# 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

## TL;DR

This paper develops and validates HPLC methods to measure B1, B2, and B6 vitamins in gummies and digestive fluids, and tests how different drinks affect vitamin release.

## Contribution

The study introduces validated HPLC methods with DAD and FLD for simultaneous vitamin analysis and evaluates in vitro digestion under different nutritional conditions.

## Key findings

- HPLC methods achieved high linearity (R2 > 0.999) and accuracy (% recovery 100 ± 3%).
- Extraction methods for gummies and gastrointestinal fluids showed high recovery rates (>99.8%).
- In vitro digestion showed no significant differences in vitamin release, with minor variations based on the drink used.

## Abstract

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.