A simultaneous extraction/derivatization strategy coupled with liquid chromatography-tandem mass spectrometry for the determination of free catecholamines in biological fluids.

The current study presents a convenient, rapid and effective simultaneous extraction/derivatization (SEDP) strategy for effective pretreatment of catecholamines (CAs). Commercial zirconium oxide (ZrO2) nanoparticles were employed for the selective capturing of cis-diol containing CAs to remove the biological interferences and phenyl isothiocyanate (PITC) was used for derivatization to improve the ionization and to improve the chromatographic separation. The extraction and derivatization procedures were integrated into one step to simplify the sample pretreatment. Excessive derivatization reagents were removed as well, reducing the degree of contaminations in mass spectrometry. The factors affecting the SEDP process were optimized and the results showed that the detection sensitivity and chromatographic separation of CAs greatly improved compared with underivatized CAs, during LC-MS/MS analysis. Combined with ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS), quantifying the concentration of norepinephrine (NE), epinephrine (E) and dopamine (DA) in biological fluids was validated in ranges of 1-200.0 ng/mL with a satisfactory correlation coefficient (R2 > 0.997). The obtained recoveries were in the range of 91.0-109.5% with RSDs less than 9.4%. Finally, significant changes in CAs levels in urine samples of healthy people and pheochromocytoma patients were detected. The developed method offers comparative advantages in terms of sensitivity, specificity and selectivity.

In Matrix Derivatization Combined with LC-MS/MS Results in Ultrasensitive Quantification of Plasma Free Metanephrines and Catecholamines.

Plasma-free metanephrines and catecholamines are essential markers in the biochemical diagnosis and follow-up of neuroendocrine tumors and inborn errors of metabolism. However, their low circulating concentrations (in the nanomolar range) and poor fragmentation characteristics hinder facile simultaneous quantification by liquid chromatography and tandem mass spectrometry (LC-MS/MS). Here, we present a sensitive and simple matrix derivatization procedure using propionic anhydride that enables simultaneous quantification of unconjugated l-DOPA, catecholamines, and metanephrines in plasma by LC-MS/MS. Dilution of propionic anhydride 1:4 (v/v) in acetonitrile in combination with 50 µL of plasma resulted in the highest mass spectrometric response. In plasma, derivatization resulted in stable derivatives and increased sensitivity by a factor of 4-30 compared with a previous LC-MS/MS method for measuring plasma metanephrines in our laboratory. Furthermore, propionylation increased specificity, especially for 3-methoxytyramine, by preventing interference from antihypertensive medication (ß-blockers). The method was validated according to international guidelines and correlated with a hydrophilic interaction LC-MS/MS method for measuring plasma metanephrines (R2 > 0.99) and high-performance liquid chromatography with an electrochemical detection method for measuring plasma catecholamines (R2 > 0.85). Reference intervals for l-DOPA, catecholamines, and metanephrines in n = 115 healthy individuals were established. Our work shows that analytes in the subnanomolar range in plasma can be derivatized in situ without any preceding sample extraction. The developed method shows improved sensitivity and selectivity over existing methods and enables simultaneous quantification of several classes of amines.

Nanosized cation exchanger for the electrophoretic separation and preconcentration of catecholamines and amino acids.

The first example of application of nanosized polystyrene-based cation exchanger (NSCE) with sulfo groups as a dynamic coating of capillary walls was demonstrated. The conditions of dynamic coating formation were optimized and ensured the long-term stability of the coating. Capillary-to-capillary and day-to-day repeatabilities were 4% and 3%, correspondingly. The NSCE coating stability at various pH and influence of pH on the EOF mobility were investigated. The developed NSCE-modified coated capillaries provided improved resolution (Rs = 0.9-3.2 for catecholamines and Rs = 1.7-2.8 for amino acids) and efficiencies (330-520 ×103 t.p./m) of basic analytes, which are 1.5 times higher compared to untreated capillary. The optimized conditions were as follows: 50 mM phosphate buffer solution at pH 2.2 with 5 µM NSCE. The effect of the NSCE concentration in BGE on the electrophoretic mobilities of the analytes was investigated. The various online concentration techniques were tested in order to decrease the LODs. The simultaneous application of NSCE capillaries and field-amplified sample stacking provided the lowest LODs of catecholamines and amino acids and allowed to determine these analytes in human urine.

Automated on-line packed fiber solid phase extraction for determination of urinary catecholamines.

Despite the development of an off-line packed fiber solid phase extraction procedure (PFSPE) for urinary catecholamines, automation remains a challenge. Here, we propose an on-line PFSPE-HPLC procedure for automated sample processing and analysis of urinary catecholamines, with good recovery and precision, to avoid manual operation errors. The on-line PFSPE-HPLC procedure has been thoroughly optimized concerning the gradient, valve switch timing, the effects of complexing reagent and buffer solution, and the stability of the nanofibers. Validation of the developed on-line PFSPE-HPLC protocol in urine yielded satisfactory accuracies of 99.6-104.2%, precision below 7.0%, as well as a linear range from 1 ng/mL to 100 ng/mL with a correlation coefficient of 0.999. The developed protocol is herein presented as a potential technology for automated sample pretreatment for the determination of urinary catecholamines.

Progress toward the development of a microchip electrophoresis separation-based sensor with electrochemical detection for on-line in vivo monitoring of catecholamines.

The development of a separation-based sensor for catecholamines based on microdialysis (MD) coupled to microchip electrophoresis (ME) with electrochemical (EC) detection is described. The device consists of a pyrolyzed photoresist film working electrode and a poly(dimethylsiloxane) microchip with a flow-gated sample injection interface. The chip was partially reversibly sealed to the glass substrate by selectively exposing only the top section of the chip to plasma. This partially reversible chip/electrode integration process not only allows the reuse of the working electrode but also greatly enhanced the reproducibility of electrode alignment with the separation channel. The developed MD-ME-EC system was then tested using l-DOPA, 3-O-MD, HVA, DOPAC, and dopamine standards, which were separated in less than 100 seconds using a background electrolyte consisting of 15 mM sodium phosphate (pH 7.4), 15 mM sodium dodecyl sulfate, and 2.5 mM boric acid. A potential of +1.0 V vs. Ag/AgCl was used for amperometric detection of the analytes. The device was evaluated for on-line monitoring of the conversion of l-DOPA to dopamine in vitro and for monitoring dopamine release in an anesthetized rat in vivo following high K+ stimulation. The system was able to detect stimulated dopamine release in vivo but not endogenous levels of dopamine.

Boronic acid-modified polyhedral oligomeric silsesquioxanes on polydopamine-coated magnetized graphene oxide for selective and high-capacity extraction of the catecholamines epinephrine, dopamine and isoprenaline.

Amino-functionalized polyhedral oligomeric silsesquioxanes (POSS-8NH2) were covalently bound to the surface of polydopamine-coated magnetized graphene oxide. It was then reacted with 4-formylphenylboronic acid to prepare a "cubic boronic acid"-bonded magnetic graphene oxide adsorbent. The new adsorbent exhibits better selectivity and much higher adsorption capacity for ortho-phenols over adsorbents where small boronic ligands are directly bound to the surface of the material. It is shown to enable selective and faster enrichment of the catecholamines epinephrine (EP), dopamine (DA) and isoprenaline (IP) with high selectivity over many potential interferents that can occur in urine. The analytes were then quantified by HPLC with fluorometric detection. Under optimal conditions, response is linear (R2 ≥ 0.9907), limits of detection are low (0.54-2.3 ng·mL-1), and reproducibility is acceptable (inter- and intra-day assay RSDs of≤10.9%). The method was successfully applied to the determination of endogenous EP and DA and exogenous IP in urine samples. Graphical abstractSchematic of boronic acid (BA)-modified polyhedral oligomeric silsesquioxanes (POSS) on polydopamine-coated magnetized graphene oxide (magGO). The material (magGO@POSS-BA) has good selectivity and higher adsorption capacity to ortho-phenols and can be applied to enrich the catecholamines in urine.

Polymethacryloyl-L-Phenylalanine [PMAPA]-Based Monolithic Column for Capillary Electrochromatography.

The ability to detect catecholamines (CAs) and their metabolites is vital to understand the mechanism behind the neuronal diseases. Neurochemistry aims to provide an improved pharmacological, molecular and physiological understanding of complex brain chemistries by analytical techniques. Capillary electrophoresis (CE) is one such analytical technique that enables the study of various chemical species ranging from amino acids and peptides to natural products and drugs. CE can easily adapt the changes in research focus and in recent years remains an applicable technique for investigating neuroscience and single cell neurobiology. The prepared phenylalanine-based hydrophobic monolithic column, Polymethacryloyl-L-phenylalanine [PMAPA], was used as a stationary phase in capillary electrochromatography to separate CAs that are similar in size and shape to each other including dopamine (DA) and norepinephrine (NE) via hydrophobic interactions. Separation carried out in a short period of 17 min was performed with the electrophoretic mobility of 5.54 × 10-6 m2 V-1 s-1 and 7.60 × 10-6 m2 V-1 s-1 for DA and NE, respectively, at pH 7.0, 65% acetonitrile ratio with 100 mbar applied pressure by the developed hydrophobic monolithic column without needing any extra process such as imprinting or spacer arms to immobilize ligands used in separation.

High-throughput and selective solid-phase extraction of urinary catecholamines by crown ether-modified resin composite fiber.

In the present study, we developed a simple and high-throughput solid phase extraction (SPE) procedure for selective extraction of catecholamines (CAs) in urine samples. The SPE adsorbents were electrospun composite fibers functionalized with 4-carboxybenzo-18-crown-6 ether modified XAD resin and polystyrene, which were packed into 96-well columns and used for high-throughput selective extraction of CAs in healthy human urine samples. Moreover, the extraction efficiency of packed-fiber SPE (PFSPE) was examined by high performance liquid chromatography coupled with fluorescence detector. The parameters affecting the extraction efficiency and impurity removal efficiency were optimized, and good linearity ranging from 0.5 to 400 ng/mL was obtained with a low limit of detection (LOD, 0.2-0.5 ng/mL) and a good repeatability (2.7%-3.7%, n = 6). The extraction recoveries of three CAs ranged from 70.5% to 119.5%. Furthermore, stable and reliable results obtained by the fluorescence detector were superior to those obtained by the electrochemical detector. Collectively, PFSPE coupled with 96-well columns was a simple, rapid, selective, high-throughput and cost-efficient method, and the proposed method could be applied in clinical chemistry.

A Convenient Method for Extraction and Analysis with High-Pressure Liquid Chromatography of Catecholamine Neurotransmitters and Their Metabolites.

The extraction and analysis of catecholamine neurotransmitters in biological fluids is of great importance in assessing nervous system function and related diseases, but their precise measurement is still a challenge. Many protocols have been described for neurotransmitter measurement by a variety of instruments, including high-pressure liquid chromatography (HPLC). However, there are shortcomings, such as complicated operation or hard-to-detect multiple targets, which cannot be avoided, and presently, the dominant analysis technique is still HPLC coupled with sensitive electrochemical or fluorimetric detection, due to its high sensitivity and good selectivity. Here, a detailed protocol is described for the pretreatment and detection of catecholamines with high pressure liquid chromatography with electrochemical detection (HPLC-ECD) in real urine samples of infants, using electrospun composite nanofibers composed of polymeric crown ether with polystyrene as adsorbent, also known as the packed-fiber solid phase extraction (PFSPE) method. We show how urine samples can be easily precleaned by a nanofiber-packed solid phase column, and how the analytes in the sample can be rapidly enriched, desorbed, and detected on an ECD system. PFSPE greatly simplifies the pretreatment procedures for biological samples, allowing for decreased time, expense, and reduction of the loss of targets. Overall, this work illustrates a simple and convenient protocol for solid-phase extraction coupled to an HPLC-ECD system for simultaneous determination of three monoamine neurotransmitters (norepinephrine (NE), epinephrine (E), dopamine (DA)) and two of their metabolites (3-methoxy-4-hydroxyphenylglycol (MHPG) and 3,4-dihydroxy-phenylacetic acid (DOPAC)) in infants' urine. The established protocol was applied to assess the differences of urinary catecholamines and their metabolites between high-risk infants with perinatal brain damage and healthy controls. Comparative analysis revealed a significant difference in urinary MHPG between the two groups, indicating that the catecholamine metabolites may be an important candidate marker for early diagnosis of cases at risk for brain damage in infants.

Complexation-mediated electromembrane extraction of highly polar basic drugs-a fundamental study with catecholamines in urine as model system.

Complexation-mediated electromembrane extraction (EME) of highly polar basic drugs (log P < -1) was investigated for the first time with the catecholamines epinephrine, norepinephrine, and dopamine as model analytes. The model analytes were extracted as cationic species from urine samples (pH 4), through a supported liquid membrane (SLM) comprising 25 mM 4-(trifluoromethyl)phenylboronic acid (TFPBA) in bis(2-ethylhexyl) phosphite (DEHPi), and into 20 mM formic acid as acceptor solution. EME was performed for 15 min, and 50 V was used as extraction voltage across the SLM. TFPBA served as complexation reagent, and selectively formed boronate esters by reversible covalent binding with the model analytes at the sample/SLM interface. This enhanced the mass transfer of the highly polar model analytes across the SLM, and EME of basic drugs with log P in the range -1 to -2 was shown for the first time. Meanwhile, most matrix components in urine were unable to pass the SLM. Thus, the proposed concept provided highly efficient sample clean-up and the system current across the SLM was kept below 50 µA. Finally, the complexation-mediated EME concept was combined with ultra-high performance liquid chromatography coupled to tandem mass spectrometry and evaluated for quantification of epinephrine and dopamine. Standard addition calibration was applied to a pooled human urine sample. Calibration curves using standards between 25 and 125 µg L-1 gave a high level of linearity with a correlation coefficient of 0.990 for epinephrine and 0.996 for dopamine (N = 5). The limit of detection, calculated as three times signal-to-noise ratio, was 5.0 µg L-1 for epinephrine and 1.8 µg L-1 for dopamine. The repeatability of the method, expressed as coefficient of variation, was 13% (n = 5). The proposed method was finally applied for the analysis of spiked pooled human urine sample, obtaining relative recoveries of 91 and 117% for epinephrine and dopamine, respectively.

Nano Secondary Ion Mass Spectrometry Imaging of Dopamine Distribution Across Nanometer Vesicles.

We report an approach to spatially resolve the content across nanometer neuroendocrine vesicles in nerve-like cells by correlating super high-resolution mass spectrometry imaging, NanoSIMS, with transmission electron microscopy (TEM). Furthermore, intracellular electrochemical cytometry at nanotip electrodes is used to count the number of molecules in individual vesicles to compare to imaged amounts in vesicles. Correlation between the NanoSIMS and TEM provides nanometer resolution of the inner structure of these organelles. Moreover, correlation with electrochemical methods provides a means to quantify and relate vesicle neurotransmitter content and release, which is used to explain the slow transfer of dopamine between vesicular compartments. These nanoanalytical tools reveal that dopamine loading/unloading between vesicular compartments, dense core and halo solution, is a kinetically limited process. The combination of NanoSIMS and TEM has been used to show the distribution profile of newly synthesized dopamine across individual vesicles. Our findings suggest that the vesicle inner morphology might regulate the neurotransmitter release event during open and closed exocytosis from dense core vesicles with hours of equilibrium needed to move significant amounts of catecholamine from the protein dense core despite its nanometer size.

Nonhydrolytic sol-gel approach to facile creation of surface-bonded zirconia organic-inorganic hybrid coatings for sample preparation. Ι. Capillary microextraction of catecholamine neurotransmitters.

Nonhydrolytic sol-gel (NHSG) route was used for the creation of novel zirconia-polypropylene oxide (ZrO2-PPO) sol-gel hybrid sorbents in the form of surface coatings for the extraction and preconcentration of catecholamine neurotransmitters and molecules structurally related to their deaminated metabolites. In comparison to other sorbents made of inorganic transition metal oxides, the presented hybrid organic-inorganic sorbents facilitated reversible sorption properties that allowed for efficient desorption of the extracted analytes by LC-MS compatible mobile phases. The presented sol-gel hybrid sorbents effectively overcame the major drawbacks of traditional silica- or polymer-based sorbents by providing superior pH stability (pH range: 0-14), and a variety of intermolecular interactions. Nonaqueous sol-gel treatment of PPO with ZrCl4 was employed for the derivatization of the terminal hydroxyl groups on PPO, providing zirconium trichloride-containing end groups characterized by enhanced sol-gel reactivity. NHSG ZrO2-PPO sorbent provided excellent microextraction performance for catecholamines, low detection limits (5.6-9.6pM), high run-to-run reproducibility (RSD 0.6-5.1%), high desorption efficiency (95.0-99.5%) and high enrichment factors (∼1480-2650) for dopamine and epinephrine, respectively, extracted from synthetic urine samples. The presented sol-gel sorbents provided effective alternative to conventional extraction media providing unique physicochemical characteristics and excellent extraction capability.

Determination of catecholamines and related compounds in mouse urine using column-switching HPLC.

We have developed an analytical method for the determination of catecholamines and related compounds in mouse urine by column-switching HPLC. Selective extraction of the catechol compounds was performed using a precolumn modified with phenylboronic acid, which has a pH dependent affinity for the catechol structures. The pretreatment buffer, which facilitated binding of the catechols to the precolumn, was optimized to ensure high analyte recoveries and good peak shapes. We found that using the same acetonitrile content in the pretreatment buffer and hydrophilic interaction liquid chromatography mobile phase was necessary to improve peak shapes. Eight catechol compounds were selectively extracted and separated using 100 mmol L(-1) ammonium formate/acetonitrile (20/80 v/v, pH 8.0) for the extraction step, and 20 mmol L(-1) ammonium formate (pH 2.5)/acetonitrile (20/80 v/v) for elution and separation. Native fluorescence of the separated catechol compounds was monitored, and the limits of detection, corresponding to a signal to noise ratio of 3, were 9-58 nmol L(-1). Five catechol compounds (dopamine, epinephrine, norepinephrine, 3,4-dihydroxyphenylglycol, and 3,4-dihydroxymandelic acid) were successfully quantified in mouse urine. Intra- and inter-day precisions were less than 10%, and performance was superior to that afforded by manual sample pretreatment.

[Development of Efficient Separation and Sensitive Analytical Methods for Biological Compounds and Their Application].

Measurement of biological compounds is important for the clarification of biological phenomena. For the quantification of trace amounts of biological compounds, efficient separation and sensitive analytical methods are necessary. The present author developed HPLC-fluorescence and chemiluminescence detection methods for biological compounds such as catecholamines, amino acids, and thiols. In this review article, two studies are summarized: one on the development of an on-chip liquid chromatography method using pillar array columns with low-dispersion turns; and another on the development of simultaneous analytical method of biothiols by HPLC with fluorescence detection under hydrophilic interaction chromatography conditions.

Facile synthesis of Fe3O4@polyethyleneimine modified with 4-formylphenylboronic acid for the highly selective extraction of major catecholamines from human urine.

The levels of catecholamines, especially dopamine, epinephrine and norepinephrine in urine and plasma have been used to assist the diagnosis and treatment of psychosis. Due to their low endogenous concentrations, the determination of the three major catecholamines is very difficult. Boronate adsorbents are often employed to extract these cis-diol compounds from complex matrices. In this work, a novel type of magnetic nanoparticles modified with 4-formylphenylboronic named Fe3O4@PEI-FPBA was synthesized by a facile two-step approach. The abundant amino groups of polyethyleneimine provided the rich binding sites for boronate ligands. Herein, the adsorption capacity of Fe3O4@PEI-FPBA is greatly improved with a value of 3.45 mg/g towards epinephrine, which is much larger than that of analogous material without polyethyleneimine. The magnetic nanoparticles also exhibited high magnetization (72.25 emu/g) and specific selectivity towards the catecholamines. Finally, a liquid chromatography tandem mass spectrometry method based on Fe3O4@PEI-FPBA nanoparticles was successfully used to determine the three catecholamines from human urine samples. The linearity, limit of quantitation, recovery and precision of the method were satisfactory. Based on the method, it is found that the levels of dopamine, epinephrine and norepinephrine in depressive patients are higher than those in healthy controls.

Selective extraction of catecholamines by packed fiber solid-phase using composite nanofibers composing of polymeric crown ether with polystyrene.

For the first time, electrospun composite nanofibers comprising polymeric crown ether with polystyrene (PCE-PS) have been used for the selective extraction of catecholamines - dopamine (DA), norepinephrine (NE) and epinephrine (E) - prior to their analysis by high-performance liquid chromatography-electrochemical detection. Using a minicartridge packed with PCE-PS composite nanofibers, the target compounds were extracted effectively from urine samples to which diphenylborinic acid 2-aminoethyl ester was added as a complexing reagent. The extracted catecholamines could be liberated from the fiber by the addition of acetic acid. A good linearity was observed for catecholamines in the range of 2.0-200 ng mL(-1) (NE, E and DA). The detection limits of catecholamines (signal-to-noise ratio = 3) were 0.5 ng mL(-1) (NE), 0.2 ng mL(-1) (E) and 0.2 ng mL(-1) (DA), respectively. Under the optimized conditions, the absolute recoveries of the above three catecholamines were 90.6% (NE), 88.5% (E) and 94.5% (DA). The repeatability of extraction performance was from 5.4 to 9.2% (expressed as relative standard deviation). Our results indicate that the proposed method could be used for the determination of NE, E and DA in urine.

Monolithic silica rods grafted with thermoresponsive anionic polymer brushes for high-speed separation of basic biomolecules and peptides.

Thermoresponsive anionic copolymer brushes, poly(N-isopropylacrylamide-co-acrylic acid-co-tert-butylacrylamide) [P(IPAAm-co-AAc-co-tBAAm)], were grafted onto a monolithic silica rod column through surface-initiated atom-transfer radical polymerization (ATRP) to prepare an effective thermoresponsive anionic chromatography matrix. An ATRP initiator was attached to the rod surface. N-Isopropylacrylamide (IPAAm), tert-butyl acrylate (tBA), tert-butylacrylamide (tBAAm), and the ATRP catalyst CuCl/CuCl2/tris[2-(N,N-dimethylamino)ethyl]amine were dissolved in 2-propanol, and the reaction mixture was pumped into the initiator-modified column. After grafting P(IPAAm-co-tBA-co-tBAAm) on the monolithic silica surfaces, deprotection of the tert-butyl group of tBA was performed. Chromatographic analysis showed that the prepared column was able to separate catecholamine derivatives and angiotensin subtypes within a shorter analysis time (5 min) than a silica-bead-packed column modified with the same copolymer brush could. These results indicated that the prepared copolymer-modified monolithic silica rod column may be a promising bioanalytical and bioseparation tool for rapid analysis of basic bioactive compounds and peptides.

Poly(triallyl isocyanurate-co-ethylene dimethacrylate-co-alkyl methacrylate) stationary phases in the chromatographic separation of hydrophilic solutes.

This study describes the ability of triallyl isocyanurate (TAIC)-co-methacrylate ester polymer monoliths as stationary phases for the separation of hydrophilic compounds (phenolic acids, amino acids and catecholamines) in capillary electrochromatography (CEC) and ultra high pressure liquid chromatography (UHPLC). Several TAIC-co-methacrylate ester polymer monoliths prepared by single-step in situ copolymerization of TAIC, ethylene dimethacrylate (EDMA) and 2-acrylamido-2-methylpropane sulfonic acid (AMPS), with or without alkyl methacrylates were characterized by examining the SEM image, surface area, contact angle, and the thermal decomposition temperature. Compared to the conventional methacrylate ester-based monoliths, these proposed monoliths possessed hydrophilic character thus increased wettability which improved chromatographic separation selectivity of polar phenolic acids. Among the proposed TAIC-co-methacrylate monoliths, poly(TAIC-co-EDMA-AMPS-co-stearyl methacrylate (SMA)) showed separation selectivity with an increased analyte resolution from 0.0 to 0.92 for 4-hydroxybenzoic acid and vanillic acid, which were consistently difficult to resolve in the reversed-phase chromatographic mechanism of these monoliths in aqueous mobile phases. Moreover, stable ionization efficiencies were observed when this monolith was combined with ESI-MS detector possibly because an organic solvent-rich sheath liquid was used in the CEC-MS. This study demonstrates the potentiality of novel TAIC-co-methacrylate polymer monoliths in hydrophilic solute separation either in CEC or UHPLC mode.

Very fast electrophoretic separation on commercial instruments using a combination of two capillaries with different internal diameters.

A capillary formed by connecting a 9.7 cm-long separation capillary with id 25 µm with an auxiliary 22.9 cm-long capillary with id 100 µm (coupled capillary) was tested for electrophoretic separation at high electric field intensities. The coupled capillary was placed in the cassette of a standard electrophoresis apparatus. It was used in the short-end injection mode for separation of a mixture of dopamine, noradrenaline, and adrenaline in a BGE of 20 mM citric acid/NaOH, pH 3.2. An intensity of 2.7 kV/cm was attained in the separation part of the capillary at a separation voltage of 30 kV, which is 2.9 times more than maximum intensity value attainable in a capillary with the same length with uniform id. At these high electric field intensities, the migration times of the tested neurotransmitters had values of 12.3-13.3 s and the attained separation efficiency was between 2350 and 2760 plates/s. It is thus demonstrated that an effective separation instrument - a coupled capillary - can be used for very rapid separation in combination with standard, commercially available instrumentation.

Selective enrichment of catecholamines using iron oxide nanoparticles followed by CE with UV detection.

This study examines the use of unmodified magnetite nanoparticles (Fe(3)O(4) NPs) for selective extraction and enrichment of the catecholamines dopamine (DA), noradrenaline (NE), and adrenaline (E), prior to analysis using capillary electrophoresis with UV detection. Coordination between Fe(3+) on-the-surface Fe(3)O(4) NPs and the catechol moiety of catecholamines enables Fe(3)O(4) NPs to capture catecholamines from an aqueous solution. We obtained maximum loading of catecholamines on the NP surface by adjusting the pH of the solution to 7.0. In addition, catecholamine loading on the Fe(3)O(4) NPs increased in conjunction with NP concentrations. H(3)PO(4) was found to be efficient for the removal of adsorbed catecholamines on the NP surface. Adding 1.2% poly(diallyldimethylammonium chloride) to the background electrolyte resulted in a baseline separation of the liberated catecholamines within 20 min. Under optimal extraction and separation conditions, the limit of detections at a S/N ratio of 3 for E, NE, and DA were 9, 8, and 10 nM, respectively. Significantly, the combination of a phenylboronate-containing spin column and the proposed method was successfully applied to the determination of NE and DA in human urine and NE in Portulaca oleracea L. leaves.