A sustainable supramolecular deep eutectic solvent system for dispersive liquid–liquid microextraction followed by HPLC for the determination of melamine in milk samples

This work presents the development and application of an innovative green supramolecular deep eutectic solvent (SUPRADES)-based DLLME approach for the extraction and quantification of melamine in milk matrices. For this purpose, thymol and nonanoic acid were combined to create a hydrophobic DES, which was then utilized as the extraction solvent. Additionally, a ternary SUPRADES based on a hydrophilic DES composed of choline chloride and ethylene glycol and doped with β-cyclodextrin (β-CD) was prepared and applied as the disperser solvent. Critical parameters were optimized systematically, yielding the following optimal conditions: Extraction solvent volume: 150 μL; disperser solvent: 600 μL; β-CD: 1% (w/v); pH: 7; extraction time: 1 min; and sample solution volume: 5 mL. Interestingly, the salting-out step did not affect the extraction efficiency. The optimized approach showed good linearity (100–900 ng/mL) with R2 = 0.998 when validated with matrix-matched calibration standards. The LOD and LOQ were 27 and 81 ng/mL, respectively, and the enrichment factor was 41. Method accuracy and precision were evaluated through recovery studies using milk samples fortified with melamine at three concentration levels (200, 500, and 800 ng/mL), and the recovery values were obtained in the range of 92–107%, with RSD < 5%. A greenness score of 79 was attained by the method, as quantified by the advanced ComplexMoGAPI. Lastly, this technique was successfully applied to extract and determine melamine in real samples of powdered milk, infant formula, and raw milk with acceptable and satisfactory results.