This work presents a developed and validated high-performance liquid chromatography with a diode array detection (HPLC–DAD) method for the separation and quantification of divanillin and 8 compounds (p-hydroxybenzyl alcohol, vanillyl alcohol, p-hydroxybenzaldehyde, p-hydroxybenzoic acid, vanillic acid, vanillin, anisyl alcohol and anisic acid) present in Vanilla planifolia Jacks. ex Andrews. Chromatographic separation was achieved in 15 min using A Zorbax Eclipse XDB-C18 column (250 mm × 4.6 mm i.d., 5 μm particle size). Gradient elution was performed using a solvent mixture of water, methanol and acidified water (10^–2 M H₃PO₄), at a flow rate of 2.25 mL/min with detection at 230, 254 and 280 nm. The method was comprehensively validated according to the International Conference of Harmonization (ICH) Q2 (R1) guidelines. The method was linear in 0.1–200 mg/L concentration range with coefficient of determination (r²) higher than 0.99. The percentage recovery ranged from 98.04 to 101.83% with a relative standard deviation of less than 2%, confirming the method's accuracy and precision for the analysis of nine compounds. Existing methods for analyzing aromatic compounds in vanilla, such as HPLC–DAD, GC–MS, and NMR, often overlook divanillin or lack validated protocols for its quantification alongside other compounds. This study presents a significant advancement by developing a robust, validated HPLC–DAD method that enables the simultaneous quantification of divanillin, vanillin, and other key phenolic compounds with evidence of divanillin presence in all analyzed samples in cured pods of Vanilla planifolia Jacks. ex Andrews, with concentrations ranging from 0.002 to 0.02 g/100g dry weight.

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.