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

Abstract

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.

Elimination kinetic of recombinant somatotropin in bovine

“…This protocol has been used to determine the elimination kinetic of reST in horse after treatment with EquiGen-5 ® . ReST was then already detectable 8 min after administration and a maximum of reST in plasma was observed after 4 h. The same method has been adapted and successfully applied to the detection of rbST in plasma from a goat treated with Lactatropin ® [23]. Maximum concentrations of rbST around 40 ng mL −1 in goat plasma have been found.…”

“…An attempt of purification and analysis of somatotropin in samples was reported by Blockland et al, but it was limited to the detection of porcine somatotropin in a spiked sample [29]. It is only recently that the direct detection of reST and rbST in incurred serum and plasma has succeeded [22,23]. This can only be achieved thanks to an efficient purification procedure coupled to mass spectrometry detection using a last generation instrument.…”

“…A sample volume of 20 L was loaded onto the column using the autosampler. Mass spectrometric acquisition was performed using the following working parameters: capillary voltage was set at 5000 V, nebulizer at 55 psi, gas flow at 13 L min −1 and gas temperature at 300 • C. The triple quadrupole was used on SRM mode and the different parameters were set according to a fragmentation study of the peptide 1 20 performed previously [23]. These different parameters are described in Table 1.…”

“…In this context, the aim of this study was to apply the analytical method recently developed [22,23] to bovine serum, in order to describe the elimination kinetic of rbST in serum consecutive to an injection of Lactatropin ® .…”

Use of benchtop exactive high resolution and high mass accuracy orbitrap mass spectrometer for screening in horse doping control

“…Since the increase in terms of sensitivity of new generation of mass spectrometers, new molecules can now be confirmed in biological samples such as protein based drugs, particularly recombinant human erythropoietin (EPO) [2][3][4] and recombinant equine [5] or bovine growth hormone [6]. Nevertheless, due to the constant release of new drugs on the market, the screening must be improved in order to fulfill the detection of whole molecules.…”