Structural Elucidation of Cisplatin and Hydrated cis-Diammineplatinum(II) Complex Conjugated with Cyanocobalamin by Liquid Chromatography with Electrospray Ionization–Mass Spectrometry and Multistage Mass Spectrometry

[Image: see text] Pt(II)-based derivatives bearing a cyanocobalamin (CNCbl) unit were synthesized in aqueous solutions, and the reaction mixtures were examined by reversed-phase liquid chromatography with electrospray ionization and linear ion trap mass spectrometry (MS). Isotopic pattern analysis, multistage mass-spectra (MS/MS and MS(3)) interpretation, and differential isotopic labeling were used to establish the chemical composition and to suggest the chemical structures of reaction products. When cisplatin (cis-[PtCl(2)(NH(3))(2)]) was used as a Pt(II) drug derivative, a coordination bond between diamminemonochloroplatinum(II) and the cyano group of CNCbl, in turn linked covalently to the vitamin Co(III) ion, occurred. The resulting conjugate with a Co(III)–CN–Pt(II) bridge was MS detected as a doubly positive charged ion with the prevailing isotopologue at m/z 810.26 (empirical formula [C(63)H(95)ClCo(III)N(16)O(14)PPt](2+)). Likewise, a peak signal centered at m/z 811.26 was observed when (15)N-labeled cisplatin cis-[PtCl(2)((15)NH(3))(2)] was used as Pt(II) complex, thus confirming the presence of both the cisplatin amino groups in the conjugate. A bifunctional conjugate was obtained between CNCbl and the cis-diamminediaquaplatinum(II), that is, cis-[Pt(NH(3))(2)(H(2)O)(2)](2+); in this case, the planar coordination complex of Pt(II) was also involved in a covalent bond with the oxygen atom of one of the CNCbl amide moieties. The peak signal detected at m/z 792.26 (empirical formula [C(63)H(94)Co(III)N(16)O(14)PPt](2+)) changed to m/z 793.26 when the labeled cis-[Pt((15)NH(3))(2)(H(2)O)(2)](2+) complex was adopted for conjugation. Comparison between MS/MS spectra allowed an extended structural characterization of both conjugates, as such or (15)N-labeled. Two-dimensional heteronuclear ((1)H–(15)N) single quantum correlation NMR spectroscopy, applied to (15)N-labeled conjugates, supported the hypotheses made on the Pt(II) coordination in both cases.