Pyrrolizidine Alkaloids in Honey: Comparison of analytical methods
Résumé
Abstract: Pyrrolizidine alkaloids (PAs) are a structural diverse group of toxicologically relevant secondary plant metabolites. Currently, two analytical methods are used to determine PA-content in honey. To achieve reasonably high sensitivity and selectivity mass spectrometry detection is demanded. One method is a HPLC-ESI-MS-MS approach, the other a sum parameter method utilizing HRGC-EI-MS operated in the selected ion monitoring mode (SIM). To date, no fully validated or standardized method exists to measure the PA- content in honey. To establish a LC-MS method, several hundred standard pollen analysis results of raw honey were analyzed. Possible PA-plants were identified and typical commercially available marker-PAs and corresponding PANOs were assigned. Three distinct honey sets were analyzed with both methods. Set A consisted of pure Echium honey (61 to 80% Echium pollen). Echium is an attractive bee plant. It is quite common in all temperate zones world wide and one of the major reasons for PA-contamination in honey. Although, only echimidine/echimidine-N-oxide were available as reference for the LC-MS target approach, the results for both analytical techniques matched very well (n=8; PA-content ranging from 311 to 520 µg kg-1). The second batch (B) consisted of a set of randomly picked raw honeys, mostly originated from Eupatorium spp. (0 to 15%), another common PA-plant, usually characterized by the occurrence of lycopsamine-type PA. Again, the results showed good consistency in terms of PA-positive samples and quantification results (n= 8; ranging from 0 to 625 µg kg-1 retronecine equivalents). The last set (C) was obtained by consciously placing bee hives in areas with high abundance of Jacobaea vulgaris (ragwort) from the Veluwe region (The Netherlands). J. vulgaris increasingly infests country sides in Central Europe, especially areas with reduced farming or sites with natural restorations. Honey from two seasons (2007 and 2008) was sampled. While only trace amounts of ragwort pollen were detected (0-6.3%) in some cases extremely high PA-values were detected (n= 31; ranging from 0 to 13019 µg kg-1, average 1261 or 76 µg kg-1 for GC-MS and LC-MS, respectively). Here the result showed significant different quantification results. The GC-MS sum parameter showed in average higher values (in average differing by a factor 17). The main reason for the discrepancy is most likely the incomplete coverage of the J. vulgaris PA-pattern. Major J. vulgaris PAs like jacobine-type PAs or erucifoline/acetylerucifoline were not available as reference compounds for the LC-MS target approach. Based on the direct comparison both methods are considered from various perspectives and the respective individual strengths and weaknesses for each method are presented in detail.
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