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

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) interpretation, and differential isotopic labeling were used to establish the chemical composition and to suggest the chemical structures of reaction products. When cisplatin (-[PtCl(NH)]) 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-CN-Pt bridge was MS detected as a doubly positive charged ion with the prevailing isotopologue at / 810.26 (empirical formula [CHClCoNOPPt]). Likewise, a peak signal centered at / 811.26 was observed when N-labeled cisplatin -[PtCl(NH)] 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 -diamminediaquaplatinum(II), that is, -[Pt(NH)(HO)]; 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 / 792.26 (empirical formula [CHCoNOPPt]) changed to / 793.26 when the labeled -[Pt(NH)(HO)] complex was adopted for conjugation. Comparison between MS/MS spectra allowed an extended structural characterization of both conjugates, as such or N-labeled. Two-dimensional heteronuclear (H-N) single quantum correlation NMR spectroscopy, applied to N-labeled conjugates, supported the hypotheses made on the Pt(II) coordination in both cases.