Even in aqueous samples, the determination of pesticide residues can be a challenge. Then how about complex foodstuffs that contain an abundance of interfering matrix components? Honey, for example, is known for its challenging matrix. In the case of neonicotinoids, the high sugar content does not prove to be a problem, however, but rather it is part of the solution.

By Guido Deußing

On April 27, 2018, a qualified majority of the European Union (EU) member states voted to ban the neonicotinoids Clothianidin, Thiamethoxam and Imidacloprid from use as active substances in plant protection products for outdoor use. These synthetically produced insecticides were previously used for seed coatings, protecting the seeds as well as the plants at early stages of growth, from being eaten by insects.  Neonicotinoids were not only applied directly to seeds, but also sprayed or distributed as granulate on fields, or added to irrigation water. Neonicotinoids have met and exceeded expectations for  their effectiveness. In terms of persistence, neonicotinoids degrade very slowly.

The wake-up buzz

The decision to ban three neonicotinoids, the most commonly used insecticides in the European Union, was greeted with mixed reactions: While beekeepers and environmental organizations were jubilant, representatives for agriculture, as well as seed companies and producers of crop protection solutions were rather less than enthusiastic. As it became clear that the game was over, further attempts at keeping the insecticides on the menu were quickly abandoned. The approval of clothianidin expired on January 31, 2019, that of thiamethoxam on April 30 of the same year, and that of imidacloprid on December 1, 2020. Finally, the approval of the neonicotinoid thiacloprid was withdrawn [1]. The only thing that remains in the running until February 28, 2033, is the neonicotinoid acetamiprid. For the sake of completeness: The neonicotinoids dinotefuran and nitenpyram never received approval for use in the EU.
Seven years after the initial approval in 2005, the European Food Safety Authority (EFSA) concluded that application of clothianidin, thiamethoxam and imidacloprid was accompanied by unacceptable side effects, not outweighed by the benefits. Neonicotinoids kill not only crop eating insects, but also beneficial insects such as the honeybee, the third most important animal in agriculture after cattle and pigs. Bees are  important pollinators, of course, it is not about the sweet byproduct. The EU Commission severely restricted the use of clothianidin, imidacloprid and thiamethoxam in 2013, particularly in bee-attractive crops including corn/maize, oilseed rape and sunflower. This was an important step that benefited not only honeybees, but also other pollen and nectar-collecting insects such as bumblebees, butterflies, and hoverflies.

Lethal effect

The term neonicotinoid indicates structural similarity to nicotine, a plant alkaloid found naturally in the leaves of the tobacco plant and, to a lesser extent, in other nightshade plants. Nicotine is a cholinergic neurotoxin; plants use it to defend themselves against herbivores. Nicotine is also a fast-acting drug. When smoking tobacco, for example, it is absorbed into the blood primarily via the lungs and it crosses the blood-brain barrier within seconds. It docks onto nicotinic receptors in the brain and, in addition to its addictive effect, triggers a series of physiological reactions.
The new nicotine-like neonicotinoids (Greek neos = new) have a similar effect. Unlike contact pesticides, which fulfill their task in direct contact with the surface of the harmful insect, the systemic neonicotinoids act from inside the plants. The agent is absorbed via the roots, the stomata of the leaves or the plant stems and distributed throughout the plant with the sap flow – without endangering the plant itself. On the other hand, insects that dig their mandibles into plant tissue or dip their proboscis into flowers to suck nectar or collect pollen are poisoned.
Once ingested, the active ingredient quickly affects the animals' nervous system, where it causes a permanent stimulus to the nerve cells, which leads to convulsions and ultimately to the death of the insects. Even small amounts, that do not kill the animals directly, can harm the bees. “A large part of the neonicotinoids can lead to an impairment of the brain processes of the bees, limiting their communication and orientation skills. The result is that the animals collect less pollen and need longer to return to the hive," according to the German Federal Information Center for Agriculture.

Far reaching consequences

By now, there is growing recognition that neonicotinoids are harmful to more than the health of insects. They are also suspected of endangering the lives of birds. Researchers in the Netherlands claim to have found an indirect effect between the concentration of pesticides in the environment and declining bird numbers. Last, but not least, neonicotinoids affect human health, and not just by killing the very animals that help us to generate agricultural yields through their pollination, above all the bees. Except for the joy of a wagging tail, what do we get back when we touch the fur of a dog that has a flea and tick repellent collar around its neck dispensing "low doses of the active ingredients imidacloprid and flumethrin" for months “. Researchers from the Universities of Tübingen and Konstanz (Loser et al.)[3] report that the breakdown products of certain neonicotinoids bind to receptors on dopamine-sensitive nerve cells and disrupt their function. Health consequences are to be expected from the amounts ingested when consuming fruit and vegetables sprayed with neonicotinoids [9].

State of affairs

As previously mentioned, the neonicotinoids clothianidin, thiamethoxam and imidacloprid were banned for use as active ingredients in plant protection products for use in outdoor crops in the EU as of May 17, 2018. The European Court of Justice (ECJ) confirmed the decision on May 6, 2021 in the second and last instance [7]. The ECJ drew a final line under the matter at the beginning of this year, more precisely on January 19, 2023, when it announced that from now on in the EU temporary exceptions - so-called emergency authorizations - for banned neonicotinoid pesticides can no longer be granted [8]. The exemption rule had certainly been used before then. The two neonicotinoids that were detected in honey in recent years, i.e. since the ban on the three neonicotinoids mentioned above came into force, were acetamiprid and thiacloprid, according to the Honey Association, which is based in Hamburg, Germany. Until it was banned in 2020, thiacloprid topped the list of the most commonly found neonicotinoids. Acetamiprid, which is said to be much less toxic to bees, then moved into the first position. Acetamiprid is mainly used in rapeseed cultivation, and residues are therefore mainly found in rapeseed honey.
Other neonicotinoids such as imidacloprid and thiamethoxam have not been detected recently or only detected in traces. Does this mean that these active ingredients are actually no longer used? "If the bees ingest these substances," the honey association states, "they die and therefore do not fly back to the hive. Accordingly, no residues would be detectable in the honey."

Positive developments

Below are highlights of the results of a study of honeys produced in Germany by the Federal Agency for Consumer Protection and Food Safety (BVL) [5], covering the years from 2018 to 2021 ...

  • between 258 and 359 honey samples were checked for pesticide residues, the majority came from Germany. The proportion of samples without pesticide residues increased from just over 50 percent in 2018 and 2019 to around 75 percent in 2020 and 2021.
  • In 2018, the active substance thiacloprid was detected in 67 honey samples, the active substance acetamiprid was detected in 16 samples, and in one case the maximum residue level (MRL) of Regulation (EU) No. 396/2005 was exceeded.
  • In 2019, thiacloprid was found in 91 honey samples. No other neonicotinoids were detected. 
  • In 2020, thiacloprid was determined in 40 honey samples. Acetamiprid was found in seven samples of honey, in three of those above the maximum residue limit (MRL).
  • In 2021, the active substance thiacloprid was detected in 33 honey samples, one of which had a value above the MRL. Residues of the active ingredient acetamiprid were found in 16 samples.

According to the BVL, the results presented here were published as part of the national reporting on pesticide residues in food. The evaluated samples were all taken and analyzed by official state agencies for food control. The figures refer only to Germany and do not allow any conclusions to be drawn about the situation in other EU member states. The BVL refers to the "European Union reports on pesticide residues in food" from the European Food Safety Authority (EFSA) [9] for more information.)

Consumer Protection first 

German beekeepers produced around 20,000 tons of honey in 2021, meeting 28 percent of domestic demand. In the same year, 78,000 tons of honey were imported to Germany from EU countries, which are required to implement EU regulations at a national level), but also from countries like Ukraine, Mexico, and Argentina that are not. To assume that honey-exporting countries refrain from using neonicotinoids, the world's most widely used insecticides, would be naïve and negligent. In the end, all products must be analyzed for potentially harmful residues in the interest of consumer protection. 
In 2019, researchers from the College of Apiary Sciences at Fujian University in China, used a highly efficient approach to determining neonicotinoids in honey [10]. The procedure most commonly used [7] involves a QuEChERS cleanup of the sample extract followed by HPLC-MS analysis [17]. Chen et al. reduced the sample preparation procedure to just a few steps, enabling the researchers to simplify and speed up the analysis of banned neonicotinoids in honey [10].

Sugaring up, sugaring out

Chen et al. relied on sugaring-out assisted liquid-liquid extraction (SULLE) for their sample preparation followed by LC-MS/MS determination of three neonicotinoids: Clothianidin, thiamethoxam and imidacloprid. This form of phase separation was described in a Chemical Engineering Science article in 2008 [9]. In it, Wang et al. report that acetonitrile (ACN), which is infinitely soluble in water, can be separated quickly and efficiently from an ACN-water solution by introducing monomeric sugars or disaccharides [19]. As an aside: The process of sugar assisted extraction (SULLE) is related to salting out assisted extraction (SALLE); a method for determining neonicotinoid pesticides using salting-out assisted liquid-liquid extraction (SALLE) was described by Wenbin Chen and colleague Xijuan Tu in the journal Molecules [10] (see also [11]). ACN is used as a solvent in the extraction of neonicotinoids due to its unlimited solubility in water and its suitability for HPLC.
Regarding the use of SULLE-HPLC/MS for determination of neonicotinoid levels in honey, Chen W et al. [10] describe the process as follows: "Due to the high sugar concentration in the honey matrix, when a sample is mixed with an ACN-water solution, it immediately triggers phase separation. Analytes are extracted into the supernatant ACN phase, which is injected into the HPLC-MS system for analysis. In their method optimization process, Chen et al. determined that two grams of honey mixed with four milliliters of an ACN-water mixture (v/v, 60:40) provided optimal extraction results, even compared with established procedures. The SULLE method was validated with detection limits of 21 to 27 μg/kg and limits of quantification between 70 and 90 μg/kg, values that can be significantly improved, for example, by using introducing an automated evaporative concentration step. The recovery rates ranged from 91.5 to 97.7 percent, with relative standard deviations in interday and intraday analysis of less than five percent. Eight real honey samples were successfully tested for the presence of banned neonicotinoids: the target analytes were not detected in any of the eight samples. As Chen W et al. report[10], the SULLE two-phase system provides fast phase separation, is environmentally friendly, and provides significant cleanup benefits.

Efficient Automation

When working to automate the determination of neonicotinoids in honey, application experts at GERSTEL, Inc. near Baltimore, Maryland decided to increase the efficiency of the SULLE method developed by Chen et al.  

The method was transferred to an autosampler (GERSTEL Multi-Purpose Sampler, MPS robotic pro) and fully automated [12]. The only manual operations that the laboratory staff need to perform are to weigh two grams of the honey sample into a 10 mL autosampler vial, add 10 μL of the internal standard (14.1 μg/mL d3-clothianidin and d4-imidacloprid) to the sample, seal the vial with a magnetically transportable cap and place it in the analysis system. All further steps, are performed by the autosampler (MPS robotic pro), including all necessary steps such as mixing and centrifuging the sample and loading it onto the connected HPLC-MS/MS system (Agilent Technologies 1260 HPLC, Agilent Ultivo Triple Quadrupole Mass spectrometer with jet stream electrospray source).  

The application scientists were fully satisfied with the results: Recoveries of the neonicotinoid compounds extracted from honey samples were 104 percent for acetamiprid, 81.5 percent for clothianidin, 94.1 percent for imidacloprid, 82.4 percent for thiamethoxam and 92.3 percent for thiacloprid. The accuracy was between 99.3 and 108 percent, the standard deviation and thus the precision of the analysis between 1.64 and 3.60 percent for all neonicotinoids extracted from honey samples.” Foster FD et al. report the limits of determination of all compounds at 2.8 μg/L. As needed, the limits of detection and quantification could be significantly reduced by using a more sensitive mass spectrometer, and/or by using the autosampler to evaporate the extract to dryness before taking up the residue in a much smaller volume of solvent and injecting it.


The platform used by Foster et al. offers a wide range of options for reacting quickly and flexibly to new application challenges, not least for tracking down possible substitutes for banned neonicotinoids and residues of other pesticides in various matrices. The neonicotinoids imidacloprid, thiamethoxam, clothianidin and thiacloprid are poisonous to bees and are banned in the EU, but not worldwide.

Sales figures for insecticides that are intended to replace neonicotinoids but are also known to have considerable negative environmental impact, have risen sharply. Substitutes for neonicotinoids include cyantraniliprole, chlorantraniliprole and sulfoxaflor. According to the EU Commission, the use of sulfoxaflor has been restricted to applications in greenhouses in Germany and Europe in 2022, as it was proven to be harmful to wild bees [13,14].


[1] Commission Implementing Regulation (EU) 2020/23 of 13 January 2020 concerning the non-renewal of the approval of the active substance thiacloprid [...], https://eur-lex.europa.eu/legal-content/EN/TXT/?uri=CELEX:32020R0023  (22.6.2023)
[2] European Commission, Neonicotinoids, https://food.ec.europa.eu/plants/pesticides/approval-active-substances/renewal-approval/neonicotinoids_en  (22.06.2023)
[3] Loser D et al. (2021), Acute effects of the imidacloprid metabolite desnitroimidacloprid on human nACh receptors relevant for neuronal signaling, Archiv of Toxicology 95:3695-3716, https://doi.org/10.1007/s00204-021-03168-z 
[4] https://curia.europa.eu/juris/document/document.jsf?text=&docid=240844&pageIndex=0&doclang=EN&mode=req&dir=&occ=first&part=1&cid=199835  (22.06.2023)
[5] https://curia.europa.eu/juris/document/document.jsf?text=&docid=269405&pageIndex=0&doclang=EN&mode=req&dir=&occ=first&part=1&cid=893  (15.05.2023)
[6] European Food Safety Agency (EFSA), Publications, https://www.efsa.europa.eu/en/publications  (15.05.2023)
[7] Chen W et al. (2019), Matrix-Induced Sugaring-Out: A Simple and Rapid Sample Preparation Method for the Determination of Neonicotinoid Pesticides in Honey, Molecules 24,15:2761, https://doi.org/10.3390/molecules24152761 
[8] Anastassiades M et al. (2003), Fast and easy multiresidue method employing acetonitrile extraction/partitioning and „dispersive solidphase extraction“ for the determination of pesticide residues in produce, J AOAC Int 86,2:412-431, https://doi.org/10.1093/jaoac/86.2.412 
[9] Wang B et al. (2008), Sugaring-out: A novel phase separation and extraction system, Chem. Eng. Sci. 2008,63: 2595–2600, https://doi.org/10.1016/j.ces.2008.02.004 
[10] Xijuan Tu und Wenbin Chen (2020), Miniaturized Salting-Out Assisted Liquid-Liquid Extraction Combined with Disposable Pipette Extraction for Fast Sample Preparation of Neonicotinoid Pesticides in Bee Pollen, Molecules 52,23:5703, https://doi.org/10.3390/molecules25235703 
[11] GERSTEL AppNote No. 230 (2022), Automated Salting-out Assisted Liquid-Liquid Extraction and Determination of Bisphenol A in Beverage Samples using a Robotic Autosampler and LC-MS/MS Platform, https://gerstel.com/en/Automated-Salting-out-Assisted 
[12] GERSTEL AppNote No. 248 (2023) Automated Sugaring-Out Assisted Liquid-Liquid Extraction and Determination of Neonicotinoids in Honey Samples using a Robotic Autosampler and LC-MS/MS Platform. https://gerstel.com/en/applications/food-beverages 
[13] The EU Commission restricts the use of sulfoxaflor to indoor use only […]  https://www.bee-life.eu/post/the-eu-commission-restricts-the-use-of-sulfoxaflor-to-indoor-use-only  
[14] COMMISSION IMPLEMENTING REGULATION (EU) 2022/686 of 28 April 2022 amending Implementing Regulations (EU) 2015/1295 and (EU) No 540/2011 as regards the conditions of approval of the active substance sulfoxaflor, […]  https://eur-lex.europa.eu/legal-content/EN/TXT/PDF/?uri=CELEX:32022R0686  (22.06.2023)