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

Summary

We developed a modified QuEChERS-HPLC method to assess the internal levels of 16 PAHs in zebrafish embryos.

Abstract

Maternal exposure to fine particulate matter (PM_2.5), 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 PM_2.5 but measuring the internal levels of polycyclic aromatic hydrocarbons (PAHs), the primary toxic constituents of PM_2.5, 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 PM_2.5 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 PM_2.5. This method is crucial for investigating the mechanisms of PM_2.5-induced developmental toxicity.

Introduction

Fine particulate matter (PM_2.5), with an aerodynamic diameter of 2.5 µm or less. is a leading environmental risk factor. Due to its small size, PM_2.5 is able to enter the circulation system and permeate the maternal-fetal barrier. Epidemiological studies have linked maternal exposure to PM_2.5 with spontaneous abortion and various congenital diseases^1,2,3,4. However, the underlying mechanisms of the embryonic toxicity of PM_2.5 are not well understood. PAHs, the primary toxic component of PM

Protocol

1. Sample treatment

1. PM_2.5 collection
   1. Collect PM_2.5 on 47 mm quartz membrane filters.
      NOTE: PM_2.5 was collected in urban Suzhou, China.
2. EOM extraction
   1. Cut the filter into pieces and place in a conical glass flask with 150 mL of acetonitrile.
   2. Ultrasonicate the flask at 35 °C for 1 h and transfer the extract to four 50 mL tubes.
   3. Centrifuge at 7,000 × g for 20 min, then transfer the supernatant to a rotary evaporator and concentrate it to approximately 5 mL.
   4. Evaporate to dryness under nitrogen flow in a 3....

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.......

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.......

Materials

List of materials used in this article

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.