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 Solutions Needed for the Extraction and Determination of Catecholamines
<ol>
	<li>Prepare the PFSPE column. Divide 1-2 mg of PCE-PS nanofibers into 5-6 aliquots, and use a fine steel rod with 0.5 mm diameter to compress them orderly into the end of a pipette tip with a volume of 200 &#181;L.</li>
	<li>Prepare the artificial urine by weighing 2.427 g urea, 0.034 g uric acid, 0.09 g creatinine, 0.297 g trisodium citrate, 0.634 g sodium chloride, 0.45 g potassium chloride, 0.161 g ammonium chloride, 0.089 g calcium chloride dihydrate, 0.1 g magnesium sulfate heptahydrate, 0.034 g sodium bicarbonate, 0.003 g sodium oxalate, 0.258 g sodium sulfate, 0.1 g sodium dihydrogen phosphate, and 0.011 g disodium hydrogen phosphate, and dissolve the above chemicals into 200 mL deionized water.</li>
	<li>Prepare 2 mg/mL stock solution of diphenylborate (DPBA) solution by dissolving 2 mg of the compound into 1 mL of distilled water. Store the solution in the dark at 4 &#176;C.</li>
	<li>Analyte standard 
	NOTE: The chemical structure and properties of catecholamines are instable, and they easily decompose. The preparation process of standards has to be very fast and must prevent exposure to direct sunlight.
	<ol>
		<li>Weigh 1.0 mg of NE, E, DA, MHPG, DOPAC and internal standard 3,4-dihydroxybenzylamine hydrobromide DHBA in separate 1.5 mL microcentrifuge tubes. Dilute the DHBA solution in water to 100 ng/mL before use.</li>
		<li>Oscillate the prepared standards in the dark at a high speed until analytes dissolve completely. This is the primary stock; store at -20 &#176;C for up to several weeks.</li>
		<li>Prepare the secondary 1,000 ng/mL analyte stocks. For NE, E, DA, DOPAC and MHPG, transfer 5 &#181;L of each primary analyte stock into 4,975 &#181;L of distilled water in a 5 mL centrifuge tube, and store it in the dark at 4 &#176;C until use. Prepare these solutions fresh daily. For DHBA, transfer 5 &#181;L of primary stock into 4,995 &#181;L of distilled water in a 5 mL centrifuge tube, and store it in the dark separately at 4 &#176;C.</li>
		<li>Make further dilutions with the secondary analyte stock to create a standard curve (e.g., Supplementary Table 2). Store solutions in the dark at 4 &#176;C and prepare fresh daily.</li>
		<li>Test the optimal voltage of ECD detector using the standard stock with proper concentration. Vary the voltage to find a value where the analytes have the best peak appearance.</li>
	</ol>
	</li>
	<li>Prepare eluant containing 30% phosphoric acid, 15% acetonitrile, and 55% distilled water. For 10 mL of eluant solvent, use 5.5 mL of distilled water, and add 1.5 mL of acetonitrile and 3 mL of phosphoric acid drop by drop into the water.</li>
</ol>
2. Preparation of Real Urine Samples and Mobile Phase
<ol>
	<li>Have mothers collect the first morning urine of their infants using aseptic urine cups. Transfer the samples into polypropylene tubes and label immediately. Then, store the samples in a -20 &#176;C freezer.</li>
	<li>Vortex and centrifuge the urinary samples at 1,510 x g for 10 min at room temperature (RT) to get rid of most particulate interference. Discard the sediment and gather the supernatants for further experiments. In order to extract analytes effectively, proceed to PFSPE pretreatment (step 3) immediately after centrifuging.</li>
	<li>Prepare the mobile phase
	<ol>
		<li>Prepare a clean bottle, at least 1 L. The composition of mobile phase is listed in Supplementary Table 1; for 1 L mobile phase, measure 6.7242 g of citric acid, 93.06 mg of ethylene diamine tetra acetic acid (EDTA) disodium salt, 7.02 g of monometallic sodium orthophosphate, 404.5 mg of 1-heptanesulfonic acid sodium salt, and 3.5 g of sodium hydrate into the bottle. Add 40 mL acetonitrile and distilled water to 1,000 mL. Agitate and vibrate ultrasonically for 15 min until the matter in the solution is all dissolved.</li>
		<li>Using a pH meter with a glass electrode, adjust the pH value of the mobile phase to 4.21 with a saturated sodium hydroxide solution.</li>
		<li>Filter the mobile phase with a 0.45 &#181;m polyvinylidene fluoride microporous membrane and a vacuum suction device to get rid of impurities.</li>
		<li>Use ultrasonic vibration for 15 min to degas the mobile phase each time before use.</li>
	</ol>
	</li>
</ol>
3. PFSPE Extraction and HPLC Analysis
<ol>
	<li>Activate the nanofibers. Press 100 &#181;L of methanol and 100 &#181;L of water sequentially through the PFSPE column using a 5 mL syringe in a slow, dropwise manner.</li>
	<li>Mix 100 &#181;L urine sample with 100 &#181;L 2 mg/ml DPBA solution and 30 &#181;L 100 ng/ml of DHBA solution (IS, internal standard) in a 0.5 mL EP tube, then transfer the mixed solution to the PFSPE column. Press the mixed sample solution through the PFSPE column with a 5 mL gastight plastic syringe using the force of the air pressure.</li>
	<li>Leach the column three times by loading 100 &#181;L of DPBA solution (2 mg/mL) into the SPE column, and push the solution slowly through the cartridge with air pressure using a 5 mL gastight plastic syringe.</li>
	<li>Load 50 &#181;L of the eluant onto the PFSPE column, and push it through the column, collecting the eluate with a 0.5 mL EP tube.</li>
	<li>Turn on the HLPC degasser to degas the air in the system. Prior to sample analyses, the system should run for more than 0.5 h with the mobile phase to equilibrate and reduce baseline noise. See Supplementary Table 1 showing the setup parameters of the HPLC system.</li>
	<li>Sample 20 &#181;L of the eluate using an automatic sampler, and then inject it into the HPLC-ECD system.</li>
	<li>When the runs are complete, turn off the detector cell using the detector interface. DO NOT turn off the cell with the switch at the back of the detector, as this could damage the instrument.</li>
	<li>Manually change the mobile phase composition to 10% methanol and 90% water. Run for at least 30 min. Then, manually change the mobile phase to HPLC-grade methanol. Run for about 15 min to protect the system in methanol. Failure to run this step following the recommended running time could result in damage to the column and the detector. Turn off the flow, then turn off the degasser.</li>
</ol>
4. Phenylboronic Acid Cartridges (PBA) Extraction
PBA cartridge extraction procedures were similar to the scheme in Kumar et al. (2011)25. All solutions are pushed through the PBA cartridge (100 mg, 1 mL) with air forced by a syringe.
<ol>
	<li>Condition the cartridge with 1 mL 80:20 acetonitrile-water (v/v) containing 1% formic acid and 1 mL of 50 mM phosphate buffer (pH 10) sequentially.</li>
	<li>Press the buffered urine sample (1 mL urine and 2 mL phosphate buffer, pH 8.5) through the PBA cartridge.</li>
	<li>Wash the cartridge with 1 mL 50:50 v/v acetonitrile-phosphate buffer (10 mM, pH 8.5).</li>
	<li>Elute the cartridge with 1 mL acetonitrile-water (80:20 v/v) containing 1% formic acid.</li>
</ol>
5. Identification and Quantification of Catecholamines
<ol>
	<li>Linearity
	<ol>
		<li>Dilute the secondary analytes stock with artificial urine to six concentrations (1.5, 3, 12, 25, 50, and 100 ng/mL); the dilution volume of artificial urine follows Supplementary Table 2. Make three parallel samples with each concentration to get 18 analyte experimental solutions for constructing calibration curves.</li>
		<li>Dilute DHBA secondary stock with artificial urine 10-fold to get 100 ng/mL experimental solution.</li>
		<li>Pretreat all the analyte solutions from 5.1.1, according to step 3 (PFSPE extraction procedures). As in Step 3, inject 20 &#181;L of each corresponding eluate into the HPLC-ECD system to get an HPLC chromatogram.</li>
		<li>Construct calibration curves of the five analytes by plotting the ratio of peak area (targets/IS) as the Y axis against the ratio of concentrations (targets/IS) as the X axis, as shown in Supplementary Figure 1.</li>
	</ol>
	</li>
	<li>LOD and LOQ value for sensitivity
	<ol>
		<li>Inject 20 &#181;L of the blank artificial urine into the HPLC-ECD system (as in Step 3), to obtain the HPLC chromatogram of the sample.</li>
		<li>In the chromatogram from 5.2.1, collect 11 blank signal values, and calculate the mean value Xb and standard deviation Sb. Calculate the minimum signal of a substance that can be detected at a certain level of confidence, XL, as XL = Xb+K*Sb (K is the coefficient determined by confidence level, Sb reflects noise level of the measuring method and the level of machine noise). Thus, LOD = (XL-Xb)/S = (K*Sb)/S (S stands for the slope value of the working curve).</li>
		<li>Define an S/N of 3:1 (K=3) as the limit of detection (LOD), and an S/N of 10:1 (K=10) as the limit of quantification (LOQ).</li>
	</ol>
	</li>
	<li>Evaluate the recoveries
	<ol>
		<li>Prepare real and spiked urine samples. Dilute the secondary analytes stock with real urine to three concentrations (5, 50, 100 ng/mL) to obtain the spiked urine samples. Prepare three parallel samples for each analyte solution. Count the spiked concentration as the quantity of target compounds spiked into the urine sample. Define this value as As.</li>
		<li>Dilute DHBA stock to 100 ng/mL, as in step 5.1.2.</li>
		<li>Process each sample solution from 5.3.1 according to step 3 (PFSPE extraction procedures) and inject the 20 &#181;L of each corresponding eluant into the HPLC-ECD system to get the chromatogram result. The value of analytes will be counted as a quantity of target compounds quantified in the spiked urine sample. Define this value as At.</li>
		<li>Inject 20 &#181;L of urine sample into the HPLC-ECD (as in step 3) system to get the chromatogram result. The value of analytes will be counted as an initial quantity of target compounds quantified in the urine sample. Define this value as Ai.</li>
		<li>Calculate the quantity of target compounds in the samples from the standard curve equation. The percentage recovery is estimated as methodological recovery % = (At - Ai) &#215; 100/(As). Mean values are shown in Table 1.</li>
	</ol>
	</li>
	<li>Evaluate the imprecision
	<ol>
		<li>Prepare spiked artificial urine samples to 5, 50, and 100 ng/mL concentrations as in step 5.3.1. Prepare six parallel samples for each analyte solution. Prepare fresh experimental samples every day.</li>
		<li>Dilute DHBA stock to 100 ng/mL as in step 5.1.2.</li>
		<li>Evaluate the intra-day precision (n=6). Process each sample solution in 5.4.1 according to step 3 and inject the 20 &#181;L of each correspondent eluant into the HPLC-ECD system to get the chromatogram. Do the same operation six times in the same day.</li>
		<li>Calculate the quantity of target compounds in the samples from the standard curve equation. Under the same concentration of the same compound, the relative standard deviation (RSD) of the six assays in one day is determined as intra-day precision. Mean values are shown in Table 1.</li>
		<li>Evaluate the inter-day precision (n=6). At the same time each day in the six sequential days, prepare spiked artificial urine samples for three concentrations of 5, 50, 100 ng/mL, as in 5.4.1 and 5.4.2., and process each analyte sample solution according to step 3.</li>
		<li>Inject the 20 &#181;L of each corresponding eluate from 5.4.5 into the HPLC-ECD system to get chromatogram results each day. Calculate the quantity of target compounds in the samples from the standard curve equation. Inter-day precision is expressed by the RSD of the assays quantity from the spiked artificial urine samples at the three concentrations in six sequential days. Mean values are shown in Table 1.</li>
	</ol>
	</li>
</ol>

The authors certify that there is no conflict of interest with any financial organization regarding the material discussed in this article.

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><tbody><tr><td>200 &amp;micro;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&amp;reg; ODS-3 5 &amp;micro;m 4.6&amp;times;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&amp;nbsp;</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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Chemistry

14.5K Views.  Laboratory of Environment and Biosafety Research Institute of Southeast University in Suzhou. We presenteren een handige solid-phase extraction gekoppeld aan hogedruk-vloeistofchromatografie (HPLC) met elektrochemische detectie (ECD) voor gelijktijdige bepaling van drie monoamino neurotransmitters en twee van hun metabolieten in de urine van zuigelingen. Wij identificeren ook de metaboliet MHPG als een potentiële biomarker voor de vroege diagnose van hersenschade voor zuigelingen.

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

Rapid High-throughput Species Identification of Botanical Material Using Direct Analysis in Real Time High Resolution Mass Spectrometry

We demonstrate that direct analysis in real time-high resolution mass spectrometry can be used to produce mass spectral profiles of botanical material, and that these chemical fingerprints can be used for plant species identification. The mass spectral data can be acquired rapidly and in a high throughput manner without the need for sample extraction, derivatization or pH adjustment steps. The use of this technique bypasses challenges presented by more conventional techniques including lengthy chromatography analysis times and resource intensive methods. The high throughput capabilities of the direct analysis in real time-high resolution mass spectrometry protocol, coupled with multivariate statistical analysis processing of the data, provide not only class characterization of plants, but also yield species and varietal information. Here, the technique is demonstrated with two psychoactive plant products, Mitragyna speciosa (Kratom) and Datura (Jimsonweed), which were subjected to direct analysis in real time-high resolution mass spectrometry followed by statistical analysis processing of the mass spectral data. The application of these tools in tandem enabled the plant materials to be rapidly identified at the level of variety and species.

A method for species identification of botanical material by direct analysis in real time-high resolution mass spectrometry and multivariate statistical analysis is presented.

We demonstrate that direct analysis in real time-high resolution mass spectrometry can be used to produce mass spectral profiles of botanical material, ...

Integration of Miniaturized Solid Phase Extraction and LC-MS/MS Detection of 3-Nitrotyrosine in Human Urine for Clinical Applications

Free 3-nitrotyrosine (3-NT) has been extensively used as a possible biomarker for oxidative stress. Increased levels of 3-NT have been reported in a wide variety of pathological conditions. However, existing methods lack the sufficient sensitivity and/or specificity necessary to measure the low endogenous level of 3-NT reliably and are too cumbersome for clinical applications. Hence, analytical improvement is urgently needed to accurately quantify the levels of 3-NT and verify the role of 3-NT in pathological conditions. This protocol presents the development of a novel liquid chromatography tandem mass spectrometry (LC-MS/MS) detection combined with a miniaturized solid phase extraction (SPE) for the rapid and accurate measurement of 3-NT in human urine as a non-invasive biomarker for oxidative stress. SPE using a 96-well plate markedly simplified the process by combining sample cleanup and analyte enrichment without tedious derivatization and evaporation steps, reducing solvent consumption, waste disposal, risk of contamination and overall processing time. The employment of 25 mM ammonium acetate (NH4OAc) at pH 9 as the SPE elution solution substantially enhanced the selectivity. Mass spectrometry signal response was improved through adjustment of the multiple reaction monitoring (MRM) transitions. Use of 0.01% HCOOH as additive on a pentafluorophenyl (PFP) column (150 mm x 2.1 mm, 3 &#181;m) improved signal response another 2.5-fold and shortened the overall run time to 7 min. A lower limit of quantitation (LLOQ) of 10 pg/mL (0.044 nM) was achieved, representing a significant sensitivity improvement over the reported assays. This simplified, rapid, selective and sensitive method allows two plates of urine samples (n = 192) to be processed in a 24 h time-period. Considering the markedly improved analytical performance, and non-invasive and inexpensive urine sampling, the proposed assay is beneficial for pre-clinical and clinical studies.

A selective and sensitive liquid chromatography tandem mass spectrometry (LC-MS/MS) method coupled with an efficient solid phase extraction on a mixed-mode cation-exchange (MCX) 96-well microplate was developed for the measurement of free 3-nitrotyrosine (3-NT) in human urine with high throughput, which is suitable for clinical applications.

Free 3-nitrotyrosine (3-NT) has been extensively used as a possible biomarker for oxidative stress. Increased levels of 3-NT have been reported in a wide ...

Multi-step Preparation Technique to Recover Multiple Metabolite Compound Classes for In-depth and Informative Metabolomic Analysis

Metabolomics is an emerging field which enables profiling of samples from living organisms in order to obtain insight into biological processes. A vital aspect of metabolomics is sample preparation whereby inconsistent techniques generate unreliable results. This technique encompasses protein precipitation, liquid-liquid extraction, and solid-phase extraction as a means of fractionating metabolites into four distinct classes.&#160;Improved enrichment of low abundance molecules with a resulting increase in sensitivity is obtained, and ultimately results in more confident identification of molecules. This technique has been applied to plasma, bronchoalveolar lavage fluid, and cerebrospinal fluid samples with volumes as low as 50 &#181;l. &#160;Samples can be used for multiple downstream applications; for example, the pellet resulting from protein precipitation can be stored for later analysis. The supernatant from that step undergoes liquid-liquid extraction using water and strong organic solvent to separate the hydrophilic and hydrophobic compounds. Once fractionated, the hydrophilic layer can be processed for later analysis or discarded if not needed. The hydrophobic fraction is further treated with a series of solvents during three solid-phase extraction steps to separate it into fatty acids, neutral lipids, and phospholipids. This allows the technician the flexibility to choose which class of compounds is preferred for analysis. It also aids in more reliable metabolite identification since some knowledge of chemical class exists.

The reliability of results in metabolomics experiments depends on the effectiveness and reproducibility of the sample preparation. Described is a rigorous and in-depth method that enables extraction of metabolites from biological fluids with the option of subsequently analyzing up to thousands of compounds, or just the compound classes of interest.

Metabolomics is an emerging field which enables profiling of samples from living organisms in order to obtain insight into biological processes. A vital ...

Bioengineering

Comprehensive Profiling of Dopamine Regulation in Substantia Nigra and Ventral Tegmental Area

Dopamine is a vigorously studied neurotransmitter in the CNS. Indeed, its involvement in locomotor activity and reward-related behaviour has fostered five decades of inquiry into the molecular deficiencies associated with dopamine regulation. The majority of these inquiries of dopamine regulation in the brain focus upon the molecular basis for its regulation in the terminal field regions of the nigrostriatal and mesoaccumbens pathways; striatum and nucleus accumbens. Furthermore, such studies have concentrated on analysis of dopamine tissue content with normalization to only wet tissue weight. Investigation of the proteins that regulate dopamine, such as tyrosine hydroxylase (TH) protein, TH phosphorylation, dopamine transporter (DAT), and vesicular monoamine transporter 2 (VMAT2) protein often do not include analysis of dopamine tissue content in the same sample. The ability to analyze both dopamine tissue content and its regulating proteins (including post-translational modifications) not only gives inherent power to interpreting the relationship of dopamine with the protein level and function of TH, DAT, or VMAT2, but also extends sample economy. This translates into less cost, and yet produces insights into the molecular regulation of dopamine in virtually any paradigm of the investigators' choice.
We focus the analyses in the midbrain. Although the SN and VTA are typically neglected in most studies of dopamine regulation, these nuclei are easily dissected with practice. A comprehensive readout of dopamine tissue content and TH, DAT, or VMAT2 can be conducted. There is burgeoning literature on the impact of dopamine function in the SN and VTA on behavior, and the impingements of exogenous substances or disease processes therein 1-5. Furthermore, compounds such as growth factors have a profound effect on dopamine and dopamine-regulating proteins, to a comparatively greater extent in the SN or VTA 6-8. Therefore, this methodology is presented for reference to laboratories that want to extend their inquiries on how specific treatments modulate behaviour and dopamine regulation. Here, a multi-step method is presented for the analyses of dopamine tissue content, the protein levels of TH, DAT, or VMAT2, and TH phosphorylation from the substantia nigra and VTA from rodent midbrain. The analysis of TH phosphorylation can yield significant insights into not only how TH activity is regulated, but also the signaling cascades affected in the somatodendritic nuclei in a given paradigm.
We will illustrate the dissection technique to segregate these two nuclei and the sample processing of dissected tissue that produces a profile revealing molecular mechanisms of dopamine regulation in vivo, specific for each nuclei (Figure 1).

Dopamine is distinctly regulated in the midbrain nuclei, which contain the cell bodies and dendrites of the dopamine neurons. Here we describe a dissection and sample-handling approach to maximize results, and thus conclusions and insights, on dopamine regulation in the midbrain nuclei of the substantia nigra (SN) and ventral tegmental area (VTA) in rodents.

Dopamine is a vigorously studied neurotransmitter in the CNS. Indeed, its involvement in locomotor activity and reward-related behaviour has fostered five ...

Neuroscience

Cellular Membrane Affinity Chromatography Columns to Identify Specialized Plant Metabolites Interacting with Immobilized Tropomyosin Kinase Receptor B

Chemicals synthesized by plants, fungi, bacteria, and marine invertebrates have been a rich source of new drug hits and leads. Medicines such as statins, penicillin, paclitaxel, rapamycin, or artemisinin, commonly used in medical practice, have been first identified and isolated from natural products. However, the identification and isolation of biologically active specialized metabolites from natural sources is a challenging and time-consuming process. Traditionally, individual metabolites are isolated and purified from complex mixtures, following the extraction of biomass. Subsequently, the isolated molecules are tested in functional assays to verify their biological activity. Here we present the use of cellular membrane affinity chromatography (CMAC) columns to identify biologically active compounds directly from complex mixtures. CMAC columns allow for the identification of compounds interacting with immobilized functional transmembrane proteins (TMPs) embedded in their native phospholipid bilayer environment. This is a targeted approach, which requires knowing the TMP whose activity one intends to modulate with the newly identified small molecule drug candidate. In this protocol, we present an approach to prepare CMAC columns with immobilized tropomyosin kinase receptor B (TrkB), which has emerged as a viable target for drug discovery for numerous nervous system disorders. In this article, we provide a detailed protocol to assemble the CMAC column with immobilized TrkB receptors using neuroblastoma cell lines overexpressing TrkB receptors. We further present the approach to investigate the functionality of the column and its use in the identification of specialized plant metabolites interacting with TrkB receptors.

The protocol describes the preparation of cell membrane affinity chromatography (CMAC) columns with immobilized cell membrane fragments containing functional transmembrane tropomyosin kinase receptor B proteins. The use of CMAC columns in the identification of specialized plant metabolites interacting with these receptors and present in complex natural mixtures is also explained.

Chemicals synthesized by plants, fungi, bacteria, and marine invertebrates have been a rich source of new drug hits and leads. Medicines such as statins, ...

Biochemistry

Classification of Tropane Alkaloids from Plant Metabolites Using LC-MS/MS

Applications of Liquid-Chromatography Tandem Mass Spectrometry in Natural Products Research: Tropane Alkaloids as a Case Study

Although many drugs utilized today are synthetic in origin, natural products still provide a rich source of novel chemical diversity and bioactivity, and can yield promising leads for resistant or emerging diseases. The challenge, however, is twofold: not only must researchers find natural products and elucidate their structures, but they must also identify what is worth isolating and assaying (and what is already known - a process known as dereplication). With the advent of modern analytical instrumentation, the pace of natural product discovery and dereplication has accelerated. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) has become an especially valuable technique for identifying and classifying chemical structures. Tropane alkaloids (TAs) are plant-derived compounds of great medicinal and toxicological significance. In this study, we developed an LC-MS/MS-based screening workflow utilizing the multiple MS/MS configurations available on a triple-quadrupole (QQQ) mass spectrometer to annotate and classify TA structures based on their distinct fragmentation patterns. By using a combination of data-dependent (DD) product ion scans, precursor ion scans (PrIS), and neutral loss scans (NLS), we applied this method to TA-rich extracts of the nightshades Datura stramonium and Datura metel. This method is rapid, sensitive, and was successfully employed for both preliminary dereplication of complex TA-containing samples and for the discovery of a novel candidate for isolation, purification (and eventual bioassay).

Author Spotlight: Discovering New Alkaloids in Plants with Advanced Mass Spectrometry Techniques

We present a method for rapid mass spectrometry (MS)/mass spectrometry (MS)-based annotation and classification of tropane alkaloids, useful for both preliminary dereplication of tropane-containing samples and discovery of novel alkaloids for isolation.

Although many drugs utilized today are synthetic in origin, natural products still provide a rich source of novel chemical diversity and bioactivity, and ...

Assessment of Dopaminergic Homeostasis in Mice by Use of High-performance Liquid Chromatography Analysis and Synaptosomal Dopamine Uptake

Dopamine (DA) is a modulatory neurotransmitter controlling motor activity, reward processes and cognitive function. Impairment of dopaminergic (DAergic) neurotransmission is strongly associated with several central nervous system-associated diseases such as Parkinson&#39;s disease, attention-deficit-hyperactivity disorder and drug addiction1,2,3,4. Delineating disease mechanisms involving DA imbalance is critically dependent on animal models to mimic aspects of the diseases, and thus protocols that assess specific parts of the DA homeostasis are important to provide novel insights and possible therapeutic targets for these diseases.
Here, we present two useful experimental protocols that when combined provide a functional read-out of the DAergic system in mice. Biochemical and functional parameters on DA homeostasis are obtained through assessment of DA levels and dopamine transporter (DAT) functionality5. When investigating the DA system, the ability to reliably measure endogenous levels of DA from adult brain is essential. Therefore, we present how to perform high-performance liquid chromatography (HPLC) on brain tissue from mice to determine levels of DA. We perform the experiment on tissue from dorsal striatum (dStr) and nucleus accumbens (NAc), but the method is also suitable for other DA-innervated brain areas.
DAT is essential for reuptake of DA into the presynaptic terminal, thereby controlling the temporal and spatial activity of released DA. Knowing the levels and functionality of DAT in the striatum is of major importance when assessing DA homeostasis. Here, we provide a protocol that allows to simultaneously deduce information on surface levels and function using a synaptosomal6 DA uptake assay.
Current methods combined with standard immunoblotting protocols provide the researcher with relevant tools to characterize the DAergic system.

Synaptosomal dopamine uptake and high-performance liquid chromatography analysis represent experimental tools to investigate dopamine homeostasis in mice by assessing the function of the dopamine transporter and levels of dopamine in striatal tissue, respectively. Here we present protocols to measure dopamine tissue content and assess the functionality of the dopamine transporter.

Dopamine (DA) is a modulatory neurotransmitter controlling motor activity, reward processes and cognitive function. Impairment of dopaminergic (DAergic) ...

Microdialysis of Excitatory Amino Acids During EEG Recordings in Freely Moving Rats

Microdialysis is a well-established neuroscience technique that correlates the changes of neurologically active substances diffusing into the brain interstitial space with the behavior and/or with the specific outcome of a pathology (e.g., seizures for epilepsy). When studying epilepsy, the microdialysis technique is often combined with short-term or even long-term video-electroencephalography (EEG) monitoring to assess spontaneous seizure frequency, severity, progression and clustering. The combined microdialysis-EEG is based on the use of several methods and instruments. Here, we performed in vivo microdialysis and continuous video-EEG recording to monitor glutamate and aspartate outflow over time, in different phases of the natural history of epilepsy in a rat model. This combined approach allows the pairing of changes in the neurotransmitter release with specific stages of the disease development and progression. The amino acid concentration in the dialysate was determined by liquid chromatography. Here, we describe the methods and outline the principal precautionary measures one should take during in vivo microdialysis-EEG, with particular attention to the stereotaxic surgery, basal and high potassium stimulation during microdialysis, depth electrode EEG recording and high-performance liquid chromatography analysis of aspartate and glutamate in the dialysate. This approach may be adapted to test a variety of drug or disease induced changes of the physiological concentrations of aspartate and glutamate in the brain. Depending on the availability of an appropriate analytical assay, it may be further used to test different soluble molecules when employing EEG recording at the same time.

Here, we describe a method for in vivo microdialysis to analyze aspartate and glutamate release in the ventral hippocampus of epileptic and non-epileptic rats, in combination with EEG recordings. Extracellular concentrations of aspartate and glutamate may be correlated with the different phases of the disease.

Microdialysis is a well-established neuroscience technique that correlates the changes of neurologically active substances diffusing into the brain ...

A Plate-Based Assay for the Measurement of Endogenous Monoamine Release in Acute Brain Slices

Monoamine neurotransmitters are associated with numerous neurologic and psychiatric ailments. Animal models of such conditions have shown alterations in monoamine neurotransmitter release and uptake dynamics. Technically complex methods such as electrophysiology, Fast Scan Cyclic Voltammetry (FSCV), imaging, in vivo&#160;microdialysis, optogenetics, or use of radioactivity are required to study monoamine function. The method presented here is an optimized two-step approach for detecting monoamine release in acute brain slices using a 48-well plate containing tissue holders for examining monoamine release, and high-performance liquid chromatography coupled with electrochemical detection (HPLC-ECD) for monoamine release measurement. Briefly, rat brain sections containing regions of interest, including prefrontal cortex, hippocampus, and dorsal striatum were obtained using a tissue slicer or vibratome. These regions of interest were dissected from the whole brain and incubated in an oxygenated physiological buffer. Viability was examined throughout the experimental time course, by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The acutely dissected brain regions were incubated in varying drug conditions that are known to induce monoamine release through the transporter (amphetamine) or through the activation of exocytotic vesicular release (KCl). After incubation, the released products in the supernatant were collected and analyzed through an HPLC-ECD system. Here, basal monoamine release is detected by HPLC from acute brain slices. This data supports previous in vivo and in vitro results showing that AMPH and KCl induce monoamine release. This method is particularly useful for studying mechanisms associated with monoamine transporter-dependent release and provides an opportunity to screen compounds affecting monoamine release in a rapid and low-cost manner.

This method introduces a simple technique for the detection of endogenous monoamine release using acute brain slices. The setup uses a 48-well plate containing a tissue holder for monoamine release. Released monoamine is analyzed by HPLC coupled with electrochemical detection. Additionally, this technique provides a screening method for drug discovery.

Monoamine neurotransmitters are associated with numerous neurologic and psychiatric ailments. Animal models of such conditions have shown alterations in ...

A Plate-Based Assay to Measure Monoamine Release from Brain Slices on Test Drug Exposure

Source: Pino, J. A. et al. <a href="https://app.jove.com/v/62127">A Plate-Based Assay for the Measurement of Endogenous Monoamine Release in Acute Brain Slices.</a> J. Vis. Exp. (2021)
In this video, we demonstrate an assay that investigates monoamine release mechanisms from the neurons of metabolically active ex vivo rat brain slices, when treated with test drugs. The assay demonstrates that neurostimulant drugs induce monoamine release via the cellular monoamine transporters, whereas high KCl concentration induces synaptic vesicle exocytosis-mediated vesicular monoamine release.

Source: Pino, J. A. et al. A Plate-Based Assay for the Measurement of Endogenous Monoamine Release in Acute Brain Slices. J. Vis. Exp. (2021)
In this ...

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

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