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The use of recombinant bovine somatotropin (rbST) to enhance milk production is approved in several countries, but it is prohibited in the European Union. According to EU legislation, it is necessary to confirm positive screening results prior to enforcement. Although adequate screening assays are available nowadays, development of liquid chromatography tandem mass spectrometry (LC-MS/MS) confirmatory methods to detect low levels of rbST is still a challenge. Here, we present a novel approach using immuno-affinity enrichment on monolithic micro-columns in combination with state-of-the-art ultra-high pressure LC-MS/MS (UHPLC-MS/MS) detection. The developed approach enables detection and confirmation of rbST in serum at a decision limit (CCα) concentration of 0.8 ng mL−1. Furthermore, the method is easy to handle, robust and reproducible. We successfully applied the confirmatory method to serum samples from rbST treated cows that were found suspect after immunoassay-based screening. The use of rbST could be confirmed over 1 week after treatment, and the developed method demonstrated the sensitivity needed for effective control.Graphical AbstractGraphical summary of the workflow, for serum preparation, enrichment with monolith microcolumns and LC-MS/MS measurement of rbST

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In this study we have investigated the use of liposomes as a drug carrier system for cis-diamminedichloroplatinum(II) (cis-DDP) in order to reduce the nephrotoxicity with preservation of antitumor activity. Liposomes (PC/PS/Chol 10:1:4) were prepared using hydration media containing no or a relatively low concentration of NaCl. It was found that cis-DDP containing liposomes (lip cis-DDP) injected i.v. to IgM immunocytoma-bearing LOU/M rats at a dose of 1 mg cis-DDP/kg (cumulative dose 7 mg cis-DDP/kg) showed less antitumor activity than the free drug. The optimal cumulative dose of free cis-DDP for induction of antitumor activity in this tumor system is 7 mg/kg (7 X 1 mg/kg). At a dose of 2 mg lip cis-DDP/kg (cumulative dose 14 mg cis-DDP/kg) the antitumor activity could not be increased by choosing another phospholipid composition of the liposomes [DPPC/DPPG/Chol (10:1:10)]. cis-DDP incorporated in DPPC/DPPG/Chol liposomes showed a similar antitumor activity to cis-DDP incorporated in PC/PS/Chol liposomes. After an i.v. dose of 2 mg lip cis-DDP/kg (PC/PS/Chol) kidney damage was less compared to the treatment with free cis-DDP (1 mg/kg). However, after a single dose of 2 mg cis-DDP/kg or a cumulative dose of 8 or 16 mg cis-DDP/kg, kidneys of rats treated with lip cis-DDP contained twice as much Pt as after treatment with free cis-DDP. Moreover, after treatment with lip cis-DDP, a twofold increase of the amount of Pt in tumor tissue was measured. In vitro studies with Pt recovered from spleens obtained from rats treated with lip cis-DDP i.v. showed that based on the equal amounts of Pt recovered the antitumor activity of the recovered Pt was reduced, indicating inactivation of cis-DDP in vivo. As during treatment with free cis-DDP, recurrence of the tumor was observed during the continued treatment with lip cis-DDP. It was found that these recurrent tumors were resistant to further therapy with cis-DDP. In conclusion, cis-DDP encapsulation into liposomes decreased the nephrotoxicity. The antitumor activity of cis-DDP is preserved by liposome encapsulation when it was used at a dose of 2 mg/kg, but it was reduced in terms of earlier onset of regrowth.

A simple procedure is described for the enzymic digestion of intact solid tissue samples. The samples are digested in an enzyme suspension containing collagenase and subtilisin Carlsberg without additional grinding or homogenizing. Bacterial growth during digestion is reduced by addition of an antibiotic solution. The resulting digests contain only very small tissue fragments. The total digestion is carried out at physiological conditions of temperature and pH, which makes the procedure suitable for inclusion in extraction procedures in a broad pH range. Since a minimum of sample manipulation is required, the procedure can be appropriately included in pharmacokinetic studies in which large series of samples are involved.