Development of a fast isocratic LC-MS/MS method for the high-throughput analysis of pyrrolizidine alkaloids in Australian honey

Abstract

Honey samples originating from Australia were purchased and analysed for targeted pyrrolizidine alkaloids (PAs) using a new and rapid isocratic LC-MS/MS method. This isocratic method was developed from, and is comparable with, a gradient elution method and resulted in no loss of sensitivity or reduction in chromatographic peak shape. Isocratic elution allows for significantly shorter run times (6 min), eliminates the requirement for column equilibration periods and, thus, has the advantage of facilitating a high-throughput analysis which is particularly important for regulatory testing laboratories. In excess of two hundred injections are possible, with this new isocratic methodology, within a 24-h period which is more than 50% improvement on all previously published methodologies. Good linear calibrations were obtained for all 10 PAs and four PA N-oxides (PANOs) in spiked honey samples (3.57–357.14 µg l⁻¹; R² ≥ 0.9987). Acceptable inter-day repeatability was achieved for the target analytes in honey with % RSD values (n = 4) less than 7.4%. Limits of detection (LOD) and limits of quantitation (LOQ) were achieved with spiked PAs and PANOs samples; giving an average LOD of 1.6 µg kg⁻¹ and LOQ of 5.4 µg kg⁻¹. This method was successfully applied to Australian and New Zealand honey samples sourced from supermarkets in Australia. Analysis showed that 41 of the 59 honey samples were contaminated by PAs with the mean total sum of PAs being 153 µg kg⁻¹. Echimidine and lycopsamine were predominant and found in 76% and 88%, respectively, of the positive samples. The average daily exposure, based on the results presented in this study, were 0.051 µg kg⁻¹ bw day⁻¹ for adults and 0.204 µg kg⁻¹ bw day⁻¹ for children. These results are a cause for concern when compared with the proposed European Food Safety Authority (EFSA), Committee on Toxicity (COT) and Bundesinstitut für Risikobewertung (BfR – Federal Institute of Risk Assessment Germany) maximum daily PA intake limit of 0.007 µg kg⁻¹ bw day⁻¹.

Graphical Abstract

Keywords:

• pyrrolizidine alkaloids (PAs)
• isocratic elution
• liquid chromatography-mass spectrometry (LC-MS/MS)
• honey
• food safety
• hazard analysis and critical control point (HACCP)

Additional information

Funding

This research work was funded by the Food for Health Research Initiative (FHRI) by the Irish Department of Agriculture, Fisheries and Food (DAFF) and the Health research Board (HRB); Project title ‘Safe and Healthy Foods’ [contract number 07FHRITAFRC5]. The Higher Education Authority (Programme for Research in Third-Level Institutions, Cycle 4 (PRTLI IV)) National Collaboration Programme on Environment and Climate Changes: Impacts and Responses is acknowledged for the capital investment in LC-MS. The authors would also like to the Department of Agriculture, Food & the Marine for granting an import licence (June 2012) for the purpose of importing Australian honey into Ireland for this research [licence number S10862]. No potential conflict of interest was reported by the author(s).