Selective Separation and Analysis of Catecholamines in Urine Based on Magnetic Solid Phase Extraction by Mercaptophenylboronic Acid Functionalized Fe 3 O 4 -NH 2 @Au Magnetic Nanoparticles Coupled with HPLC on by

: A novel magnetic solid phase extraction based on mercaptophenylboronic acid (MPBA)-functionalized Fe 3 O 4 -NH 2 @Au nanomaterial (Fe 3 O 4 -NH 2 @Au-MPBA) was developed for selective separation and enrichment of catecholamines (including dopamine, norepinephrine and adrenaline). Fe 3 O 4 -NH 2 @Au-MPBA nanoparticles were achieved by self-assembly-anchoring MPBA molecules on the surface of Fe 3 O 4 -NH 2 @Au nanocomposites, which were synthesized via a facial ultrasonic auxiliary in situ reduction process. The interaction between cis-diol from catecholamines and boronic acid was reversible and could be ﬂexibly controlled by adjusting pH value. The catecholamines could be quickly adsorbed by Fe 3 O 4 -NH 2 @Au-MPBA in weak alkaline solution (pH 8.0–9.0) and subsequently released in acid solution (pH 1.0–2.0). The process of adsorption and dissociation was very fast. Furthermore, the three catecholamines could be detected in urine from children by high performance liquid chromatography (HPLC) with electrochemical detector. Under optimal conditions, norepinephrine (NE), epinephrine (EP) and dopamine (DA) were separated very well from internal standard and exhibited a good linearity in the range of 2.5–500.0 ng mL − 1 , with correlation coefﬁcients of r 2 > 0.9907. Limits of detection (LOD) (signal to noise = 3) were 0.39, 0.27 and 0.60 ng mL − 1 for NE, EP and DA, respectively. Recoveries for the spiked catecholamines were in the range of 85.4–105.2% with the relative standard deviation (RSD) < 11.5%.


Introduction
Catecholamines (CAs), which include norepinephrine (NE), epinephrine (EP) and dopamine (DA), are important neurotransmitters and hormones released by the adrenal glands and sympathetic nervous system [1]. Currently, it is known that catecholamines are involved in some of the most prevalent human pathologies, such as neurological disorders such as Parkinson's [2], pheochromocytoma [3] and schizophrenia [4]. Hence, monitoring the concentration of catecholamines in biological fluids including blood, urine and specific tissue has attracted considerable interest in diagnostic analysis and biological systems [5,6].
In spite of the great diversity of analytical approaches that have been developed in recent years, the detection of catecholamines in biological samples remains a hot spot in analytical fields [7,8]. High-performance liquid chromatography (HPLC) is an analytical method routinely used for the separation and quantification of catecholamines in clinical laboratories [9,10], usually coupled with electrochemical [11], fluorescence [12] or mass spectrometry detection [13]. Among the detectors, electrochemical detectors [14,15] attract more preference due to good selectivity, highly sensitive characteristics, a lack of need for derivatization and low detection cost. However, matrix effects, extremely low concentrations and chemical instability of catecholamines in biological samples are major difficulties encountered in their analysis.
To solve these problems, optimization of the sample pretreatment process plays an important role in the enrichment and separation of targets. The common sample preparation methods for extracting catecholamines in chromatographic analysis involves liquidliquid extraction (LLE) [16] and solid phase extraction (SPE) [17,18]. Nevertheless, LLE is labor-intensive and time-consuming [19] and encounters low selectivity and extraction yields. SPE is widely adopted for the extraction and concentration of catecholamines, possibly due to the high extraction recoveries and selectivity [20].
Magnetic solid phase extraction (MSPE) is a flexible solid phase extraction technology. The magnetic adsorbents can be recycled and reused easily, which is cost-effective and environmentally friendly. Fe 3 O 4 magnetic nanoparticles (NPs), commonly used magnetic cores, are easily oxidized in air and have a tendency to aggregate [21,22]. To overcome these problems, different materials such as metals [23], metal oxides [24], mesoporous [25], polymers [26], graphene [27] and dendrimers [28] have been developed to hybridize with Fe 3 O 4 NPs. In recent years, gold-coated Fe 3 O 4 (Fe 3 O 4 @Au) magnetic NPs have drawn intense scientific and technological interest for potential applications in disease diagnosis and therapy [29], biological detection [30], catalytic application and separation science [31].
In recent years, phenylboronic acid (PBA) and its derivatives containing boronic acid functional groups have been employed for selective capture and enrichment of cisdiol molecules (carbohydrates [32], mucin [33], nucleosides and glycoconjugates) from biological samples. Owing to the ability to form a five-or six-membered boronate ester [34] via reversible covalent interactions with the position at cis-1, 2-or 1, 3-diol, they exhibit a high selectivity for targets. The formation and dissociation of boronic esters are controlled by appropriately adjusting the pH value. 4-mercaptophenylboronic acid (4-MPBA) is a thiol-containing boronic acid compound which has been used to modify gold NPs for the selective interactions with cis-diol structure on sugars [35], sialic acid, protein kinase and alpha-fetoprotein [36][37][38][39]. However, to the best of our knowledge, we have not seen this idea used for the selective separation of catecholamines.
In this report, a novel magnetic nanocomposite, 4-MPBA-functionalized Fe 3 O 4 -NH 2 @Au (Fe 3 O 4 -NH 2 @Au-MPBA), was prepared and used for the pretreatment of catecholamines in urine and followed by HPLC-electrochemical detection. The cis-diol structures of catecholamines were able to form ring boronate ester structures with boronic acid groups of Fe 3 O 4 -NH 2 @Au-MPBA nanocomposite by covalent bond interaction. The morphologies and magnetization saturation values of the prepared magnetic material were explored by scanning electron microscope (SEM) and vibrating sample magnetometer (VSM). The analytical performances of the method were evaluated before determining catecholamines in urine samples.

Reagents and Chemicals
Dopamine (DA) and norepinephrine (NE) were purchased from Yuanye biotechnology Co., Ltd. (Shanghai, China). Epinephrine (EP) was bought from Chromadex (Irvine, CA, USA). 3,4-dihydroxybenzylamine hydrobromide (DHBA) acting as internal standard (IS) was purchased from Aladdin Reagent (Shanghai, China). 4-mercaptophenylboronic acid (MPBA) was bought from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Molecular structures of these compounds are shown in Figure 1.  Stock solutions (1 mg mL −1 ) of NE, DA and IS were prepared by dissolving 5 mg of standard substance in 5 mL of deionized water. Stock solution (1 mg mL −1 ) of EP was prepared by dissolving 5 mg of standard substance in 100 μL 0.01 mol L −1 HCl solution before diluting to 5 mL by deionized water. All solutions were stored at −20 °C until use.
The artificial urine reported in the reference [40] was used in recovery tests. The artificial urine consisted of 19.4 mg mL −1 of urea, 8.0 mg mL −1 of NaCl, 1.1 mg mL −1 of magnesium sulfate and 0.6 mg mL −1 of CaCl2, and the solution was adjusted to pH 8.0 with 1 mol L −1 NaOH. Spiked artificial urine solution was prepared containing CAs (NE, EP and DA) in the concentrations of 10, 50 and 200 ng mL −1 , and then 2 mL of the spiked solution was mixed with 100 μL of IS (1000 ng mL −1 ).

Instruments
A pH meter (Shanghai, China) with a composite electrode was used for the pH values. Special indicator papers indicating pH 6.9-8.4 were bought from SSS Reagent Company (Shanghai, China). An analytical balance (Shanghai, China) was used for weight measurement. A Hitachi S-3400N scanning electron microscope (SEM, Hitachi, Tokyo, Japan) was used at an acceleration voltage of 20.0 kV. Magnetic measurement was carried out using a 7407 vibrating sample magnetometer (Lakeshore, Columbus, OH, USA) at room temperature.

Chromatography Conditions
A Thermo Scientific UltiMate 3000 ECD UHPLC system based on a glass carbon electrode detector (Thermo Fisher Scientific, Waltham, MA, USA) equipped with a Kromasil C18 column (5 μm, 250 mm × 4.6 mm) was used. The mobile phase was isocratic elution and consisted of acetonitrile and 0.7% NaH2PO4 in water (5.5:94.5, v/v) with addition of citric acid (35 mmol L −1 ), EDTA (0.25 mmol L −1 ) and 1-heptane sulfonic acid sodium salt (2 mmol L −1 ). The pH of the mobile phase was adjusted to pH 4.0 by adding saturated NaOH solution. The run was performed at 1.0 mL min −1 with the column oven temperature at 30 °C . The injection volume was 20 μL and the working potential was 700 mV.

Preparation of Phenylboronic Acid
The Fe3O4 NPs were prepared through reducing the solution of Fe (II) and Fe (III) chlorides of the molar ratio 1:2 with 30% ammonia solution. Appropriate Fe3O4 NPs were modified by APTES through stirring in 10% (v/v) APTES solution in absolute ethyl alcohol for 12 h. The obtained Fe3O4 NPs modified by -NH2 groups (Fe3O4-NH2) were magnetically separated, washed with ethanol and dispersed in ethanol.
Next, 500 mg Fe3O4-NH2 NPs, 50 mL of 2% HAuCl4 and 300 mL distilled water were mixed for over 10 min by sonication, and then 50 mmol L −1 KBH4 solution was dropped into the above mixture under ultrasound until the color changed to purple. Another 5 min Stock solutions (1 mg mL −1 ) of NE, DA and IS were prepared by dissolving 5 mg of standard substance in 5 mL of deionized water. Stock solution (1 mg mL −1 ) of EP was prepared by dissolving 5 mg of standard substance in 100 µL 0.01 mol L −1 HCl solution before diluting to 5 mL by deionized water. All solutions were stored at −20 • C until use.
The artificial urine reported in the reference [40] was used in recovery tests. The artificial urine consisted of 19.4 mg mL −1 of urea, 8.0 mg mL −1 of NaCl, 1.1 mg mL −1 of magnesium sulfate and 0.6 mg mL −1 of CaCl 2 , and the solution was adjusted to pH 8.0 with 1 mol L −1 NaOH. Spiked artificial urine solution was prepared containing CAs (NE, EP and DA) in the concentrations of 10, 50 and 200 ng mL −1 , and then 2 mL of the spiked solution was mixed with 100 µL of IS (1000 ng mL −1 ).

Instruments
A pH meter (Shanghai, China) with a composite electrode was used for the pH values. Special indicator papers indicating pH 6.9-8.4 were bought from SSS Reagent Company (Shanghai, China). An analytical balance (Shanghai, China) was used for weight measurement. A Hitachi S-3400N scanning electron microscope (SEM, Hitachi, Tokyo, Japan) was used at an acceleration voltage of 20.0 kV. Magnetic measurement was carried out using a 7407 vibrating sample magnetometer (Lakeshore, Columbus, OH, USA) at room temperature.

Chromatography Conditions
A Thermo Scientific UltiMate 3000 ECD UHPLC system based on a glass carbon electrode detector (Thermo Fisher Scientific, Waltham, MA, USA) equipped with a Kromasil C 18 column (5 µm, 250 mm × 4.6 mm) was used. The mobile phase was isocratic elution and consisted of acetonitrile and 0.7% NaH 2 PO 4 in water (5.5:94.5, v/v) with addition of citric acid (35 mmol L −1 ), EDTA (0.25 mmol L −1 ) and 1-heptane sulfonic acid sodium salt (2 mmol L −1 ). The pH of the mobile phase was adjusted to pH 4.0 by adding saturated NaOH solution. The run was performed at 1.0 mL min −1 with the column oven temperature at 30 • C. The injection volume was 20 µL and the working potential was 700 mV.

Preparation of Phenylboronic Acid-Functionalized Fe 3 O 4 -NH 2 @Au (Fe 3 O 4 -NH 2 @Au-MPBA)
The Fe 3 O 4 NPs were prepared through reducing the solution of Fe (II) and Fe (III) chlorides of the molar ratio 1:2 with 30% ammonia solution. Appropriate Fe 3 O 4 NPs were modified by APTES through stirring in 10% (v/v) APTES solution in absolute ethyl alcohol for 12 h. The obtained Fe 3 O 4 NPs modified by -NH 2 groups (Fe 3 O 4 -NH 2 ) were magnetically separated, washed with ethanol and dispersed in ethanol.
Next, 500 mg Fe 3 O 4 -NH 2 NPs, 50 mL of 2% HAuCl 4 and 300 mL distilled water were mixed for over 10 min by sonication, and then 50 mmol L −1 KBH 4 solution was dropped into the above mixture under ultrasound until the color changed to purple. Another 5 min of sonication was performed. The Fe 3 O 4 -NH 2 @Au NPs were obtained by magnetic separation.
An amount of 500 mg Fe 3 O 4 -NH 2 @Au NPs was added to 100 mL 1 mg mL −1 of MPBA solution under stirring to complete the covalent binding between the Au shell and sulfydryl (-SH). Fe 3 O 4 -NH 2 @Au-MPBA composites were obtained, then separated and washed with distilled water based on external magnetic field. After that, the magnetic nanocomposites were dried in a vacuum oven at 40 • C for 24 h.

Sample Collection
To investigate the suitability of the proposed method, a pilot study was conducted by analyzing urinary samples from healthy volunteers. The urine samples from 6 healthy children aged 4-6 years (3 boys and 3 girls). Their morning urine samples were collected into sterile urine cups. They were then immediately transferred to polypropylene tubes and stored in a -20 • C refrigerator.
The study was carried out according to the principles of the Declaration of Helsinki (World Medical Association 2008). Written informed consent was obtained from the legal guardians of the volunteers. This study was approved by the Ethics Committee of Zhongda Hospital Affiliated to Southeast University.

Sample Pretreatment
When analyzing samples, the stored 2.5 mL urine samples were thawed by incubation at 37 • C for 5 min and centrifuged for 5 min at 10,000 rpm. Then, 2 mL of supernatant, 100 µL of 1 µg mL −1 DHBA and 5 mg Fe 3 O 4 -NH 2 @Au-MPBA NPs were added into a glass spawn bottle, and the pH was adjusted to 8.0 by adding moderate amounts of 0.01 mol L −1 NaOH. After that, the mixture was stirred for about 10 min at room temperature, and the supernatant was removed by external magnetic field. An amount of 200 µL of 0.5% H 3 PO 4 solution (pH = 2.0) was added for desorbing the analytes. 20 µL of eluent was injected into HPLC for analysis. The process schematic for the preparation of the magnetic Fe 3 O 4 -NH 2 @Au-MPBA NPs and the pretreatment of samples are shown in Figure 2.
An amount of 500 mg Fe3O4-NH2@Au NPs was added to 100 mL 1 mg mL −1 of MPBA solution under stirring to complete the covalent binding between the Au shell and sulfydryl (-SH). Fe3O4-NH2@Au-MPBA composites were obtained, then separated and washed with distilled water based on external magnetic field. After that, the magnetic nanocomposites were dried in a vacuum oven at 40 °C for 24 h.

Sample Collection
To investigate the suitability of the proposed method, a pilot study was conducted by analyzing urinary samples from healthy volunteers. The urine samples from 6 healthy children aged 4-6 years (3 boys and 3 girls). Their morning urine samples were collected into sterile urine cups. They were then immediately transferred to polypropylene tubes and stored in a -20 °C refrigerator.
The study was carried out according to the principles of the Declaration of Helsinki (World Medical Association 2008). Written informed consent was obtained from the legal guardians of the volunteers. This study was approved by the Ethics Committee of Zhongda Hospital Affiliated to Southeast University.

Sample Pretreatment
When analyzing samples, the stored 2.5 mL urine samples were thawed by incubation at 37 °C for 5 min and centrifuged for 5 min at 10,000 rpm. Then, 2 mL of supernatant, 100 μL of 1μg mL −1 DHBA and 5 mg Fe3O4-NH2@Au-MPBA NPs were added into a glass spawn bottle, and the pH was adjusted to 8.0 by adding moderate amounts of 0.01 mol L −1 NaOH. After that, the mixture was stirred for about 10 min at room temperature, and the supernatant was removed by external magnetic field. An amount of 200 μL of 0.5% H3PO4 solution (pH = 2.0) was added for desorbing the analytes. 20 μL of eluent was injected into HPLC for analysis. The process schematic for the preparation of the magnetic Fe3O4-NH2@Au-MPBA NPs and the pretreatment of samples are shown in Figure 2.

Characterization
The morphologies of Fe 3 O 4 -NH 2 @Au NPs were examined by SEM at 20.0 kV (8.2 mm, 40.0k SE) ( Figure 3A). The size distribution of Fe 3 O 4 -NH 2 @Au NPs is shown in Figure 3B, where we can see that the size of Fe 3 O 4 -NH 2 @Au NPs was uniform, and the average particle size was about 150 nm. The energy-dispersive spectroscopy (EDS) results showed the existence of Fe, C, Si and O elements in Fe 3 O 4 -NH 2 samples ( Figure 3C). Moreover, the Au elements could be found in Fe 3 O 4 -NH 2 @Au material ( Figure 3D), which indicated that Fe 3 O 4 -NH 2 @Au NPs were successfully prepared. The morphologies of Fe3O4-NH2@Au NPs were examined by SEM at 20.0 kV (8.2 mm, 40.0k SE) ( Figure 3A). The size distribution of Fe3O4-NH2@Au NPs is shown in Figure 3B, where we can see that the size of Fe3O4-NH2@Au NPs was uniform, and the average particle size was about 150 nm. The energy-dispersive spectroscopy (EDS) results showed the existence of Fe, C, Si and O elements in Fe3O4-NH2 samples ( Figure 3C). Moreover, the Au elements could be found in Fe3O4-NH2@Au material ( Figure 3D), which indicated that Fe3O4-NH2@Au NPs were successfully prepared. The profile of magnetization loops in Figure 4 revealed that the Fe3O4-NH2 and Fe3O4-NH2@Au NPs are superparamagnetic [29]. The magnetization saturation (Ms) values of Fe3O4-NH2 and Fe3O4-NH2@Au NPs were determined as 32.1 and 22.0 emu/g, respectively. The Fe3O4-NH2@Au NPs showed slightly lower Ms values than that of Fe3O4-NH2 due to the surface conjugation of Au NPs on Fe3O4-NH2.
In the inset, the two bottles are both Fe3O4-NH2@Au NPs suspension solution. The composite NPs were able to be magnetically separated by an external magnetic field, which indicated that the enrichment and separation of Fe3O4-NH2@Au NPs during the extraction process can be regulated by an external magnetic field.

Optimizing the Mass Ratio of Au 3+ and Fe3O4-NH2
The effect of the mass ratios of Fe3O4-NH2 and Au 3+ on magnetic separation time was studied. As shown in Figure 5A, when the mass ratio of Fe3O4-NH2 and Au 3+ was 3:1, the magnetic separation time was less than 2 minutes; however, too little Au 3+ may reduce the functionalized molecule numbers of MPBA, which would reduce the sites of interaction for targets. With the increase in Au 3+ , the magnetic separation time was gradually prolonged. However, when the mass ratio was 1:2, the gold nanoparticles were not completely wrapped on the surface of Fe3O4-NH2 NPs, which was very wasteful for Au 3+ . When the mass ratio was 1:1.5, the magnetic separation time was 15 minutes, which was too long for rapid pretreatment for samples. Therefore, the mass ratio of 1:1 between In the inset, the two bottles are both Fe 3 O 4 -NH 2 @Au NPs suspension solution. The composite NPs were able to be magnetically separated by an external magnetic field, which indicated that the enrichment and separation of Fe 3 O 4 -NH 2 @Au NPs during the extraction process can be regulated by an external magnetic field.

Optimizing the Mass Ratio of Au 3+ and Fe 3 O 4 -NH 2
The effect of the mass ratios of Fe 3 O 4 -NH 2 and Au 3+ on magnetic separation time was studied. As shown in Figure 5A, when the mass ratio of Fe 3 O 4 -NH 2 and Au 3+ was 3:1, the magnetic separation time was less than 2 minutes; however, too little Au 3+ may reduce the functionalized molecule numbers of MPBA, which would reduce the sites of interaction for targets. With the increase in Au 3+ , the magnetic separation time was gradually prolonged. However, when the mass ratio was 1:2, the gold nanoparticles were not completely wrapped on the surface of Fe 3 O 4 -NH 2 NPs, which was very wasteful for Au 3+ . When the mass ratio was 1:1.5, the magnetic separation time was 15 minutes, which was too long for rapid pretreatment for samples. Therefore, the mass ratio of 1:1 between Fe 3 O 4 -NH 2 NPs and Au 3+ was chosen.

The pH Value of Desorption Solvent
The pH value of the eluent has an important effect on the formation of boronate ester, because the reversible covalent interactions could be adjusted by pH between the positions of the cis-diol moieties of the targets and the hydroxyl groups of the boronic acid ligand. Acidic environments can break the ester bond and make the target dissociate from Fe3O4-NH2@Au-MPBA NPs. The pH value (1.0-7.0) was investigated for the eluent. With the decrease in pH value of the eluent, the extraction recovery of the targets increased gradually. When the pH value was 2.0, the extraction recovery of the CAs reached the maximum. As the pH value continued to decrease, the recoveries almost remained unchanged. Therefore, pH 2.0 was chosen as the acidity value for the eluent ( Figure 5D).

Exploring the Elution Time
In order to obtain the best extraction performance, different elution time from 2 to 30 min was optimized. Figure 5E demonstrated the relationship between the elution time and the recoveries. When elution time was shorter than 10 min, the extraction recovery increased with the extraction time increasing. When elution time was longer than 10 min, the recovery of CAs was almost no longer changed. Therefore, 10 min was chosen as the final elution time.

Method Evaluation
To test the linearity, we analyzed the mixed solutions of NE, EP and DA at the concentrations of 2.5, 5.0, 10.0, 20.0, 100.0 and 500.0 ng/mL for each target, which were prepared using artificial urine. Quantification was worked out using internal standard (IS) method. The calibration equation was calculated by the HPLC-ECD peak areas ratio and

Optimizing the pH Value of the Adsorption Solution
The pH value of adsorption solution was a critical factor for boronate affinity. Most boronic acids are generally weak acids, having a pKa values of 8.0-9.0. With pH less than the pKa value, most of them still exist in trigonal form [-B(OH) 2 ], which cannot react with cis-diol groups [41]. For phenylboronic acids, when the pH is greater than the pKa value of the phenylboronic acid ligand, the phenylboronic acid group is transformed to tetrahedral anionic form [-B(OH) 4 − ] under alkaline conditions [35]. Subsequently, [-B(OH) 4 − ] bonds with cis-1,2-diol units to form a stable 5-membered cyclic ester. This is in good agreement with the results obtained from MPBA functionalized Fe 3 O 4 -NH 2 @Au. In this work, we studied the effect of adsorption pH value (5.0-9.0) on adsorption performance, and the results are shown in Figure 5B. As described above, the phenylboronic acids were the sole functional group on Fe 3 O 4 -NH 2 @Au nanoparticles, and thus the adsorption rates of adsorbents for the three catecholamines were pH-dependent. The adsorption rates improved when the pH value increased from 5.0 to 8.0 and then remained almost unchanged. Thus, pH 8.0 was selected for adsorption.

Optimizing Reaction Time of MPBA and Fe 3 O 4 -NH 2 @Au
The interaction time between MPBA and Fe 3 O 4 -NH 2 @Au NPs is very important, as it could impact the adsorption performance of the magnetic NPs for catecholamines. The relationships between reaction time (2-7 h) and extraction efficiency were explored. As shown in Figure 5C, when reaction time was more than 3h, the extraction efficiency remained nearly unchanged. Thus, 3 h was chosen as the reaction time of MPBA and Fe 3 O 4 -NH 2 @Au NPs.

The pH Value of Desorption Solvent
The pH value of the eluent has an important effect on the formation of boronate ester, because the reversible covalent interactions could be adjusted by pH between the positions of the cis-diol moieties of the targets and the hydroxyl groups of the boronic acid ligand. Acidic environments can break the ester bond and make the target dissociate from Fe 3 O 4 -NH 2 @Au-MPBA NPs. The pH value (1.0-7.0) was investigated for the eluent. With the decrease in pH value of the eluent, the extraction recovery of the targets increased gradually. When the pH value was 2.0, the extraction recovery of the CAs reached the maximum. As the pH value continued to decrease, the recoveries almost remained unchanged. Therefore, pH 2.0 was chosen as the acidity value for the eluent ( Figure 5D).

Exploring the Elution Time
In order to obtain the best extraction performance, different elution time from 2 to 30 min was optimized. Figure 5E demonstrated the relationship between the elution time and the recoveries. When elution time was shorter than 10 min, the extraction recovery increased with the extraction time increasing. When elution time was longer than 10 min, the recovery of CAs was almost no longer changed. Therefore, 10 min was chosen as the final elution time.

Method Evaluation
To test the linearity, we analyzed the mixed solutions of NE, EP and DA at the concentrations of 2.5, 5.0, 10.0, 20.0, 100.0 and 500.0 ng/mL for each target, which were prepared using artificial urine. Quantification was worked out using internal standard (IS) method. The calibration equation was calculated by the HPLC-ECD peak areas ratio and the concentration ratios of analytes and IS. The analysis performances of the established method for the detection of catecholamines are summarized in Table 1. The intra-day and inter-day precision and accuracy were evaluated for artificial urine samples spiked with catecholamines at the concentrations of 10, 50 and 200 ng mL −1 based on the above pretreatment method. The limits of detection (LOD), defined as the signal-tonoise ratio of 3:1, and limits of quantification (LOQ), defined as a signal-to-noise ratio of 10:1, were calculated; the results are listed in Table 1. Results showed that good linearity of the targets was achieved in the range of 2.5-500.0 ng mL −1 with the correlation coefficients (r 2 ) of 0.9911, 0.9935 and 0.9907 for NE, EP and DA, respectively. The LOQ were 1.3, 0.9 and 2.0 ng mL −1 (S/N = 10), and LOD (S/N = 3) were 0.39, 0.27 and 0.60 ng mL −1 for NE, EP and DA, respectively. The RSDs (relative standard deviations) for the catecholamines were from 6.1% to 10.7% for intra-day determination (n = 5) and from 7.4% to 11.5% for inter-day determination (n = 5).
The comparison of recoveries obtained by the present method and a reference method [42] has been added in Table S1 in the Supplementary. The results showed that the prepared method has good extraction recoveries for catecholamines.

Reproducibility and Stability
The relative standard deviation (RSD, %) of adsorption quantities was 8.1% for six batches of Fe 3 O 4 -NH 2 @Au-MPBA NPs, and 7.3% for one batch used 10 times. The adsorption capacities of Fe 3 O 4 -NH 2 @Au-MPBA NPs for catecholamines decreased by approximately 7.5% after one month. The data above showed that the prepared magnetic composite nanoparticles were a reliable and stable adsorbent for catecholamines.

Application of the Method to Real Samples
To evaluate the application of the prepared magnetic Fe 3 O 4 -NH 2 @Au-MPBA NPs, urine samples were analyzed using internal standard method. As shown in Figure 6, A was the liquid chromatogram of CAs at 20 ng mL −1 in artificial urine with the extraction by magnetic NPs. The liquid chromatogram of a real urine sample ( Figure 6B) without the pretreatment by Fe 3 O 4 -NH 2 @Au-MPBA NPs exhibited many impurity peaks. Figure 6C was the liquid chromatogram of a real urine sample with the pretreatment by Fe 3 O 4 -NH 2 @Au-MPBA NPs, where we can find fewer impurity peaks compared with Figure 6B. The results showed that Fe 3 O 4 -NH 2 @Au-MPBA NPs have a highly selective adsorption for catecholamines. First morning urinations, free of interference of medications and foods, were collected. Free catecholamines were measured in the morning urine samples from six healthy children using our developed method. The concentrations of CAs (NE, EP and DA) of the samples detected by the prepared method are shown in Table 2. The results showed that Fe3O4-NH2@Au-MPBA NPs coupled with HPLC-ECD could be successfully used for the detection of CAs.  First morning urinations, free of interference of medications and foods, were collected. Free catecholamines were measured in the morning urine samples from six healthy children using our developed method. The concentrations of CAs (NE, EP and DA) of the samples detected by the prepared method are shown in Table 2. The results showed that Fe 3 O 4 -NH 2 @Au-MPBA NPs coupled with HPLC-ECD could be successfully used for the detection of CAs.

Conclusions
Fe 3 O 4 -NH 2 @Au-MPBA magnetic nanocomposites were synthesized in a simple method first and employed as adsorbents for the selective extraction of NE, EP and DA based on external magnetic field. 4-MPBA was modified on the surface of Fe 3 O 4 -NH 2 @Au NPs through Au-S bond. The interaction between catecholamines and Fe 3 O 4 -NH 2 @Au-MPBA magnetic NPs is based on the covalent bond of borate ester. The adsorption and desorption of catecholamines could be easily achieved through flexibly adjusting the pH value of solution. The SPE based on magnetic nanocomposite coupled with HPLCelectrochemical detection for the separation and analysis of catecholamines in human urine exhibited good sensitivity and selectivity.
Supplementary Materials: The following are available online at https://www.mdpi.com/article/10 .3390/separations8110196/s1, Figure S1: The SEM graph of PS-PCE, Table S1: The comparison of the extraction recoveries for the mixed catecholamines standard in artificial urine at the concentration of 100 ng/mL extracted by the present method and a reference method.  Informed Consent Statement: Informed consent was obtained from all subjects and guardians involved in the study. Written informed consent has been obtained from the patient(s) and the guardians to publish this paper.

Data Availability Statement:
The data presented in this study is available in supplementary material.