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

Koesukwiwat, Urairat, Vaclavik, Lukas, Mastovska, Katerina
Analytical and bioanalytical chemistry 2018 v.410 no.22 pp. 5521-5528
acetonitrile, alkaline hydrolysis, cleaning, emulsifiers, esters, haloxyfop, infant formulas, ingredients, liquid chromatography, magnesium sulfate, models, separation, sodium chloride, sodium hydroxide, solid phase extraction, sorbents, tandem mass spectrometry
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