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

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 C16 column with buffer potassium dihydrogenphosphate and acetonitrile as the mobile phases in linear gradients elution mode. Cobalamins were previously preconcentrated in C18 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.

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► We optimized a HPLC method for the quantification of four cobalamins in seawater.
► The method was successfully applied to coastal seawater samples from Mallorca.
► Four cobalamins were separated and quantified simultaneously in a single injection.
► Cobalamin concentration ranged 44-99 ng mL-1 in the seawater samples.