We developed a modified QuEChERS-HPLC method to assess the internal levels of 16 PAHs in zebrafish embryos.
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Method Article
A Modified QuEChERS-HPLC Method for Detection of Polycyclic Aromatic Hydrocarbons in Zebrafish Embryos Exposed to Fine Particulate Matter
* These authors contributed equally
In This Article
Summary
Abstract
Maternal exposure to fine particulate matter (PM2.5), with an aerodynamic diameter of 2.5 µm or less, has been linked to spontaneous abortion and various congenital diseases in humans. However, the underlying mechanisms remain unclear. Zebrafish embryos are widely used to study the developmental toxicity of PM2.5 but measuring the internal levels of polycyclic aromatic hydrocarbons (PAHs), the primary toxic constituents of PM2.5, poses a challenge. In this study, we developed a Quick, Easy, Cheap, Effective, Rugged, and Safe High-performance Liquid Chromatography (QuEChERS-HPLC) method to assess the internal levels of the 16 PAHs listed in the US Environmental Protection Agency (EPA) priority pollutant list. Zebrafish embryos were treated with extractable organic matter (EOM) from PM2.5 for 2-72 h post fertilization (hpf). The QuEChERS method was employed for sample preparation, and HPLC-FLD/DAD (HPLC with fluorescence detector and diode array detector) was employed for the qualitative and quantitative analyses of PAHs. We successfully detected six PAHs (Phenanthrene, Anthracene, Fluorene, Acenaphthene, Fluoranthene, and Pyrene) in zebrafish embryos exposed to PM2.5. This method is crucial for investigating the mechanisms of PM2.5-induced developmental toxicity.
Introduction
Fine particulate matter (PM2.5), with an aerodynamic diameter of 2.5 µm or less. is a leading environmental risk factor. Due to its small size, PM2.5 is able to enter the circulation system and permeate the maternal-fetal barrier. Epidemiological studies have linked maternal exposure to PM2.5 with spontaneous abortion and various congenital diseases1,2,3,4. However, the underlying mechanisms of the embryonic toxicity of PM2.5 are not well understood. PAHs, the primary toxic component of PM2.5, are highly toxic to humans due to their mutagenic and carcinogenic properties5,6. Among the hundreds of PAHs, 16 have been assigned as high-priority pollutants by the US Environmental Protection Agency (EPA).
Zebrafish, Danio rerio, is a powerful vertebrate model organism due to its genetic similarity to humans, rapid development, high reproductive rate, and ease of maintenance. The transparency of zebrafish embryos allows direct observation of the entire body under a microscope, enabling detailed studies. Dissolved PAHs can enter zebrafish embryos by diffusion7. Many studies have used zebrafish embryos to explore the toxic effects and mechanisms of Extractable Organic Matter (EOM) from PM2.58,9,10,11. However, most of these studies have only characterized the 16 priority PAHs in EOM, without detecting the internal levels of PAHs in zebrafish embryos12. This challenge may be due to the small size of zebrafish embryos and the complexity of the embryonic matrix.
The Quick, Easy, Cheap, Effective, Rugged, and Safe (QuEChERS) method is an innovative sample preparation technique used for the analysis of pesticide residues, veterinary drugs, and other contaminants in food and environmental samples13,14. When coupled with high-performance liquid chromatography (HPLC), this method has been used to detect pesticides in zebrafish15. PAHs, which contain two or more benzene rings, have strong fluorescence properties, making them particularly suitable for detection using fluorescence detectors. In this study, we developed a modified QuEChERS-HPLC method to assess the levels of 16 PAHs in zebrafish embryos exposed to EOM.
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Protocol
1. Sample treatment
- PM2.5 collection
- Collect PM2.5 on 47 mm quartz membrane filters.
NOTE: PM2.5 was collected in urban Suzhou, China.
- Collect PM2.5 on 47 mm quartz membrane filters.
- EOM extraction
- Cut the filter into pieces and place in a conical glass flask with 150 mL of acetonitrile.
- Ultrasonicate the flask at 35 °C for 1 h and transfer the extract to four 50 mL tubes.
- Centrifuge at 7,000 × g for 20 min, then transfer the supernatant to a rotary evaporator and concentrate it to approximately 5 mL.
- Evaporate to dryness under nitrogen flow in a 35 °C water bath and dissolve the residue in dimethyl sulfoxide (DMSO).
- Zebrafish embryo treatment
- Maintain zebrafish in a recirculating water system on a 14 h light and 10 h dark cycle at 28 °C16.
- Collect fertilized eggs (200 embryos per group) and transfer them to glass Petri dishes (soda-lime glass, 80 x 15 mm) with 30 mL of water from the recirculating water system.
- Add 30 µL of EOM (10 mg/mL) to a glass Petri dish containing 200 embryos at 2 h post fertilization (hpf). Untreated groups serve as blank controls.
NOTE: The final concentration of EOM is 10 mg/L, based on the cardiac malformation rate (Supplemental Figure S1). - Put the dishes in a 28 °C incubator.
2. QuEChERS sample preparation
- Remove the dishes with embryos at 72 hpf from the incubator. Wash the embryos 3x with pure water.
- Transfer the embryos to 1.5 mL tubes (according to different groups) and remove excess water.
- Place the tubes into a -80 °C freezer for 30 min to lyophilize the embyros17 and lyophilize them. Then, grind the embryos using a glass rod.
- Add 25 µL of 16 PAH mixed standard solution (2.0 µg/mL) to the DMSO control group to test spike recovery.
- Add 1 mL of n-hexane and vortex for 30 s, then ultrasonicate at 35 °C for 30 min.
- Centrifuge at 7,000 × g for 5 min and transfer the supernatant into a fresh tube.
- Repeat steps 2.5 and 2.6.
- Evaporate the collected supernatants to dryness under nitrogen flow in a 35 °C water bath.
- Add 1 mL of acetonitrile and vortex for 30 s.
- Add 10 mg of primary secondary amine (PSA), 45 mg of magnesium sulfate, and 15 mg of C18 packing, then vortex for 30 s.
- Centrifuge at 7,000 × g for 5 min and transfer the supernatant to a fresh tube.
- Repeat step 2.9 and centrifuge at 7,000 × g for 5 min. Transfer the supernatant to a fresh tube.
- Evaporate the supernatant to 0.5 mL under nitrogen.
- Filter through a 0.22 µm microporous membrane.
3. Determination of PAHs using HPLC
- Prepare a series of working solutions (ranging from 1 to 500 ng/mL) for the 16 PAH mixed standard with acetonitrile and inject them into a liquid chromatograph using acetonitrile/water as the mobile phase.
- Inject 20 µL and use the following elution procedure: 0-18.5 min, 40% acetonitrile to 100% acetonitrile; 18.5-28 min, 100% acetonitrile; 28-29.5 min, 100% to 40% acetonitrile; stop time: 32 min; column temperature: 25 °C; flow rate: 1.8 mL/min. 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 5 ng/mL to 500 ng/mL.
NOTE: Excitation/emission wavelengths of PAHs can be found in Supplemental Table S1.
- Inject 20 µL and use the following elution procedure: 0-18.5 min, 40% acetonitrile to 100% acetonitrile; 18.5-28 min, 100% acetonitrile; 28-29.5 min, 100% to 40% acetonitrile; stop time: 32 min; column temperature: 25 °C; flow rate: 1.8 mL/min. 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 5 ng/mL to 500 ng/mL.
- Inject the purified sample (from step 2.14.) into the liquid chromatograph with a Fluorescence detector (FLD) and diode array detector (DAD).
- Identify the presence of PAHs by comparing retention times and measure the associated peak areas.
NOTE: The mass concentrations of the PAHs were determined using the previously generated standard curve from step 3.1.1. - Calculate the internal concentrations of the 16 PAHs based on the measured mass concentrations of the samples.
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Results
The entire procedure for EOM exposure, sample preparation, and PAH detection is illustrated in Figure 1. For HPLC-FLU/DAD, a signal-to-noise ratio of 3:1, with a detection range from 0.3 to 5.0 ng/mL, was considered acceptable. The method limits of detection ranged from 3.0 × 10-4 to 5.0 × 10-3 ng/organism. All calibration curves exhibited high linearity (r2 > 0.999). Precision values determined using extracts obtained from matrix samples were under 5%. Th...
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Discussion
Zebrafish embryos are a powerful vertebrate model for developmental toxicity studies, but their small size complicates the detection of semi-volatile organic compounds (SVOCs) such as PAHs. During sample preparation, preventing contamination of zebrafish embryos is crucial. Proper cleaning of samples is a key step to avoid the carryover of compounds from the exposure medium into the sample extract. Incomplete washing can lead to overestimation, while excessive washing can cause the loss of substances from within the embr...
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Disclosures
The authors declare no conflicts of interest.
Acknowledgements
This work was financially supported by the National Natural Science Foundation of China (Grant number: 81972999) and Suzhou Science and Technology Development Plan (Basic Research on Medical Applications, SKY2023036 and SKY2023115).
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Materials
| Name | Company | Catalog Number | Comments |
|---|---|---|---|
| 16 PAHs mixed standard solutions | o2si Smart Solutions (South Carolina, USA) | 110124-06 | 16 PAHs mixed standard solutions includes naphthalene (Nap), acenaphthylene (Acy), ace-naphthene (Ace), fluorene (Flo), phenanthrene (Phe), anthracene (Ant),fluoranthene (Flu), pyrene (Pyr), benzo [a] anthracene (BaA), chrysene(Chr), benzo [b] fluoranthene (BbF), benzo [k] fluoranthene (BkF),benzo [a] pyrene (BaP), indeno [1,2,3-cd] pyrene (IcdP), dibenz [a,h]anthracene (DahA), benzo [g,h,i] perylene (BghiP). |
| 1260 Infinity II LC System | Agilent Technologies (Santa Clara, USA) | FLD(G1321A);DAD SL (G1315C) | |
| Acetonitrile | Scharlab (Barcelona,Spain) | AC03661000 | solvent |
| CNWBOND HC-C18 SPE | ANPEL Laboratory Technologies (Shanghai) Inc.(Shanghai, CHINA) | SBEQ-CA0801 | C18 packing is a reversed-phase packing with a good balance between hydrophilic and hydrophobic, which can adsorb and separate polar and non-polar organic compounds well. |
| Diatomaceous earth | Agilent Technologies, Inc (Santa Clara, USA) | 12214013 | For separation, purification and removal of organic impurities |
| Dispersive SPE 15 mL, EN | Agilent Technologies, Inc (Santa Clara, USA) | 5982-5056 | Sample purification |
| Filter | ANPEL Laboratory Technologies (Shanghai) Inc.(Shanghai, CHINA) | SCAA-104 | Organic phase needle filter (nylon),0.22µm |
| Hexane | Scharlab (Barcelona,Spain) | HE02481000 | solvent |
| Millipore | Merck (New Jersey, USA) | Prepare ultrapure water | |
| Organomation - N-EVAP Nitrogen Evaporators | Organomation (Massachusetts,USA) | Evaporate solvents | |
| Sorvall ST4 Plus | Thermo Fisher Scientific (Waltham, USA) | centrifugation | |
| Vortex- Genie 2 (G560E) | Scientific Industries (New York,USA) | Vortex | |
| ZORBAX Eclipse PAH | Agilent Technologies, Inc (Santa Clara, USA) | 959990-918 | This column is commonly used in HPLC systems to achieve precise and reliable analysis of PAHs. |
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