Vitamin B12 Quantification Protocol

Cyanocobalamin (vitamin B₁₂, vit. B₁₂) was purchased from Sigma-Aldrich Inc., St. Louis, MO, USA. FeCl₃ × 6H₂O, FeSO₄ × 4H₂O, and potassium dihydrogen phosphate (KH₂PO₄) were purchased from Merck (Darmstadt, Germany). Ammonia solution (25% wt.%) was obtained from POCH S. A. (Gliwice, Poland). Methanol was purchased from E. Merck (Darmstadt, Germany). Trifluoroacetic acid (TFAA) suitable for HPLC, ≥99.0%, trichloroacetic acid (TCAA) ACS reagent, ≥99.0%, and heptafluorobutyric acid (HFBA, perfluorobutyric acid PFBA), ≥99.5% were purchased from Sigma-Aldrich Inc., St. Louis, MO, USA. Water with a resistivity of 18.2 MΩ cm was obtained from an ULTRAPURE Millipore Direct-Q 3UV-R (Merck, Darmstadt, Germany).