Extraction and Analysis of Neonicotinoid Pesticides from Flower Blossoms Using Supel™ QuE and Ascentis® Express

By: Katherine K. Stenerson, Reporter US, Volume 32.1

Introduction
Neonicotinoids are systemic insecticides that are toxic to insects but do not affect mammals. They are water soluble and have the ability to migrate from treated soil into plant tissue, including fluids such as the nectar in flowers. These characteristics make them very useful for crop protection[1]. One neonicotinoid, imidacloprid, is currently the most widely used pesticide in the world[2]. The advent of the die-off of honey bees due to colony collapse disorder (CCD) has spawned investigation into pesticide exposure as the cause. Recent studies have indicated neonicotinoid pesticides as having detrimental effects on the brain cells of honey bees[3]. Bees can come into contact with these pesticides in several ways. Seeds that are treated with these pesticides can generate contaminated dust when using equipment that incorporates air for planting[4]. Also, some crops directly pollinated by honeybees such as cantaloupe and cucumber are treated with these pesticides[5]. Recently the European Union adopted a regulation restricting the use of three of these pesticides: clothianidin, thiamethoxam and imidacloprid[6].

In this application, the Quick, Easy, Cheap, Effective, Rugged, Safe (QuEChERS) approach was used to develop an extraction and cleanup method for the analysis of seven neonicotinoid pesticides in flower blossoms. Analysis of the final extracts was done by LC-MS/ MS on an Ascentis® Express C18 column. This column was chosen due to the high efficiency associated with its Fused-Core® particle structure, and its relatively low backpressure as compared to sub-2 micron UHPLC columns. This allowed the column to be used in a standard pressure HPLC system with a relatively high concentration of aqueous at the start of the mobile phase gradient.

Experimental
The blossoms used represent several types pollinated by honeybees: dandelion, sweet cherry and crab apple. The blossoms were picked fresh and then frozen prior to use. Prior to extraction, they were pulverized and spiked at 50 ng/g with a mixture of neonicotinoid pesticides. After 1 hour of wait time, extraction and cleanup proceeded as described in Table 1. Three replicate spikes and one unspiked sample were processed for each blossom type.

LC-MS/MS Analysis was performed using the conditions in Table 2. The MRMs used for each pesticide are listed in Table 3. Quantitation was performed against a matrix-matched calibration curve. Separate curves were prepared for each blossom type.

Table 1. Extraction and Cleanup Procedure

 

Table 2. LC-MS/MS Analysis Conditions
column:
mobile phase:
gradient:

flow rate:
column temp.:
detector:
injection:
Ascentis Express C18, 10 cm x 3.0 mm I.D., 2.7 µm (53814-U)
(A) 0.1% formic acid in water; (B) 0.1% formic acid in methanol
30% B from 0 to 5 min; to 100% B in 0.2 min; held at 100% B for 5.3 min; to 30% B in 0.5 min; held at 30% B for 5 min
0.5 mL/min
ambient
MS, ESI(+), MRM, see Table 3
3 µL

 

Table 3. MRMs Used
Name m/z
Acetamiprid 223.2/126.0
Clothianidin 250.0/132.0
Dinotefuran 203.2/129.2
Imadicloprid 256.0/175.2
Nitenpyram 271.2/225.0
Thiacloprid 253.0/125.8
Thiamethoxam 292.1/211.0

Results and Discussion
For QuEChERS extraction, sodium acetate (NAOAc) was chosen as the salt based on previously published findings[5]. For cleanup, several different sorbents were evaluated. It was found that PSA/C18 yielded the best recoveries while still reducing pigmentation in the sample extract. PSA/C18/ENVI-Carb™ yielded the cleanest extracts (no pigmentation), but very poor recovery for all the target compounds. The zirconia-based sorbents Z-Sep and Z-Sep+ were found to be incompatible with these pesticides, yielding very low recoveries. PSA alone yielded an extract with more pigmentation than PSA/C18, and recoveries were very poor.

A summary of the recoveries from the three blossom types obtained after extraction and PSA/C18 cleanup is summarized in Table 4. None of the target pesticides were detected in the unspiked blossoms. Excellent recoveries and low %RSD values were obtained for each target compound for all blossom types. The extract color after cleanup appeared slightly yellow for the dandelion and crab apple, and pale green for the cherry, and all were significantly lighter in color than uncleaned extract. The TICs appeared very clean for dandelion and crab apple (Figures 1 and 2). The cherry had the most matrix present (Figure 3), although it did not interfere with quantitation. The use of blossom specific matrix-matched standards compensated for any additional ion suppression that may have occurred.

Table 4. Average Percent Recoveries from Flower Blossoms Spiked at 50 ng/g, QuEChERS Extraction and Cleanup with PSA/C18.
n=3 Dandelion Sweet Cherry Crab Apple
Acetamiprid 99 (1) 96 (2) 105 (4)
Clothianidin 99 (6) 106 (2) 106 (7)
Dinotefuran 101 (2) 103 (4) 89 (4)
Imadicloprid 93 (4) 97 (4) 90 (3)
Nitenpyram 105 (1) 107 (2) 97 (6)
Thiacloprid 94 (2) 99 (1) 104 (2)
Thiamethoxam 98 (1) 99 (3) 93 (8)

 

Figure 1. TIC of Dandelion Extract, Spiked at 50 ng/g Pesticides; PSA/C18 Cleanup

 

Figure 2. TIC of Crab Apple Extract, Spiked at 50 ng/g Pesticides, PSA/C18 Cleanup

 

 

Figure 3. TIC of Sweet Cherry Blossom Extract, Spiked at 50 ng/g Pesticides, PSA/C18 Cleanup

Conclusions
The QuEChERS approach can be used in the extraction of neonicotinoid pesticides from various species of flower blossoms. For sample cleanup, this same approach can also be used. Of the various cleanup sorbents evaluated, PSA/C18 was found to yield the best recoveries for these pesticides, while providing adequate matrix removal. The Ascentis Express C18 column provided for an efficient separation with backpressure compatible with a standard HPLC system.

References

  1. The Latest Buzz on Honeybee Decline. www.chromatographonline.com
  2. Richter, K.R., What Would the World Be(e) Without Any Honeybees? Analytix Issue 3, 2013.
  3. Drahl, C., Erickson, B. Bad News Bees. Chemical and Engineering News, April 1, 2013, pp 13.
  4. Erickson, B.E., Curtailing Honey Bee Losses. Chemical and Engineering News, March 25, 2013, pp 30-31.
  5. Kamel, A. Refined Methodology for the Determination of Neonicotinoid Pesticides and Their Metabolites in Honey Bees and Bee Products by Liquid Chromatography- Tandem Mass Spectrometry (LC-MS/MS). J. Agric. & Food Chem. 2010, 58, 5926-5931.
  6. EU Commission Regulation No. 485/2013; Official Journal of the European Union; May 5, 2013.
Featured Products  
Description Cat. No.
Ascentis Express C18 Column, 10 cm x 3.0 mm, 2.7 µm 53814-U
Supel™ QuE Acetate Tube, 12 mL, pk. of 50 55234-U
Supel QuE PSA/C18 Tube, 2 mL, pk. of 100 55288-U
Centrifuge Tube, 50 mL 55248-U
Acetamiprid, PESTANAL® 33674
Thiacloprid, PESTANAL 37905
Imidacloprid, PESTANAL 37894
Clothianidin, PESTANAL 33589
Nitenpyram, PESTANAL 46077
Dinotefuran, PESTANAL 32499
Thiamethoxam, PESTANAL 37924
Formic Acid, Eluent Additive for LC-MS 14265
Low Adsorption QSertVial, 300 µL, with PTFE/silicone septa (w/slit) 29662-U

Trademarks
Supel, Ascentis, and ENVI-Carb are trademarks of Sigma-Aldrich Co. LLC.
PESTANAL is a registered trademarks of Sigma-Aldrich Laborchemikalien GmbH.
Fused-Core is a trademark of Advanced Materials Technology Inc.

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