Development and Validation of HPLC-DAD/FLD Methods for the Determination of Vitamins B₁, B₂, and B₆ in Pharmaceutical Gummies and Gastrointestinal Fluids—In Vitro Digestion Studies in Different Nutritional Habits

Two HPLC-based analytical methods, one with DAD and the other with an FLD detector, were developed and validated for the simultaneous analysis of B₁, B₂, and B₆ vitamins, both in pharmaceutical gummies and in gastric and intestinal fluids (with water or milk or orange juice). For the detection of B₁ by fluorometry, a pre-column oxidation/derivatization process was accomplished in the presence of B₂ and B₆ vitamins. The methods were performed on an Aqua column (250 mm × 4.6 mm, 5 mm) at 40 °C, with isocratic elution (70% NaH₂PO₄ buffer pH 4.95 and 30% methanol) and a flow rate of 0.9 mL/min. Both were validated according to ICH specifications in terms of linearity (R² > 0.999), accuracy (% Mean Recovery 100 ± 3%) and precision (%RSD < 3.23). For the analysis of the samples, a stability study (in diluents, pH and fluids) was conducted, while for their purification two different extraction procedures, a liquid/solid for the gummies (%Recovery > 99.8%) and a Solid Phase Extraction (SPE) for the Gastrointestinal (G.I.) fluids, (%Recovery 100 ± 5%) were developed. Finally, to investigate whether the co-administration of B-complex with water, orange juice or milk plays a significant role in their release from gummies, a three-phase in vitro digestion protocol was applied. The results did not show significant differences with a slight superiority in the release of B₂ and B₆ with water, while B₁ with orange juice.