Determination of Fipronil and Fipronil Sulfone in Food Using Purospher® STAR RP-18 Endcapped Column and UV Detection

By: Anita Piper
Merck KGaA, Darmstadt, Germany

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
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