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

Abstract

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 ...

In vitro reactions of a cyanocobalamin–cisplatin conjugate with nucleoside monophosphates

“…[C 63 H 88 CoN 14 O 14 P + 2H] 2+ ), monoprotonated CNCbl‐mCP conjugate (i.e. [C 63 H 94 ClCoN 16 O 14 PPt + H] 2+ ) and a previously described by‐product here labelled as 2′ , 25 respectively. The mass spectra of each chromatographic peak (i.e.…”

Tandem mass spectrometry characterization of a conjugate between oleuropein and hydrated cis‐diammineplatinum(II)

“…CP ([Pt II (NH 3 ) 2 Cl 2 ]) exhibits a great affinity for cyano groups after aquation, which involves the facile loss of one or two chloride labile ligands, and the formation of monohydrated species (i.e., [Pt II (NH 3 ) 2 Cl(OH 2 )] + and Pt II (NH 3 ) 2 Cl(OH)) and a dihydroxylated complex ([Pt II (NH 3 ) 2 (OH) 2 ] + ) which are considered to be the active reaction intermediates . Methanol was used instead of acetonitrile as organic modifier of the mobile phase to separate the in vitro reaction mixture products, in combination with a reversed‐phase C18 amide‐embedded column . Figure shows the base peak chromatogram obtained in ESI positive ion mode by RPLC/ESI‐MS analysis.…”

“…26,24 Methanol was used instead of acetonitrile as organic modifier of the mobile phase to separate the in vitro reaction mixture products, in combination with a reversedphase C18 amide-embedded column. 26,32,33 Figure 2 shows the base peak chromatogram obtained in ESI positive ion mode by RPLC/ESI-MS analysis. Two main chromatographic peaks can be observed, with retention times 7.2 and 9.8 min, peaks 1 and 2, respectively; peak 2 was easily recognized as due to unreacted Ole, since a peak at the same retention time and an identical MS spectrum was obtained after injection of an Ole standard solution (data not shown).…”

Effect of pH and mobile phase additives on the chromatographic behaviour of an amide‐embedded stationary phase: Cyanocobalamin and its diaminemonochloro‐platinum(II) conjugate as a case study

“…The m/z values listed in the text refer to the most intense peak of each isotopic cluster. Extracted ions current (XIC) chromatograms were generated from ionic currents recorded at m/z 678.3 of the doubly protonated CNCbl (i.e., [C 63 H 90 CoN 14 O 14 P] 2+ ) and at m/z 810.3 of the monoprotonated doubly charged CNCbl ‐ cis ‐DAMCP conjugate, [C 63 H 95 CoN 16 O 14 PClPt] 2+ .…”

“…Extracted ions current (XIC) chromatograms were generated from ionic currents recorded at m/z 678. 3 [11].…”