A Modified QuEChERS-HPLC Method for Detection of Polycyclic Aromatic Hydrocarbons in Zebrafish Embryos Exposed to Fine Particulate Matter

This research aims to develop a modified QuEChERS-HPLC method to quantify PAHs in zebrafish embryos exposed to EOM, addressing the current gap caused by technical challenges in measuring internal PAHs levels due to the embryos' mass size and the complexity of their biological matrix. Obtaining reliable results requires 200 embryos per group, presenting a practical challenge. Combining HPLC with fluorescence detector could further enhance the sensitivity of the method. Compared to other techniques, this modified QuEChERS-HPLC method significantly reduce sample hangtime and provides a rapid and efficient approach for analyzing the levels of 16 PAHs in zebrafish embryos exposed to EOM.

[Narrator] To begin, prepare the QuEChERS sample by taking out the dishes containing the embryos from the incubator at 72 hours post-fertilization. Wash the embryos three times with pure water. Transfer the embryos to 1.5-milliliter tubes and remove excess water. Place the tubes in a minus 80 degrees Celsius freezer for 30 minutes to lyophilize the embryos. After lyophilization, grind the embryos using a glass rod. Add 25 microliters of 16 polycyclic aromatic hydrocarbon, or PAH, mix standard solution to the DMSO control group to test spike recovery. Then, add one milliliter of n-hexane and vortex for 30 seconds. Ultrasonicate the mixture at 35 degrees Celsius for 30 minutes. Now, centrifuge the sample at 7,000g for five minutes. Transfer the supernatant into a fresh tube. Evaporate the collected supernatants to dryness under nitrogen flow in a 35 degrees Celsius water bath. Add one milliliter of acetonitrile and vortex for 30 seconds. Add 10 milligrams of primary secondary amine, 45 milligrams of magnesium sulfate, and 15 milligrams of C18 packing to the sample, then vortex for 30 seconds. Centrifuge at 7,000g for five minutes and transfer the supernatant into a fresh tube. Evaporate the supernatant to 0.5 milliliters under nitrogen and filter the sample through a 0.22-micrometer micro-porous membrane. Prepare a series of working solutions ranging from one to 500 nanograms per milliliter for the 16 PAH mix standard using acetonitrile. Inject 20 microliters of the sample solutions into a liquid chromatograph using an acetonitrile or water-mobile phase and follow the elution procedure shown on the screen. Plot a standard curve with mass concentrations of the standard working solutions on the x-axis and the corresponding peak areas on the y-axis, covering a range from five nanograms per milliliter to 500 nanograms per milliliter. Inject the purified sample into the liquid chromatograph equipped with a fluorescence detector, or FLD, and diode array detector, or DAD. Identify the presence of PAHs by comparing retention times. Measure peak areas to determine the concentration. Finally, calculate the internal concentrations of the 16 PAHs based on the measured mass concentrations. The concentrations of polycyclic aromatic hydrocarbons, or PAHs, in zebrafish embryos at 72 hours post-fertilization are given in this table. Six PAHs were successfully detected in extractable organic matter, or EOM, exposed zebrafish embryos. In contrast, only two PAHs were identified in the DSMO control samples, at levels significantly lower than those in EOM samples.