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Title: Determination of catecholamines in urine using aminophenylboronic acid functionalized magnetic nanoparticles extraction followed by high-performance liquid chromatography and electrochemical detection

Liwei Jianga, Yibang Chena, Yanmei Luoa, Yueming Tana, Ming Maa,*, Bo Chena, Qingji Xiea, Xubiao Luob a

Key Laboratory of Chemical Biology and Traditional Chinese Medicine Research (Ministry

of Education of China), Key Laboratory of Phytochemical R&D of Hunan Province, Hunan Normal University, Changsha 410081, PR China b

Key Laboratory of Jiangxi Province for Persistent Pollutants Control and Resources Recycle

(Nanchang Hangkong University), Nanchang 330063, PR China Correspondence: Professor Ming Ma, Key Laboratory of Chemical Biology and Traditional Chinese Medicine Research (Ministry of Education of China), Key Laboratory of Phytochemical R&D of Hunan Province, Hunan Normal University, Changsha 410081, P. R. China. E-mail: [email protected]; Tel & Fax: +86-0731-88872531 Keywords: Catecholamines / Boronate affinity interaction / Electrochemical detection /Magnetic nanoparticles / Sample preparation / Abbreviations: MNT, monoamine neurotransmitter; CA, catecholamine; NE, norepinephrine; E, epinephrine; DA, dopamine; 5-HT, 5-hydroxytryptamine; ECD, electrochemical detection; FLD, fluorescence detection; APBA, 3-aminophenylboronic acid monohydrate; Fe3O4@ APBA NP, aminophenlyboronic acid functionalized magnetic particle; Fe3O4@DM NP, dextran functionalized magnetic particle

Received: 21-Aug-2014; Revised: 08-Nov-2014; Accepted: 18-Nov-2014 This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1002/jssc.201400920. This article is protected by copyright. All rights reserved.

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Abstract A new method was developed for the simultaneous determination of three catecholamines in urine using aminophenylboronic acid functionalized magnetic nanoparticles extraction followed by high-performance liquid chromatography with electrochemical detection. Novel aminophenylboronic acid functionalized magnetic nanoparticles were prepared by multi-step covalent

modification,

and

characterized

by

transmission

electron

microscopy,

Fourier-transformed infrared spectroscopy, X-ray diffraction and vibrating sample magnetometry. With the help of the high affinity between the boronate and cis-diol group, the particles were used for the highly selective separation and enrichment of three major catecholamines, norepinephrine, epinephrine, and dopamine. Effects of the pH of the feed solution, the extraction time, the composition of the buffer solution, the amount of the magnetic particles, the elution conditions, and the recycling of aminophenylboronic acid functionalized magnetic nanoparticles were explored. Under the optimized conditions, 13–17-fold enrichment factors were obtained. The linear ranges were 0.01–2.0 g/mL for the studied analytes. The limits of detection and quantification were in the range of 2.0–7.9 and 6.7–26.3 ng/mL, respectively. The relative recoveries were in the range of 92–108%, with intraday and interday relative standard deviations lower than 6.8%. This method was successfully applied to analysis of catecholamines in real urine.

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1 Introduction Monoamine neurotransmitters (MNTs), including catecholamines (CAs) (norepinephrine (NE), epinephrine (E), and dopamine (DA)) and indole amine (5-hydroxytryptamine (5-HT)) (chemical structures are shown in Supporting Information Fig. S1), play significant functional roles in the central nervous system [1]. The content of MNTs in plasma and urine are commonly used for the clinical diagnosis of various neurodegenerative diseases and the evaluation of hypotension or hypertension [2,3]. Hence, it is significant to determine the MNTs in urine. Combined with different detectors, such as MS, electrochemical detection (ECD), and fluorescence detection (FLD), HPLC is the most commonly used technique for the determination of MNTs [4–10]. Although MS is the state-of-the-art in bioanalytical determination due to the robustness and selectivity required for valid results, ECD is also remarkable in the determination of electrochemical active compounds (such as MNTs) due to its excellent selectivity, high sensitivity, and cheap price. Up to now, analysis of MNTs in urine sample is still a challenging work because of the complexity of biological samples and low concentrations of analytes. Classical pretreatment techniques employed in MNTs analysis are LLE [11], ion exchange resin separation [12], activated alumina adsorption [13], and packing column separation [7,14]. Because boronic acid group can reversibly form cyclic esters with cis-diol containing compounds [15,16], agarose column derivatized with m-aminophenylboronic acid [17], boron affinity chromatography column [18,19], in-tube boronate affinity SPME [20], and boric acid-functionalized silica SPE [21] have been employed successfully in CAs extraction and separation. Recently, magnetic adsorbents have been widely applied in different fields because of their rapidness and simplicity in separating and enriching procedures [22–25]. Lin et al. used This article is protected by copyright. All rights reserved.

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unmodified magnetite nanoparticles (Fe3O4 NPs) to adsorb CAs [26]. Qiao et al. synthesized a

novel

magnetic

nanoparticle

coated

with

sulfonated

complex

of

meso-2,3-dimercaptosuccinic acid and chitosan, which exhibited high CA conjugation efficiency through electrostatic interactions [27]. Bouri et al. developed a facile and selective approach for the extraction of CAs based on magnetic molecularly imprinted polymers which used DA as template molecule [28]. It has been demonstrated that functionalized magnetic particles are alternative adsorption materials. Boronic acid functionalized magnetic absorbents based on the interaction between boronate and some cis-diol groups have already been successfully applied in the purification of glycoproteins [29–31], glycopeptides [29,32], and carbohydrates [33]. Despite the fact that boronic acid groups can specifically interact with ortho phenolic hydroxyl groups of CAs, boronic acid functionalized magnetic nanoparticles has not been used for the selective separation and enrichment of CAs up to now. The aim of the present study is to explore the feasibility of selective separation and enrichment of CAs in urine using aminophenylboronic acid functionalized magnetic nanoparticles (Fe3O4@APBA NPs). Combined with HPLC–ECD analysis, the developed method was applied to urine samples analysis after the extraction of catecholamines with Fe3O4@APBA NPs.

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2 Materials and methods 2.1 Chemicals and reagents Ferrous sulfate (FeSO47H2O), ferric nitrate (Fe(NO3)39H2O), methanol, ethanol, ammonium hydroxide solution (25 wt%, NH3H2O), sodium acetate, citric acid, and glacial acetic acid (CH3COOH) were all purchased from Tianjin Damao Chemical Reagent Factory (Tianjin, China). Dextran, potassium dihydrogen phosphate, dipotassium phosphate, and sodium hydroxide were obtained from Sinopharm Chemical Reagent (Shanghai, China). Sodium periodate, octanesulfonic acid sodium, and sodium metabisulfite were purchased from Tianjin Kermel Chemical Reagent (Tianjin, China). 3-aminophenylboronic acid monohydrate (APBA) and sodium cyanoborohydride (NaBH3CN) were supplied by Aladdin (Shanghai, China). All above chemicals were of analytical grade. 5-Hydroxytryptamine hydrochloride, epinephrine, and dopamine hydrochloride were reference substances (purity > 98%) purchased from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). L-Norepinephrine (purity > 98%) was supplied by Aladdin (Shanghai, China). The water used was purified with a Milli-Q system from Millipore (Bedford, MA, USA).

2.2 Standard solution preparation, sample collection and preservation Individual stock solutions of E, NE, DA, and 5-HT at concentration of 500 μg/mL, containing 20 mmol/L antioxidant sodium metabisulfite to prevent the oxidation of analytes, were prepared in HCl solution (pH=3) and stored at 4°C in darkness. A mixed standard solution containing 25 μg/mL of E, NE, DA, and 5-HT was prepared daily by pipetting 250 L of each stock solution into 5 mL volumetric flask, diluting to the mark with HCl solution (pH=3) and stored at 4°C in darkness. Fresh feed solutions containing 5 μg/mL of E, NE, DA, and 5-HT were prepared by diluting the mixed standard solution with phosphate buffer (25 mmol/L, pH 8.0) unless otherwise specified, and used in the optimization of experimental This article is protected by copyright. All rights reserved.

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conditions. The pH of the feed solution was measured with a PHS-3C digital pH meter manufactured by INESA Scientific Instrument (Shanghai, China). Twenty-four hours of urine samples were collected from four healthy volunteers (one male, 23 years old, and three females, 22, 23, and 24 years old, respectively) and sodium metabisulfite was added to avoid the oxidation of CAs until the sodium metabisulfite concentration was 0.8 mmol/L. To investigate the feasibility of the developed method, the blank urine was prepared as follows: Firstly, the pH of the urine sample (the female volunteer of 22 years old) was adjusted to 8.0 with 0.1 mol/L NaOH or HCl solution. Secondly, 50 mg of as-prepared Fe3O4@ APBA NPs were added in 50 mL of the above urine sample and vibrated vigorously for 7 min. The procedure was repeated three times. Then the supernatant was used as the blank urine sample. The spiked urine samples containing E, NE, and DA of 0.01, 0.05, 0.1, 0.5, 1, and 2 μg/mL, were prepared by adding a certain amount of CAs mixed standard solution to the blank urine sample, respectively, and then the pH was adjusted to 8.0 with 0.1 mol/L NaOH or HCl solution. The subsequent steps, including the extraction and the elution of CAs, were the same as those used in the extraction procedures of the mixed standard solution. All urine samples were stored at 4°C and tested within 48 h except for the inter-day reproducibility test, for which the spiked urine samples were stored at –18°C before testing.

2.3 Synthesis of Fe3O4@ APBA NPs The ferromagnetic particles were prepared according to a modified chemical coprecipitation method [34]. Briefly, 5 g of dextran, 2.3 g of FeSO47H2O, and 5.8 g of Fe(NO3)3∙9H2O were dissolved in 100 mL of deionized water under nitrogen atmosphere. The resulting solution was transferred into a three-necked round-bottomed flask, stirred for 10 min in a 60°C water bath under nitrogen atmosphere. Afterwards, 10 mL of 25% NH3H2O was injected quickly. A black solution was resulted immediately and the stirring was This article is protected by copyright. All rights reserved.

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continued for 1 h to form dextran-functionalized magnetic particles (Fe3O4@DM NPs). The as-prepared Fe3O4@DM NPs were separated with a magnet and washed adequately to be neutral with deionized water for further surface functionalization. To obtain oxidized dextran functionalized magnetic particles, the above Fe3O4@DM NPs were dispersed in 100 mL of acetate buffer (pH=4.6) containing 0.21 g sodium periodate, and then the mixture was vigorously stirred at 4°C for 3 h in darkness. The particles after reaction were washed 5 times with deionized water. Thereafter, these products were dispersed in 100 mL of phosphate buffer (pH=7.5) with stirring at room temperature. 0.5 g of APBA and 0.2 g of NaBH3CN were added to the above dispersion under stirring. The dispersion was continuously stirred for 5 h under ambient temperature. Finally, the as-prepared Fe3O4@APBA NPs were washed with deionized water and ethanol several times, and vacuum-dried at 60°C for 24 h.

2.4 Characterization of the nanoparticles The morphology of the particles was determined with a transmission electron microscope (TEM, JEOL-1230, Kyoto, Japan). FTIR spectroscopy (FTIR, Bruker, Vecter 22, Germany) was used to detect functional groups in magnetic particles. Powder X-ray diffraction (XRD, Bruker D8 Advance, Germany) was carried out to investigate the internal array of the composite. Magnetization curves were determined with a vibrating sample magnetometer (VSM; Lake Shore, 7410, USA).

2.5 Extraction procedures Five milligrams of Fe3O4@APBA NPs were put into a 5 mL vial. 1.0 mL 5 μg/mL of CAs feed solution (pH=8.0) was added into the vial and the as-prepared mixture was vibrated vigorously for 7 min at 25°C. Then, the Fe3O4@APBA NPs were collected by magnetic separation. The supernatant was kept for HPLC analysis. The magnetic particles were rinsed by 1.0 mL deionized water. Thereafter, 1.0 mL of eluent (acidic solution prepared in methanol) This article is protected by copyright. All rights reserved.

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was introduced to the vial and vortexed for 7 min to desorb the analytes. Finally, the supernatant was collected and concentrated to 30 μL by a mild nitrogen stream at room temperature for HPLC–ECD analysis. The recycle of Fe3O4@APBA NPs was investigated by repeating the extraction procedures for five times.

2.6 HPLC–ECD analysis The

analysis

of

CAs

was

performed

by

HPLC–ECD.

The

self-fabricated

wall-jet/thin-layer ECD system electrochemical detection cell was made in our laboratory [35]. The HPLC separation was completed on a Spherigel ODS-C18 column (250 mm4.6 mm, i.d., 5 μm) at the column temperature of 30°C. Isocratic elution employed with the mobile phase of (A) a mixture aqueous solution of 45 mmol/L citric acid, 50 mmol/L sodium acetate, and 0.23 mmol/L octanesulfonic acid sodium and (B) methanol (A/B=92:8, v/v). The flow rate was 1.0 mL/min. The injection volume was 10 μL. The samples were filtered through a 0.45 μm mixed cellulose ester membrane before injection. Electrochemical detection was performed with a CHI760E electrochemical system (CH Instrument, USA). The three-electrode system consisted of a glassy carbon electrode as the working electrode, a saturated calomel electrode as the reference electrode and a carbon electrode as the counter electrode. The detection potential was +0.70 V.

2.7 Related calculation formulas The extraction efficiency and the elution efficiency are defined as follows: Extraction efficiency = (nf,ini –nf,fin) / nf,ini (1) Elution efficiency = ne / (nf,ini –nf,fin) (2) where nf, ini , nf, fin and ne are the initial molar quantity of the analyte in the feed solution before extraction, the final molar quantity of the analyte in the feed solution after extraction and in the eluent, respectively. This article is protected by copyright. All rights reserved.

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The enrichment factor was calculated according to the following equation: Enrichment factor = ce / cf,ini (3) where ce and cf, ini represent the concentration of the analyte in the eluent and the initial concentration of the analyte in the feed solution, respectively. The method relative recovery was calculated according to the following formula: Relative recovery = cf,det / cf,ini (4) where cf, det is the determined concentration of the analyte in the feed solution with the present method.

3 Results and discussion 3.1 Characterization of materials FTIR, TEM, XRD and VSM studies were employed to characterize the synthesized Fe3O4@APBA NPs. In the FTIR spectra, the strong adsorption peak at 550 cm-1 was observed, which can be ascribed to the Fe–O vibration. The presence of APBA in the magnetic particles is proven by the peak at 1639 cm-1. The peak at 1557 cm-1 was assigned to the aromatic rings and peak at 1340 cm-1 was ascribed to the characteristic C–B stretches. These results suggest that APBA is successfully immobilized on the surface of Fe3O4 particles via both physical and chemical adsorption. The typical TEM image of Fe3O4@APBA NPs (Supporting Information Fig. S2) showed that the magnetic nanoparticles were nearly monodisperse and sphere-like, with an average diameter of about 8 nm. The crystal phases of Fe3O4@DM NPs and Fe3O4@APBA NPs were contrastively analyzed by XRD (Supporting Information Fig. S3). In the 2θ range of 10–90°, six characteristic peaks (2θ=30.2, 35.5, 43.4, 54.2, 57.4, 63.1°) were observed for two samples, which matched well with those from the JCPDS card (no.: 75–1610) for magnetite, revealing This article is protected by copyright. All rights reserved.

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that both of Fe3O4@DM NPs and Fe3O4@APBA NPs particles are well crystallized in a cubic system [36]. To characterize the magnetic properties, a vibrating sample magnetometer was used to record hysteresis loops of the as-prepared Fe3O4@DM NPs and Fe3O4@APBA NPs at 300 K (Supporting Information Fig. S4). The magnetization curves indicated that these magnetic particles were of super paramagnetism as well as no hysteresis in low fields. The corresponding saturation magnetizations were 64.6 and 47.5 emu/g for Fe3O4@DM NPs and Fe3O4@APBA NPs, respectively. The results demonstrate that the saturation magnetization decreased evidently due to the modification of APBA. However, the saturation magnetization is high enough for the magnetic particles separated rapidly and efficiently from matrix solutions by an external magnet.

3.2 Investigation of the extraction conditions To investigate the applicability of the as-synthesized Fe3O4@APBA NPs for the determination of CAs in urine, extraction conditions including the pH of the feed solution, the extraction time, the composition of the buffer solution, the amount of the magnetic particles, the elution conditions and the recycle of Fe3O4@APBA NPs were explored carefully to achieve the optimal extraction efficiency for CAs.

3.2.1 pH of the feed solution The pH of the feed solution is a significant factor affecting the extraction of analytes because the existence forms of the analytes and the functional groups modified on the magnetic particles are different under different pH conditions [37]. The acidity affects the reaction mechanism between the analyte and the magnetic particle. The effect of the pH of the feed solution on the extraction efficiency at room temperature (shown in Fig. 1A) indicates that the analytes are hardly adsorbed by the magnetic particles under higher acidity (pH

Determination of catecholamines in urine using aminophenylboronic acid functionalized magnetic nanoparticles extraction followed by high-performance liquid chromatography and electrochemical detection.

A new method was developed for the simultaneous determination of three catecholamines in urine using aminophenylboronic acid functionalized magnetic n...
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