Simultaneous Determination of the Main Aromatic Com-pounds and Divanillin in Vanilla planifolia Jacks. ex Andrews by HPLC-DAD

Vanilla planifolia Jacks. ex Andrews pods can lose up to 50% of their vanillin during curing. One explanation for this is the transformation of vanillin due to enzyme action, such as peroxidases, which generate the formation of dimers like divanillin. Therefore, in this work, a simultaneous high-performance liquid chromatography method (HPLC-DAD) was developed and validated for the separation and quantification of the main compounds present in Vanilla planifolia Jacks. ex Andrews and divanillin. The separation of 9 compounds of interest was achieved within 15 minutes using a Zorbax Eclipse XDB-C18 column (250 mm x 4.6 mm i.d., 5 μm particle size). The variables optimized included the mobile phase (water as solvent A, methanol as solvent B, and acidified water, 10–2 M H3PO4, as solvent C), the separation gradient, and the column tempera-ture (40-60 °C). The maximum divanillin content was 0.02 g/100 g d.w. in a sample from Papantla de Olarte. Chromatographic performance evaluation revealed excellent resolution, retention factor, and selectivity. The method was successfully validated in terms of limits of detection and quantification, linearity, and precision, as well as its application to cured pods, with evidence of divanillin presence in all analyzed sample.