Determination of cobalamins (hydroxo-, cyano-, adenosyl- and methyl-cobalamins) in seawater using reversed-phase liquid chromatography with diode-array detection.

Abstract A high-performance liquid chromatography method was developed for the separation and determination of four cobalamins in seawater. Chromatographic separation was performed on a reversed-phase discovery RP-amide C 16 column with buffer potassium dihydrogenphosphate and acetonitrile as the mobile phases in linear gradients elution mode. Cobalamins were previously preconcentrated in C 18 resins cartridges. Detection was performed using UV-diode array detector in a range of λ of 200–400 nm. The method showed to be linear over a range of 1–300 ng mL −1 with acceptable precision and accuracy. The detection limits ranged between 0.07 pg mL −1 for 5′-deoxyadenosylcobalamin and 0.5 pg mL −1 for hydroxocobalamin. The mean cobalamins recoveries for direct determination ranged between 76 and 93% for hydroxo-, cyano- and methylcobalamin, while the recovery for 5′-deoxyadenosylcobalamin was only 31% suggesting that the preconcentration method was not valid for this cobalamin. The method was successfully applied to coastal seawater where the concentrations ranged from 4.2 to 7.3 ng L −1 for hydroxo-, 1.4–3.9 ng L −1 for cyano-, 2.1–4.6 ng L −1 for 5′-deoxyadenosyl- and 33–83.5 ng L −1 for methylcobalamin.