Evaluation of oxidized buckypaper as material for the solid phase extraction of cobalamins from milk: Its efficacy as individual and support sorbent of a hydrophilic–lipophilic balance copolymer

• A LC–tandem MS method for the cobalamin speciation in milk has been developed.
• The use of buckypaper as solid phase extraction sorbent has been evaluated and tested for the first time.
• The cobalamin composition of cow's milk has been elucidated.
• Methylcobalamin has been found as prevalent form of vitamin B12 in cow's milk.

This work describes a new analytical method for the determination of four cobalamins (adenosylcobalamin (AdoCbl), methylcobalamin (MeCbl), hydroxocobalamin (OHCbl) and cyanocobalamin (CNCbl)) in cow's milk. The extraction procedure is fast and based on dilution/protein precipitation of a milk sample with 50 mM sodium acetate buffer (pH 4.6), followed by solid phase extraction (SPE) of the filtered supernatant. Relative recoveries higher than 60% have been obtained for all the cobalamins by combining two different types of sorbents in the same SPE cartridge: two disks of buckypaper (BP), a nanoporous felt composed of oxidized multiwalled carbon nanotubes (MWCNTs), separated by a Teflon frit from OASIS HLB (500 mg), a hydrophilic–lipophilic balance copolymer. Before its use as sorbent, BP was characterized in terms of porosity, permeability, surface area, specific adsorption capacity and tested for a potential reuse after adequate chemical regeneration. The analysis of the extracts was performed by liquid chromatography (LC) coupled to tandem mass spectrometry (MS/MS) on an analytical C18 column in less than 10 min. After validation, the method was applied to the determination of the natural content of the four B12 homologues in cow's milk samples, providing data lacking in the literature.