Detection of methyl cobalamin and adenosyl cobalamin using a nitroxide radical trap

Abstract

Methyl cobalamin and adenosyl cobalamin, two of the coenzymes of vitamin B₁₂ (cyanocobalamin) which perform vital metabolic functions, have been detected using photolytic derivatisation with a nitroxide radical trap. This technique makes the important distinction between these two active coenzymes of vitamin B₁₂ and other inactive cobalamins and cobalamin analogues, such as cobinamides or cobamides. Methods currently employed for routine vitamin B₁₂ analysis do not make this distincion. The derivatisation reaction is based on the light induced homolytic fission of the cobalt–carbon bond present in the coenzymes which results in the formation of carbon centred radical species. A nitroxide radical trap, derivatised with the fluorescent agent fluorescamine, is used to trap the radical species and the products are separated using reverse phase HPLC with fluorescence detection. Characterisation of the derivatised material is achieved using negative ion electrospray mass spectrometry. Derivatisation of methyl cobalamin results in a chromatogram with one unsymmetric peak, whereas adenosyl cobalamin results in a chromatogram with one resolved and two unresolved peaks. Calibration curves are linear in the range 75 to 0.075 µM for both methyl cobalamin and adenosyl cobalamin, with a detection limit of 7.5 pmoles injected (signal:noise 5:1) for both compounds.