigations.

Conclusions and future perspectives

The current literature surrounding nLC-nESI clearly demonstrates that significant gains in sensitivity for metabolites can be achieved compared to conventional LC-ESI platforms (see Table 4 for a summary of advantages and drawbacks of nLC-nESI platforms). While in the past there has been concern about nLC retention time reproducibility, the review of existing data shows that the improvements made since the introduction of nanoflow, such as the implementation of direct nanoflow platforms, have greatly contributed to improve the reproducibility of these platforms since the majority of these studies fall within the guidelines proposed within the metabolomic community.

As mentioned in this review, one key factor is sample preparation, which is required to extend column and emitter lifetimes by reducing blockages. While this process is indeed more time consuming and may be seen as selective, several studies have shown that more extensive sample preparation can increase the coverage of the metabolome as it allows injection of more concentrated extracts and, on the other hand, improve the repeatability of the method. Hence, more automated sample preparation methods should be developed in the future to implement metabolomic methods based on nLC-nESI platforms for high throughput applications. Taking the current nLC-nESI methods and potential future advances into account, new and exciting possibilities exist to expand impact of metabolomics on human and environmental health and disease. The increased sensitivity of these nLC-nESI platforms offer the potential to uncover the roles played by low abundant signalling metabolites in health and disease studies, and would also enable an improvement in the detection of xenobiotics present at trace levels in biological matrices to better characterise the chemical exposome and uncover potential health outcomes associated. While many aspects of metabolomics are still under progress (e.g., development of more automated annotation work-flows to overcome problems linked to unidentified features), the development of analytical methods of higher sensitivity is also a priority to be as comprehensive as possible. detection and quantification of trace pharmaceuticals in benthic invertebrates, Talanta 132(0) (2015) 796-802.

[141] M. Sordet, A. Berlioz-Barbier, A. Bulete, J. Garric, E. Vulliet, Quantification of emerging micropollutants in an amphipod crustacean by nanoliquid chromatography coupled to mass spectrometry using multiple reaction monitoring cubed mode, J Chromatogr A 1456 (2016) 217-25. Positive ESI mode base peak intensity (BPI) of pooled HIV patient urine run using nLC-nESI-TOFMS (A) and extracted ion chromatograms of protease inhibitors and their metabolites (B) as described in [START_REF] Chetwynd | Nanoflow-Nanospray Mass Spectrometry Metabolomics Reveals Disruption of the Urinary Metabolite Profiles of HIV-Positive Patients on Combination Antiretroviral Therapy[END_REF]. D= darunavir, DM1-3 = darunavir metabolites 1-3, A= atazanavir, AM1-5 = atazanavir metabolites 1-5, R=ritonavir, RM1-2 = ritonavir metabolites 1-2). Samples run on a Waters nanoAcquity-nanoESI-XEVO G2 TOFMS with a 100 mm x 100 µm x 2.8 µm x 100 Å HSS T3 column.

Figure captions Figure 1 :

 captions1 Figure captionsFigure1: Base peak intensity (BPI) chromatograms of a standard mixture of compounds analysed by BEH C18 300 and 130 Å and HSS T3 100 Å nUHPLC columns. The base peak intensity peaks of selected standards are labelled to demonstrate the different retention characteristics of each column. TIC = total ion count. 1, unretained polar compounds, 2, metropolol; 3, venlafaxine; 4, propranolol; 5, carbamazepine; 6, testosterone; 7, androstenedione; 8, sphingosine; 9, diazinon; 10, tris(2-butoxyethyl) phosphate. Reproduced with permission from Ref[START_REF] Chetwynd | Evaluation of analytical performance and reliability of direct nanoLC-nanoESI-high resolution mass spectrometry for profiling the (xeno)metabolome[END_REF].

Figure 2 :

 2 Figure 2:Positive ESI mode base peak intensity (BPI) of pooled HIV patient urine run using nLC-nESI-TOFMS (A) and extracted ion chromatograms of protease inhibitors and their metabolites (B) as described in[START_REF] Chetwynd | Nanoflow-Nanospray Mass Spectrometry Metabolomics Reveals Disruption of the Urinary Metabolite Profiles of HIV-Positive Patients on Combination Antiretroviral Therapy[END_REF]. D= darunavir, DM1-3 = darunavir metabolites 1-3, A= atazanavir, AM1-5 = atazanavir metabolites 1-5, R=ritonavir, RM1-2 = ritonavir metabolites 1-2). Samples run on a Waters nanoAcquity-nanoESI-XEVO G2 TOFMS with a 100 mm x 100 µm x 2.8 µm x 100 Å HSS T3 column.

Figure 3 .

 3 Figure 3.Example of base peak intensity (BPI) chromatograms (A) of testis extracts from fish exposed to wastewater effluent in positive (+ESI) and negative (-ESI) modes. Chromatograms B show overlaid signals of selected xenobiotics extracted using their exact masses. Chemical identity was confirmed from accurate mass, isotopic fit and fragmentation data obtained from high energy collisional induced dissociation and from comparison with standard compounds. Testis samples were extracted by Strata-X-C solid-phase extraction after phospholipid removal and profiled in -ESI or +ESI modes by nUHPLC-nESI-TOFMS as described in[START_REF] David | Disruption of the Prostaglandin Metabolome and Characterization of the Pharmaceutical Exposome in Fish Exposed to Wastewater Treatment Works Effluent As Revealed by Nanoflow-Nanospray Mass Spectrometry-Based Metabolomics[END_REF].1=10 Hydroxyamitriptyline;2=Norclozapine;3=citalopram;4=Amitriptyline;5=norsertraline;6=sertraline;7=c lopidogrel

Figure 2 .

 2 Figure 2.

Table 1 : Overview of existing nLC-nESI instrumentations and associated parameters (nanocolumns characteristics, flow rate, injection volume, run time and matrices) used in metabolomic studies.

 1

	nLC	MS	Flow	Colum	Lengt	Particl	Phase Injectio	Run	Matrice	Refs
			rate	n ID	h	e		n	time
			(nL/min	(µm)	(mm)	size		volume	(min
			)			(µm)		(nL)	)
	Thermo Ultimate 3000 nano	Thermo LTQ-Orbitrap	1000	150	150	5	HILIC	Not defined	60	CSF	[69]
	Eksigent nanoLC Ultra	Thermo LTQ-Orbitrap	500	100	100	5	C18	1000	45	In vitro cell lines	[84]
	Bruker EASY-nLC	Bruker MicroTO F	300	75	100	3	C18	5000	47	In vitro cell lines	[106 ]
	Agilent 1100 Series	Bruker F MicroTO	312	75	150	3	C18	6.25	23	Urine	[65]
	Waters nanoAcquit y	Orbitrap Elite	300	75	100	1.7	BEH C18	2000	60	In vitro cell lines	[70]
	Waters nanoAcquit y	Waters XEVO G2 QTOF	700	100	100	1.8	HSS-T3 500	50	Urine/Plasm a	[73]
	Waters nanoAcquit y	Waters XEVO G2 QTOF	700	100	100	1.7	BEH C18	500	50	Plasma	[11]
	Waters	Waters
	nanoAcquit	XEVO G2	700	100	100	1.8	HSS-T3 500	50	Urine	[62]
	y	QTOF
	Waters nanoAcquit y	Waters QTOF Premier	350	75	200	1.7	Acclaim Pepma x C18	5000	45	Urine and sweat	[68]
	Waters	Waters
	nanoAcquit	XEVO G2	700	100	100	1.8	HSS-T3 500	50	Urine	[63]
	y	QTOF
	Eksigent 415 -nanoflex cHiPLC	Sciex 5600 TOF Triple	300	200	150	Not d define	C18	5000	24	Faeces	[66]

Table 2 :

 2 Relative standard deviation (RSD) of retention time (RT) for small molecules (<1000 Da) analysed in different matrices using direct (D) and split (S) flow nLC platforms.

	Analyte		Matrix		Flow	RT	nLC system		Flow	Ref
					rate	(%RS			type
					(nL/	D)
					min)
	6	Deuterated	Plasma	and	700	<0.20	Waters nanoAcquity UPLC	D	[7
	standards	urine			%				3]
	100		Plasma		700	<1.9% Waters nanoAcquity UPLC	D	[1
	(Xeno)metabolites								1]
	100 Metabolites	Urine		700	<1.9% Waters nanoAcquity UPLC	D	[6
										2]
	8 Amino acids	Urine	and	350	<1.1% Waters nanoAcquity UPLC	D	[6
			sweat							8]
	Tryptic digests	Bovine serum	325	0.73% Proxeon EASY-nLC	D	[1
			albumin							07
										]
	Tryptic digests	Bovine serum	325	0.66% Waters nanoAcquity UPLC	D	[1
			albumin							07
										]
	Tryptic digests	Bovine serum	325	0.87% Eksigent nanoLC-Ultra	D	[1
			albumin							07
										]
	Tryptic digests	Bovine serum	325	2.2%	Eksigent nanoLC-2D,	D	[1
			albumin							07
										]
	Pharmaceuticals	Standard	300	<0.50	Agilent 1200 HPLC	D	[1
			solution			%				08
										]
	Biogenic amines	Wine		634	<1.2% Dionex Ultimate Capillary HPLC	D	[1
										09
										]
	Nonglycosylated	Plasma		450-	<1.6% Waters nanoAcquity UPLC	D	[1
	tryptic peptides			500					10
										]
	Phosphoproteins	Semen		300	2.0%	Waters nanoAcquity UPLC	D	[1
										11
										]
	Penicillin antibiotics Standard	200	<0.35	Dionex Ultimate 3000 nano LC	D	[1
			solution			%				12
										]
	Pharmaceuticals	Standard	300	<0.50	Dionex Ultimate	D	[1
			solution			%				13
	Phenolic		Standard	300	≤0.80	Bruker Easy-nLC	TM	D	] [1
	compounds	solution			%				14
										]
	Peptides		HeLa	protein	20-	≤0.30	Thermo Ultimate 3000 RSLC nano	D	[1
			standard		50	%	LC			15
										]
	Glycans		Plasma		500	<0.50 Eksigent Technologies nanoLC 2D	D	[1

% 16 ]

Table 3 :

 3 Relative standard deviation (RSD) of peak area for small molecules (<1000 Da) analysed in different matrices using nLC-nESI platforms

	Analyte		Matrix	Peak area	Emitter	Emitter	Ref
				(%RSD)	material	ID
	Deuterated and normal standards Standard solution/ urine	<18%	Fused silica	10 µm	[73
			and plasma				]
	Cationic metabolites	Cerebrospinal fluid	<20%	Fused silica	8 µm	[69
							]
	8 Amino acids		Urine and sweat	<7%	Fused silica	5 µm	Z.
							L[6
							8]
	Tryptic digests		Cellular protein extract	<15%	Fused silica	5 µm	[13
							1]
	Ceramides		Cerebral spinal fluid	<15%	Fused silica	10 µm	[13
							2]
	Pharmaceuticals		Standard solution	<11%	Fused silica	10 µm	[11
							3]
	Anthocyanins		Fruit juice	<16%	Fused silica	25 µm	[12
							7]
	Pharmaceuticals		Standard solution	10%	Fused silica	25 µm	[38
							]
	Perfluorooctanoic	River water	<20%	Fused silica	30 µm	[12
	acid/perfluorooctane sulfonate					3]
	Aloe-based	phytotherapeutic	Standard solution and	<12.5%	Fused silica	25 µm	[12
	products		leaves				8]
	Ketamine and metabolites	Human hair	≤4%	Tapered	20 µm	[13
					fused silica		3]
	Plant hormones		Tobacco seeds	<11%	Tapered	8-9 µm	[12
					fused silica		0]
	Phenolics		Olive oil and cell culture	<4%	Tapered	Not	[10
					fused silica	defined	6]
	Phenolic compounds	Standard solution	<7%	Tapered	Not	[13
					fused silica	defined	4]
	Phenolic compounds	Standard solution	<15%	Tapered	Not	[11
					fused silica	defined	4]
	Metabolite standards	Cell extracts	<23%	Silica	10 µm	[84
							]
	Pharmaceuticals		Standard solution and	<10%	Not defined	75 µm	[10
			plasma				8]
	Phytohormones		Leaves	<12%	Not defined	8 µm	[13
							5]
	Peptides		Serum	≤25%	Not defined	5 µm	[13
							0]
	50 Glycans		Serum standard	<10%	Not defined	Not	[13
						defined	6]
	Polyphenols		Urine	≤13%	Not defined	Not	[13
						defined	7]
	Penicillin antibiotics	Milk, liver, kidney	<8%	Not defined	Not	[11
						defined	2]
	Wax esters		Seed oil extracts	<5%	Not defined	Not	[13
						defined	8]
	Oligosaccharides	Standard solution	<5%	Not defined	Not	[11
						defined	9]
	Environmental contaminants	Benthic tissue extracts	<13%	Not defined	Not	[13
						defined	9]

Table 4 :

 4 Advantages and drawbacks of nLC-nESI compared to conventional LC-ESI platforms for metabolomic analysis Advantages Drawbacks Increased ionisation efficiency in the source and transfer efficiency in the mass spectrometer More care to be done for sample preparation Reduced chromatographic dilution Less sample throughput due to longer LC run time More comprehensive analysis due to the detection of trace level metabolites More prone to column/emitter blockages Reduced cost and environmental impact by using less solvent Less array of column chemistries readily available Reduce need for desolvation gas and temperature in the source

	More expertise needed

Acknowledgment

AC received funding from the EPSRC and University of Sussex as part of his PhD. AD was supported by a Marie Curie Intra European Fellowship within the European Community Seventh Framework Programme ([FP7/2007-2013]) under grant agreement no: 302097.