Pyrrolizidine alkaloids in honey: comparison of analytical methods.

Pyrrolizidine alkaloids (PAs) are a structurally 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 an HPLC-ESI-MS-MS approach, the other a sum parameter method utilising HRGC-EI-MS operated in the selected ion monitoring mode (SIM). To date, no fully validated or standardised method exists to measure the PA content in honey. To establish an LC-MS method, several hundred standard pollen analysis results of raw honey were analysed. Possible PA plants were identified and typical commercially available marker PA-N-oxides (PANOs). Three distinct honey sets were analysed with both methods. Set A consisted of pure Echium honey (61-80% Echium pollen). Echium is an attractive bee plant. It is quite common in all temperate zones worldwide and is 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 originating from Eupatorium spp. (0-15%), another common PA plant, usually characterised 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 beehives in areas with a high abundance of Jacobaea vulgaris (ragwort) from the Veluwe region (the Netherlands). J. vulgaris increasingly invades countrysides 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 results showed significantly different quantification results. The GC-MS sum parameter showed in average higher values (on 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.

Determination of patulin by online-SPE-LC.

The mycotoxin patulin mainly occurs in fruits and fruit-derived products. For its determination a newly developed method employing a simplified liquid-liquid partitioning step followed by an online-SPE-LC analysis with UV detection is presented. The sample is diluted with phosphate buffer and extracted with ethyl acetate. The extract is evaporated and re-dissolved. The online-SPE-LC analysis employs a styrene-divinylbenzene copolymere phase for the SPE step, on which the carbonate washing step is carried out as well. The final LC analysis with UV detection uses a Polaris C18A column. This method reaches a limit of quantification of 15 µg/kg (clear apple juice), with a standard deviation of 10.7% (matrix calibration, n=5; c (patulin)=50 µg/kg).

Ribonucleosides as minor milk constituents.

Ribonucleosides are minor milk constituents and show a typical pattern which is assumed to be species-specific. As well as the unmodified components adenosine, cytidine, guanosine, inosine, and uridine, modified compounds such as Nl-methyladenosine and N6-carbamoylthreonyladenosine--products of the transfer RNA catabolism--have been identified and quantified in individual and bulk herd (race: German black pied) milk samples throughout a whole lactation period. The results of our longitudinal study have shown that--with the exception of the colostral phase--the levels of these minor constituents vary only slightly throughout lactation. These findings imply that ribonucleosides are useful for characterizing milk of different species and technological treatment. Ribonucleosides were determined and balanced, for example, in the course of the churning process, showing that the pattern of these minor milk constituents is useful as a "fingerprint" that allows differentiation between the three butter types defined in the German Federal Butter Ordinance.

[Ribonucleosides in milk: characterization and determination of the concentration profile of these minor components throughout a lactation period]. / Ribonucleoside in Milch: Charakterisierung und Bestimmung des Konzentrationsprofils dieser minoren Komponenten über eine Laktationsperiode.

The ribonucleosides adenosine, cytidine, guanosine, inosine and uridine as well as the modified components N1-methyladenosine and N6-carbamoylthreonyladenosine were characterized and determined quantitatively as minor constituents in raw bovine milk by use of an automated high performance liquid chromatography system. The studies have shown that except for the colostral phase the ribonucleoside levels are constant throughout the whole lactation period. That means, there is a typical ribonucleoside pattern which is assumed to be species-specific.