Development and validation of a QuEChERS method for the analysis of 5-nitroimidazole traces in infant milk-based samples by ultra-high performance liquid chromatography-tandem mass spectrometry

Abstract

QuEChERS (Quick, Easy, Cheap, Effective, Rugged and Safe) methodology in combination with UHPLC–MS/MS is proposed for the determination of 5-nitroimidazole (5-NDZ) residues in infant milk-based products. Chromatographic separation was accomplished in a C18 Zorbax Eclipse Plus RRHD (50 × 2.1 mm, 1.8 μm) column under gradient elution conditions. Mobile phase consisted of 0.025% (v/v) aqueous formic acid (eluent A) and MeOH (eluent B), and was supplied at a flow rate of 0.5 mL/min. Under these conditions, eleven 5-NDZs including three metabolites were separated in less than 4 min. A novel QuEChERS method was optimized, and primary-secondary amine (PSA) sorbent was selected as clean-up agent. In addition, the proposed QuEChERS procedure was compared with other sample preparation methodologies, which are usually used in the analysis of 5-NDZs, namely solid phase extraction (SPE) using mixed cation exchange (MCX) cartridges and molecularly imprinted solid phase extraction (MISPE). Method comparison was carried out in terms of process efficiency, which includes matrix effect and extraction recovery. Higher process efficiency was generally achieved for QuEChERS and MISPE than for SPE. However, matrix effect was more significant for the non-selective QuEChERS method than for the selective MISPE. Higher extraction recoveries (63.2–94.1%) were observed for QuEChERS. The studied methods were validated in terms of linearity, detection limits (CCα), detection capabilities (CCβ) and precision, observing similar performance characteristics independently of the proposed sample treatment. CCα and CCβ ranged between 0.05 and 1.69 μg/L for all analytes and extraction techniques. QuEChERS-UHPLC–MS/MS method was also validated in terms of precision (relative standard deviations <10.3%), trueness (recovery>70.2%) and selectivity according to Regulation 2002/657/EC. It is proposed as a good alternative for the monitoring of 5-NDZ residues in enriched infant-milk based products and other similar milk based-products.