Optimized ethyl chloroformate derivatization using a box–behnken design for gas chromatography–mass spectrometry quantification of gallic acid in wine

Gallic acid, a major phenolic compound in wine, significantly influences its sensory profile and health-related properties, making its accurate measurement essential for both enological and nutritional studies. In this context, a derivatization protocol for gallic acid using ethyl chloroformate (ECF) was developed and optimized for GC-MS analysis, with experimental conditions refined through a Box-Behnken Design (BBD). The BBD systematically investigated the effects of three critical reagent volumes: ethyl chloroformate, pyridine, and ethanol. This approach elucidated complex interactions and quadratic effects, leading to a predictive second-order polynomial model and identifying the optimal derivatization conditions for maximum yield (137 mu L of ethyl chloroformate, 51 mu L of pyridine, and 161 mu L of ethanol per 150 mu L of wine). The BBD-optimized GC-MS method was validated and successfully applied to quantify gallic acid in diverse commercial wine samples (white, red, conventional, natural). A key finding was the method's wide dynamic range, enabling accurate quantification from 5 up to over 600 mu g/mL without sample dilution. This work represents, to our knowledge, the first application of a BBD for optimizing the ethyl chloroformate derivatization of gallic acid, providing a robust, efficient, and widely applicable analytical tool for routine quality control and enological research.