Analytical and Bioanalytical Chemistry

, Volume 410, Issue 22, pp 5521–5528 | Cite as

Method development and validation for total haloxyfop analysis in infant formulas and related ingredient matrices using liquid chromatography-tandem mass spectrometry

  • Urairat Koesukwiwat
  • Lukas Vaclavik
  • Katerina MastovskaEmail author
Research Paper
Part of the following topical collections:
  1. Food Safety Analysis


According to the European Commission directive 2006/141/EC, haloxyfop residue levels should not exceed 0.003 mg/kg in ready-to-feed infant formula, and the residue definition includes sum of haloxyfop, its esters, salts, and conjugates expressed as haloxyfop. A simple method for total haloxyfop analysis in infant formula and related ingredient matrices was developed and validated using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The sample preparation consisted of an alkaline hydrolysis with methanolic sodium hydroxide to release haloxyfop (parent acid) from its bound forms prior to the extraction with acetonitrile. A mixture of magnesium sulfate (MgSO4) and sodium chloride (NaCl) (4:1, w/w) was added to the extract to induce phase separation and force the analyte into the upper acetonitrile-methanol layer and then a 1-mL aliquot was subsequently cleaned up by dispersive solid phase extraction with 150 mg of MgSO4 and 50 mg of octadecyl (C18) sorbent. The analytical procedure was developed and carefully optimized to enable low-level, total haloxyfop analysis in a variety of challenging matrices, including infant formulas and their important high-carbohydrate, high-protein, high-fat, and emulsifier ingredients. The final method was validated in two different laboratories by fortifying samples with haloxyfop and haloxyfop-methyl, which was used as a model compound simulating bound forms of the analyte. Mean recoveries of haloxyfop across all fortification levels and evaluated matrices ranged between 92.2 and 114% with repeatability, within-lab reproducibility, and reproducibility RSDs ≤ 14%. Based on the validation results, this method was capable to convert the haloxyfop ester into the parent acid in a wide range of sample types and to reliably identify and quantify total haloxyfop at the target 0.003 mg/kg level in infant formulas (both powdered and ready-to-feed liquid forms).

Graphical abstract

LC-MS/MS-based workflow for the determination of the total haloxyfop in infant formula and related ingredients


Pesticides Phenoxy acid herbicides Alkaline hydrolysis QuEChERS Infant formula Liquid chromatography-tandem mass spectrometry 



The authors wish to thank Zdenka Veprikova, Hana Novotna, and Lucie Drabova from the University of Chemistry and Technology in Prague, Czech Republic, for their contribution to the method development. Jean-Francois Halbardier, Erika Deal, and Shi Ting Ong from Covance Food Solutions are acknowledged for their contribution and support to the method validation.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.


  1. 1.
    Anastassiades M, Roux D, Mack D, Sigalov I. Advance in the analysis of acidic pesticides. EPRW 2010 Poster presentation; 2010. ( Accessed Sep 2015.
  2. 2.
    European Food Safety Authority (EFSA). Reasoned opinion on the review of the existing maximum residue levels (MRLs) for haloxyfop-P according the Article 12 of Regulation (EC) No 396/2005. EFSA J. 2014;12:8–60.Google Scholar
  3. 3.
    FAO Manual on the submission and evaluation of pesticide residues data for the estimation of maximum residue levels in food and feed, 1st Edition 2009. ( Accessed Oct 2017.
  4. 4.
    Commision Regulation No. 2006/141/EC of 22 December 2006 on infant formulae and follow-on formulae and amending Directive 1999/21/EC. Off J Eur Union, 30.12.2006, L 401/1.Google Scholar
  5. 5.
    EU Reference Laboratory for Residues of Pesticides. Analysis of acidic pesticides using QuEChERS (EN15662) and acidified QuEChERS method (reported by EURL-SRM, version 1, last update). 2015;20:05.Google Scholar
  6. 6.
    EU Reference Laboratory for Residues of Pesticides. Phenoxyalkanoic acids in milk using modified QuEChERS method (version 1, last update). 2014;10:04.Google Scholar
  7. 7.
    EU Reference Laboratory for Residues of Pesticides. Analysis of phenoxyalkanoic acids in milk using QuEChERS method and LC-MS/MS (version 2, last update) 05.05.2014.Google Scholar
  8. 8.
    Santilio A, Stefanelli P, Dommarco R. Fast determination of phenoxy acid herbicides in carrots and apples using liquid chromatography coupled triple quadrupole mass spectrometry. J Environ Sci Health B. 2009;44:584–90.CrossRefPubMedGoogle Scholar
  9. 9.
    Crosby DG. Metabolism of herbicides, metabolites of 2,4-dichlorophenoxyacetic acid (2,4-D) in bean plants. J Agric Food Chem. 1964;12:3–6.CrossRefGoogle Scholar
  10. 10.
    Bristol DW, Cook LW, Koterba MT, Nelson DC. Determination of free and hydrolyzable residues of 2, 4-dichlorophenoxyacetic acid and 2, 4-dichlorophenol in potatoes. J Agric Food Chem. 1982;30:137–44.CrossRefGoogle Scholar
  11. 11.
    Steinwandter H. Contributions to the analysis of chlorophenoxy acids – I. A simple method for the determination of five chlorophenoxyalkane carboxylic acids in cereals. Fresenius J Anal Chem. 1989;334:133–5.CrossRefGoogle Scholar
  12. 12.
    Schaner A, Konecny J, Luckey L, Hickes H. Determination of chlorinated acid herbicides in vegetation and soil by liquid chromatography/electrospray-tandem mass spectrometry. J AOAC Int. 2007;90:1402–10.PubMedGoogle Scholar
  13. 13.
    EU Reference Laboratory for Residues of Pesticides. Analysis of acidic pesticides in wheat flour samples by LC-MS/MS using the QuEChERS method, incl. optional alkaline hydrolysis to release covalently bound compounds (last update) 07.05.2007).Google Scholar
  14. 14.
    Santilio A, Stefanelli P, Girolimetti S, Dommarco R. Determination of acidic herbicides in cereals by QuEChERS extraction and LC/MS/MS. J Environ Sci Health B. 2011;46:535–43.PubMedGoogle Scholar
  15. 15.
    Sack C, Vonderbrink J, Smoker M, Smith RE. Determination of acid herbicides using modified QuEChERS with fast switching ESI+/ESI LC-MS/MS. J Agric Food Chem. 2015;63:9657–65.CrossRefPubMedGoogle Scholar
  16. 16.
    Steinborn A, Alder L, Spitzke M, Dork D, Anastassiades M. Development of a QuEChERS-based method for the simultaneous determination of acidic pesticides, their esters, and conjugates following alkaline hydrolysis. J Agric Food Chem. 2017;65:1296–305.CrossRefPubMedGoogle Scholar
  17. 17.
    Løkke H. Analysis of free and bound chlorophenoxy acids in cereals. Bull Environ Contam Toxicol. 1975;13:730–6.CrossRefPubMedGoogle Scholar
  18. 18.
    Anastassiades M, Lehotay SJ, Stajnbaher D, Schenck FJ. Fast and easy multiresidue method employing acetonitrile extraction/partitioning and “dispersive solid-phase extraction” for the determination of pesticide residues in produce. J AOAC Int. 2003;86:412–31.PubMedGoogle Scholar
  19. 19.
    Guidance document on analytical quality control and method validation procedures for pesticides residues analysis in food and feed. SANTE/11945/2015 Supersedes SANCO/12571/2013 Implemented by 01/01/2016.Google Scholar
  20. 20.
    Lehotay SJ, Mastovska K, Yun SJ. Evaluation of two fast and easy methods for pesticide residue analysis in fatty food matrixes. J AOAC Int. 2005;88:630–8.PubMedGoogle Scholar
  21. 21.
    Mastovska K, Zulkoski J, Zweigenbaum J. Triggered MRM LC/MS/MS method development-practical considerations for MRM optimization using Agilent MassHunter Optimizer software. In: Agilent Technologies publication number 5991-7195EN; 2017.Google Scholar
  22. 22.
    Commission Regulation No. 2006/125/EC of 5 December 2006 on processed cereal-based foods and baby foods for infants and young children. Off J Eur Union, 6.12.2006, L 339/16.Google Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  • Urairat Koesukwiwat
    • 1
  • Lukas Vaclavik
    • 2
  • Katerina Mastovska
    • 3
    Email author
  1. 1.Covance (Asia) Pte. Ltd., Covance Food SolutionsSingaporeSingapore
  2. 2.Covance Laboratories Inc., Covance Food SolutionsNorth YorkshireUK
  3. 3.Covance Laboratories Inc.Covance Food SolutionsMadisonUSA

Personalised recommendations