Validation of an ion-pair liquid chromatography–electrospray-tandem mass spectrometry method for the determination of heterocyclic aromatic amines in meat-based infant foods

Abstract

A method based on ion-pair liquid chromatography–electrospray tandem mass spectrometry (LC–MS/MS) is reported for determining heterocyclic aromatic amines (HAAs) in meat-based infant foods. The HAAs encompassed quinoline (IQ, MeIQ), quinoxaline (MeIQx), pyridine (PhIP), and carboline derivatives (AαC, Harman, Norharman) with d₃-IQ, ¹³C₂-MeIQx, and d₃-PhIP used as labelled internal standards. The method used extraction into acetone followed by a clean-up on an SCX solid-phase extraction column. LC separation was performed on a TSKgel ODS-80TS column (250 × 2.0 mm, 5 µm), the mobile phase being an ammonium formate–formic acid buffer (3.03 mM ammonium formate, pH = 2.8) aqueous solution–acetonitrile gradient at a flow rate of 0.2 ml min⁻¹. For unequivocal identification of each analyte, three ions were detected and chosen for selected reaction monitoring (SRM). Validation was carried out on lyophilized meat samples. Mean recoveries ranged between 78 ± 4% and 98 ± 2% for different analytes. Limits of quantification generally lower than 8 ng g⁻¹ were demonstrated in meat samples for the analytes investigated. The method exhibited a good linearity and repeatability. Robustness testing identified those factors which were statistically significant in influencing chromatographic separation and response, and indicated which parameters have to be strictly controlled for a reliable analysis of HAAs. In particular, the mobile-phase flow rate was found to be statistically significant (α = 0.05) for the capacity factor (k′) of all analytes except for AαC peak, whereas the mobile-phase pH resulted to be a critical parameter for the k′ values of IQ, MeIQ, and Norharman. The method was proved to be robust vs. resolution between IQ and MeIQ peaks. Among mass-spectrometric parameters, collision energy was found to significantly affect quantitative response of all analytes except that of IQ. The applicability of the method to the analysis of meat-based infant food samples was demonstrated.

Keywords:

• Heterocyclic aromatic amines
• ion-pair liquid chromatography–mass spectrometry
• validation
• robustness

Acknowledgements

This work was financially supported by MIUR (Ministero dell’Istruzione, dell’Università e della Ricerca, Italy), COFIN 2002 Project “Food safety. New analytical methods for the control of natural trace contaminants”.