In this study, solid phase extraction and QuEChERS was been compared follow by LC/MS/MS. This method was used to established the detection of fipronil in Egg sample under the multiple reaction monitoring (MRM) mode. The target compounds were extracted by 1% acetic acid ACN, clean up by Cleanert PEP Plus solid-phase extraction column also QuEChERS method, separated by Unisol C18 HPLC Column (3.0*50mm,3 μm) and quantitated by external standard method with water and acetonitrile as mobile phase in gradient elution. The results show that the recovery rate higher than 70% at concentration of 4 µg/kg and RSD lower than 5%, lower than the EU MRL 5 µg/kg.
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Fipronil; Egg; Cleanert PEP Plus;QuEChERS;Unisol C18; LC/MS/MS;
Contaminated eggs have been found in 16 European countries and millions of chicken eggs have been pulled from European supermarket shelves because of the risk of fipronil contamination. 
Fipronil is a popular insecticide to treat pets for fleas. The World Health Organization considers fipronil to be moderately toxic and says very large quantities can cause organ damage.
Take too much of Fipronil can have dangerous effects on people's kidneys, liver and thyroid glands. National Food Safety Standard has announced the Max pesticide residues limit in food should be lower than 5 µg/kg such as grains, fruits and vegetables.
 quote the statement from Tencent news, Aug. 13rd, 2017.
Weight 5.0 g egg sample into the extraction tube and 5 mL deionized water for dilution, then add 10 mL 1% acetic acid ACN（v/v）, supersonic extraction for 10 min then add 3 g NaCl, shake vigorously by hand for 5 mins then centrifuge at 8000 r/min for 5 min, take all the supernatant for further clean up.
Transfer 1.5 mL supernatant into Cleanert Lipono QuEChERS tube, vertex for 1 min and keep standing for 30s. Take 0.5 mL supernatant and dilute with 0.5 mL deionized water, filter the sample with 0.22 µm Nylon syringe filter and then for analysis.
Cleanert PEP Plus 60mg/3mL
Note: No need to do condition and equilibration
Load 2 mL supernatant onto PEP Plus, and throw away about 0.5 mL at the beginning and then collect the elution, dilute the elution with 0.5 mL deionized water and filtered by 0.22 µm Nylon syringe filter and then for analysis.
Column：Unisol C18, 3 µm, 100 Å, 3.0 × 50 mm; (P/N：UO930503-0)
Mobil Phase: A: Water; B: ACN;
Temperature: 30 °C;
Injection Volume: 5 µL;
Flow rate: 0.4 mL/min.
Tab 1. HPLC Gradient Condition
Flow rate mL/min
Mass spectrometer: API 4000 PLUS;
Ion source: ESI-;
Scan mode: positive;
Electrospray voltage: -4500 V;
Atomizer pressure: 45 psi;
Curtain Gas pressure: 15 psi;
Aux Gas Pressure: 50 psi;
Ion source temperature: 450 °C;
Detection mode: multiple reaction monitoring (MRM).
Tab 2. Mass spectrum parameters
Two sample preparation methods were used in the method, and the results show that the recovery rate was 78.8 % at concentration of 4 µg/kg by QuEChERS method and RSD lower than 5%. The recovery rate was 92.2 % at concentration of 4 µg/kg by SPE method and RSD lower than 5%.
By spiked fipronil into the blank egg sample matrix extraction, by compare with the standard solution response value. The recovery increase to 100% from 46.8% by using both two kinds of method. The result indicated that matrix effect was decreased significantly by both two kinds of sample preparation method and ensure the method repeatability and result accuracy.
Extraction Solution without of sample preparation
Tab 3. Recovery of blank Egg sample spiked with fipronil standard at Concentration of 4 µg/kg
Ave. Recovery /%
Ave. Recovery /%
Fig 1. Chromatography of Fipronil Standard solution at concentration of 0.001 µg/mL
Fig 2. Chromatography of blank Egg sample spiked with fipronil standard at Concentration of 4 µg/kg, SPE PEP Plus method.
Fig 3. Chromatography of blank Egg sample spiked with fipronil standard at Concentration of 4 µg/kg, QuEChERS method.
Cleanert® LipoNo QuEChERS kit: P/N: MS-LN0202;
Cleanert® PEP Plus (60 mg/3 mL): P/N: PE0603X;
Unisol C18, 3 µm, 100 Å, 3.0 × 50 mm; P/N：UO930503-0;
Syringe Filter (Nylon), 0.22 mm, diameter 13 mm, 200/pk; P/N:AS021320.