Abstract
In this study, a simple and efficient solid phase extraction procedure was developed for the simultaneously extraction of Fipronil and its three metabolites in egg and chicken samples. Chromatographic separation of the analytes using conditions compatible with LC-MS/MS was also developed. The target compounds were extracted by 1% acetic acid ACN, clean up by Cleanert PEP Plus, separated by Unisol C18 HPLC Column with water and acetonitrile as mobile phase in gradient elution. The results show that the recoveries were higher than 90% at concentration of 4 µg/kg and 10 µg/kg for egg and chicken respectively, together with RSD lower than 10%.
Bonna-Agela introduced PEP Plus Pre-Conditioned SPE Cartridges in 2010, which was a revolutionary product in Sample Preparation providing convenience to scientists working in analytical labs by eliminating the CONDITIONING and EQUILIBRATION steps in traditional SPE process resulting in overall cost reduction in solvents usage and sample processing time.
Keywords
Fipronil; Metabolites; Egg; Chicken; Cleanert PEP Plus; LC-MS/MS;
Introduction
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. [1]
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.
Sample Extraction
Egg samples
Weigh homogenized 5.0 g egg sample into the extraction tube and add 5 mL deionized water for dilution. Then add 10 mL 1% acetic acid ACN (v/v), sonicate extracted for 10 min. Add 3 g NaCl, into the sample and shake vigorously by hand for 5 min then centrifuge at 8000 r/min for 5 min, take all the supernatant for further cleanup.
Chicken samples
Weigh homogenized Sample 2.0 g and follow the same extraction process as Egg.
SPE Process
Load 2 mL supernatant from extraction step onto PEP Plus cartridge, note that there is no need to do condition and equilibration as PEP Plus was Pre-conditioned product. Throw away about 0.5 mL at the beginning and then collect all the elution. Take 0.5 mL elution and diluted with 0.5 mL deionized water and filtered through 0.22 µm Nylon syringe filter followed by LC-MS/MS analysis.
Chromatography Condition
Column：Unisol C18, 3 µm, 100 Å, 3.0 × 50 mm; (P/N：UO930503-0)
Mobil Phase: A: Water; B: ACN;
Column Temperature: 30 °C;
Injection: 5 µL;
Flow Rate: 0.4 mL/min.
Table 1 HPLC Gradient Condition
RT/min | Flow rate mL/min | A% | B% |
0.5 | 0.4 | 45 | 55 |
1.0 | 0.4 | 20 | 80 |
4.0 | 0.4 | 20 | 80 |
4.5 | 0.4 | 45 | 55 |
7.0 | 0.4 | 45 | 55 |
MS/MS Condition
Mass Spectrometer: API 4000 PLUS;
Ion Source: ESI-;
Electrospray Voltage: -4500 V;
Nebulizer Pressure: 45 psi;
Curtain Gas Pressure: 15 psi;
Aux. Gas Pressure: 50 psi;
Ion Source Temperature: 450 °C;
Scan Mode: MRM.
Table 2 Mass spectrum parameters
Analytes | Q1 | Q3 | DP/V | CE/V |
Fipronil | 435.0 | 330 | -60 | -22 |
399 | -60 | -15 | ||
Fipronil-desulfinyl | 387 | 351 | -65 | -16 |
282 | -65 | -45 | ||
Fipronil-sulfone | 451 | 282 | -75 | -37 |
244 | -75 | -61 | ||
Fipronil-sulfide | 419 | 383 | -64 | -18 |
262 | -64 | --39 |
Result
This application note describes the analysis of fipronil and its metabolites residues in egg and chicken samples using a simple extraction procedure coupled to LC-MS/MS detection. Fipronil and its main Metabolites been tested together, Unisol C18 HPLC column was used in this application and the results show satisfied resolution. The recoveries are higher than 95% and precision lower than 8% at concentration level of 4 µg/kg for egg sample and 10 µg/kg for chicken sample.
Cleanert PEP Plus Cartridge which is a kind of high cross link polymer material were used in the method, Pre-conditioned cartridge make the SPE process more simple and fast, which could be a good choice for fast analysis or screening.
Table 3 Accuracy and repeatability of fipronil and its metabolites residues in egg and chicken, spiked level of 4 µg/kg for egg and 10 µg/kg for chicken
Analytes | Retention time /min | Egg | Chicken | ||
Average Recovery/% | RSD/% | Average Recovery/% | RSD/% | ||
Fipronil | 3.58 | 101.6 | 7.1 | 97.5 | 1.1 |
Fipronil-desulfinyl | 3.75 | 104.0 | 7.2 | 101.6 | 2.6 |
Fipronil-sulfone | 3.96 | 112.8 | 7.0 | 100.4 | 1.8 |
Fipronil-sulfide | 3.99 | 111.4 | 5.1 | 105.4 | 2.8 |
Figure 1 Chromatogram of egg sample spiked with 4 µg/kg Fipronil and metabolites standard
Figure 2 Chromatogram of chicken sample spiked with 10 µg/kg Fipronil and metabolites
Ordering Information
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