Name: a convenient method for extraction and analysis with high-pressure liquid chromatography of catecholamine neurotransmitters and their metabolites
Uploaded: 2018-03-01T13:00:00
Description: Watch this Scientific Journal Video about A Convenient Method for Extraction and Analysis with High-Pressure Liquid Chromatography of Catecholamine Neurotransmitters and Their Metabolites at JoVE.com

This study was supported by the National Science Foundation of China (No.81172720, No. 81673230), the Social Development Research Program of Jiangsu Province Science and Technology department (No. BE2016741), Science &#38; Technology Project of China General Administration of Quality Supervision, Inspection and Quarantine (2015QK055), The Open Project Program of Key Laboratory of Child Development and Learning Science of Ministry of Education, Southeast University (CDLS-2016-04). We sincerely acknowledge Yuan Song and Ping Liu who assisted us in samples collection.

Informed consent from the parents was obtained, and institutional review board approval was obtained for the study. The study was performed in accordance with the code of Ethics of the World Medical Association (Declaration of Helsinki) for experiments involving humans. Caregivers of all participants provided written consent for being enrolled in the study. Ethical committee approval from Zhongda Hospital, affiliate with Southeast University, was also obtained.
1. Preparation of the Columns and ...

The proposed PFSPE method in this paper may be significant and meaningful with respect to its rapidity, simplicity, and convenience. The adsorbents used in the protocol are electrospun nanofibers, which have high surface area-to-volume ratios, and adsorb the analytes with high efficiency. The procedure only needs a few milligrams of nanofiber and a small volume of eluant solvent, and does not require an evaporation step to concentrate the analytes. Here, we have presented a detailed overview of an HPLC-ECD based protocol...

Catecholamine neurotransmitters and their metabolite contents in body fluids can affect neural function and affect the balance of response-to-stimulus states to a large extent1. Abnormities may cause a variety of diseases, such as pheochromacytoma, ganglioneuroma, neuroblastoma, and neurological disorders1,2. The extraction and determination of catecholamines in body fluids is meaningful to the diagnosis of the relevant diseases. However, catecholamines in biological samples exist in low concentrations and are easily oxidized. Furthermore, they are very difficult to elute because of the large amount of interference in the medium3. Thus, simultaneous detection of catecholamines in biological fluids is still a challenge.
There have been reviews showing that urinary catecholamines can be a measure of stress, and that their levels are important biological markers responding to tactile stimulation processing in newborns5. According to the research, all infants who have suffered from premature incidents are at risk for brain injury4,5,6, and injury may cause abnormal release of catecholamines and related matters into the fluids. There do exist advanced magnetic resonance techniques that can detect brain damage in earlier phases7,8. However, within the first 48 h, an abnormal neurodevelopmental process will cause permanent brain injury that won&#39;t be evident in medical images11. Besides, the high cost and scarce instrument resources, along with other factors, makes it impossible for all neonatal units to have access to these specialized neuro-imaging techniques. However, the use of an easily approachable and practical biomarker (such as catecholamines and their metabolites) could overcome these shortcomings, and the screening of a biomarker in human fluids may help in the early diagnosis of brain injury and lead to prompt identification of new-born infants needing neuroprotection9. The catecholamines in urine can be an easy and obvious index, because of the direct correlation between the amount of them released into fluids and neuroactivity function.
Among biological fluids, cerebrospinal fluid (CSF) and plasma samples are not easy to get via existing traumatic procedures, and it is also very difficult to get rid of interference due to adhesive protein and other impurities, leading to a troublesome and time-consuming sampling process that is unsuited for repeated detection. Also, for children, it is almost impossible to get the samples in a traumatic fashion. Therefore, urinary sampling is better than the other forms of sampling, as it is non-invasive, easy to operate, and can be repeatedly done. Urine samples are abundant and easy to store, and show great advantages over the other forms of biological samples.
The main methods to quantify catecholamines in biological fluids include radioenzymic assays10, enzyme-linked immune-sorbent assays11, voltammetry12 and thermal lens spectrometry13. But shortcomings exist, such as complicated operations and hard-to-detect multiple targets. Today, the dominant analysis technique is high-performance liquid chromatography (HPLC)14, coupled with sensitive electrochemical15 or fluorimetric detection16, because of its high sensitivity and good selectivity. With tandem mass spectrometry technology, such as liquid chromatography /mass spectrometry (LC/MS) and liquid chromatography/mass spectrometry/mass spectrometry (LC/MS/MS), the analysis and quantification of the neurotransmitters can achieve high accuracy and specificity17,18. However, the MS technique requires expensive instrumentation as well as substantially qualified manpower, making the method difficult to apply universally in most conventional laboratories. HPLC-ECD systems are commonly equipped in most conventional and clinical laboratories, and have thus become a common and good choice for research groups to use for chemical determination, but they require the sample introduced into the system to be clean and of microscale volume19. Thus, it is of great importance to purify and condense the sample prior to the analysis. The classical method for the purification step is liquid-liquid extraction14,15,20 and off-line solid-phase extraction, including activated alumina column21,22 and diphenylborate (DPBA) complexation23,24,25,26.
Myeongho Lee et al. have been using polymer resin chemically modified with crown ether as the adsorbent to selectively extract catecholamines from human urine since 200727. Also, in 2006, Haibo He et al. demonstrated a facile synthesis approach for boronate affinity extraction sorbent byutilizing a functionalizable nanomagnetic polyhedral oligomeric silsesquioxane (POSS) based nanomagnetic composite, and applying it to the enrichment of catecholamines in human urine (noradrenaline, epinephrine and isoprenaline)28. They also took advantage of the nanomaterials to fulfill the work, using a technology called nano-electrospinning and forming the polymer fibrous material in the nanoscale. The electrospinning process can adjust the diameter, morphology, and spatial alignment of the product by controlling the working voltage and changing the content of the spinning solution along with other parameters29. Compared with the conventional SPE cartridge, electrospun nanofibers are highly suitable to extract and enrich target analytes from a complex matrix, as they are equipped with high surface-area-to-volume ratios to adsorb the analytes with high efficiency, and exhibit more easily-controlled surface chemical properties, allowing handy attachment of the target compounds. These properties make them good choices for SPE adsorbents, greatly reducing the solid phase and desorption solvent amount30,31,32,33. For catecholamines in urine samples, electrospun nanofibers composed of apolymeric crown ether with polystyrene (PCE-PS) were used to selectively extract three catecholamines (NE, E, and DA)34. The paper indicated that the selective crown ether adsorbed the targets of NE, E, and DA, which was based on its correct geometry for binding catecholamines via forming hydrogen bonds. The results displayed the material crown ether effectively, removing other interfering compounds contained in biological samples. Inspired by this report, a novel method was developed for the selective extraction of the catecholamines by use of electrospun composite nanofibers composed of PCE-PS.
In this paper, the method reported previously34&#160; was improved and employed not only to successfully analyze E, NE, and DA, but also their metabolites, MHPG and DOPAC, in urine. We also explore new possibilities for the mechanism of the adsorption process. The method shows satisfying extraction efficiency and selectivity for the five analytes, and the method was verified in the analysis of urine from high-risk infants with perinatal brain damage and healthy controls.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>200 &#181;L pipette tip</td>
			<td></td>
			<td></td>
			<td>column to contain nanofibers</td>
		</tr>
		<tr>
			<td>PCE-PS nanofibers</td>
			<td></td>
			<td></td>
			<td>material for PFSPE extraction</td>
		</tr>
		<tr>
			<td>steel rod (about 0.5 mm diameter)</td>
			<td></td>
			<td></td>
			<td>fill the nanofibres into the column</td>
		</tr>
		<tr>
			<td>gastight plastic syringe (5 ml)</td>
			<td></td>
			<td></td>
			<td>compress solution into the end of the tip</td>
		</tr>
		<tr>
			<td>methanol</td>
			<td>Sinopharm Chemical ReagentCo., Ltd</td>
			<td>67-56-1</td>
			<td></td>
		</tr>
		<tr>
			<td>diphenylborinic acid 2-aminoethyl ester(DPBA)</td>
			<td>Sigma-Aldrich.Inc</td>
			<td>A-106408</td>
			<td>complex reagent</td>
		</tr>
		<tr>
			<td>norepinephrine(NE)</td>
			<td>Sigma-Aldrich.Inc</td>
			<td>A-9512</td>
			<td>analyte</td>
		</tr>
		<tr>
			<td>3-Methoxy-4-hydroxyphenylglycol(MHPG)</td>
			<td>Sigma-Aldrich.Inc</td>
			<td>H1377</td>
			<td>analyte</td>
		</tr>
		<tr>
			<td>epinephrine(E)</td>
			<td>Sigma-Aldrich.Inc</td>
			<td>100154-200503</td>
			<td>analyte</td>
		</tr>
		<tr>
			<td>3, 4-Dihydroxyphenylacetic acid(DOPAC)</td>
			<td>Sigma-Aldrich.Inc</td>
			<td>D-9128</td>
			<td>analyte</td>
		</tr>
		<tr>
			<td>dopamine(DA)</td>
			<td>Sigma-Aldrich.Inc</td>
			<td>H-8502</td>
			<td>analyte</td>
		</tr>
		<tr>
			<td>3, 4-dihydroxybenzylamine hydrobromide(DHBA)</td>
			<td>Sigma-Aldrich.Inc</td>
			<td>858781</td>
			<td>interior label</td>
		</tr>
		<tr>
			<td>acetonitrile</td>
			<td>Sigma-Aldrich.Inc</td>
			<td>75-05-8</td>
			<td>eluriant and mobile phase</td>
		</tr>
		<tr>
			<td>phosphoric acid</td>
			<td>Sinopharm Chemical ReagentCo., Ltd</td>
			<td>7664-38-2</td>
			<td>eluriant</td>
		</tr>
		<tr>
			<td>uric acid</td>
			<td>Sinopharm Chemical ReagentCo., Ltd</td>
			<td>69-93-2</td>
			<td>artifical urine</td>
		</tr>
		<tr>
			<td>creatinine</td>
			<td>Sinopharm Chemical ReagentCo., Ltd</td>
			<td>60-27-5</td>
			<td>artifical urine</td>
		</tr>
		<tr>
			<td>trisodium citrate</td>
			<td>Sinopharm Chemical ReagentCo., Ltd</td>
			<td>6132-04-3</td>
			<td>artifical urine</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Sinopharm Chemical ReagentCo., Ltd</td>
			<td>7447-40-7</td>
			<td>artifical urine</td>
		</tr>
		<tr>
			<td>NH4Cl</td>
			<td>Sinopharm Chemical ReagentCo., Ltd</td>
			<td>12125-02-9</td>
			<td>artifical urine</td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>Sinopharm Chemical ReagentCo., Ltd</td>
			<td>SWC0140326</td>
			<td>artifical urine</td>
		</tr>
		<tr>
			<td>C2Na2O4</td>
			<td>Sinopharm Chemical ReagentCo., Ltd</td>
			<td>62-76-0</td>
			<td>artifical urine</td>
		</tr>
		<tr>
			<td>NaSO4</td>
			<td>Sinopharm Chemical ReagentCo., Ltd</td>
			<td>7757-82-6</td>
			<td>artifical urine</td>
		</tr>
		<tr>
			<td>disodium hydrogen phosphate</td>
			<td>Sinopharm Chemical ReagentCo., Ltd</td>
			<td>10039-32-4</td>
			<td>artifical urine</td>
		</tr>
		<tr>
			<td>urea</td>
			<td>Sinopharm Chemical ReagentCo., Ltd</td>
			<td>57-13-6</td>
			<td>artifical urine</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sinopharm Chemical ReagentCo., Ltd</td>
			<td>7647-14-5</td>
			<td>artifical urine</td>
		</tr>
		<tr>
			<td>MgSO4.7H2O</td>
			<td>Sinopharm Chemical ReagentCo., Ltd</td>
			<td>10034-99-8</td>
			<td>artifical urine</td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>Sinopharm Chemical ReagentCo., Ltd</td>
			<td>10035-04-8</td>
			<td>artifical urine</td>
		</tr>
		<tr>
			<td>HCl</td>
			<td>Sinopharm Chemical ReagentCo., Ltd</td>
			<td>7647-01-0</td>
			<td>artifical urine</td>
		</tr>
		<tr>
			<td>citric acid</td>
			<td>Sinopharm Chemical ReagentCo., Ltd</td>
			<td>77-92-9</td>
			<td>artifical urine and mobile phase</td>
		</tr>
		<tr>
			<td>EDTA disodium salt</td>
			<td>Sinopharm Chemical ReagentCo., Ltd</td>
			<td>34124-14-6</td>
			<td>mobile phase</td>
		</tr>
		<tr>
			<td>monometallic sodium orthophosphate</td>
			<td>Sinopharm Chemical ReagentCo., Ltd</td>
			<td>7558-80-7</td>
			<td>artifical urine and mobile phase</td>
		</tr>
		<tr>
			<td>1-heptanesulfonic acid sodium salt</td>
			<td>Sinopharm Chemical ReagentCo., Ltd</td>
			<td>22767-50-6</td>
			<td>mobile phase</td>
		</tr>
		<tr>
			<td>sodium hydroxide</td>
			<td>Sinopharm Chemical ReagentCo., Ltd</td>
			<td>1310-73-2</td>
			<td>mobile phase</td>
		</tr>
		<tr>
			<td>phenylboronic acid column(PBA column)</td>
			<td>Aglilent</td>
			<td>12102018</td>
			<td>PBA extraction</td>
		</tr>
		<tr>
			<td>Inertsil&#174; ODS-3 5 &#181;m 4.6&#215;150 mm column</td>
			<td>Dikma</td>
			<td>5020-06731</td>
			<td>HPLC column for seperation</td>
		</tr>
		<tr>
			<td>SHIMADZU SIL-20AC prominence AUTO SAMPLER</td>
			<td>Shimadzu Corporation, Japan</td>
			<td>SIL-20AC</td>
			<td>auto injection for eluriant</td>
		</tr>
		<tr>
			<td>SHIMADZU LC-20AD High Performance Liquid Chromatography</td>
			<td>Shimadzu Corporation, Japan</td>
			<td>LC-20AD</td>
			<td>HPLC pump</td>
		</tr>
		<tr>
			<td>SHIMADZU L-ECD-60A electrochemical detector</td>
			<td>Shimadzu Corporation, Japan</td>
			<td>L-ECD-60A</td>
			<td>detector for the analytes</td>
		</tr>
		<tr>
			<td>ASAP 2020 Accelerated Surface Area and Porosimetry System</td>
			<td>Micromeritics, USA</td>
			<td></td>
			<td>surface and porosity analyzer&#160;</td>
		</tr>
	</tbody>
</table>

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.

This protocol is a simple and convenient PFSPE method to pretreat urine samples and enrich five catecholamines for detection via an HPLC-ECD system; a diagram of the process is shown in Figure 1. The protocol mainly includes four steps-activating, loading, rinsing, and eluting- coupled with a small quantity of PCE-PS nanofibers and a simple solid-phase extraction device. The morphology of PCE-PS nanofibers was assessed using a surface and porosity analyzer (s...

Watch this Scientific Journal Video about A Convenient Method for Extraction and Analysis with High-Pressure Liquid Chromatography of Catecholamine Neurotransmitters and Their Metabolites at JoVE.com

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

We present a convenient solid-phase extraction coupled to high-pressure liquid chromatography (HPLC) with electrochemical detection (ECD) for simultaneous determination of three monoamine neurotransmitters and two of their metabolites in infants' urine. We also identify the metabolite MHPG as a potential biomarker for the early diagnosis of brain damage for infants.

The extraction and analysis of catecholamine neurotransmitters in biological fluids is of great importance in assessing nervous system function and ...

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