Selective Determination of Fourteen Nitroimidazoles in Honey by High-Performance Liquid Chromatography–Tandem Mass Spectrometry

Abstract

A selective method for the determination of fourteen nitroimidazoles and their hydroxy-metabolites in honey was developed based on improved molecularly imprinted solid-phase extraction followed by liquid chromatography–tandem mass spectrometry. The separation of analytes was performed on a C₁₈ column using a mobile phase of 0.1% formic acid in acetonitrile and 0.1% formic acid in water with gradient elution. The method was suitable for metronidazole, hydroxymetronidazole, dimetridazole, ronidazole, hydroxydimetridazole, ipronidazole, hydroxyipronidazole, carnidazole, menidazole, nimorazole, ornidazole, secnidazole, ternidazole, and tinidazole. The procedure was evaluated according to EU Commission Decision 2002/657/EC requirements by determining linearity, specificity, recovery, repeatability, within-laboratory reproducibility, decision limit, detection capability, matrix effects, and stability. The method determined nitroimidazoles and their hydroxy-metabolites below the recommended concentration level of 3 µg kg⁻¹. The decision limits and detection capabilities ranged from 0.110 µg kg⁻¹ to 0.387 µg kg⁻¹ and from 0.179 µg kg⁻¹ to 0.508 µg kg⁻¹, respectively. The results from stability tests indicated that all analyzed nitroimidazoles were stable in honey stored at 4°C for at least 28 weeks and that elevated temperature and exposure to light exposure accelerated their degradation. The method was successfully applied to the analysis of a wide variety of honey samples.

Keywords:

• Honey
• HPLC
• Liquid chromatography tandem mass spectrometry
• Molecularly imprinted solid-phase extraction
• Nitroimidazoles

ACKNOWLEDGMENTS

The authors are grateful to Mrs. Janina Wrotniak for her technical assistance in this study.

Notes

^a ion transition used for quantification with metronidazole-d₃ as internal standard.

^b ion transition used for quantification with hydroxymetronidazole-d₂ as internal standard.

^c ion transition used for quantification with dimetridazole-d₃ as internal standard.

^d ion transition used for quantification with ronidazole-d₃ as internal standard.

^e ion transition used for quantification with hydroxydimetridazole-d₃ as internal standard.

^f ion transition used for quantification with ipronidazole-d₃ as internal standard.

^g ion transition used for quantification with hydroxyipronidazole-d₃ as internal standard.

^a A value of matrix effect > 100% indicates ionization enhancement, and a value of matrix effect < 100% indicates ionization suppression.

^a The nitroimidazoles were considered stable if measured values were within the range from 90% to 110% of nominal value as compared with freshly fortified samples.