Organic & Biomolecular Chemistry, 2016, Volume: 14, Issue: 37, Pages: 8664-8669, DOI: 10.1039/C6OB01594H
C. Bonnel, B. Legrand, J. L. Bantignies, H. Petitjean, J. Martinez, N. Masurier, L. T. Maillard
In the search of new robust and environmental-friendly anal. methods able to answer quant. issues in pharmacol., we explore liq. chromatog. (LC) assocd. with elemental mass spectrometry (ICP-MS) to monitor peptides in such complex biol. matrixes. The novelty is to use mass spectrometry to replace radiolabelling and radioactivity measurements, which represent up-to now the gold std. to measure org. compd. concns. in life science. As a proof of concept, we choose the vasopressin (AVP)/V1A receptor system for model pharmacol. assays. The capacity of ICP-MS to provide highly sensitive quantitation of metallic and hetero elements, whatever the sample medium, prompted us to investigate this technique in combination with appropriate labeling of the peptide of interest. Selenium, that is scarcely present in biol. media, was selected as a good compromise between ICP-MS response, covalent tagging ability using conventional sulfur chem. and peptide detection specificity. Applying selenium monitoring by elemental mass spectrometry in pharmacol. is challenging due to the very high salt content and org. material complexity of the samples that produces polyat. aggregates and thus potentially mass interferences with selenium detection. Hyphenation with a chromatog. sepn. was found compulsory. Noteworthy, we aimed to develop a straightforward quant. protocol that can be performed in any lab. equipped with a std. macrobore LC-ICP-MS system, in order to avoid time-consuming sample treatment or special implementation of instrumental set-up, while allowing efficient suppression of all mass interferences to reach the targeted sensitivity. Significantly, a quantification limit of 57 ng Se L-1 (72 fmol of injected Se) was achieved, the samples issued from the pharmacol. assays being directly introduced into the LC-ICP-MS system. The established method was successfully validated and applied to the measurement of the vasopressin ligand affinity for its V1A receptor through the detn. of the dissocn. const. (Kd) which was compared to the one recorded with conventional radioactivity assays.