Quantitative analysis of penicillins in porcine tissues, milk and animal feed using derivatisation with piperidine and stable isotope dilution liquid chromatography tandem mass spectrometry

Penicillins are used universally in both human and veterinary medicine. The European Union (EU) has established maximum residue levels (MRLs) for most ß-lactam antibiotics in milk and animal tissues and included them in the National Residue Monitoring Programs. In this study, a novel method is described for the determination and confirmation of eight penicillins in porcine tissues, milk and animal feed by liquid chromatography–tandem mass spectrometry (LC–MS/MS). To prevent degradation of penicillin residues during workup, a derivatisation procedure was developed, by which penicillins were converted to stable piperidine derivatives. Deuterated piperidine derivatives were synthesised for all relevant penicillins, enabling the use of isotope dilution for accurate quantification. Penicillin residues were derivatised in the crude extract with piperidine and isolated using solid-phase extraction. The penicillin piperidine derivatives were determined by LC–MS/MS. The method was validated at the current MRLs, which range from 25–300 µg kg−1 in muscle and kidney to 4–30 µg kg−1 in milk as well as at the target value of 100 µg kg−1 chosen for animal feed, according to the EU requirements for a quantitative confirmatory method. Accuracy ranged from 94–113% (muscle), 83–111% (kidney) and 87–103% (milk) to 88–116% (animal feed). Intra-day precision (relative standard deviation (RSD)r) ranged from 5–13% (muscle, n = 18), 4–17% (kidney, n = 7) and 5–18% (milk, n = 7) to 11–32% (animal feed, n = 18). Inter-day precision (RSDRL, n = 18) ranged from 6–23% (muscle) to 11–36% (animal feed). From the results, it was concluded that the method was fit for purpose at the target MRLs in animal tissue and target levels for animal feed

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

In 2013 the Dutch authorities issued a warning against a dietary supplement that was linked to 11 reported adverse reactions, including heart problems and in one case even a cardiac arrest. In the UK a 20-year-old woman, said to have overdosed on this supplement, died. Since according to the label the product was a herbal mixture, initial LC-MS/MS analysis focused on the detection of plant toxins. Yohimbe alkaloids, which are not allowed to be present in herbal preparations according to Dutch legislation, were found at relatively high levels (400–900 mg kg–1). However, their presence did not explain the adverse health effects reported. Based on these effects the supplement was screened for the presence of a β-agonist, using three different biosensor assays, i.e. the validated competitive radioligand β2-adrenergic receptor binding assay, a validated β-agonists ELISA and a newly developed multiplex microsphere (bead)-based β-agonist assay with imaging detection (MAGPIX®). The high responses obtained in these three biosensors suggested strongly the presence of a β-agonist. Inspection of the label indicated the presence of N-isopropyloctopamine. A pure standard of this compound was bought and shown to have a strong activity in the three biosensor assays. Analysis by LC-full-scan high-resolution MS confirmed the presence of this ‘unknown known’ β3-agonist N-isopropyloctopamine, reported to lead to heart problems at high doses. A confirmatory quantitative analysis revealed that one dose of the preparation resulted in an intake of 40–60 mg, which is within the therapeutic range of this compound. The case shows the strength of combining bioassays with chemical analytical techniques for identification of illegal pharmacologically active substances in food supplements