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

The overall goal of this procedure is to determine three monoamine neurotransmitters and two of their metabolites in infant's urine simultaneously via a convenient solid-phase extraction coupled to high-pressure liquid chromatography with electrochemical detection. This method can predict and determine unstable target compounds in a complete sample with easy operation in a short time. Your examples can be easily pre-cleaned by a narrow fiber-packed solid-face quorum, and the analytes in the sample can be readily enriched, dissolved, and detect on a electrochemical-detection system.

We first had the idea for this method when we realized that extraction and analysis of catecholamine neurotransmitters in biological fluids is critical in assessing the resistant function and the related diseases. Individuals new to this method will struggle because light exposure must be avoided, and the pretreatment time should be as short as possible. Demonstrating the procedure, we have Lanlan Wei and Qing Han, two PhD students from our laboratory.

To begin, prepare two milligrams per milliliter stock solution of DPBA solution by dissolving two milligrams of the compound into one milliliter of distilled water. Store the solution in the dark at four degrees Celsius. When preparing the analyte extenders keep in mind the chemical structure and the properties of catecholamines are instable, and as they easily decompose, the preparation process extenders must be very fast, and exposure to thin sunlight must be avoided.

Weigh 1.0 milligrams of norepinephrine, epinephrine, dopamine, MHPG, DOPAC, and internal-standard DHBA in separate 1.5-milliliter microcentrifuge tubes. Add 1.0 milliliters of distilled water to dissolve each of the compounds, except epinephrine, which should be dissolved in 0.01-moles-per-liter hydrochloric-acid solution. Oscillate the prepared standards in the dark at high speed until analytes dissolve completely.

These are the primary stocks, and can be stored at minus 20 degrees Celsius for up to several weeks. To prepare the secondary analyte stocks of norepinephrine, epinephrine, dopamine, DOPAC, and MHPG, transfer five microliters of each primary analyte stock into 4, 975 microliters of distilled water in separate five-milliliter centrifuge tubes. Prepare these solutions fresh daily, and store the solutions in the dark at four degrees Celsius until use.

For DHBA, transfer five microliters of primary stock into 4, 995 microliters of distilled water in a five-milliliter centrifuge tube, and store it in the dark separately at four degrees Celsius. Make further dilutions with the secondary analyte stock to create a standard curve. Store the solutions in the dark at four degrees Celsius, and prepare fresh daily.

Next, test the optimal voltage of the ECD detector, using the standard stock with the proper concentration. Vary the voltage to find a value where the analytes have the best peak appearance. To prepare 10 milliliters of eluent solvent, use 5.5 milliliters of distilled water, then add 1.5 milliliters of acetonitrile and three milliliters of phosphoric acid, drop by drop, into the water.

To prepare the mobile phase, measure the components listed in the text protocol into a clean one-liter bottle. Add 40 milliliters of acetonitrile and distilled water to 1, 000 milliliters. Agitate and vibrate ultrasonically for 15 minutes until the matter in the solution is all dissolved.

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. Filter the mobile phase with a 0.45-micron polyvinylidene fluoride microporous membrane and a vacuum suction device to remove impurities. Use ultrasonic vibration for 15 minutes to degas the mobile phase each time before use.

Then, vortex and centrifuge the urinary samples at 1, 510 times G for 10 minutes at room temperature to remove most particulate interference. Gather the supernatants, and discard the sediment for further experiments. To extract analytes effectively, proceed to Packed Fiber Solid Phase Extraction, or PFSPE pretreatment, immediately after centrifuging.

To activate the nanofibers, press 100 microliters of methanol and 100 microliters of water sequentially through the PFSPE column using a five-milliliters syringe in a slow drop-wise manner. The urine sample must be mixed with DPPA solution to improve its hydrophobicity and help the catecholamines and metabolites absorb onto the fibers. DHBA solution must also be added as internal standard.

Mix 100 microliters of urine sample with 100 microliters of two milligrams per milliliter DPBA solution and 30 microliters of 100 nanograms per milliliter of DHBA internal-standard solution in a 0.5 milliliter Eppendorf tube. Then press the mixed sample solution through the PFSPE column with a five-milliliter gas-type plastic syringe using the force of the air pressure. Leach the column three times by using a five-milliliter gas-type plastic syringe to load 100 microliters of the DPBA solution into the SPE column, and push the solution slowly through the cartridge with air pressure.

This rinsing step is necessary because the DPBA solution can remove the impurities while the analytes are retained, thus improving sensitivity and selectivity for the analytes. Next, load 50 microliters of the eluent onto the PFSPE column, and push it through the column, collecting the eluate with a 0.5-milliliter Eppendorf tube. Turn on the HPLC degasser to degas the air in the system.

Prior to sample analyses, the system should run for more than 0.5 hours with the mobile phase to equilibrate and reduce baseline noise. Sample 20 microliters of the eluate using an automatic sampler, and then inject it into the HPLC ECD system. 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. Manually change the mobile-phase composition to 10%methanol and 90%water. After running for at least 30 minutes, manually change the mobile phase to HPLC-grade methanol.

Run the system for about 15 minutes to protect it 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, and then turn off the degasser.

Proceed to catecholamine analysis as described in the text protocol. The chromatogram of a spiked-water sample with the targets and the internal standard is shown without extraction. Under the HPLC condition in the protocol, the five target peaks of the PFSPE method are well-separated and significantly higher than those extracted by a commercial phenylboronic-acid cartridge.

Also, the DOPAC peak did not appear in the PBA cartridge extraction result, indicating that the PBA column was not capable of extracting DOPAC. When the urine sample was extracted, the PFSPE method could not only extract the targets with good peak identification but also could get rid of most of the interference in the urine. Calibration curves were constructed for each compound by plotting the ratio of peak area, showing good linearity with low concentration.

For validation with real urine samples, the urinary samples of 28 high-risk infants and 22 healthy infants were tested. While some catecholamines were not significantly different between the high-risk and healthy groups, the high-risk infants had higher amounts of the MHPG metabolite than the control group. Therefore, the level of urinary MHPG may be a potential marker for early identification of high-risk infants.

Once mastered, this technique can be done in minutes. While attempting the procedure, it's important to remember to prepare cinders fresh, operate worry fast, and prevent exposure to direct sunlight. The diphenylborate-acid-solution addition in absorption and rinsing steps are also critical.

Following this procedure, as measures lack of termination of catecholamines and metabolites in plasma or cerebrospinal fluid. Alsliver can be performed in order to determine the universality of this protocol in testing the other biological fluids. After its development, this technique paved the way for researchers to study the relationship between neurodevelopmental deficits and the level of urinary catecholamines in children's samples.

After watching this video you should have a good understanding of how to perform the solid-phase-extraction method with nair figures and a simple device, the PCE PS air fibules corporate-waste DPBA can help clean up the catecholamine neurotransmitters in the urine samples. This technique extends toward early diagnosis of cassis at risk for brain damage in infants. Because comparative analysis revealed an abnormal differences in unary MHPG of autism children, indicating that the catecholamine metabolites might be important candidate marker.