Identification of an unknown β‐agonist in feed by liquid chromatography/bioassay/quadrupole time‐of‐flight tandem mass spectrometry with accurate mass measurement

Abstract

A new approach to the search for residues of unknown growth promoting agents such as anabolic steroids and beta-agonists in feed is presented. Following primary extraction and clean-up, samples are separated using gradient liquid chromatography (LC). The effluent is split towards two identical 96-well fraction collectors and an optional electrospray quadrupole time-of-flight mass spectrometry (QTOFMS) system for accurate mass measurement. One 96-well plate is used for a bioassay (enzyme-immuno assay, receptor assay) and will detect the bioactivity and position of the relevant peak in the chromatogram. The positive well in the second 96-well plate is used for identification by LC/QTOFMS/MS. The value of this LC/bioassay/QTOFMS/MS methodology is highlighted by the finding and structure elucidation of a new beta-agonist in a feed extract.

Evaluation of MISPE for the multi-residue extraction of β-agonists from calves urine

“…Further work is in progress to make imprints with templates expected to be key factors for effective multi-residual analysis, taking into account the described occurrence of new drugs in animal productions [22], with the aim both to improve recovery rates and to better understand the mechanisms affecting molecular recognition.…”

Screening for estrogen residues in calf urine: Comparison of a validated yeast estrogen bioassay and gas chromatography–tandem mass spectrometry

“…Consequently, targeted gas chromatography/mass spectrometry (GC/MS) and liquid chromatography-tandem mass spectrometry (LC/MS/MS) methods, which are set-up to monitor a few selected ions or MS/MS transitions, can only exert control to a limited degree (Hewitt et al 2002;Van Poucke et al 2002;Impens et al 2003;O'Keeffe et al 2003). Moreover, residues of designer steroids and b-agonists will escape control until the original illicit preparations are identified by NMR and mass spectrometry, and included in targeted residue analysis methods (Nielen et al 2003;Catlin et al 2004). …”

Dietary supplement for energy and reduced appetite containing the β-agonist isopropyloctopamine leads to heart problems and hospitalisations

“…However, effect-based assays are not able to identify the responsible compounds and ideally are combined with chemical analytical methods to confirm the presence of a regulated substance or to identify an unknown compound causing the effect. Effect-based assays and chemical methods are thus complementary and the added value of the combination has often been demonstrated Nielen et al 2003;Bovee & Pikkemaat 2009;Rijk et al 2009;Tooriaans et al 2010).…”

“…Effect-based screening methods are very useful in the area of growth promoters, where there is the incentive to develop novel compounds for the treatment of diseases, but which can also be misused to circumvent control on the illegal application of such substances (Nielen et al 2003. Besides the nuclear receptors, the β2-adrenergic receptor is the main target and receptor type that is responsive to anabolic agents that are illegally used in sports doping and meat production.…”

“…This is well below the quantities normally associated with β-agonist medicated feeds. Nielen et al used this competitive β2-adrenergic receptor binding assay to follow-up a 'false-positive' immunoassay result and reported the finding of an unknown β2-agonist (Nielen et al 2003). Many immunoassays have been developed for the screening of β2-agonists.…”