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Delphinidin-3-O-sambubioside and cyanidin-3-O-sambubioside are the main anthocyanins of Hibiscus sabdariffa calyces, traditionally used to make a bright red beverage by decoction in water. At natural pH, these anthocyanins are mainly in their flavylium form (red) in equilibrium with the quinonoid base (purple) and the hemiketal (colorless). For the first time, their acidity and hydration equilibrium constants were obtained from a pH-jump method followed by UV-vis spectroscopy as a function of temperature from 4 to 37 °C. Equilibrium constant determination was also performed by multivariate curve resolution (MCR). Acidity and hydration constants of cyanidin-3-O-sambubioside at 25 °C were 4.12 × 10(-5) and 7.74 × 10(-4), respectively, and were significantly higher for delphinidin-3-O-sambubioside (4.95 × 10(-5) and 1.21 × 10(-3), respectively). MCR enabled the obtaining of concentration and spectrum of each form but led to overestimated values for the equilibrium constants. However, both methods showed that formations of the quinonoid base and hemiketal were endothermic reactions. Equilibrium constants of anthocyanins in the hibiscus extract showed comparable values as for the isolated anthocyanins.