Introduction
Fipronil is normally used as a broad spectrum insecticide to treat dogs and cats for fleas and to combat red lice in poultry. Fipronil sulfone is a degradation product of fipronil, and is more toxic to organisms than the parent compound. Fipronil is a phenylpyrazole pesticide and its use with food producing animals is not permitted in the European Union (EU). Fipronil is classified by the World Health Organization (WHO) as moderately hazardous.
This application focusses on HPLC testing of Fipronil and Fipronil sulfone in Food, in the sum of residues. The maximum residue limit designated by the EU for fipronil in eggs and chicken meat is 5 ng/g (0.005 mg/Kg), reported as a sum of the parent compound and the sulfone degradant.
The limit of detection (LOD) is better than 5 ng/g for Fipronil and better than 3 ng/g for Fipronil sulfone, the limit of quantitation (LOQ) is better than 11 ng/g for Fipronil and better than 7 ng/g for Fipronil sulfone, using HPLC-UV detection.
Fipronil |
Fipronil sulfone |
|---|
Fipronil and Fipronil sulfone Standards
| Experimental Conditions |
|---|
| Column |
Purospher® STAR RP-18 endcapped (3µm) 100x2.1 mm |
Injection volume: |
1 µL |
| Detection |
Dionex Ultimate 3000 VWD-3400 @ VIS = 220 nm (micro flow cell; 1.4 µL/7 mm) |
Flow Rate: |
208 µL/min |
| Mobile phase A |
Ammonium acetate buffer |
Temperature: |
40°C |
| Mobile phase B |
Methanol |
Pressure Drop: |
63-248 bar (914-3596 psi) |
| Isocratic |
See table |
| Ammonium acetate buffer |
Prepare a 5 mM ammonium acetate solution by dissolving 0.385 g ammonium acetate in 1 L deionized (DI) water and add 0.05 % acetic acid. Filter trough a 0.45 µm syringe filter. |
Standard stock solutions
(100 µg/mL) |
Accurately weigh 10.0 mg of each reference standard into a 100 mL volumetric flasks, dilute to volume with acetonitrile and mix. |
Standard solution
(100 ng/mL) |
Pipette 1 mL of the stock solution into a 1000 mL volumetric flask, dilute to volume with acetonitrile and mix. |
Experimental Conditions - Gradient Table
| Time (min) |
A (%) |
B (%) |
|---|
| 0 |
90 |
10 |
| 4 |
40 |
60 |
| 15 |
10 |
90 |
| 16 |
0 |
100 |
| 20 |
0 |
100 |
| 21 |
90 |
10 |
| 26 |
90 |
10 |
Chromatographic Data [Standard Solution 1 (100 µg/mL)]
| No. |
Compound |
Retention Time (min) |
S/N |
Area (mAU*min) |
Tailing Factor |
|---|
| 1 |
t0 void volume |
1.1 |
|
|
|
| 2 |
Fipronil |
12.9 |
934.9 |
20.694 |
2.2 |
| 3 |
Fipronil sulfone |
13.8 |
896.7 |
26.738 |
2.4 |
Chromatographic Data [Standard Solution 2 (100 ng/mL)]
| No. |
Compound |
Retention Time (min) |
S/N |
Area (mAU*min) |
Tailing Factor |
|---|
| 1 |
t0 void volume |
1.1 |
|
|
|
| 2 |
Fipronil |
13.4 |
4.1 |
0.206 |
1.3 |
| 3 |
Fipronil sulfone |
13.8 |
- |
0.285 |
1.5 |
Fipronil and Fipronil sulfone in Eggs
| Experimental Conditions |
|---|
| Column |
Purospher® STAR RP-18 endcapped (3µm) 100x2.1 mm |
Injection volume: |
1 µL |
| Detection |
Dionex Ultimate 3000 VWD-3400 @ VIS = 220 nm (micro flow cell; 1.4 µL/7 mm) |
Flow Rate: |
208 µL/min |
| Mobile phase A |
Ammonium acetate buffer |
Temperature: |
40°C |
| Mobile phase B |
Methanol |
Pressure
Drop: |
63-248 bar
(914-3596 psi) |
| Isocratic |
See table |
| Ammonium acetate buffer |
Prepare a 5 mM ammonium acetate solution by dissolving 0.385 g ammonium acetate in 1 L deionized (DI) water and add 0.05 % acetic acid. Filter trough a 0.45 µm syringe filter. |
Standard stock solutions
(100 µg/mL) |
Accurately weigh 10.0 mg of each reference standard into a 100 mL volumetric flasks, dilute to volume with acetonitrile and mix. |
Standard solution
(100 ng/mL) |
Pipette 1 mL of the stock solution into a 1000 mL volumetric flask, dilute to volume with acetonitrile and mix. |
| Sample solution without clean-up |
Evaporate after sample preparation. Dissolve the residue into 2.5 mL acetonitrile.
|
| Sample fortification |
100 µL of the standard stock solution were added to the sample before sample preparation, and allowed to equilibrate with the sample for 60 minutes. |
Clean-up
with SPE |
Condition disposable 250 mg C18 SPE columns with 10 mL of acetonitrile. Transfer 1 mL sample solution to the C18 SPE and let the solution pass through by gravity. Elute by gravity with 3 x 4 mL of methanol + 0.5 % of acetic acid. With hand bulb or syringe, blow out the remaining solvent from column into glass tube. Evaporate by 20 °C bath temperature with a TurboVap system. Dissolve the residue after the extraction in 1 mL of methanol. |
Experimental Conditions - Gradient Table
| Time (min) |
A (%) |
B (%) |
|---|
| 0 |
90 |
10 |
| 4 |
40 |
60 |
| 15 |
10 |
90 |
| 16 |
0 |
100 |
| 20 |
0 |
100 |
| 21 |
90 |
10 |
| 26 |
90 |
10 |
Sample Preparation
The sample was prepared by a Matrix Solid Phase Dispersion (MSPD) technique. Therefore, accurately weigh 5 g of homogenized egg into a mortar. Add 10 g of dry sodium sulphate and 10 g of sea sand and laevigate with a pestle until a dry powder occurs. Transfer the powder into a glass extraction tube, which contains 2 g of sodium sulphate. Extract with 50 mL of cyclohexane/ethyl acetate 1/1 (v/v) (20-30 drops/30 s). Evaporate to dryness with a TurboVap system at bath temperature of 20 °C.
Chromatographic Data [Egg without clean-up]
| No. |
Compound |
Retention Time (min) |
S/N |
Area (mAU*min) |
Tailing Factor |
|---|
| 1 |
t0 void volume |
1.1 |
|
|
|
| 2 |
Fipronil |
13.2 |
3.2 |
0.015 |
0.8 |
| 3 |
Fipronil sulfone |
14.1 |
|
0.055 |
|
Chromatographic Data [Egg after clean-up]
| No. |
Compound |
Retention Time (min) |
S/N |
Area (mAU*min) |
Tailing Factor |
|---|
| 1 |
t0 void volume |
1.1 |
|
|
|
| 2 |
Fipronil |
13.2 |
1.5 |
0.092 |
1.1 |
| 3 |
Fipronil sulfone |
14.1 |
9.4 |
0.476 |
1.6 |
Chromatographic Data [Egg after clean-up spiked with 10 µg/mL standard]
| No. |
Compound |
Retention Time (min) |
S/N |
Area (mAU*min) |
Tailing Factor |
|---|
| 1 |
t0 void volume |
1.1 |
|
|
|
| 2 |
Fipronil |
13.3 |
58.1 |
2.863 |
1.9 |
| 3 |
Fipronil sulfone |
14.2 |
39.4 |
3.010 |
2.2 |
Fipronil and Fipronil sulfone in Noodles
| Experimental Conditions |
|---|
| Column |
Purospher® STAR RP-18 endcapped (3µm) 100x2.1 mm |
Injection volume: |
1 µL |
| Detection |
Dionex Ultimate 3000 VWD-3400 @ VIS = 220 nm (micro flow cell; 1.4 µL/7 mm) |
Flow Rate: |
208 µL/min |
| Mobile phase A |
Ammonium acetate buffer |
Temperature: |
40°C |
| Mobile phase B |
Methanol |
Pressure Drop: |
63-248 bar (914-3596 psi) |
| Isocratic |
See table |
| Ammonium acetate buffer |
Prepare a 5 mM ammonium acetate solution by dissolving 0.385 g ammonium acetate in 1 L deionized (DI) water and add 0.05 % acetic acid. Filter trough a 0.45 µm syringe filter. |
| Standard stock solutions 100 µg/mL |
Accurately weigh 10.0 mg of each reference standard into a 100 mL volumetric flasks, dilute to volume with acetonitrile and mix. |
Standard solution
(100 ng/mL) |
Pipette 1 mL of the stock solution into a 1000 mL volumetric flask, dilute to volume with acetonitrile and mix. |
| Sample solution without clean-up |
Evaporate after sample preparation. Dissolve the residue into 2.5 mL acetonitrile. |
| Sample fortification |
100 µL of the standard stock solution were added to the sample before sample preparation, and allowed to equilibrate with the sample for 60 minutes. |
| Clean-up with SPE |
Condition disposable 250 mg C18 SPE columns with 10 mL of acetonitrile. Transfer 1 mL sample solution to the C18 SPE and let the solution pass through by gravity. Elute by gravity with 3 x 4 mL of methanol + 0.5 % of acetic acid. With hand bulb or syringe, blow out the remaining solvent from column into glass tube. Evaporate by 20 °C bath temperature with a TurboVap system. Dissolve the residue after the extraction in 1 mL of methanol. |
Experimental Conditions - Gradient Table
| Time (min) |
A (%) |
B (%) |
|---|
| 0 |
90 |
10 |
| 4 |
40 |
60 |
| 15 |
10 |
90 |
| 16 |
0 |
100 |
| 20 |
0 |
100 |
| 21 |
90 |
10 |
| 26 |
90 |
10 |
Sample Preparation
Accurately weigh 10 g of noodles into a mortar and laevigate with a pistil. Transfer the homogenized crushed noodles into a beaker, add 50 mL of cyclohexane/ethyl acetate 1/1 (v/v) and stir for 1 h. Filter the suspension through a prepared filter, and evaporate to dryness with a TurboVap system at bath temperature of 20 °C.
Chromatographic Data [Noodles without clean-up]
| No. |
Compound |
Retention Time (min) |
S/N |
Area (mAU*min) |
Tailing Factor |
|---|
| 1 |
t0 void volume |
1.1 |
|
|
|
| 2 |
Fipronil |
N/A |
|
|
|
| 3 |
Fipronil sulfone |
N/A |
|
|
|
Chromatographic Data [Noodles after clean-up]
| No. |
Compound |
Retention Time (min) |
S/N |
Area (mAU*min) |
Tailing Factor |
|---|
| 1 |
t0 void volume |
1.1 |
|
|
|
| 2 |
Fipronil |
N/A |
|
|
|
| 3 |
Fipronil sulfone |
N/A |
|
|
|
Chromatographic Data [Noodles after clean-up spiked with 10 µg/mL standard]
| No. |
Compound |
Retention Time (min) |
S/N |
Area (mAU*min) |
Tailing Factor |
|---|
| 1 |
t0 void volume |
1.1 |
|
|
|
| 2 |
Fipronil |
13.3 |
36.6 |
2.019 |
1.5 |
| 3 |
Fipronil sulfone |
14.2 |
38.7 |
2.600 |
1.7 |
Fipronil and Fipronil sulfone in Mayonnaise
| Experimental Conditions |
|---|
| Column |
Purospher® STAR RP-18 endcapped (3µm) 100x2.1 mm |
Injection volume: |
1 µL |
| Detection |
Dionex Ultimate 3000 VWD-3400 @ VIS = 220 nm (micro flow cell; 1.4 µL/7 mm) |
Flow Rate: |
208 µL/min |
| Mobile phase A |
Ammonium acetate buffer |
Temperature: |
40°C |
| Mobile phase B |
Methanol |
Pressure Drop: |
63-248 bar (914-3596 psi) |
| Isocratic |
See table |
| Ammonium acetate buffer |
Prepare a 5 mM ammonium acetate solution by dissolving 0.385 g ammonium acetate in 1 L deionized (DI) water and add 0.05 % acetic acid. Filter trough a 0.45 µm syringe filter. |
Standard stock
solutions 100 µg/mL |
Accurately weigh 10.0 mg of each reference standard into a 100 mL volumetric flasks, dilute to volume with acetonitrile and mix. |
Standard solution
(100 ng/mL) |
Pipette 1 mL of the stock solution into a 1000 mL volumetric flask, dilute to volume with acetonitrile and mix. |
Sample solution
without clean-up |
Evaporate after sample preparation. Dissolve the residue into 2.5 mL acetonitrile. |
| Sample fortification |
100 µL of the standard stock solution were added to the sample before sample preparation, and allowed to equilibrate with the sample for 60 minutes. |
Clean-up
with SPE |
1 mL of the supernatant was added to a 2 mL Supel QuE Z-Sep + clean-up tube and shaked for 1 minute at 2250 rpm. Centrifuge at 5000 rpm for 3 minutes. The supernatant will be used for analysis. |
Experimental Conditions - Gradient Table
| Time (min) |
A (%) |
B (%) |
|---|
| 0 |
90 |
10 |
| 4 |
40 |
60 |
| 15 |
10 |
90 |
| 16 |
0 |
100 |
| 20 |
0 |
100 |
| 21 |
90 |
10 |
| 26 |
90 |
10 |
Sample Preparation
Mayonnaise must be homogenized with an UltraTurax with 5000 rpm. Accurately weigh 10 g of mayonnaise into a 50 mL extraction tube. Add 10 mL of acetonitrile and shake for 10 minutes at 2250 rpm. Add the content of a Supel QuE non-buffered extraction tube and shake for 1 minute at 2250 rpm. Centrifuge at 5000 rpm for 5 minutes. The supernatant will be used for the clean-up.
Chromatographic Data [Mayonnaise without clean-up]
| No. |
Compound |
Retention Time (min) |
S/N |
Area (mAU*min) |
Tailing Factor |
|---|
| 1 |
t0 void volume |
1.1 |
|
|
|
| 2 |
Fipronil |
|
|
|
|
| 3 |
Fipronil ulfone |
|
|
|
|
s
Chromatographic Data [Mayonnaise after clean-up]
| No. |
Compound |
Retention Time (min) |
S/N |
Area (mAU*min) |
Tailing Factor |
|---|
| 1 |
t0 void volume |
1.1 |
|
|
|
| 2 |
Fipronil |
|
|
|
|
| 3 |
Fipronil sulfone |
|
|
|
|
Chromatographic Data [Mayonnaise after clean-up spiked with 10 µg/mL standard]
| No. |
Compound |
Retention Time (min) |
S/N |
Area (mAU*min) |
Tailing Factor |
|---|
| 1 |
t0 void volume |
1.1 |
|
|
|
| 2 |
Fipronil |
13.2 |
55.3 |
2.032 |
1.2 |
| 3 |
Fipronil sulfone |
14.2 |
35.4 |
2.648 |
1.7 |
Fipronil and Fipronil sulfone in chicken Meat
| Experimental Conditions |
|---|
| Column |
Purospher® STAR RP-18 endcapped (3µm) 100x2.1 mm |
Injection volume: |
1 µL |
| Detection |
Dionex Ultimate 3000 VWD-3400 @ VIS = 220 nm (micro flow cell; 1.4 µL/7 mm) |
Flow Rate: |
208 µL/min |
| Mobile phase A |
Ammonium acetate buffer |
Temperature: |
40°C |
| Mobile phase B |
Methanol |
Pressure
Drop: |
63-248 bar (914-3596 psi) |
| Isocratic |
See table |
| Ammonium acetate buffer |
Prepare a 5 mM ammonium acetate solution by dissolving 0.385 g ammonium acetate in 1 L deionized (DI) water and add 0.05 % acetic acid. Filter trough a 0.45 µm syringe filter. |
| Standard stock solutions 100 µg/mL |
Accurately weigh 10.0 mg of each reference standard into a 100 mL volumetric flasks, dilute to volume with acetonitrile and mix. |
| Standard solution (100 ng/mL) |
Pipette 1 mL of the stock solution into a 1000 mL volumetric flask, dilute to volume with acetonitrile and mix.
|
| Sample solution without clean-up |
Evaporate after sample preparation. Dissolve the residue into 2.5 mL acetonitrile. |
| Sample fortification |
100 µL of the standard stock solution were added to the sample before sample preparation, and allowed to equilibrate with the sample for 60 minutes. |
| Clean-up with Supel QuE Z-Sep+ |
1 mL of the supernatant was added to a 2 mL Supel QuE Z-Sep + clean-up tube and shaked for 1 minute at 2250 rpm. Centrifuge at 5000 rpm for 3 minutes. The supernatant will be used for analysis. |
Experimental Conditions - Gradient Table
| Time (min) |
A (%) |
B (%) |
|---|
| 0 |
90 |
10 |
| 4 |
40 |
60 |
| 15 |
10 |
90 |
| 16 |
0 |
100 |
| 20 |
0 |
100 |
| 21 |
90 |
10 |
| 26 |
90 |
10 |
Sample Preparation
Chicken meat was partially freezed and chopped. Accurately weigh 10 g of the prepared chicken meat into a 50 mL extraction tube. Add 10 mL of acetonitrile and shake for 10 minutes at 2250 rpm. Add the content of a Supel QuE non-buffered extraction tube and shake for 1 minute at 2250 rpm. Centrifuge at 5000 rpm for 5 minutes. The supernatant will be used for the clean-up.
Chromatographic Data [Mayonnaise without clean-up]
| No. |
Compound |
Retention Time (min) |
S/N |
Area (mAU*min) |
Tailing Factor |
|---|
| 1 |
t0 void volume |
1.1 |
|
|
|
| 2 |
Fipronil |
|
|
|
|
| 3 |
Fipronil sulfone |
|
|
|
|
Chromatographic Data [Mayonnaise after clean-up]
| No. |
Compound |
Retention Time (min) |
S/N |
Area (mAU*min) |
Tailing Factor |
|---|
| 1 |
t0 void volume |
1.09 |
|
|
|
| 2 |
Fipronil |
|
|
|
|
| 3 |
Fipronil sulfone |
|
|
|
|
Chromatographic Data [Mayonnaise after clean-up spiked with 10 µg/mL standard]
| No. |
Compound |
Retention Time (min) |
S/N |
Area (mAU*min) |
Tailing Factor |
|---|
| 1 |
t0 void volume |
1.1 |
|
|
|
| 2 |
Fipronil |
13.2 |
23.3 |
1.988 |
1.9 |
| 3 |
Fipronil sulfone |
14.1 |
25.0 |
2.550 |
1.4 |
Fipronil and Fipronil sulfone in food - validation data
| 1. Specificity: Inject standard solution of Fipronil and Fipronil sulfone (each 100 ppm) and determine the retention time and content of desired analyte |
|---|
| |
|
Retention Time (min) |
Area (mAU*min) |
Tailing Factor |
Resolution |
| 1 |
Fipronil |
12.86 |
22.038 |
2.4 |
|
| 2 |
Fipronil sulfone |
13.75 |
28.422 |
2.2 |
5.0
|
| 2. Standard repeatability egg (spiked with 10 ppm of each standard) in mAU*min |
|---|
| |
Fipronil |
Fipronil sulfone |
| STD 1 |
2.8630 |
3.0100 |
| STD 2 |
2.8351 |
2.9872 |
| STD 3 |
2.8483 |
2.9908 |
| STD 4 |
2.8398 |
3.0201 |
| STD 5 |
2.8506 |
3.0059 |
| Mean |
2.8474 |
3.0028 |
| Standard Deviation |
0.0108 |
0.0137 |
| (%) RSD |
0.4 |
0.5 |
| 2. Standard repeatability noodles (spiked with 10 ppm of each standard) in mAU*min |
|---|
| |
Fipronil |
Fipronil sulfone |
| STD 1 |
2.0069 |
2.5884 |
| STD 2 |
2.0165 |
2.6565 |
| STD 3 |
2.0043 |
2.6008 |
| STD 4 |
2.0139 |
2.6125 |
| STD 5 |
2.0190 |
2.6000 |
| Mean |
2.0121 |
2.6116 |
| Standard Deviation |
0.0063 |
0.0265 |
| (%) RSD |
0.3 |
1.0 |
| 2. Standard repeatability mayonnaise (spiked with 10 ppm of each standard) in mAU*min |
|---|
| |
Fipronil |
Fipronil sulfone |
| STD 1 |
2.0320 |
2.6480 |
| STD 2 |
2.0476 |
2.6214 |
| STD 3 |
2.0357 |
2.6887 |
| STD 4 |
2.0578 |
2.7115 |
| STD 5 |
2.0394 |
2.6757 |
| Mean |
2.0425 |
2.6691 |
| Standard Deviation |
0.0103 |
0.0352 |
| (%) RSD |
0.5 |
1.3 |
| 2. Standard repeatability chicken meat (spiked with 10 ppm of each standard) in mAU*min |
|---|
| |
Fipronil |
Fipronil sulfone |
| STD 1 |
1.9880 |
2.6500 |
| STD 2 |
1.9901 |
2.6295 |
| STD 3 |
2.0041 |
2.6341 |
| STD 4 |
1.9811 |
2.6731 |
| STD 5 |
1.9974 |
2.6900 |
| Mean |
1.9921 |
2.6553 |
| Standard Deviation |
0.0089 |
0.0258 |
| (%) RSD |
0.4 |
1.0 |
| 3. LOD & LOQ Fipronil |
|---|
| Conc. (ng/g) |
Mean Area (mAU*min) |
| 1.06 |
0.0001 |
| 5.00 |
0.0005 |
| 10.60 |
0.0011 |
| 53.00 |
0.0058 |
| 106.00 |
0.0119 |
| 212.00 |
0.0243 |
| STEYEX |
0.0001239672 |
| Slope |
0.0001145495 |
| LOD (ng/g) |
3.6 |
| LOQ (ng/g) |
10.8 |
| 3. LOD & LOQ Fipronil sulfone |
|---|
| Conc. (ng/g) |
Mean Area (mAU*min) |
| 0.96 |
0.0003 |
| 4.80 |
0.0015 |
| 9.60 |
0.0029 |
| 48.00 |
0.0148 |
| 96.00 |
0.0288 |
| 192.00 |
0.0573 |
| STEYEX |
0.0002083983 |
| Slope |
0.0002980968 |
| LOD (ng/g) |
2.3 |
| LOQ (ng/g) |
7.0 |
| 4. Linearity (area mAU*min) Fipronil |
|---|
| Conc. (ng/g) |
Mean Area |
| 1.06 |
0.0001 |
| 5.00 |
0.0005 |
| 10.60 |
0.0011 |
| 53.00 |
0.0058 |
| 106.00 |
0.0119 |
| 212.00 |
0.0243 |
| 4. Linearity (area mAU*min) Fipronil sulfone |
|
|---|
| Conc. (ng/g) |
Mean Area |
| 0.96 |
0.0003 |
| 4.80 |
0.0015 |
| 9.60 |
0.0029 |
| 48.00 |
0.0148 |
| 96.00 |
0.0288 |
| 192.00 |
0.0573 |
Materials
| Product No. |
Description |
|---|
| 1.50653.0001 |
Purospher® STAR RP-18 endcapped (3µm) 100x2.1 mm |
| 1.00033.2500 |
Acetonitrile gradient grade for liquid chromatography LiChrosolv® |
| 1.15333.2500 |
Water for chromatography (LC-MS grade) LiChrosolv® |
| |
or tap fresh from an appropriate Milli-Q® system |
| Z227447 |
Millex® syringe filter units, disposable, Durapore® PVDF, pore size 0.45 μm, non-sterile |
| 1.00573.1000 |
Ammonium acetate EMSURE® |
| 1.00063.1000 |
Acetic acid glacial (100%) EMSURE® |
| 1.06007.2500 |
Methanol gradient grade for liquid chromatography LiChrosolv® |
| Grocery store |
Eggs, noodles, Mayonnaise and Chicken meat |
| 1.06649.0500 |
Sodium sulphate anhydrous for analysis EMSURE® |
| 1.07711.1000 |
Sea sand extra pure |
| 1.19847.0001 |
LiChrolut® RP-18e |
| 55294-U |
Supel QuE non-buffered extraction tube 1 |
| 55408-U |
Supel QuE Z-Sep+ 2 mL tubes |
| 1.02827.2500 |
Cyclohexane |
| 1.00868.1000 |
Ethyl acetate |
| 46451-100MG |
Fipronil reference standard |
| 32333-50MG |
Fipronil sulfone reference standard |
