*2.2. Cyanocobalamin Quantification. Development and Validation of a HPLC Method*

B12 was quantified by HPLC using a PerkinElmer® Series 200 equipped with a UV detector settled at 360nm and a Kromasil® C18 HPLC column of 5 µm particle size, pore size 100 Å, L × I.D. 150 mm × 4.6 mm. The volume injection was 50 µL, and the mobile phase consisted of a isocratic mixture of methanol:water (30:70), delivered at a flow rate of 1 mL min−<sup>1</sup> . The B12 retention time was 3.6 min, and the analysis duration was 5 min.

For the validation of the method, a stock standard solution of B12 (100 µg/mL) was prepared by dissolving 0.01 g of the drug in 100 mL of water. Ten working solutions (100, 10, 5, 2.5, 1.25, 0.625, 0.312, 0.156, 0.078, and 0.039 µg/mL) were prepared by diluting an adequate amount of stock standard solution with water. All analyses were performed in triplicate (*n* = 3).
