This study quantified, for the first time, 2-isopropylmalic and 3-isopropylmalic acids, in green, roasted and espresso coffee by UHPLC-MS/MS. Moreover, it reports the influence of postharvest processing methods (natural, washed and honey) on their content. New extraction techniques were developed and validated from three coffee matrices (green, roasted and espresso). Honey coffee exhibited levels substantially higher of 2-isopropylmalic acid than those processed by natural and washed methods (p < 0.05). Specifically, 2-isopropylmalic acid levels in honey green, roasted and espresso coffee samples were 48.24 ± 7.31 ng/g, 168.8 ± 10.88 ng/g and 177.5 ± 9.49 ng/g, respectively. This research highlights the significant impact of processing methods on the chemical profile of coffee and introduces 2-isopropylmalic and 3-isopropylmalic acids as potential quality indicators. Moreover, it suggests that the fermentation stage during processing may play a crucial role in their formation, laying the foundation for optimizing coffee processing to enhance quality.
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