Bioactivitydirected identification of emerging unknown contaminants

Ideally, the sample should be extracted and purified in such a way to maintain all relevant contaminant classes of interest, the first challenge being clearly in the field of generic sample preparation development. The sample extract is analysed for bioactivity using a suitable assay (whole cell-, receptor-, immuno-, microbiological inhibition assay, etc.). When the sample has been screened suspect in one of these assays and the cause cannot be identified using the

Figure 4 Generic set-up for bioactivity-directed identification of emerging unknown contaminants.

prescribed confirmatory analysis method targeted for the presence of known contaminants, then possibly an emerging unknown contaminant is present in the sample. Next the unknown can be identified using the generic experimental setup shown in Figure 4. In short, the suspect sample is fractionated by gradient LC and narrow fractions are collected in duplicate using a parallel 96-well plate setup. One plate is re-analysed for bioactivity using the original bioassay yielding a bioactivity chromatogram or "biogram". The duplicate well number(s) of the suspect positions in the biogram are subjected to full-scan LC/TOFMS or GC/TOFMS techniques in order to perform chemical identification of the bio-active unknown, preferably using accurate mass measurement and isotope fitting which allows elemental composition assessment of the molecular ion and its collision-induced dissociation fragments. This approach has been demonstrated for cell-, immuno- and SPR biosensor assays and applied successfully to the identification of b-agonists in feed, hormones in urine and antibiotics in chicken muscle [48-50]. A key issue in this approach is the biocompatibility between the LC fraction and the subsequent bioactivity measurement. In this respect we have good experience with wells filled with a mixture of 2 p.L dimethylsulfoxide (DMSO) and 50 p.L water as a keeper solvent prior to the fractionation. The water-acetonitrile eluate from the LC gradient yields a large number of filled wells which are simply evaporated overnight in a fume hood, leaving the substances of interest in a tiny volume of water/DMSO. Of course, an LC fractionation is also directly compatible with atmospheric pressure ionization MS. That option requires only one well plate for fraction collection and bioactivity assessment and the suspect well number is then simply correlated with the retention time in the parallel MS system [51].

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