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Method development and validation for total haloxyfop analysis in infant formulas and related ingredient matrices
Urairat Koesukwiwat1, Lukas Vaclavik2, Katerina Mastovska3
1Covance (Asia) Pte. Ltd., Covance Food Solutions, The Synergy, International Business Park, Singapore, 609917, Singapore.
Insights
A new method accurately measures total haloxyfop residues in infant formula, ensuring safety. This liquid chromatography-tandem mass spectrometry (LC-MS/MS) technique detects haloxyfop and its bound forms down to 0.003 mg/kg.
Area of Science:
- Analytical Chemistry
- Food Safety Analysis
- Environmental Chemistry
Background:
- European Commission directive 2006/141/EC sets a maximum residue limit of 0.003 mg/kg for total haloxyfop in infant formula.
- The residue definition includes haloxyfop, its esters, salts, and conjugates, requiring a method capable of analyzing all forms.
- Existing methods may not adequately address the analysis of total haloxyfop in complex infant formula matrices.
Purpose of the Study:
- To develop and validate a simple, sensitive method for total haloxyfop determination in infant formula and related ingredients.
- To ensure compliance with regulatory limits for haloxyfop residues in infant nutrition products.
- To provide a reliable analytical tool for monitoring haloxyfop contamination in challenging food matrices.
Main Methods:
- Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was employed for sensitive and selective detection.
- Sample preparation involved alkaline hydrolysis to release bound haloxyfop, followed by acetonitrile extraction.
- Dispersive solid-phase extraction (d-SPE) using magnesium sulfate and C18 sorbent was used for cleanup.
Main Results:
- The method successfully converted haloxyfop esters to the parent acid, enabling total haloxyfop analysis.
- Mean recoveries ranged from 92.2% to 114% across various infant formula matrices and fortification levels.
- Repeatability and reproducibility relative standard deviations (RSDs) were ≤14%, demonstrating method robustness.
Conclusions:
- The developed LC-MS/MS method is suitable for reliable quantification of total haloxyfop at the 0.003 mg/kg regulatory level.
- The method is effective for analyzing diverse infant formula types (powdered and liquid) and ingredient matrices.
- This validated method supports regulatory compliance and ensures the safety of infant formula regarding haloxyfop residues.
Abstract:
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.
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