Skip to main content
SpringerLink
Log in

Determination of Neonicotinoid Insecticides in Bee Products by Using Ultra-High-Performance Liquid Chromatography–Tandem Mass Spectrometry

  • Protocol
  • First Online:
Pesticide Toxicology

Part of the book series: Methods in Pharmacology and Toxicology ((MIPT))

Abstract

As a result of growing concern regarding the disappearance of pollinators, numerous studies have been carried out in different countries in an attempt to find the causes of this phenomenon. A large part of this study is aimed at justifying this concern due to the presence of pesticide residues from the crops visited by bees. In particular, neonicotinoid insecticides have been identified as one potential cause, and three of these (thiamethoxam, clothianidin, and imidacloprid) have been banned by the European Authorities. Therefore, the determination of these insecticides in different matrices, whether they be food, environmental, etc., is an important current object of study. In this chapter, we summarize the analytical methods and the most relevant findings of four recent publications devoted to developing and validating specific analytical methods for determining neonicotinoid insecticides in different bee products, namely, honey, beeswax, bee pollen, and royal jelly. In all cases, ultra-high-pressure liquid chromatography methods coupled with tandem mass spectrometry were employed, and different sample treatments were proposed depending on the bee matrix studied. These included solid-phase extraction (honey and royal jelly), Quick, Easy, Cheap, Effective, Rugged, and Safe based-methodologies (bee pollen and honey), solvent extraction combined with dispersive solid-phase extraction (beeswax), and dispersive liquid–liquid microextraction (royal jelly). All the methods were fully validated and applied to an analysis of samples from different origins, namely, experimental and commercial apiaries.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution

Subscribe and save

Springer+ Basic
$34.99 /Month
  • Get 10 units per month
  • Download Article/Chapter or eBook
  • 1 Unit = 1 Article or 1 Chapter
  • Cancel anytime
Subscribe now

Buy Now

Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 99.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 129.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 199.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

Similar content being viewed by others

Abbreviations

ACET:

Acetamiprid

ACN:

Acetonitrile

AF:

Samples spiked after sample treatment

BF:

Samples spiked before sample treatment

CAR:

Centro Apícola Regional

CLO:

Clothianidin

DAD:

Diode array detector

DLLME:

Dispersive liquid-liquid microextraction

DN:

Dinetofuran

dSPE:

Dispersive solid-phase extraction

EIC:

Extracted ion chromatogram

EMR-lipid:

Enhanced matrix removal-lipid

ESI:

Electrospray ionization

GC:

Gas chromatography

HPLC:

High-performance liquid chromatography

IMI:

Imidacloprid

IS:

Internal standard

LOD:

Limit of detection

LOQ:

Limit of quantification

MS:

Mass spectrometry

MS/MS:

Tandem mass spectrometry

NEOs:

Neonicotinoids

NT:

Nitenpyram

PSA:

Primary secondary amine

QC:

Quality control

QTOF:

Quadrupole time-of-flight

QuEChERS:

Quick, Easy, Cheap, Effective, Rugged, and Safe

SE:

Solvent extraction

THIA:

Thiacloprid

TMX:

Thiamethoxam

UHPLC:

Ultra-high-performance liquid chromatography

References

  1. Liang H, Zao CL, Huang F, Bai RE, Lü YB, Yan FM, Hao ZP (2015) Effects of imidacloprid and thiamethoxam as seed treatments on the early seedling characteristics and aphid-resistance of oilseed rape. J Integr Agric 14:2581–2589

    Article  Google Scholar 

  2. Yáñez KP, Martín MT, Bernal JL, Nozal MJ, Bernal J (2014) Trace analysis of seven neonicotinoid insecticides in bee pollen by solid–liquid extraction and liquid chromatography coupled to electrospray ionization mass spectrometry. Food Anal Methods 7:490–497

    Article  Google Scholar 

  3. Kasiotis KM, Anagnostopoulos C, Anastasiadou P, Machera K (2014) Pesticide residues in honeybees, honey and bee pollen by LC-MS/MS screening: reported death incidents in honeybees. Sci Total Environ 485-486:633–642

    Article  CAS  PubMed  Google Scholar 

  4. Yáñez KP, Bernal JL, Nozal MJ, Martín MT, Bernal J (2013) Determination of seven neonicotinoid insecticides in beeswax by liquid chromatography coupled to electrospray-mass spectrometry using a fused-core column. J Chromatogr A 1285:110–117

    Article  PubMed  Google Scholar 

  5. Chen M, Collins EM, Tao L, Lu C (2013) Simultaneous determination of residues in pollen and high-fructose corn syrup from eight neonicotinoid insecticides by liquid chromatography-tandem mass spectrometry. Anal Bioanal Chem 405:9251–9264

    Article  CAS  PubMed  Google Scholar 

  6. Parrilla-Vázquez P, Lozano A, Uclés S, Gómez-Ramos MM, Fernández-Alba AR (2015) A sensitive and efficient method for routine pesticide multiresidue analysis in bee pollen samples using gas and liquid chromatography coupled to tandem mass spectrometry. J Chromatogr A 1426:161–173

    Article  Google Scholar 

  7. Mullin CA, Frazier M, Frazier JL, Ashcraft S, Simonds R, vanEngelsdorp D, Pettis JS (2010) High levels of miticides and agrochemicals in North American apiaries: implications for honey bee health. PLoS One 5:e9754

    Article  PubMed  PubMed Central  Google Scholar 

  8. Fairbrother A, Purdy J, Anderson T, Fell R (2014) Risks of neonicotinoid insecticides to honeybees. Environ Toxicol Chem 33:719–731

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. VanEngelsdorp D, Evans C, Evans JD, Saegerman C, Mullin C, Haubruge E, Nguyen BK, Frazier M, Frazier J, Cox-Foster D, Chen Y, Underwood R, Tarpy D, Pettis J (2009) Colony collapse disorder: a descriptive study. PLoS One 4:1–17

    Article  Google Scholar 

  10. Lawal A, Wong RCS, Tan GH, Abdulra’uf LB, Alsharif AMA (2018) Recent modifications and validation of QuEChERS-dSPE coupled to LC–MS and GC–MS instruments for determination of pesticide/agrochemical residues in fruits and vegetables: review. J Chromatogr Sci 56:656–669

    Article  CAS  PubMed  Google Scholar 

  11. Garrido-Frenich AG, Romero-González R, Aguilera-Luiz MM (2014) Comprehensive analysis of toxics (pesticides, veterinary drugs and mycotoxins) in food by UHPLC-MS. TRAC-Trend Anal Chem 63:158–169

    Article  Google Scholar 

  12. Madej K, Kalenik TK, Piekoszewski W (2018) Sample preparation and determination of pesticides in fat-containing foods. Food Chem 269:527–541

    Article  CAS  PubMed  Google Scholar 

  13. Jiménez-López J, Llorent-Martínez EJ, Ortega-Barrales P, Ruiz-Medina A (2020) Analysis of neonicotinoid pesticides in the agri-food sector: a critical assessment of the state of the art. Appl Spectrosc Rev 58:613–646

    Article  Google Scholar 

  14. Tu X, Chen W (2020) Overview of analytical methods for the determination of neonicotinoid pesticides in honeybee products and honeybee. Crit Rev Anal Chem. https://doi.org/10.1080/10408347.2020.1728516

  15. Valverde S, Bernal JL, Martín MT, Nozal MJ, Bernal J (2016) Fast determination of neonicotinoid insecticides in bee pollen using QuEChERS and ultra-high performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry. Electrophoresis 37:2470–2477

    Article  CAS  PubMed  Google Scholar 

  16. Valverde S, Ares AM, Bernal JL, Nozal MJ, Bernal J (2018) Fast determination of neonicotinoid insecticides in beeswax by ultra-high performance liquid chromatography-tandem mass spectrometry using an enhanced matrix removal-lipid sorbent for clean-up. Microchem J 142:70–77

    Article  CAS  Google Scholar 

  17. Valverde S, Ibáñez M, Bernal JL, Nozal MJ, Hernández F, Bernal J (2018) Development and validation of ultra high performance-liquid chromatography–tandem mass spectrometry-based methods for the determination of neonicotinoid insecticides in honey. Food Chem 266:215–222

    Article  CAS  PubMed  Google Scholar 

  18. Valverde S, Ares AM, Arribas M, Bernal JL, Nozal MJ, Bernal J (2018) Development and validation of UHPLC–MS/MS methods for determination of neonicotinoid insecticides in royal jelly-based products. J Food Compos Anal 70:105–113

    Article  CAS  Google Scholar 

  19. Michlig MP, Merke J, Pacini A, Orellano E, Beldoménico HR, Repetti MR (2018) Determination of imidacloprid in beehive samples by UHPLC-MS/MS. Microchem J 143:72–81

    Article  CAS  Google Scholar 

  20. Hrynko I, Łozowicka B, Kaczyński P (2018) Liquid chromatographic MS/MS analysis of a large group of insecticides in honey by modified QuEChERS. Food Anal Methods 11:2307–2319

    Article  Google Scholar 

  21. Kamel A (2010) 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 58:5926–5831

    Article  CAS  PubMed  Google Scholar 

  22. Alarcón-Flores MI, Romero-González R, Martínez Vidal JL, Garrido Frenich A (2013) Multiclass determination of phytochemicals in vegetables and fruits by ultra high performance liquid chromatography coupled to tandem mass spectrometry. Food Chem 141:1120–1129

    Article  PubMed  Google Scholar 

  23. Bernal J, González D, Valverde S, Toribio L, Ares AM (2019) Improved separation of intact glucosinolates in bee pollen by using ultra-high-performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry. Food Anal Methods 12:1170–1178

    Article  Google Scholar 

  24. Glauser G, Schweizer F, Turlings TCJ, Reymond P (2012) Rapid profiling of intact glucosinolates in Arabidopsis leaves by UHPLC–QTOFMS using a charged surface hybrid column. Phytochem Anal 23:520–528

    Article  CAS  PubMed  Google Scholar 

  25. European Commission Directorate-General for Health and Food Safety, Document SANTE/12682/2019 (2019) https://www.eurl-pesticides.eu/userfiles/file/EurlALL/AqcGuidance_SANTE_2019_12682.pdf. Accessed 4 May 2021

  26. EURACHEM (2014) EURACHEM guide: the fitness for purpose of analytical methods—a laboratory guide to method validation and related topics. https://www.eurachem.org/images/stories/Guides/pdf/MV_guide_2nd_ed_EN.pdf. Accessed 4 May 2021

  27. David A, Botías C, Abdul-Sada A, Goulson D, Hill EM (2015) Sensitive determination of mixtures of neonicotinoid and fungicide residues in pollen and single bumblebees using a scaled down QuEChERS method for exposure assessment. Anal Bioanal Chem 407:8151–8162

    Article  CAS  PubMed  Google Scholar 

  28. Moreno-González D, Alcántara-Durán J, Gilbert-López B, Beneito-Cambra M, Cutillas VM, Rajski LR, Molina-Díaz A, García-Reyes AM (2019) Sensitive detection of neonicotinoid insecticides and other selected pesticides in pollen and nectar using nanoflow liquid chromatography orbitrap tandem mass spectrometry. J AOAC Int 101:367–373

    Article  Google Scholar 

  29. Hall MJ, Dang V, Bradbury SP, Coats JR (2020) Optimization of QuEChERS method for simultaneous determination of neonicotinoid residues in pollinator forage. Molecules 25:2732

    Article  CAS  PubMed Central  Google Scholar 

  30. Codling G, Al Naggar Y, Giesy JP, Robertson AJ (2016) Concentrations of neonicotinoid insecticides in honey, pollen and honey bees (Apis mellifera L.) in central Saskatchewan, Canada. Chemosphere 144:2321–2328

    Article  CAS  PubMed  Google Scholar 

  31. Li H, Yin J, Liu Y, Shang L (2012) Effect of protein on the detection of fluoroquinolone residues in fish meat. J Agric Food Chem 60:1722–1727

    Article  CAS  PubMed  Google Scholar 

  32. Niell S, Cesio V, Hepperle J, Doerk D, Kirsch L, Kolberg D, Scherbaum E, Anastassiades M, Heinzen H (2014) QuEChERS-based method for the multiresidue analysis of pesticides in beeswax by LC-MS/MS and GC×GC-TOF. J Agric Food Chem 62:3675–3683

    Article  CAS  PubMed  Google Scholar 

  33. Harriet J, Campá JP, Grajales M, Lhéritier C, Gómez Pajuelo A, Mendoza-Spina Y, Carrasco-Letelier L (2017) Agricultural pesticides and veterinary substances in Uruguayan beeswax. Chemosphere 177:77–83

    Article  CAS  PubMed  Google Scholar 

  34. Herrera López S, Lozano A, Sosa A, Hernando MD, Fernández-Alba AR (2016) Screening of pesticide residues in honeybee wax comb by LC-ESI-MS/MS. A pilot study. Chemosphere 163:44–53

    Article  PubMed  Google Scholar 

  35. Kaczyński P, Hrynko I, Łozowicka B (2017) Evolution of novel sorbents for effective clean-up of honeybee matrix in highly toxic insecticide LC/MS/MS analysis. Ecotoxicol Environ Saf 139:124–131

    Article  PubMed  Google Scholar 

  36. Al Naggar Y, Codling G, Giesy JP (2017) Human dietary intake and hazard characterization for residues of neonicotinoids and organophosphorus pesticides in Egyptian honey. Toxicol Environ Chem 99:1397–1408

    Article  Google Scholar 

  37. Tette PA, da Silva Oliveira FA, Pereira EN, Silva G, de Abreu Glória MB, Fernandes C (2016) Multiclass method for pesticides quantification in honey by means of modified QuEChERS and UHPLC–MS/MS. Food Chem 211:130–139

    Article  CAS  PubMed  Google Scholar 

  38. Sánchez-Hernández L, Hernández D, Martín MT, Nozal MJ, Higes M, Bernal JL (2016) Residues of neonicotinoids and their metabolites in honey and pollen from sunflower and maize seed dressing crops. J Chromatogr A 1428:220–227

    Article  PubMed  Google Scholar 

  39. Jovanov P, Guzsvány V, Franko M, Lazić S, Sakač M, Milovanović I, Nedeljković N (2014) Development of multiresidue DLLME and QuEChERS based LC-MS/MS methods for determination of selected neonicotinoid insecticides in honey liqueur. Food Res Int 55:11–19

    Article  CAS  Google Scholar 

  40. Hayes R, Ahmed A, Edge T, Zhang H (2014) Core-shell particles: preparation, fundamentals and applications in high performance liquid chromatography. J Chromatogr A 1357:36–52

    Article  CAS  PubMed  Google Scholar 

  41. Totti S, Fernández M, Ghini S, Picó Y, Fini F, Mañes J, Girotti S (2006) Application of matrix solid phase dispersion to the determination of imidacloprid, carbaryl, aldicarb, and their main metabolites in honeybees by liquid chromatography–mass spectrometry detection. Talanta 69:724–729

    Article  CAS  PubMed  Google Scholar 

  42. Bonmatin JM, Moineau I, Charvet R, Fleche C, Colin ME, Bengsch ER (2003) A LC/APCI-MS/MS method for analysis of imidacloprid in soils, in plants and in pollens. Anal Chem 75:2027–2033

    Article  CAS  PubMed  Google Scholar 

  43. Yáñez KP, Bernal JL, Nozal MJ, Martín MT, Bernal J (2013) Fast determination of imidacloprid in beeswax by liquid chromatography coupled to electrospray-mass spectrometry. Curr Anal Chem 9:495–503

    Article  Google Scholar 

  44. Hou J, Xie W, Zhang W, Li F, Qian Y, Sheng T, Mao R, Yao X (2019) Simultaneous determination of neonicotinoid insecticides and two metabolites in royal-jelly by LC-MS/MS. J Chinese Mass Spectr Soc 40:139–150

    CAS  Google Scholar 

  45. European Union Pesticide Database. Current MRLs values. Active substances detail. https://ec.europa.eu/food/plant/pesticides/eu-pesticides-database/active-substances/?event=search.as. Accessed 4 May 2021

Download references

Author information

Authors and Affiliations

  1. IU CINQUIMA, Analytical Chemistry Group, University of Valladolid, Valladolid, Spain

    Silvia Valverde, Ana M. Ares, José L. Bernal, María J. Nozal & José Bernal

  2. Department of Agricultural Chemistry and Food Science, Autonomous University of Madrid, Madrid, Spain

    Silvia Valverde

Authors
  1. Silvia Valverde
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Ana M. Ares
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. José L. Bernal
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. María J. Nozal
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. José Bernal
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to José Bernal .

Editor information

Editors and Affiliations

  1. Laboratory of Drug-Toxicology, Universidade da Beira Interior, Covilhã, Portugal

    Eugenia Gallardo

  2. National Institute of Legal Medicine and Forensic Sciences- South Branch, Lisbon, Portugal

    Mário Barroso

Rights and permissions

Reprints and permissions

Copyright information

© 2022 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Valverde, S., Ares, A.M., Bernal, J.L., Nozal, M.J., Bernal, J. (2022). Determination of Neonicotinoid Insecticides in Bee Products by Using Ultra-High-Performance Liquid Chromatography–Tandem Mass Spectrometry. In: Gallardo, E., Barroso, M. (eds) Pesticide Toxicology. Methods in Pharmacology and Toxicology. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1928-5_2

Download citation

  • DOI: https://doi.org/10.1007/978-1-0716-1928-5_2

  • Published:

  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-1927-8

  • Online ISBN: 978-1-0716-1928-5

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation