Recombinant bovine growth hormone identification and the kinetic of elimination in rainbow trout treated by LC-MS/MS.

The efficiency of the administration of recombinant bovine growth hormone (rbGH) to enhance fish growth has been widely reported in the literature. Although its use is probable and has been described in several countries, rbGH is prohibited in the European Union (Council Decisions 1994/936/EC, 1999/879/EC). In this context, an analytical strategy was optimised in order to identify rbGH-treated fish. Currently, one of the most difficult challenges for the detection of rbGH in fish is probably the choice of the matrix and the corresponding available quantity for analysis. Therefore, based on a previous efficient protocol developed for mammalian species, a method was adapted for very limited serum volume (50 µl) and was successfully implemented to analyse serum collected from seven trout treated with rbGH. The detection of rbGH was possible from the very first day after administration and the hormone could easily be identified at least for 1 month with levels in the range 5-10 µg ml(-1). The limits of detection (LODs) estimated around 0.5 µg ml(-1) rbGH in fish serum are far below observed concentrations in incurred samples and therefore attest to the relevance of the developed protocol.

Development and validation of an enzyme-linked immunosorbent assay for the detection of circulating antibodies raised against growth hormone as a consequence of rbST treatment in cows.

The recombinant bovine growth hormone (rbST) is used to increase lactating performances of dairy cows. Administration of rbST is banned in the European Union; nevertheless, its use is probable. Until now, efficient analytical strategies to detect such practice are based on the direct detection by mass spectrometry of the presence of rbST in biological fluids, which suits for confirmatory purposes. Current screening strategies do not offer satisfactory performances; therefore, alternative screening strategies are required. The aim of the present work is to develop and validate an ELISA to measure the production of specific antibodies upon rbST in bovine sera. In this immunoassay, rbST is absorbed onto microtiter plate. After specific purification of the antibodies in serum, samples are analysed and the presence of antibodies anti-rbST is detected by Protein G peroxidase conjugate and 2-2'-azino di-ethyl benz-thiazoline-6-sulphonic acid (ABTS). The mean reproducibility of the OD (λ=405 nm) measurement was calculated with a CV of 13%. The intra- and inter-assay CVs ranged from 0.79% to 7.91% and from 2.69% to 20% respectively. The test presents cross-reaction with other growth hormones such as the recombinant equine (reST) and porcine (pST) (100% and 80% respectively). The specificity of the test toward rbST anabolic treatment was confirmed through the analysis of sera samples collected on animals administered with other anabolic compounds (steroids). The performances of the present anti-rbST ELISA proves its efficiency as a new screening tool to highlight illegal administration of rbST in cattle up to at least 3 weeks after treatment.

Detection of recombinant bovine somatotropin in milk and effect of industrial processes on its stability.

A LC-MS/MS method has been developed for the direct detection of recombinant bovine somatotropin (rbST) in milk and dairy products. The sample preparation protocol is based on a solid phase extraction step followed by precipitation with cold methanol and enzymatic digestion. The analysis is focused on the tryptic N-terminal peptide, specific of the recombinant form of the hormone and the detection is performed by LC-ESI(+)-MS/MS. This method has been validated according to the European Union criteria described in the Directive 2002/657/EC. Acceptable performances, with a decision limit (CCalpha) of 1.24 ng mL(-1) and detection capability (CCbeta) of 1.92 ng mL(-1) were obtained. Calculation of repeatability and intermediate reproducibility of the signal at 100 ng mL(-1) lead to relative standard deviations lower than 20%, showing the robustness of the method. Samples subjected to various industrial processes namely, heating, freezing, defatting, pasteurization and spray-drying were then analysed in order to determine the consequences of these treatments on the stability of the hormone. Results showed that temperature related processes, such as pasteurization and spray-drying induce a loss of the hormone up to 95%.

Identification of cows treated with recombinant bovine somatotropin.

A method for the specific detection and quantification of recombinant bovine somatotropin (rbST) in bovine blood has been validated according to criteria described in the EU Commission Decision 2002/657/EC. The method is based on a thorough purification procedure followed with the detection by LC-ESI-MS/MS of the tryptic N-terminal peptide specific of the rbST. The recombinant equine somatotropin (reST) is used as internal standard. Performance of the method was assessed based on specificity, linearity, trueness and repeatability. Decision limit (CCalpha) and detection capability (CCbeta) were found to be 2.5 ng mL(-1) and 6.8 ng mL(-1), respectively. This method was subsequently applied to the analysis of serum and plasma collected from two different animals treated with 500 mg of rbST. No significant variations were observed when analyzing either serum or plasma, but an important difference between animals was encountered. In all cases, recombinant bovine somatotropin was still detected two weeks after administration.

Elimination kinetic of recombinant somatotropin in bovine.

Bovine somatotropin (bST), also called growth hormone is a protein hormone produced by the pituitary gland and responsible directly or indirectly for various effects on growth, development and reproductive functions. Its recombinant bovine somatotropin form (rbST) is used in dairy cattle to enhance milk production. Even if the effects of treatment with rbST have been largely studied, until now analytical methods able to detect rbST were limited to immunoassays, which suffer from the impossibility to distinguish between the endogenous and the recombinant form. In this study, a sample preparation procedure based on different precipitation steps, extraction on solid phase and enzymatic digestion was used to purify rbST from serum. The detection was performed by liquid chromatography coupled to tandem mass spectrometry in positive electrospray ionization mode (LC-ESI(+)-MS/MS) allowing the unambiguous identification and quantification of rbST in serum. Samples collected from a cow treated with recombinant bovine somatotropin were analysed and for the first time, the elimination kinetic specific to recombinant somatotropin has been characterized in serum. Detection of rbST was possible from 4h 30min to 4 days after administration and concentration was found up to 10ngmL(-1) during the kinetic.

Direct determination of recombinant bovine somatotropin in plasma from a treated goat by liquid chromatography/high-resolution mass spectrometry.

Recombinant bovine somatotropin (rbST) is used in dairy cattle to enhance milk production. Despite the ban on this hormone in some countries, especially in Europe, there is so far no method available for the direct detection of rbST either in milk or in plasma. An analytical strategy has been developed to analyze rbST in plasma, including a purification procedure based on a precipitation with ammonium sulphate, followed by a solid-phase extraction (SPE)-based clean-up on C4 sorbent and precipitation with cold methanol. The hormone was then digested with trypsin and analyzed by liquid chromatography/high-resolution mass spectrometry (LC/HRMSn) on a linear ion trap coupled with an Orbitrap. The tryptic N-terminal peptide, specific to the difference between the endogenous and recombinant form of the somatotropin, was fragmented and product ions were analyzed at high mass resolution. Applying this approach to goat plasma allowed the direct detection of 10 ng mL(-1) of rbST in fortified samples. It also showed the presence of rbST in plasma collected from a goat treated with the hormone, even 2 days after administration. These results are of a great interest in the field of somatotropin control and undoubtedly constitute a first step in the development of a method for the detection of rbST not only in bovine plasma, but also in other biological matrices such as milk.