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Perfluoroalkyl substances (PFASs) are a major contaminant class due to their ubiquitous prevalence, persistence, and putative endocrine disrupting activity that may contribute to chronic disease risk notably with exposures early in life. Herein, multisegment injection-nonaqueous capillary electrophoresis-tandem mass spectrometry (MSI-NACE-MS/MS) is introduced as a high throughput approach for PFAS screening in serum samples following a simple methyl- tert -butyl ether (MTBE) liquid extraction. Separation and ionization conditions were optimized to quantify low nanomolar concentration levels of perfluorooctanoic acid (PFOA) and perfluorooctanesulfonic acid (PFOS) from serum extracts when using multiple reaction monitoring under negative ion mode conditions. Multiplexed separations of PFOA and PFOS were achieved with excellent throughput (<3 min/sample), adequate concentration sensitivity (LOD ∼ 20 nM, S/N = 3) and good technical precision over three consecutive days of analysis (mean CV = 9.1%, n = 84). Accurate quantification of PFASs was demonstrated in maternal serum samples (n = 16) when using MSI-CE-MS/MS following pre-column sample enrichment with median concentrations of 3.46 nM (0.7-9.0 nM) and 3.29 nM (1.5-6.6 nM) for PFOA and PFOS, respectively. This was lower than average PFAS exposures measured in pregnant women who had serum collected prior to 2009 likely due to subsequent phase out of their production. Overall, this method offers a convenient approach for large-scale biomonitoring of environmental exposures to legacy PFASs and their emerging replacements that is relevant to maternal health and chronic disease risk assessment in children., Competing Interests: The authors declare no conflict of interest., (© 2020 The Authors. Analytical Science Advances published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Introduction: Exclusive enteral nutrition (EEN) and corticosteroids (CS) are effective induction therapies for pediatric Crohn's Disease (CD). CS are also therapy for ulcerative colitis (UC). Host-microbe interactions may be able to explain the effectiveness of these treatments. This is the first prospective study to longitudinally characterize compositional changes in the bacterial community structure of pediatric UC and CD patients receiving EEN or CS induction therapy., Methods: Patients with diagnoses of CD or UC were recruited from McMaster Children's Hospital (Hamilton, Canada). Fecal samples were collected from participants aged 5-18 years old undergoing 8 weeks of induction therapy with EEN or CS. Fecal samples were submitted for 16S rRNA sequencing. The Shannon diversity index and the relative abundance of specific bacterial taxa were compared using a linear mixed model., Results: The clustering of microbiota was the highest between patients who achieved remission compared to patients still showing active disease (p = 0.029); this effect was independent of the diagnosis or treatment type. All patients showed a significant increase in Shannon diversity over the 8 weeks of treatment. By week 2, a significant difference was seen in Shannon diversity between patients who would go on to achieve remission and those who would not., Conclusion: The gut microbiota of pediatric UC and CD patients was most influenced by patients' success or failure to achieve remission and was largely independent of the choice of treatment or disease type. Significant differences in Shannon diversity indices occurred as early as week 2 between patients who went on to achieve remission and those who continued to have active disease., Competing Interests: The authors declare no conflict of interest.

Nutritional studies rely on various biological specimens for FA determination, yet it is unclear how levels of serum NEFAs correlate with other circulating lipid pools. Here, we used a high-throughput method (<4 min/sample) based on multisegment injection-nonaqueous capillary electrophoresis-mass spectrometry (MSI-NACE-MS) to investigate whether specific serum NEFAs have utility as biomarkers of dietary fat intake in women. We first identified circulating NEFAs correlated with long-term/habitual food intake among pregnant women with contrasting dietary patterns ( n = 50). Acute changes in serum NEFA trajectories were also studied in nonpregnant women ( n = 18) following high-dose (5 g/day) fish oil (FO) supplementation or isoenergetic sunflower oil placebo over 56 days. In the cross-sectional study, serum ω-3 FAs correlated with self-reported total ω-3 daily intake, notably EPA as its NEFA ( r = 0.46; P = 0.001), whereas pentadecanoic acid was associated with full-fat dairy intake ( r = 0.43; P = 0.002), outcomes consistent with results from total FA serum hydrolysates. In the intervention cohort, serum ω-3 NEFAs increased 2.5-fold from baseline within 28 days following FO supplementation, and this increase was most pronounced for EPA ( P = 0.0004). Unlike for DHA, circulating EPA as its NEFA also strongly correlated to EPA concentrations measured from erythrocyte phospholipid hydrolysates ( r = 0.66; P = 4.6 × 10 -10 ) and was better suited to detect dietary nonadherence. We conclude that MSI-NACE-MS offers a rapid method to quantify serum NEFAs and objectively monitor dietary fat intake in women that is complementary to food-frequency questionnaires., (Copyright © 2020 Azab et al.)

Expanded newborn screening of inborn errors of metabolism (IEM) based on tandem mass spectrometry (MS/MS) technology is one of the most successful preventative healthcare initiatives for presymptomatic diagnosis and treatment of rare yet treatable genetic diseases in the population. However, confirmatory testing of presumptive screen-positive cases is required using high efficiency separations for improved specificity in order to improve the positive predictive value (PPV) for certain classes of IEMs. Here, we describe recent advances using capillary electrophoresis-mass spectrometry (CE-MS) for reliable second-tier screening or confirmatory testing based on targeted analysis of amino acids, acylcarnitines, nucleosides, and other classes of polar metabolites associated with IEMs. Additionally, nontargeted metabolite profiling enables the identification of unknown biomarkers of clinical significance for other genetic diseases that are currently screened by bioassays and/or mutation panels, such as cystic fibrosis (CF). Noteworthy, CE-MS allows for resolution of isobaric/isomeric interferences without complicated sample handling that is ideal when analyzing volume-restricted biospecimens from neonates/infants, including dried blood spots and sweat specimens. New developments to improve concentration sensitivity, as well as enhance sample throughput and quality control for unambiguous confirmatory testing of IEMs will also be discussed when using multiplexed separations based on multisegment injection-CE-MS.

Capillary electrophoresis-mass spectrometry (CE-MS) offers a high efficiency microseparation platform for amino acid profiling when analyzing volume-restricted biological samples, such as a dried blood spot punch. Direct analysis of amino acids and their analogs is routinely achieved using strongly acidic buffer conditions under positive-ion mode detection with a coaxial sheath liquid interface for electrospray ionization (ESI). New advances in online sample preconcentration, pre-column chemical derivatization, and/or low flow/sheathless CE-MS interface designs can further improve sensitivity while allowing for resolution of amino acid stereoisomers and labile aminothiols with low nanomolar detection limits. Additionally, multiplexed separations in CE-MS based on serial injection of seven or more samples within a single run greatly boosts sample throughput (<2-3 min/sample) without added infrastructure costs while allowing for stringent quality control and signal batch correction. Accurate prediction of the electromigration behavior of amino acids and their analogs offers a convenient approach for structural elucidation that is complementary to high-resolution MS and MS/MS. Simultaneous analysis of amino acids together with other classes of ionic metabolites by CE-MS allows for comprehensive metabolomic screening as required for new advances in clinical medicine, nutritional sciences, and population health.

High quality assays are needed in drug discovery to reduce the high attrition rate of lead compounds during primary screening. Capillary electrophoresis (CE) represents a versatile micro-separation technique for resolution of enzyme-catalyzed reactions, including substrate(s), product(s), cofactor(s) and their stereoisomers, which is needed for reliable characterization of biomolecular interactions in free solution. This review article provides a critical overview of new advances in CE for drug screening over the past five years involving biologically relevant enzymes of therapeutic interest, including transferases, hydrolases, oxidoreductases, and isomerases. The basic principles and major configurations in CE, as well as data processing methods needed for rigorous characterization of enzyme inhibition are described. New developments in functional screening of small molecules that modulate the activity of disease-related enzymes are also discussed. Although inhibition is a widely measured response in most enzyme assays, other important outcomes of ligand interactions on protein structure/function that impact the therapeutic potential of a drug will also be highlighted, such as enzyme stabilization, activation and/or catalytic uncoupling. CE offers a selective platform for drug screening that reduces false-positives while also enabling the analysis of low amounts of complex sample mixtures with minimal sample handling., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Expanded newborn screening of inborn errors of metabolism (IEM) based on tandem mass spectrometry technology has emerged as one of the most successful preventative healthcare initiatives for presymptomatic diagnosis and treatment of rare yet treatable genetic diseases. However, confirmatory testing using methods with improved specificity is required in clinical laboratories to improve the positive predictive value for certain classes of IEMs due to their high rates of false positives. Here, we describe recent advances for comprehensive profiling of amino acids and acylcarnitines derived from dried blood spot extracts or plasma using capillary electrophoresis-electrospray ionization-mass spectrometry (CE-ESI-MS) that allows for resolution of major isobaric/isomeric interferences without complicated sample handling. The integration of online sample preconcentration together with desalting in CE-ESI-MS enables the direct analysis of hydrophilic amino acids, surface-active acylcarnitines, as well as labile thiols under a single format when using a simple aqueous buffer electrolyte system.

Capillary electrophoresis-electrospray ionization-mass spectrometry (CE-ESI-MS) offers a selective, sensitive yet robust approach for amino acid profiling in complex biological samples with minimal sample pretreatment. Direct analysis of amino acids and their analogs is routinely performed using strongly acidic buffer conditions under positive-ion mode ESI-MS with a coaxial sheath liquid interface. New advances in online sample preconcentration, chemical derivatization, and/or ESI interface designs can further improve assay performance allowing for resolution of amino acid stereoisomers and labile aminothiols with low nanomolar detection limits. Accurate prediction of the electromigration behavior of amino acids offers a convenient approach for their qualitative identification complementary to ESI-MS. Simultaneous analysis of amino acids together with other classes of cationic metabolites can be realized by CE-ESI-MS for comprehensive metabolite profiling applications relevant to disease prognosis, drug efficacy, and food safety/quality control.

Pharmacological chaperones (PCs) represent a promising therapeutic strategy for treatment of lysosomal storage disorders based on enhanced stabilization and trafficking of mutant protein upon orthosteric and/or allosteric binding. Herein, we introduce a simple yet reliable enzyme assay using capillary electrophoresis (CE) for inhibitor screening of PCs that target the lysosomal enzyme, β-glucocerebrosidase (GCase). The rate of GCase-catalyzed hydrolysis of the synthetic substrate, 4-methylumbelliferyl-β-D: -glucopyranoside was performed using different classes of PCs by CE with UV detection under standardized conditions. The pH and surfactant dependence of inhibitor binding on recombinant GCase activity was also examined. Enzyme inhibition studies were investigated for five putative PCs including isofagomine (IFG), ambroxol, bromhexine, diltiazem, and fluphenazine. IFG was confirmed as a potent competitive inhibitor of recombinant GCase with half-maximal inhibitory concentration (IC(50)) of 47.5 ± 0.1 and 4.6 ± 1.4 nM at pH 5.2 and pH 7.2, respectively. In contrast, the four other non-carbohydrate amines were demonstrated to function as mixed-type inhibitors with high micromolar activity at neutral pH relative to acidic pH conditions reflective of the lysosome. CE offers a convenient platform for characterization of PCs as a way to accelerate the clinical translation of previously approved drugs for oral treatment of rare genetic disorders, such as Gaucher disease.

Thiol homeostasis plays an important role in human health and aging by regulation of cellular responses to oxidative stress. Due to major constraints that hamper reliable plasma thiol/disulfide redox status assessment in clinical research, we introduce an improved strategy for comprehensive thiol speciation using capillary electrophoresis-electrospray ionization-mass spectrometry (CE-ESI-MS) that overcomes sensitivity, selectivity and dynamic range constraints of conventional techniques. This method integrates both specific and nonspecific approaches toward sensitivity enhancement for artifact-free quantification of labile plasma thiols without complicated sample handling. A multivariate model was developed to predict increases in ionization efficiency for reduced thiols when conjugated to various maleimide analogs based on their intrinsic physicochemical properties. Optimization of maleimide labeling in conjunction with online sample preconcentration allowed for simultaneous analysis of nanomolar levels of reduced thiols and free oxidized thiols as their intact symmetric or mixed disulfides. Identification of low-abundance thiols and various other polar metabolites detected in plasma was supported by prediction of their relative migration times using CE as a qualitative tool complementary to ESI-MS. Plasma thiol redox status determination together with untargeted metabolite profiling offers a systemic approach for elucidation of the causal role of dysregulated thiol metabolism in the etiology of human diseases.

Metabolomics is a rapidly emerging field of functional genomics research whose aim is the comprehensive analysis of low molecular weight metabolites in a biological sample. Capillary electrophoresis-electrospray ionization-mass spectrometry (CE-ESI-MS) represents a promising hyphenated microseparation platform in metabolomics, since a majority of primary metabolites are intrinsically polar. CE-ESI-MS offers a convenient format for the separation of complex mixtures of cationic, anionic, and/or zwitterionic metabolites, as well as their isobaric/isomeric ions without complicated sample handling. Moreover, online sample preconcentration with desalting is readily integrated during separation prior to ionization, where the migration behavior and ionization response of metabolites can be predicted based on their fundamental physicochemical properties. Herein, we describe recent developments in CE-ESI-MS with emphasis on practical protocols necessary for realizing reliable analyses as applied to targeted metabolite profiling and untargeted metabolomic studies in various biological samples.

The design of boronic acid sensors for photometric detection of carbohydrates has relied on exploiting differences in the thermodynamic stability of complex formation for molecular recognition. Herein, we introduce a direct method for analysis of sugar alcohols using 3-nitrophenylboronic acid (NPBA) as an electrokinetic probe in capillary electrophoresis (CE). Dynamic complexation of neutral polyols by NPBA during electromigration allows for their simultaneous resolution and UV detection based on formation of an anionic ternary boronate ester complex in phosphate buffer. Unlike conventional boronic acid sensors, thermodynamic and electrokinetic processes in CE allow for improved selectivity for the resolution of sugar alcohol stereoisomers having different vicinal polyol chain lengths even in cases when binding affinity is similar due to differences in their complex mobility. Three complementary approaches were investigated to compare the thermodynamics of polyol chelation with NPBA, namely direct binding assays by CE, UV absorbance spectroscopy and an indirect pK (a) depression method. Overall, CE offers a convenient platform for characterization of reversible arylboronic acid interactions in free solution while allowing for direct analysis of complex mixtures of neutral/UV-transparent polyols without complicated sample handling.

Glutathione metabolism plays a fundamental role in maintaining homeostasis and regulating the redox environment of a cell. Despite the widespread interest in quantifying glutathione metabolites in oxidative stress research, conventional techniques are hampered by complicated sample handling procedures to prevent significant oxidation artifacts generated during sample collection, sample pretreatment, and/or chemical analysis. In this report, a simple and validated method for glutathione analysis from filtered red blood cell (RBC) lysates was developed using capillary electrophoresis-electrospray ionization-mass spectrometry (CE-ESI-MS) in conjunction with fingerprick microsampling and ultrafiltration. About a 3-fold improvement in precision with nanomolar detection limits was achieved when using online sample preconcentration with CE-ESI-MS via a modified injection sequence, which permitted accurate determination of the intracellular reduced/oxidized glutathione ratio (GSH/GSSG), as well as other glutathione species, including protein-bound glutathione mixed disulfide (PSSG), free glutathione mixed disulfides (GSSR) and glutathione thioether conjugates (GSX). In this work, the redox status of filtered hemolysates was determined by the equilibrium half-cell reduction potential for glutathione (E(GSSG/2GSH)), whereas its intrinsic antioxidant capacity was assessed by the apparent rate of metal-catalyzed oxidation of glutathione. In-vitro incubation studies of intact RBCs with 1-chloro-2,4-dinitrobenzene (CDNB) and N-acetyl-L-cysteine (NAC) were found to significantly alter E(GSSG/2GSH) and/or glutathione oxidation kinetics (e.g., k(GSSG)) relative to normal controls based on their function as a toxic electrophilic compound and a competitive free radical scavenging/reducing agent, respectively. Differential rates of glutathione oxidation (DIRGO) using CE-ESI-MS offers a novel strategy for global assessment of the impact of intrinsic metabolite constituents (i.e., metabolome) and/or extrinsic perturbants on cellular redox status that is relevant to improved understanding of aging and the pathogenesis of acute or chronic disease states.

The advent of electrospray-ionization mass spectrometry (ESI-MS) has given rise to expanded newborn screening programs for the early detection of inborn errors of metabolism (IEM). Despite the benefit of high-throughput screening for disease prognosis, conventional ESI-MS methods are limited by inadequate specificity, complicated sample handling, and low positive predictive outcome that can contribute to a high rate of false-positives. Herein, we report a robust strategy for neonatal screening based on capillary electrophoresis-electrospray ionization-mass spectrometry (CE-ESI-MS) that offers a convenient platform for the direct analysis of amino acids, acylcarnitines, and their stereoisomers from dried blood spot (DBS) extracts without chemical derivatization. On-line sample preconcentration with desalting by CE-ESI-MS allowed for improved concentration sensitivity when detecting poorly responsive metabolites in complex biological samples without ionization suppression or isomeric/isobaric interferences. Method validation demonstrated that accurate yet precise quantification can be achieved for 20 different amino acid and acylcarnitine biomarkers associated with IEMs when using a single non-deuterated internal standard. CE-ESI-MS represents a promising second-tier method in newborn screening programs that is compatible with ESI-MS/MS technology in cases when improved specificity and sensitivity is warranted for diagnosis confirmation and subsequent monitoring.

Characterization of unknown low-abundance metabolites in biological samples is one the most significant challenges in metabolomic research. In this report, an integrative strategy based on capillary electrophoresis-electrospray ionization-ion trap mass spectrometry (CE-ESI-ITMS) with computer simulations is examined as a multiplexed approach for studying the selective nutrient uptake behavior of E. coli within a complex broth medium. On-line sample preconcentration with desalting by CE-ESI-ITMS was performed directly without off-line sample pretreatment in order to improve detector sensitivity over 50-fold for cationic metabolites with nanomolar detection limits. The migration behavior of charged metabolites were also modeled in CE as a qualitative tool to support MS characterization based on two fundamental analyte physicochemical properties, namely, absolute mobility (muo) and acid dissociation constant (pKa). Computer simulations using Simul 5.0 were used to better understand the dynamics of analyte electromigration, as well as aiding de novo identification of unknown nutrients. There was excellent agreement between computer-simulated and experimental electropherograms for several classes of cationic metabolites as reflected by their relative migration times with an average error of <2.0%. Our studies revealed differential uptake of specific amino acids and nucleoside nutrients associated with distinct stages of bacterial growth. Herein, we demonstrate that CE can serve as an effective preconcentrator, desalter, and separator prior to ESI-MS, while providing additional qualitative information for unambiguous identification among isobaric and isomeric metabolites. The proposed strategy is particularly relevant for characterizing unknown yet biologically relevant metabolites that are not readily synthesized or commercially available.

Dynamic pH junction is an on-line preconcentration method in capillary electrophoresis (CE) based on electrokinetic focusing of weakly ionic analytes with in large sample volumes in a multisection electrolyte system. In this report, experiments and computer simulations were performed to gain a better insight of the analyte focusing mechanism when a dynamic pH junction was used. A computer program, SIMUL, was used to simulate the band-narrowing process of a group for phenol derivatives under optimized buffer conditions, which were compared with experimental results. Computer simulations revealed the formation of a sharp moving pH boundary within the sample zone causing efficient focusing of long plugs of weakly acidic analytes based on their pKa. These studies offered useful information for understanding the band-narrowing process by control of the depth and lifetime of the moving pH boundary as a function of analyte pKa, sample pH, and injection length. The change in pH of the sample within the capillary was also estimated by measuring the absorbances of an analyte at two different wave-lengths. Optimization of analyte focusing resulted in enhanced detection responses of about 60-450-fold in terms of peak heights for some phenol derivatives' relation to conventional injections. Dynamic pH junction represents a novel approach to control band dispersion (peak width) and selectivity (mobility) of specific analytes for high-resolution CE separations.

On-line sample preconcentration by a dynamic pH junction in conjunction with multiplexed capillary electrophoresis (CE) and UV detection represents a sensitive and high-throughput format for future metabolomic research, such as purine analysis. The optimization of purine focusing can be rapidly assessed by systematically altering the sample matrix properties, such as the buffer co-ion, pH and ionic strength using a 96-capillary array format. This method permits focusing of large sample injection volumes, resulting in over a 50-fold improvement in the concentration sensitivity. The limit of detection (S/N = 3) for purine metabolites was less than 8.0 x 10(-8) M under optimum conditions when using UV absorbance. Dynamic pH junction multiplexed CE demonstrated excellent linearity over a hundred-fold concentration range, as well as low inter-capillary precision in terms of normalized migration times and peak areas. The potential for clinically relevant high-throughput analyses of micromolar amounts of purine metabolites in urine was also demonstrated.

Simple yet effective methods to enhance concentration sensitivity is needed for capillary electrophoresis (CE) to become a practical method to analyze trace levels of analytes in real samples. In this report, the development of a novel on-line preconcentration technique combining dynamic pH junction and sweeping modes of focusing is applied to the sensitive and selective analysis of three flavin derivatives: riboflavin, flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD). Picomolar (pM) detectability of flavins by CE with laser-induced fluorescence (LIF) detection is demonstrated through effective focusing of large sample volumes (up to 22% capillary length) using a dual pH junction-sweeping focusing mode. This results in greater than a 1,200-fold improvement in sensitivity relative to conventional injection methods, giving a limit of detection (S/N = 3) of approximately 4.0 pM for FAD and FMN. Flavin focusing is examined in terms of analyte mobility dependence on buffer pH, borate complexation and SDS interaction. Dynamic pH junction-sweeping extends on-line focusing to both neutral (hydrophobic) and weakly acidic (hydrophilic) species and is considered useful in cases when either conventional sweeping or dynamic pH junction techniques used alone are less effective for certain classes of analytes. Enhanced focusing performance by this hyphenated method was demonstrated by greater than a 4-fold reduction in flavin bandwidth, as compared to either sweeping or dynamic pH junction, reflected by analyte detector bandwidths <0.20 cm. Novel on-line focusing strategies are required to improve sensitivity in CE, which may be applied toward more effective biochemical analysis methods for diverse types of analytes.