Determination of Neonicotinoids in Honey using a Chromolith RP-18 HPLC column and UV Detection

Introduction

Neonicotinoids are water soluble insecticides. They are used for crop protection and can migrate from treated soil into plant tissue and flower nectar. Neonicotinoids have a negative effect on brain cells of honey bees, and colony collapse can occur. [Clothianidin, Imidacloprid and Thiamethoxam are banned in Germany,  (EU) No. 485/2013].

This application focuses on HPLC testing of neonicotinoids in honey. The limit of detection (LOD) and limit of quantitation (LOQ) for neonicotinoids is better than 30 µM and 90 µM, respectively, when using HPLC combined with UV detection.

Experimental Conditions
Column: Chromolith® Performance RP-18 endcapped 100x2.0 mm  
Detection: Dionex Ultimate 3000 VWD-3400;
Thiamethoxam, Acetamiprid and Thiacloprid @ UV = 254 nm;
Dinotefuran, Nitenpyram, Imidacloprid and Clothianidin @ UV = 270 nm
(micro flow cell; 1.4 μL/7 mm)
 
Mobile phase A: Water + 0.1% formic acid  
Mobile Phase B: Acetonitrile + 0.1% formic acid  
Gradient: See table
 
Standard solution (200 μM): Pipette 500 μL of each of the stock solutions into a 25 mL volumetric flask, dilute to volume with acetonitrile to obtain a 200 μMol standard solution.  
Sample solution: Place 3 g of homogenized honey in a 50 mL extraction tube, add 10 mL deionized water and homogenize the sample. Add 15 mL acetonitrile and shake for 2 minutes at 2250 rpm.
Combine with Supel QuE acetate tube and mix for 1 minute. Centrifuge at 3,400 rpm for 5 minutes. Add 1 mL supernatant to a 2 mL extraction tube, containing cleanup sorbent.
Shake for 1 minute at 2250 rpm. Centrifuge for 3 minutes at 3,400 rpm. Place 1.5 mL of the supernatant into an eppendorf vial end evaporate to dryness under nitrogen at 30 °C.
Reconstitute the residue in 200 μL acetonitrile.
 
Sample fortification: Place 3 g of homogenized honey in a 50 mL extraction tube and spike with 5 μL of 200 μM neonicotinoid pesticide standard solution.
After 1 hour, proceed with extraction and cleanup as described in sample preparation part.
 
Injection volume: 1 μL  
Flow Rate: 250 μL/min  
Temperature: Ambient  
Pressure Drop: 27-32 bar (392-464 psi)  



Time %A %B
0 100 0
3 90 10
5 70 30
12 60 40
12.4 100 0
16 100 0

Chromatographic Data - blank run

  

Chromatographic Data - Standard Solution

Standard Solution (200 µM @ 254 nm)

Compound Retention Time (min) S/N Area (mAU*min) Tailing Factor
t0 void volume 1.20      
Dinotefuran 4.84 167.5 9.44 0.71
Nitenpyram 5.63 207.4 7.00 0.99
Thiamethoxam 6.27 544.6 18.48 0.99
Imidacloprid 6.87 458.6 13.87 1.04
Clothianidin 7.04 385.8 11.64 1.13
Acetamiprid 7.31 601.3 19.28 1.15
Thiacloprid 7.91 470.8 18.71 0.96

   


Standard Solution (200 µM @ 270 nm)

Compound Retention Time (min) S/N Area (mAU*min) Tailing Factor
t0 void volume 1.20      
Dinotefuran 4.84 258.8 13.85  0.70
Nitenpyram 5.63 428.7 6.36  0.96
Thiamethoxam 6.27 1061.5 10.07  1.11
Imidacloprid 6.87 640.3 16.05  0.99
Clothianidin 7.04 939.6 22.53  1.09
Acetamiprid 7.31 327.9 7.98  1.08
Thiacloprid 7.91 253.2 7.72  1.07

Chromatographic Data - Sample Solution

Sample Solution @ 254 nm

Compound Retention Time (min) S/N Area (mAU*min) Tailing Factor
t0 void volume 1.20      
Dinotefuran 4.84 167.5 9.44 0.71
Nitenpyram 5.63 207.4 7.00 0.99
Thiamethoxam 6.27 544.6 18.48 0.99
Imidacloprid 6.87 458.6 13.87 1.04
Clothianidin 7.04 385.8 11.64 1.13
Acetamiprid 7.31 601.3 19.28 1.15
Thiacloprid 7.91 470.8 18.71 0.96

   


Sample Solution @ 270 nm

Compound Retention Time (min) S/N Area (mAU*min) Tailing Factor
t0 void volume 1.20      
Dinotefuran 4.84 258.8 13.85  0.70
Nitenpyram 5.63 428.7 6.36  0.96
Thiamethoxam 6.27 1061.5 10.07  1.11
Imidacloprid 6.87 640.3 16.05  0.99
Clothianidin 7.04 939.6 22.53  1.09
Acetamiprid 7.31 327.9 7.98  1.08
Thiacloprid 7.91 253.2 7.72  1.07

Chromatographic Data - Sample Solution spiked

Sample Solution Spiked @ 254 nm

Compound Retention Time (min) S/N Area (mAU*min) Tailing Factor
t0 void volume 1.20      
Dinotefuran 4.77 7.2 0.0633  1.76
Nitenpyram 5.13 3.6 0.0562  0.97
Thiamethoxam 6.29 20.0 0.0857  1.05
Imidacloprid 6.77 9.2 0.0515  1.02
Clothianidin 6.94 12.3 0.0546  1.03
Acetamiprid 7.23 22.9 0.1139  1.09
Thiacloprid 7.82 1.6 0.0623  0.91

   


Sample Solution Spiked @ 270 nm

Compound Retention Time (min) S/N Area (mAU*min) Tailing Factor
t0 void volume 1.20      
Dinotefuran 4.77 28.1 0.1194  1.30
Nitenpyram 5.13 3.6 0.0557  0.89
Thiamethoxam 6.29 1.9 0.0480  1.10
Imidacloprid 6.77 2.8 0.0631  0.91
Clothianidin 6.94 16.1 0.1101  1.06
Acetamiprid 7.23 5.4 0.0305  1.00
Thiacloprid 7.82 0.5 0.0168  0.82


1. Specificity: Inject standard solution of neonicotinoids (200 µM) and determine the retention time and content of desired analyte @254 nm

    Retention Time (min) Area (mAU*min) Tailing Factor S/N
1 Dinutefuran 4.84 9.4436 0.71 167.5
2 Nitenpyram 5.63 6.9950 0.99 207.4
3 Thiamethoxam 6.27 18.4814 0.99 544.6
4 Imidacloprid 6.87 13.8702 1.04 458.6
5 Clothianidin 7.04 11.6378 1.13 385.8
6 Acetamiprid 7.31 19.2818 1.15 601.3
7 Thiacloprid 7.91 18.7140 0.96 470.8


1. Specificity: Inject standard solution of neonicotinoids (200 µM) and determine the retention time and content of desired analyte @270 nm

    Retention Time (min) Area (mAU*min) Tailing Factor S/N
1 Dinutefuran 4.84 13.8532  0.70 258.8
2 Nitenpyram 5.63 6.3631  0.96 428.7
3 Thiamethoxam 6.27 10.0740  1.11 1061.5
4 Imidacloprid 6.87 16.0465  0.99 640.3
5 Clothianidin 7.04 22.5338  1.09 939.6
6 Acetamiprid 7.31 7.9812  1.08 327.9
7 Thiacloprid 7.91 7.7241  1.07 253.2


2. Standard repeatability honey (spiked with 5 µL of a 200 µM standard solution) in mAU*min

  Dinutefuran @270 nm Nitenpyram @270 nm Thiamethoxam @254 nm Imidacloprid @270 nm Clothianidin @270 nm Acetamiprid @254 nm Thiacloprid @254 nm
STD 1 0.1133 0.0557 0.0857 0.0631 0.1101 0.1139 0.0623
STD 2 0.1143 0.0552 0.0868 0.0634 0.1135 0.1127 0.0632
STD 3 0.1128 0.0551 0.0864 0.0629 0.1115 0.1130 0.0633
STD 4 0.1136 0.0547 0.0850 0.0639 0.1112 0.1134 0.0625
STD 5 0.1139 0.0553 0.0859 0.0638 0.1116 0.1123 0.0629
Mean 0.1137 0.0552 0.0860 0.0634 0.1116 0.1131 0.0628
Standard Deviation 0.0006 0.0004 0.0007 0.0004 0.0012 0.0006 0.0004
(%) RSD 0.6 0.7 0.8 0.7 1.1 0.5 0.7


3. LOD & LOQ

  Dinutefuran @270 nm Nitenpyram @270 nm Thiamethoxam @254 nm Imidacloprid @270 nm Clothianidin @270 nm Acetamiprid @254 nm Thiacloprid @254 nm
Conc. (µM) Mean Area (mAU*min) Mean Area (mAU*min) Mean Area (mAU*min) Mean Area (mAU*min) Mean Area (mAU*min) Mean Area (mAU*min) Mean Area (mAU*min)
0.010 0.015 0.009 0.006 0.006 0.009 0.006 0.006
0.050 0.061 0.041 0.024 0.033 0.042 0.033 0.034
5.000 0.104 0.083 0.045 0.060 0.081 0.063 0.057
0.250 0.268 0.233 0.117 0.159 0.201 0.153 0.157
0.500 0.496 0.410 0.222 0.312 0.402 0.312 0.324
1.000 1.048 0.857 0.464 0.632 0.812 0.632 0.606
2.500 2.570 2.089 1.176 1.542 2.036 1.554 1.447
STEYEX 0.981245406 0.798553563 0.448929151 0.589925112 0.777408272 0.594302962 0.553751358
Slope 0.111210209 0.089714772 0.051411713 0.066058565 0.088907887 0.067687526 0.060146902
LOD (µM) 29.1 29.4 28.8 29.5 28.9 29.0 30.4
LOQ (µM) 88.2 89.0 87.3 89.3 87.4 87.8 92.1

4. Linearity (area mAU*min)

Dinutefuran @270 nm

Conc. (µM) Mean Area
0.01 0.015
0.05 0.061
0.10 0.104
0.25 0.268
0.50 0.496
1.00 1.048
2.50 2.570

Nitenpyram @270 nm

Conc. (µM) Mean Area
0.01 0.009
0.05 0.041
0.10 0.083
0.25 0.233
0.50 0.410
1.00 0.857
2.50 2.089

Thiamethoxam @254 nm

Conc. (µM) Mean Area
0.01 0.006
0.05 0.024
0.10 0.045
0.25 0.117
0.50 0.222
1.00 0.464
2.50 1.176

Imidacloprid @270 nm

Conc. (µM) Mean Area
0.01 0.006
0.05 0.033
0.10 0.060
0.25 0.159
0.50 0.312
1.00 0.632
2.50 1.542

Clothianidin @270 nm

Conc. (µM) Mean Area
0.01 0.009
0.05 0.042
0.10 0.081
0.25 0.201
0.50 0.402
1.00 0.812
2.50 2.036

Acetamiprid @254 nm

Conc. (µM) Mean Area
0.01 0.006
0.05 0.033
0.10 0.063
0.25 0.153
0.50 0.312
1.00 0.632
2.50 1.554

Thiacloprid @254 nm

Conc. (µM) Mean Area
0.01 0.006
0.05 0.034
0.10 0.057
0.25 0.157
0.50 0.324
1.00 0.606
2.50 1.447

Materials List

     
Related Links