Dispersive Liquid-Liquid Microextraction Method for the Simultaneous Determination of Four Isomers of Hexachlorocyclohexane and Six Pyrethroid Pesticides in Milk by Gas Chromatography Electron Capture Detector

  • Yuanling Zhao
  • Xi’ai Hou
  • Dongmei Qin
  • Dan LiuEmail author


In this study, a simple and rapid dispersive liquid-liquid microextraction method was established, and the residuals of four isomers of hexachlorocyclohexane and six kinds of pyrethroid pesticides in milk were simultaneously determined by gas chromatography electron capture detector (GC-ECD). The milk sample was first extracted with acetonitrile and cleaned with primary secondary amine (PSA). Then 0.5 mL of acetonitrile was mixed with 140 μL of cyclohexane and rapidly injected into 3 mL of pure water. After vortexing and centrifugation, the floating phase was removed with a 0.1-mL pipette into the GC-ECD. The type and volume of extraction solvent, volume of disperser solvent, volume of water, vortex time, and amount of salt were optimized. Under optimal extraction conditions, the ten pesticides showed a good linear relationship in a certain concentration range in milk matrix, and the correlation coefficients were greater than 0.99. The limits of detection ranged from 0.07 to 2 μg/kg, and the limits of quantitation ranged from 0.2 to 5 μg/kg. The average recovery rates were between 70.1% and 106.3%, and the relative standard deviations were less than 15.2%. This method can be used for the determination of hexachlorocyclohexane and pyrethroid pesticides in milk and for subsequent research.


Dispersive liquid-liquid microextraction Floating organic drop Milk Hexachlorocyclohexane and pyrethroid pesticides 


Compliance with Ethical Standards

Conflicts of Interest

Yuanling Zhao declares no conflict of interest. Xi’ai Hou declares no conflict of interest. Dongmei Qin declares no conflict of interest. Dan Liu declares no conflict of interest.

Ethical Approval

This article does not contain any studies involving animals and human participants performed by any of the authors.


  1. Abhilash PC, Singh V, Singh N (2009) Simplified determination of combined residues of lindane and other HCH isomers in vegetables, fruits, wheat, pulses and medicinal plants by matrix solid-phase dispersion (MSPD) followed by GC–ECD. Food Chem 113:267–271. CrossRefGoogle Scholar
  2. Aguinaga N, Campillo N, Viñas P, Hernández-Córdoba M (2007) Determination of 16 polycyclic aromatic hydrocarbons in milk and related products using solid-phase microextraction coupled to gas chromatography-mass spectrometry. Anal Chim Acta 596:285–290. CrossRefPubMedGoogle Scholar
  3. Anand N, Kundu A, Ray S (2018) A validated method for the determination of neonicotinoid, pyrethroid and organochlorine residues in human milk. Chromatographia 81:315–325. CrossRefGoogle Scholar
  4. Asensio-Ramos M, Ravelo-Pérez LM, González-Curbelo MÁ, Hernández-Borges J (2011) Liquid phase microextraction applications in food analysis. J Chromatogr A 1218:7415–7437. CrossRefPubMedGoogle Scholar
  5. Berger M, Löffler D, Ternes T, Heininger P, Ricking M, Schwarzbauer J (2016) The effect of distribution processes on the isomeric composition of hexachlorocyclohexane in a contaminated riverine system. Int J Environ Sci Technol 13:995–1008. CrossRefGoogle Scholar
  6. Berijani S, Assadi Y, Anbia M, Milani Hosseini MR, Aghaee E (2006) Dispersive liquid-liquid microextraction combined with gas chromatography-flame photometric detection. Very simple, rapid and sensitive method for the determination of organophosphorus pesticides in water. J Chromatogr A 1123:1–9. CrossRefPubMedGoogle Scholar
  7. Bordet F, Inthavong D, Fremy JM (2002) Interlaboratory study of a multiresidue gas chromatographic method for determination of organochlorine and pyrethroid pesticides and polychlorobiphenyls in milk, fish, eggs, and beef fat. J AOAC Int 85:1398–1409PubMedGoogle Scholar
  8. CAC (Codex Alimentarius Commission) (2018) Codex Online Database Pesticides Residues in Food. Link:
  9. Caldas SS, Costa FP, Primel EG (2010) Validation of method for determination of different classes of pesticides in aqueous sample by dispersive liquid-liquid microextraction with liquid chromatography-tandem mass spectrometric detection. Anal Chim Acta 665:55–62. CrossRefPubMedGoogle Scholar
  10. Campillo N, Peñalver R, Hernández-Córdoba M (2010) Determination of dimethylselenide and dimethyldiselenide in milk and milk by-products by solid-phase microextraction and gas chromatography with atomic emission detection. Talanta 80:1856–1861. CrossRefPubMedGoogle Scholar
  11. Campillo N, Viñas P, Férezmelgarejo G, Hernández-Córdoba M (2013) Dispersive liquid-liquid microextraction for the determination of macrocyclic lactones in milk by liquid chromatography with diode array detection and atmospheric pressure chemical ionization ion-trap tandem mass spectrometry. J Chromatogr A 1282:20–26. CrossRefPubMedGoogle Scholar
  12. Cao H, Chen XZ, Zhu Y, Li ZG, Wu XG, Zhu Y (2013) Determination of sulfonamides and quinolones in milk by QuEChERS and ultra performance liquid chromatography-tandem mass spectrometry. Food Sci Technol 38:323–329.
  13. Cao JP, Xie QL, Zhou JM, Yi ZH (2015) Application progress of dispersive liquid-liquid microextraction in food analysis. J Instrum Anal 34:616–624. CrossRefGoogle Scholar
  14. Cao JP, Di HW, Zhou JM, Liang YF, Xie QL (2016) A review on dispersive liquid-liquid microextraction method. J Instrum Anal 35:913–921. CrossRefGoogle Scholar
  15. Chung TL, Liao CJ, Chen MF (2010) Comparison of liquid–liquid extraction and solid-phase extraction for the determination of polycyclic aromatic hydrocarbons in the milk of Taiwan. J Taiwan Inst Chem E 41:178–183. CrossRefGoogle Scholar
  16. Dagnac T, Garcia-Chao M, Pulleiro P, Garcia-Jares C, Llompart M (2009) Dispersive solid-phase extraction followed by liquid chromatography-tandem mass spectrometry for the multi-residue analysis of pesticides in raw bovine milk. J Chromatogr A 1216:3702–3709. CrossRefPubMedGoogle Scholar
  17. Di Muccio A, Pelosi P, Barbini DA, Generali T, Ausili A, Vergori F (1997) Selective extraction of pyrethroid pesticide residues from milk by solid-matrix dispersion. J Chromatogr A 765:51–60. CrossRefPubMedGoogle Scholar
  18. Farajzadeh MA, Djozan D, Mogaddam MRA, Bamorowat M (2011) Extraction and preconcentration technique for triazole pesticides from cow milk using dispersive liquid-liquid microextraction followed by GC-FID and GC-MS determinations. J Sep Sci 34:1309–1316. CrossRefPubMedGoogle Scholar
  19. Farajzadeh MA, Djozan D, Mogaddam MRA, Norouzi J (2012) Determination of phthalate esters in cow milk samples using dispersive liquid-liquid microextraction coupled with gas chromatography followed by flame ionization and mass spectrometric detection. J Sep Sci 35:742–749. CrossRefPubMedGoogle Scholar
  20. Farajzadeh MA, Mogaddam MRA, Ghorbanpour H (2014) Development of a new microextraction method based on elevated temperature dispersive liquid–liquid microextraction for determination of triazole pesticides residues in honey by gas chromatography-nitrogen phosphorus detection. J Chromatogr A 1347:8–16. CrossRefPubMedGoogle Scholar
  21. Farajzadeh MA, Mogaddam MRA (2016) Low-density-solvent-based air-assisted liquid-liquid microextraction followed by gas chromatography with flame ionization detection for the determination of synthetic phenolic antioxidants in milk samples. J Sep Sci 39:1160–1167. CrossRefPubMedGoogle Scholar
  22. Gao XS, Zhang Y, Wang SX, Hua R, Zhang XM (2010) Determination of pyrethroid pesticide residues in milk by QuEChERS-gas chromatography. China Dairy Cattle 8:56–60. CrossRefGoogle Scholar
  23. Gao YL, Xu C, Liu SB, Sun P (2018) Determination of seven pyrethroid pesticides in liquid milk by dispersive liquid–liquid microextraction based on the solidification of a floating organic droplet followed by GC. Chromatographia 81:539–544. CrossRefGoogle Scholar
  24. Jank L, Hoff RB, Tarouco PC, Barreto F, Pizzolato TM (2012) β-Lactam antibiotics residues analysis in bovine milk by LC-ESI-MS/MS: a simple and fast liquid-liquid extraction method. Food Addit Contam Part A 29:497–507. CrossRefGoogle Scholar
  25. Karaseva NM, Amelin VG, Tret’yakov AV (2014) QuEChERS coupled to dispersive liquid-liquid microextraction for the determination of aflatoxins B1 and M1 in dairy foods by HPLC. J Anal Chem 69:461–466. CrossRefGoogle Scholar
  26. Khay S, El-Aty AMA, Choi JH, Shin EH, Shin HC, Kim JS, Chang BJ, Lee CH, Shin SC, Jeong JY, Shim JH (2009) Simultaneous determination of pyrethroids from pesticide residues in porcine muscle and pasteurized milk using GC. J Sep Sci 32:244–251. CrossRefPubMedGoogle Scholar
  27. Knobel G, Calimag-Williams K, Campiglia AD (2013) Analysis of polycyclic aromatic hydrocarbon metabolites in cow's milk by liquid–liquid extraction and synchronous room-temperature fluorescence spectroscopy. Anal Methods 5:1577–1582. CrossRefGoogle Scholar
  28. Kokosa JM (2013) Advances in solvent-microextraction techniques. Trends Anal Chem 43:2–13. CrossRefGoogle Scholar
  29. Liang P, Xu J, Li Q (2008) Application of dispersive liquid-liquid microextraction and high-performance liquid chromatography for the determination of three phthalate esters in water samples. Anal Chim Acta 609:53–58. CrossRefPubMedGoogle Scholar
  30. Li XJ, Yu H, Peng RF, Gan PS (2017) Determination of 19 sulfonamides residues in pork samples by combining QuEChERS with dispersive liquid–liquid microextraction followed by UHPLC–MS/MS. J Sep Sci 40:1377–1384. CrossRefPubMedGoogle Scholar
  31. Liu D, Min SG, Ping H, Song XZ (2016) The application of directly suspended droplet microextraction for the evaluation of phthalic acid esters in cow's milk by gas chromatography mass spectrometry. J Chromatogr A 1443:66–74. CrossRefPubMedGoogle Scholar
  32. Liu HL (2014) Research progress of overview on sample pretreatment of pesticides residue in milk. J Food Saf Qual 5:1419–1426 Google Scholar
  33. Liu XJ, Zhao AJ, Zhang AN, Liu HQ, Xiao WJ, Wang CJ, Wang XD (2011) Dispersive liquid-liquid microextraction and gas chromatography-mass spectrometry determination of polychlorinated biphenyls and polybrominated diphenyl ethers in milk. J Sep Sci 34:1084–1090. CrossRefPubMedGoogle Scholar
  34. Mahmoudpour M, Mohtadinia J, Ansarin M, Nemati M (2016) Dispersive liquid–liquid microextraction for HPLC-UV determination of PAHs in milk. J AOAC Int 99:527–533. CrossRefPubMedGoogle Scholar
  35. Maragou NC, Lampi EN, Thomaidis NS, Koupparis MA (2006) Determination of bisphenol A in milk by solid phase extraction and liquid chromatography-mass spectrometry. J Chromatogr A 1129:165–173. CrossRefPubMedGoogle Scholar
  36. Miao XX, Liu DB, Wang YR, Yang YY, Yang XY, Gong HR (2015) Modified QuEChERS in combination with dispersive liquid–liquid microextraction based on solidification of the floating organic droplet method for the determination of organophosphorus pesticides in milk samples. J Chromatogr Sci 53:1813–1820. CrossRefPubMedGoogle Scholar
  37. Morelli-Cardoso MHW, Cardozo RTM, Mello JL, Abrantes S, Menezes KMP (1999) Extraction and clean-up method for the determination of twenty organochlorine pesticide residues in tomatoes by GLC-ECD. J Sep Sci 22:619–622.<619::AID-JHRC619>3.0.CO;2-0 CrossRefGoogle Scholar
  38. Rezaee M, Yamini Y, Faraji M (2010) Evolution of dispersive liquid–liquid microextraction method. J Chromatogr A 1217:2342–2357. CrossRefPubMedGoogle Scholar
  39. Rúbies A, Guo LL, Centrich F, Granados M (2016) Analysis of non-steroidal anti-inflammatory drugs in milk using QuEChERS and liquid chromatography coupled to mass spectrometry: triple quadrupole versus Q-Orbitrap mass analyzers. Anal Bioanal Chem 408:5769–5778. CrossRefPubMedGoogle Scholar
  40. Salas JH, González MM, Noa M, Pérez NA, Díaz G, Gutiérrez R, Zazueta H, Osuna I (2003) Organophosphorus pesticide residues in Mexican commercial pasteurized milk. J Agric Food Chem 51:4468–4471. CrossRefPubMedGoogle Scholar
  41. Singh S, Nelapati K (2017) Effect of food processing on degradation of hexachlorocyclohexane and its isomers in milk. Vet World 10:270–275. CrossRefPubMedPubMedCentralGoogle Scholar
  42. Shamsipur M, Najmeh Y, Ghambarian M (2016) Combination of solid-phase extraction with dispersive liquid–liquid microextraction followed by GC–MS for determination of pesticide residues from water, milk, honey and fruit juice. Food Chem 204:289–297. CrossRefPubMedGoogle Scholar
  43. Tuncel SG, Şenlik D (2016) Determination of phthalates in milk by ultrasound-assisted dispersive liquid–liquid microextraction and gas chromatography–mass spectrometry. Anal Lett 49:1334–1343. CrossRefGoogle Scholar
  44. Wang S, Yang S, Liu F, Xue J, You X (2012) Review on the application of liquid phase microextraction in pesticide residue analysis. Chinese J Pestic Sci 14:461–474. CrossRefGoogle Scholar
  45. Yu X, Ang HC, Yang HS, Zheng C, Zhang YQ (2016) Low temperature cleanup combined with magnetic nanoparticle extraction to determine pyrethroids residue in vegetables oils. Food Control 74:112–120. CrossRefGoogle Scholar
  46. Zgoła-Grześkowiak A, Grześkowiak T (2011) Dispersive liquid-liquid microextraction. Trends Anal Chem 30:1382–1399. CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Department of Applied Chemistry, College of ScienceChina Agricultural UniversityBeijingPeople’s Republic of China
  2. 2.Institute Control of AgrochemicalsMinistry of AgricultureBeijingPeople’s Republic of China

Personalised recommendations