Spatially confined CuFe2O4 nanosphere in N/O-codoped porous carbon mimetics for triple-mode sensing of antibiotics and visual detection of neurotransmitters in biofluids.

BACKGROUND: Carbon-based nanozymes have recently received enormous concern, however, there is still a huge challenge for inexpensive and large-scale synthesis of magnetic carbon-based "Two-in-One" mimics with both peroxidase (POD)-like and laccase-like activities, especially their potential applications in multi-mode sensing of antibiotics and neurotransmitters in biofluids. Although some progresses have been made in this field, the feasibility of biomass-derived carbon materials with both POD-like and laccase-like activities by polyatomic doping strategy is still unclear. In addition, multi-mode sensing platform can provide a more reliable result because of the self-validation, self-correction and mutual agreement. Nevertheless, the use of magnetic carbon-based nanozyme sensors for the multi-mode detection of antibiotics and neurotransmitters have not been investigated. RESULTS: We herein report a shrimp shell-derived N, O-codoped porous carbon confined magnetic CuFe2O4 nanosphere with outstanding laccase-like and POD-like activities for triple-mode sensing of antibiotic d-penicillamine (D-PA) and chloramphenicol (CPL), as well as colorimetric detection of neurotransmitters in biofluids. The magnetic CuFe2O4/N, O-codoped porous carbon (MCNPC) armored mimetics was successfully fabricated using a combined in-situ coordination and high-temperature crystallization method. The synthesized MCNPC composite with superior POD-like activity can be used for colorimetric/temperature/smartphone-based triple-mode detection of D-PA and CPL in goat serum. Importantly, the MCNPC nanozyme can also be used for colorimetric analysis of dopamine and epinephrine in human urine. SIGNIFICANCE: This work not only offered a novel strategy to large-scale, cheap synthesize magnetic carbon-based "Two-in-One" armored mimetics, but also established the highly sensitive and selective platforms for triple-mode monitoring D-PA and CPL, as well as colorimetric analysis of neurotransmitters in biofluids without any tanglesome sample pretreatment.

Machine-learning assisted multiplex detection of catecholamine neurotransmitters with a colorimetric sensor array.

Catecholamine neurotransmitters (CNs), such as dopamine (DA), epinephrine (EP), norepinephrine (NEP), and levodopa (LD), are recognized as the primary biomarkers of a variety of neurological illnesses. Therefore, simultaneous monitoring of these biomarkers is highly recommended for clinical diagnosis and treatment. In this study, a high-performance colorimetric artificial tongue has been proposed for the multiplex detection of CNs. Different aggregation behaviors of gold nanoparticles in the presence of CNs under various buffering conditions generate unique fingerprint response patterns. Under various buffering conditions, the distinct acidity constants of CNs, and consequently their predominant species at a given pH, drive the aggregation of gold nanoparticles (AuNPs). The utilization of machine learning algorithms in this design enables classification and quantification of CNs in various samples. The response profile of the array was analyzed using the linear discriminant analysis algorithm for classification of CNs. This colorimetric sensor array is capable of accurately distinguishing between individual neurotransmitters and their combinations. Partial least squares regression was also applied for quantitation purposes. The obtained analytical figures of merit (FOMs) and linear ranges of 0.6-9 µM (R2 = 0.99) for DA, 0.1-10 µM (R2 = 0.99) for EP, 0.1-9 µM (R2 = 0.99) for NEP and 1-70 µM (R2 = 0.99) for LD demonstrated the potential applicability of the developed sensor array in precise and accurate determination of CNs. Finally, the feasibility of the array was validated in human urine samples as a complex biological fluid with LODs of 0.3, 0.5, 0.2, and 1.9 µM for DA, EP, NEP, and LD, respectively.

Improved LC-MS/MS method for the determination of 42 neurologically and metabolically important molecules in urine.

Simultaneous determination of kynurenines, neurotransmitters, pterins and steroids linked to various neurological and metabolic diseases have important diagnostic significance for related pathology and drug monitoring. An improved, sensitive and selective ultra-high performance liquid chromatography coupled to electrospray ionization triple quadrupole mass spectrometric (UHPLC-MS/MS) method, based on our earlier publication, has been proposed for the quantitative measurement of 42 metabolites in human urine. The assay covers a larger number of analytes, uses an advanced, Waters Atlantis T3 chromatographic column and similarly meets the guideline of European Medicines Agency (EMA) on bioanalytical method validation. Analytical performance met all the EMA requirements and the assay covered the relevant clinical concentrations. Linear correlation coefficients were all > 0.998. Intra-day and inter-day accuracy and precision were 87-118%, 81-120% and 2-20%, respectively including the lower limit of quantification (LLOQ). The assay is expected to facilitate the diagnosis and allows drug level monitoring from urine.

A metabolomics approach to investigate urine levels of neurotransmitters and related metabolites in autistic children.

Since recently metabolic abnormalities in autistic children have been associated with ASD disturbs, the aim of this study is to determine the neurotransmitter levels in urine samples of autistic children and to analyse the altered metabolic pathway involved in their production. Thus, ASD-specific urinary metabolomic patterns were explored in 40 ASD children and 40 matched controls using untargeted metabolomics through UHPLC-mass spectrometry (Q-exactive analyser), and by using XCMS Metlin software for data interpretation. Through this new advanced technique, a more considerable number of urinary altered metabolites were recorded in autistic children, than in the previous investigations, which allowed us to collect metabolites involved in neurotransmitter production. In these subjects, a high amount of dopamine was revealed and an increased amount of homovanillic acid, to the detriment of noradrenaline and adrenaline production, as well as MHPG and vanillylmandelic acid, which were found lower. This indicates that the accumulation of dopamine is not due to its greater production, but its lesser biotransformation into noradrenaline, due to the blockage of the dopamine ß-hydroxylase enzyme by 4-cresol and vitamin C, both found in high quantities in autistic subjects. Finally, a decreased amount of the active form of vitamin B6, pyridoxal phosphate (P5P), implicated in biotransformation of glutamate into γ-aminobutyric acid (GABA), was also detected, justifying the lower levels of latter. All of these alterations are correlated with a peculiar intestinal microbiome in autistic subjects, supporting the idea of a microbiota-gut-brain axis, then altered levels of neurotransmitters and altered neuronal transmission exist.

Identification of markers of depression and neurotoxicity in pesticide exposed agriculture workers.

Earlier, we reported that chronic exposure to pesticides causes a reduction in the acetylcholinesterase activity and hematological and biochemical alterations in agriculture workers. In continuation with that, the present study aimed to investigate the pesticide-induced neurochemical imbalance and its association with behavior alterations in agricultural workers. A significant increase in depressive symptoms, assessed by the Beck Depression Inventory-II was observed in pesticide exposed workers as compared to the unexposed. A decrease in the level of dopamine in plasma and levels of dopamine, 3,4-dihydroxyphenylacetic acid, homovanillic acids, norepinephrine, serotonin, and hydroxyindoleacetic acid in urine was also observed. An increase in the levels of MAO-A and MAO-B has also been observed in these individuals. The decreased levels of neurotransmitters in the blood and urine have been linked with increased levels of MAO and pesticide residues in plasma and urine. Furthermore, these changes were associated with a higher incidence of depression in agricultural workers.

Hydrophilic interaction chromatography combined with ultrasound-assisted ionic liquid dispersive liquid-liquid microextraction for determination of underivatized neurotransmitters in dementia patients' urine samples.

A sensitive, rapid, precise and specific analytical method of hydrophilic interaction ultra-performance liquid chromatography coupled with triple-quadrupole linear ion-trap tandem mass spectrometry (HILIC-UHPLC-QTRAP®/MS2) combined with a high-efficiency and easy sample preparation technology of ultrasound-assisted ionic liquid dispersive liquid-liquid microextraction (UA-IL-DLLME) was developed to investigate neurotransmitters (NTs) in mild cognitive impairment, mild dementia and moderate dementia patients' urine samples. Firstly, the UA-IL-DLLME parameters were optimized using Plackett-Burman screening and rotatable central composite design, and the main optimal conditions were obtained: ultrasound power of 307 W, ultrasound time of 4.3 min and agitation time of 4.8 min. Secondly, HILIC-UHPLC-QTRAP®/MS2 method was developed to simultaneously determine 15 underivatized NTs in urine samples. The analysis results of clinical samples showed that some NTs such as γ-aminobutyric acid (GABA), acetylcholine (Ach) and glutamic acid (Glu) presented significant differences in different dementia stages. Finally, multivariate analysis based on the combination of principal component analysis and supervised counter propagation artificial neural network was developed for comprehensive analysis of the obtained clinical data sets. As a result, GABA and Glu were simultaneously presented meaningful contribution for classification of samples, and might be considered as potential differential compounds to the urine samples from cluster patients with different dementia stages. In summary, the presented strategy of preparation, analysis and statistics might be used to investigate NTs in different clinical biological fluids.

Long-term blood pressure lowering and cGMP-activating actions of the novel ANP analog MANP.

Based on the cardiac hormone atrial natriuretic peptide (ANP) and its seminal role in blood pressure (BP) homeostasis, we investigated the chronic BP lowering actions of a novel ANP analog currently entering clinical trials for hypertension. Previous reports demonstrate that this analog MANP activates the guanylyl cyclase A receptor (GC-A) and results in more potent biological actions compared with ANP; thus, it may represent a new therapeutic drug for hypertension. A major goal of this study was to establish that chronic subcutaneous delivery of MANP is feasible and hypotensive together with cGMP effects. We investigated the BP-lowering and cGMP-activating actions of acute and chronic subcutaneous delivery in normal and hypertensive rats. Furthermore, we explored vascular mechanisms of MANP in human aortic smooth muscle cells (HASMC) and ex vivo in isolated arteries. In normal rats with a single subcutaneous injection, MANP promoted robust dose-dependent BP-lowering actions and natriuresis, together with cGMP activation. Most importantly in hypertensive rats, once-a-day subcutaneous injection of MANP for 7 days induced cGMP elevation and long-term BP reduction compared with vehicle. Mechanistically, in HASMC, MANP activated cGMP and attenuated angiotensin II-mediated increases in intracellular Ca2+ levels while directly vasorelaxing arterial rings. Our study demonstrates for the first time the effectiveness of subcutaneous administration of MANP for 7 days and provides innovative, vascular mechanisms of BP regulation supporting its continued development as a novel therapeutic for hypertension.

Amino bearing core-shell structured magnetic covalent organic framework nanospheres: Preparation, postsynthetic modification with phenylboronic acid and enrichment of monoamine neurotransmitters in human urine.

Covalent organic frameworks (COFs) have been increasingly employed in separation science, including sample preparation. Herein we fabricated the amino bearing core-shell structured COFs nanospheres [Fe3O4@TpBD(NH2)2], and a novel magnetic boronate affinity adsorbent was synthesized by postsynthetic modification of the Fe3O4@TpBD(NH2)2 with 2-formylphenylboronic acid. The magnetic boronate affinity adsorbent possesses fast magnetic response and high binding capacity up to 1037 µmol g-1 for dopamine. Besides, it was used as an adsorbent for extraction of urinary monoamine neurotransmitters at neutral pH. A method for detection of the monoamine neurotransmitters was developed by coupling the magnetic solid phase extraction with high-performance liquid chromatography-fluorescence detection. Under the optimized conditions, a good analytical method was obtained in the linear dynamic range of 2-200 ng mL-1 with R2 between 0.9917 and 0.9966, with low limit of detection (0.31-0.54 ng mL-1) and limit of quantification (1.04-1.80 ng mL-1). The recoveries of the monoamine neurotransmitters were in the range of 86.3-115%, with relative standard deviations of 2.34-10.5% (intra-day) and 2.84-14.4% (inter-day). The method was successfully applied to the determination of the monoamine neurotransmitters in human urine samples. This work is of great importance for preparing functionalized core-shell structured magnetic covalent organic framework nanospheres, it also demonstrates the feasibility of the functionalized magnetic COFs as adsorbents in sample pretreatment.

[Simultaneous rapid detection of eight neurotransmitters in rat tissues,blood and urine samples by UPLC-MS/MS].

Ultra performance liquid chromatography-tandem mass spectrometry( UPLC-MS/MS) was used to establish the simultaneous determination method of eight neurotransmitters in brain,liver,kidney,adrenal gland,serum and urine,including serotonin,5-hydroxyindole acetic acid,epinephrine,norepinephrine,dopamine,glutamic acid,γ-aminobutyric acid,and acetylcholine,and then investigate the distribution characteristics of neurotransmitters in rat tissues,blood and urine. Waters ACQUITY UPLC BEH C_(18) column( 2. 1 mm×100 mm,1. 7 µm) was used,with 0. 3% formic acid solution-acetonitrile as the mobile phase for gradient elution.Multiple reaction monitoring( MRM) scanning method under positive mode by atmospheric pressure electrospray ion source( ESI) was also performed to establish the detection method of neurotransmitters for methodological research. The plasma,urine and tissues of normal rats were pre-treated including homogenization,centrifuging,and protein removal,then the 2 µL supernatant was injected for analysis. The results showed that eight kinds of neurotransmitters could be accurately determined within 7 min,with linear correlation coefficients all greater than 0. 99. This method showed high accuracy and good precision,with specificity,stability,extraction recovery and matrix effects all complying with the biological sample analysis requirements. The most abundant transmitters in the brain,liver,kidney,kidney gland,blood and urine were γ-aminobutyric acid,glutamic acid,glutamic acid,adrenaline,glutamic acid and dopamine.The method is sensitive,rapid,precise,accurate and specific,and can be used for simultaneous quantitative analysis of eight neurotransmitters in biological samples. The investigation of the distribution ratio of transmitters in rats is of important significance to disease prevention and treatment.

Development of an underivatized LC-MS/MS method for quantitation of 14 neurotransmitters in rat hippocampus, plasma and urine: Application to CUMS induced depression rats.

Sensitive and comprehensive measurement of systemic metabolites of tryptophan, phenylalanine and glutamate metabolism in biological samples is effective for understanding the pathogenesis of depression and other neurological diseases. Therefore, this study developed an underivatized liquid chromatography tandem mass spectrometry (LC-MS/MS) method for simultaneous monitoring the 3 components of glutamate metabolism in rat hippocampus and 11 components of tryptophan and phenylalanine metabolism in rat hippocampus, plasma and urine, and applied it to investigate their changes in rats induced by chronic unpredictable mild stress (CUMS). The investigated analytes are as follows: tryptophan, serotonin, 5-hydroxyindoleacetic acid, kynurenine, kynurenic acid, xanthurenic acid, 3-hydroxyanthranilic acid, quinolinic acid, phenylalanine, tyrosine, tyramine, glutamate, glutamine and gamma-aminobutyric acid. The method was verified to be sensitive and effective with satisfactory linearity, accuracies in the range of 78.2%-120.4%, and precisions less than 17.8% for all identified analytes. A series of significant changes in CUMS-induced rats can be detected: tryptophan, serotonin and tyrosine levels decreased and quinolinic acid increased in both hippocampus and plasma. In addition, the kynurenine/tryptophan ratios increased in hippocampus and plasma, the kynurenic acid/quinolinic acid ratios of plasma and urine were significantly reduced. These findings demonstrated that the CUMS procedure could lead to the central and peripheral imbalances of tryptophan and phenylalanine metabolism. In conclusion, a LC-MS/MS method for simultaneous measurement of several neurotransmitters in rat hippocampus, plasma and urine was developed and successfully applied to investigation of the central and peripheral changes in CUMS-induced rats. The method would be expected to provide applicability to the study of the mechanisms of depression and other related diseases associated with these neurotransmitters.

Electrochemical simultaneous analysis of dopamine and epinephrine using double imprinted One MoNomer acryloylated graphene oxide-carbon black composite polymer.

A novel One MoNomer dual imprinted graphene oxide/carbon black composite polymer was developed applying 'surface-grafting from' approach on the screen printed carbon electrode for the electrochemical sensing of dopamine and epinephrine. Acryloylated-graphene oxide/carbon black was synthesized for the first time. This served both as a crosslinker and monomer leading to the fast electron transfer from the redox centre to the electrode. The oxidation peak potentials of both the targets were found separated by 200 mV which enabled their simultaneous analysis in real world samples, without any cross reactivity, interferences, and false-positives. The detection limits realized by the proposed sensor, under optimized analytical conditions, were found to be as low as 0.028, 0.028,0.061 and 0.029 ng mL-1 for dopamine and 0.017, 0.018, 0.019 and 0.020 ng mL-1 for epinephrine (S/N = 3) in aqueous, blood serum, urine and pharmaceutical samples. Such sensor could be considered suitable for the primitive diagnosis of several chronic diseases, manifested at ultra-trace level.

Detection of Neurotransmitters by Three-Dimensional Laser-Scribed Graphene Grass Electrodes.

Carbon nanomaterials possess superb properties and have contributed considerably to the advancement of integrated point-of-care chemical and biological sensing devices. Graphene has been widely researched as a signal transducing and sensing material. Here, a grass-like laser-scribed graphene (LSG) was synthesized by direct laser induction on common polyimide plastics. The resulting LSG grass was employed as a disposable electrochemical sensor for the detection of three neurotransmitters, dopamine (DA), epinephrine (EP), and norepinephrine (NE), and in the presence of uric acid and ascorbic acid as potential interferants, using differential pulse voltammetry and cyclic voltammetry. The LSG grass sensor achieved sensitivities of 0.243, 0.067, and 0.110 µA µM-1 for DA, EP, and NE, respectively, whereas the limits of detection were 0.43, 1.1, and 1.3 µM, respectively. The selectivity of LSG grass was excellent for competing biomarkers with high structural similarity (EP vs NE and EP vs DA). The exceptional performance of LSG grass for DA, EP, and NE detection holds a promising future for carbon nanomaterial sensors with unique surface morphologies.

Facile synthesis of gold nanoparticles using carbon dots for electrochemical detection of neurotransmitter, dopamine in human serum and as a chemocatalyst for nitroaromatic reduction.

Herein, the authors reported a carbon dots mediated synthesis of gold nanoparticles (AuNPs) at room temperature. Transmission electron microscopy revealed that the AuNPs are spherical in shape with a size of 10 nm. As-prepared AuNPs was immobilised on carbon paste electrode and subjected to electrochemical sensing of an important neurotransmitter dopamine. Differential pulse voltammetry studies revealed sensitive and selective determination of dopamine in the presence of commonly interfering ascorbic acid and uric acid. The linear detection range was 10-600 µM and the limit of detection was 0.7 ± 0.18 µM. The practical application was demonstrated by measuring dopamine in human blood serum and urine samples. The catalytic activity of AuNPs was evaluated by sodium borohydride mediated reduction of nitroaromatic compounds. The reduction kinetics was found to be pseudo-first-order kinetics. All the tested nitroaromatics reduced to corresponding amines in <10 min.

Dual-Signal Luminescent Detection of Dopamine by a Single Type of Lanthanide-Doped Nanoparticles.

Detection of dopamine, an important neurotransmitter, is vital for understanding its roles in mammals and disease diagnosis. However, commonly available methods for dopamine detection typically rely on a single signal readout, which can be susceptible to interference by internal or external factors. Here, we report a dual-signal detection of dopamine based on label-free luminescent NaGdF4:Tb nanoparticles. In the presence of dopamine, the NaGdF4:Tb nanoparticles exhibit luminescence quenching under the excitation of 272 nm, while they give enhanced luminescence under 297 nm excitation, realizing both turn off and turn on detection of dopamine. The nanoparticle-based dual-signal sensors exhibit high sensitivity, with a detection limit of ∼30 nM, and good selectivity, which offers the possibility to identify potential interferents in the samples. We further demonstrate that the dual-signal response results from different energy-transfer processes within the nanoparticles under the excitation of different light. The new strategy demonstrated here should pave the way for the development of multiresponse nanosensors based on lanthanide-doped luminescent nanomaterials.

Development and validation of a simple, rapid and sensitive LC-MS/MS method for the measurement of urinary neurotransmitters and their metabolites.

Neurotransmitters play crucial roles in physiological functions and their imbalances have demonstrated association in the pathology of several diseases. The measurement of neurotransmitters possesses a great potential as a significant clinical tool. This study presents the development and validation of an LC-MS/MS method for simultaneous quantification of multi-class neurotransmitters associated with dopamine, tryptophan and glutamate-γ-aminobutyric acid pathways. A total of ten neurotransmitters and their metabolites (dopamine, epinephrine, metanephrine, tryptophan, serotonin, kynurenic acid, kynurenine, anthranilic acid, GABA, glutamic acid) were determined based on a simple and rapid 'dilute and shoot' method using minimal urine volume. The chromatographic separation was achieved using a Poroshell 120 Bonus-RP LC Column in combination with a gradient elution within an 8.5-min time frame. The method exhibited good sensitivity as the limits of quantification ranged between 0.025 and 0.075 µg/mL with acceptable matrix effects (< ± 14.5%), no carryover and good linearity (r 2 > 0.98). The accuracy and precision for all analytes were within tolerances, at < ± 9.9% mean relative error (MRE) and < 8.6% relative standard deviation (RSD), respectively. The method was successfully applied in measuring the neurotransmitter concentrations in urine of healthy donors. Furthermore, the undertaken stability experiments indicated that acidified urine specimens allowed the analytes to be stable for prolonged durations in comparison to those untreated. The study also reveals the performance of the method is unaffected by the absence of expensive deuterated reference standards under the experimental conditions employed which further simplifies the analytical procedures and provides a significant cost saving for running the assay. Graphical abstract The quantification of multi-class neurotransitters associated with the dopamine, tryptophan and GABA-glutamate pathways using a simple 'dilute and shoot' LC-MS/MS method.

Colorimetric Fingerprints of Gold Nanorods for Discriminating Catecholamine Neurotransmitters in Urine Samples.

Catecholamine neurotransmitters, generally including dopamine (DA), epinephrine (EP) and norepinephrine (NE) are known as substantial indicators of various neurological diseases. Simultaneous detection of these compounds and their metabolites is highly recommended in early clinical diagnosis. To this aim, in the present contribution, a high performance colorimetric sensor array has been proposed for the detection and discrimination of catecholamines based on their reducing ability to deposit silver on the surface of gold nanorods (AuNRs). The amassed silver nanoshell led to a blue shift in the longitudinal localized surface plasmon resonance (LSPR) peak of AuNRs, creating a unique pattern for each of the neurotransmitters. Hierarchical cluster analysis (HCA) and linear discriminate analysis (LDA) pattern recognition techniques were employed to identify DA, EP and NE. The proposed colorimetric array is able to differentiate among individual neurotransmitters as well as their mixtures, successfully. Finally, it was shown that the sensor array can identify these neurotransmitters in human urine samples.

Multiple metal exposures and their correlation with monoamine neurotransmitter metabolism in Chinese electroplating workers.

Excessive metal exposure has been recognized as one of the detrimental factors for brain damage. However, the potential adverse effects induced by heavy metals on monoamine neurotransmitter pathways remains poorly understood. Our study aimed to investigate the possible association between metal exposure and neurotransmitter metabolism. By a cross-sectional investigation, 224 electroplating workers and 213 non-electroplating exposure workers were recruited in the exposure and control groups. Metal exposure levels were analyzed using inductively-coupled plasma mass spectrometry and monoamine neurotransmitter pathway metabolites were measured by ultra-performance liquid chromatography tandem mass spectrometry in human urine samples. Multivariate linear regression model was used to assess the dose-response relationships of urinary metals and neurotransmitter pathway metabolites. Significant dose-dependent trends of urinary vanadium quartiles with all metabolites were observed, and the trends demonstrated significance after multiple testing correction. It also showed that urinary chromium levels were significantly associated with decreased serotonin level and cadmium was positively associated with norepinephrine and epinephrine. In addition, arsenic was positively associated with tryptophan, serotonin, dopamine and norepinephrine. Iron was positively associated with increased homovanillic acid (HVA) and epinephrine while nickel was negatively associated with increased epinephrine levels. Zinc was positively related to tryptophan, kynurenin (KYN), 5-hydroxyindole acetic acid (5-HIAA), dopamine, HVA and norepinephrine. There was no significant association between urinary copper with any other metabolites after adjusting of multiple metal models. Metal exposure may be associated with neurotransmitter metabolism disturbances. The present work is expected to provide some support in the prevention and management of metal-associated neurological diseases.

Development and application of a new in-line coupling of a miniaturized boronate affinity monolithic column with capillary zone electrophoresis for the selective enrichment and analysis of cis-diol-containing compounds.

An integrated, miniaturized and fully automated system was developed for the analysis (preconcentration/purification, separation and detection) of cis-diol containing molecules in complex matrices. This innovative in-line coupling system was achieved via the in-situ and localized synthesis of a short segment of silica-based monolith at the inlet of a 75-µm inner diameter fused silica capillary. The monolithic segment was locally functionalized with an acrylamide derivative of phenylboronic acid by free radical photopolymerization within 10min of irradiation time. Efficiency of the photopolymerization reaction was followed by frontal affinity chromatography of 1,2-dihydroxybenzene (catechol) as cis-diol model solute. An active-site amount of 0.43nmolcm-1 (9.8nmolµL-1) of phenylboronic acid moieties was obtained, with a Kd value of about 290µM close to reported value for the phenyl boronate-catechol complex. The optimal conditions of use of the miniaturized boronate affinity monolithic column (µBAMC) were determined and adapted to the in-line coupling with capillary electrophoresis. Catechol was specifically preconcentrated in a pH 8.5 phosphate buffer/MeOH (80/20, v/v) mixture. A volume up to 20 times the monolith volume can be percolated with a quantitative recovery yield. Three catecholamines were purified, preconcentrated and in-line separated. Elution from the µBAMC was performed with a small plug of acidic solution, allowing field amplified sample stacking of solutes within the plug before their in-line electrophoretic separation at pH 8.75. This unique in-line coupling was successfully used for the fully automated analysis of catecholamines neurotransmitters in urine samples, highlighting the purification efficiency of the µBAMC and the potential of such a fully integrated approach. In addition to the low sample volume required (less than 2µL), the limits of detection (LOD) accomplished with this coupling were estimated at 9.0, 9.5 and 4.8ngmL-1 for dopamine, adrenaline and noradrenaline respectively, which improves the LOD of theses solutes compared to other CE methods.

Simultaneous extraction and determination of monoamine neurotransmitters in human urine for clinical routine testing based on a dual functional solid phase extraction assisted by phenylboronic acid coupled with liquid chromatography-tandem mass spectrometry.

The major monoamine neurotransmitters, serotonin (5-HT) and catecholamines (i.e., norepinephrine (NE), epinephrine (E), and dopamine (DA)), are critical to the nervous system function, and imbalances of the neurotransmitters have been connected to a variety of diseases, making their measurement useful in a clinical setting. A simple, rapid, robust, sensitive, and specific LC-MS/MS method has been developed and validated for the simultaneous quantitation of urinary serotonin and catecholamines with low cost, which is ideal for routine clinical applications. A simple extraction from complex urine was accomplished using tailored solid phase extraction incorporating phenylboronic acid complexation on a 96-well HLB microplate for the sample extraction and resulted in significantly improved throughput, selectivity, and extraction recovery. Compared to 1-10 mL of urine typically used, this method required only 10 µL. A rapid chromatographic elution with a total cycle time of 6 min per sample compared to reported run times of 19-75 min was achieved on a PFP column. The sensitivity of l and 2 ng mL-1 for the detection of low abundant E and NE combined with the high coverage of 1024 ng mL-1 for DA enabled the multi-analyte detection of these biogenic amines in a single run. Good linearity (2.0-512, 1.0-512, 4.0-1024, and 4.0-1024 ng mL-1 for NE, E, DA, and 5-HT, respectively), accuracy (87.6-104.0%), precision (≤8.0%), extraction recovery (69.6-103.7%), and matrix effect (87.1-113.1% for catecholamines and 63.6-71.4% for 5-HT) were obtained. No autosampler carryover was observed. The analytes were stable for 5 days at 20 °C, 14 days at 4 °C, and 30 days at -20 °C and five freeze-thaw cycles. The easy sample preparation, rapid LC, and multi-analyte MS detection allow two 96-well plates of samples to be extracted within 2 h and analyzed on an LC-MS/MS system within 24 h. The applicability and reliability of the assay were demonstrated by assessment of the reference interval for authentic urine specimens from 90 healthy individuals. Graphical abstract A simple, rapid, robust, sensitive and specific LC-MS/MS method combined with a dual functional solid phase extraction has been developed and validated for the simultaneous extraction and quantitation of monoamine neurotransmitters in human urine with low cost.

Analysis of amino acid and monoamine neurotransmitters and their metabolites in rat urine of Alzheimer's disease using in situ ultrasound-assisted derivatization dispersive liquid-liquid microextraction with UHPLC-MS/MS.

Neurotransmitters (NTs) may play an important role in neurodegenerative disorders such as Alzheimer's disease (AD). In order to investigate the potential links, a new simple, fast, accurate and sensitive analytical method, based on in situ ultrasound-assisted derivatization dispersive liquid-liquid microextraction (in situ UA-DDLLME) coupled with ultra high-performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS), has been developed and validated. The quantitation of amino acid neurotransmitters (AANTs) and monoamine neurotransmitters (MANTs) in urine of AD rats were performed in this work. The in situ UA-DDLLME procedure involved the rapid injection of the mixture of low toxic 4-bromoanisole (extractant) and acetonitrile (dispersant), which containing the new designed and synthesized 4'-carbonyl chloride rosamine (CCR) as derivatization reagent, into the aqueous phase of real sample and buffer. Under the selected conditions, the derivatization and microextraction of analytes were simultaneously completed within 1min. Good linearity for each analyte (R>0.992) was observed with low limit of detections (LODs, S/N>3). Moreover, the proposed method was compared with direct detection or other reported methods, and the results showed that low matrix effects and good recoveries results were obtained in this work. Taken together, in situ UA-DDLLME coupled with UHPLC-MS/MS analysis was demonstrated to be a good method for sensitive, accurate and simultaneous monitoring of AANTs and MANTs. This method would be expected to be highly useful in AD diseases' clinical diagnostics and may have potential value in monitoring the efficacy of treatment.