Optimized Ethyl Chloroformate Derivatization Using a Box–Behnken Design for Gas Chromatography–Mass Spectrometry Quantification of Gallic Acid in Wine

Botta, Sofia; Piacentini, Roberta; Cappelletti, Chiara; Incocciati, Alessio; Boffi, Alberto; Bonamore, Alessandra; Macone, Alberto

doi:10.3390/separations12070183

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Optimized Ethyl Chloroformate Derivatization Using a Box–Behnken Design for Gas Chromatography–Mass Spectrometry Quantification of Gallic Acid in Wine

by

Sofia Botta

^†,

Roberta Piacentini

^†

,

Chiara Cappelletti

,

Alessio Incocciati

,

Alberto Boffi

,

Alessandra Bonamore

^*

and

Alberto Macone

Department of Biochemical Sciences, Sapienza University of Rome, P.le A. Moro 5, 00185 Rome, Italy

^*

Author to whom correspondence should be addressed.

^†

These authors contributed equally to this work.

Separations 2025, 12(7), 183; https://doi.org/10.3390/separations12070183

Submission received: 18 June 2025 / Revised: 2 July 2025 / Accepted: 8 July 2025 / Published: 9 July 2025

(This article belongs to the Special Issue Innovative Analytical Strategies Based on Mass Spectrometry in Food Analysis)

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Abstract

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 µL of ethyl chloroformate, 51 µL of pyridine, and 161 µL of ethanol per 150 µL 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 µg/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.

Keywords: gallic acid; ethyl chloroformate; derivatization; Box–Behnken design; GS-MS; wine analysis

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MDPI and ACS Style

Botta, S.; Piacentini, R.; Cappelletti, C.; Incocciati, A.; Boffi, A.; Bonamore, A.; Macone, A. Optimized Ethyl Chloroformate Derivatization Using a Box–Behnken Design for Gas Chromatography–Mass Spectrometry Quantification of Gallic Acid in Wine. Separations 2025, 12, 183. https://doi.org/10.3390/separations12070183

AMA Style

Botta S, Piacentini R, Cappelletti C, Incocciati A, Boffi A, Bonamore A, Macone A. Optimized Ethyl Chloroformate Derivatization Using a Box–Behnken Design for Gas Chromatography–Mass Spectrometry Quantification of Gallic Acid in Wine. Separations. 2025; 12(7):183. https://doi.org/10.3390/separations12070183

Chicago/Turabian Style

Botta, Sofia, Roberta Piacentini, Chiara Cappelletti, Alessio Incocciati, Alberto Boffi, Alessandra Bonamore, and Alberto Macone. 2025. "Optimized Ethyl Chloroformate Derivatization Using a Box–Behnken Design for Gas Chromatography–Mass Spectrometry Quantification of Gallic Acid in Wine" Separations 12, no. 7: 183. https://doi.org/10.3390/separations12070183

APA Style

Botta, S., Piacentini, R., Cappelletti, C., Incocciati, A., Boffi, A., Bonamore, A., & Macone, A. (2025). Optimized Ethyl Chloroformate Derivatization Using a Box–Behnken Design for Gas Chromatography–Mass Spectrometry Quantification of Gallic Acid in Wine. Separations, 12(7), 183. https://doi.org/10.3390/separations12070183

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Optimized Ethyl Chloroformate Derivatization Using a Box–Behnken Design for Gas Chromatography–Mass Spectrometry Quantification of Gallic Acid in Wine

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