title: Determination of hydroxyalkyl derivatives of cobalamin (vitamin B12) using reversed phase high performance liquid chromatography with electrospray tandem mass spectrometry and ultraviolet diode array detection.
abstract: Electrospray ionization tandem mass spectrometry (ESI-MS/MS) and ultraviolet diode array detection (UV-DAD), coupled on-line to reversed phase high performance liquid chromatography (HPLC), was used for the characterization of hydroxyalkyl derivatives of cob(I)alamin. The reduced form of vitamin B12, cob(I)alamin, denoted a supernucleophile due to its high nucleophilic strength, has shown promise as an analytical tool in studies of electrophilically reactive compounds in vitro and in vivo. A method for analysis of DNA-phosphate adducts was developed earlier utilizing the supernucleophilicity of cob(I)alamin to transfer alkyl groups from the phosphotriester configuration in DNA, with the formation of a Co-substituted alkyl-cobalamin (alkyl-Cbl) complex. For the purpose of identification and quantification of alkyl-Cbls at high sensitivity, an MS/MS method has been developed with application to a number of 2-hydroxyalkyl-cobalamins (OHalkyl-Cbls). The precursor oxiranes were reacted with cob(I)alamin, followed by clean-up and mass spectrometric analysis of the resulting OHalkyl-Cbls. It was found that ionization was highly dependent on solvent composition. By using acetonitrile/water/trifluoroacetic acid (TFA) (eluent I), the base peak was the doubly protonated molecule [M + 2H](2+), whereas acetonitrile/water/1-methylpiperidine (eluent II) yielded the singly protonated molecule [M + H](+) as the base peak. Excellent separation was obtained with eluent II, with good separation between stereoisomers, thus enabling the characterization of these by means of UV spectra. Limits of quantitation for 2-hydroxypropyl-cobalamin (OHPr-Cbl) were 0.2 and 2 pg/microL (or 0.1 and 1 fmol/microL) using selected ion recording (SIR) with eluent I and II, respectively. The obtained detection level should be sufficient for analysis of alkyl-Cbls from a wide range of toxicological applications.
authors.affiliation: Laboratory for Analytical Environmental Chemistry, Institute of Applied Environmental Research (ITM), Stockholm University, SE-106 91 Stockholm, Sweden.
authors.forename: T
authors.initials: T
authors.lastname: Alsberg
authors.forename: J
authors.initials: J
authors.lastname: Minten
authors.forename: J
authors.initials: J
authors.lastname: Haglund
authors.forename: M
authors.initials: M
authors.lastname: Törnqvist
mesh_terms: D002851:Chromatography, High Pressure Liquid; D007202:Indicators and Reagents; D012015:Reference Standards; D021241:Spectrometry, Mass, Electrospray Ionization; D013056:Spectrophotometry, Ultraviolet; D014805:Vitamin B 12 / Q000031:analogs & derivatives* / Q000032:analysis*
chemical_list: D007202:Indicators and Reagents; D014805:Vitamin B 12