*3.1. Spectral Characterization of NADES Standard Solutions*

The spectra of OHTyr and Tyr in NADES were acquired and analyzed. Figure 1 reports the mean spectra (a), and the second derivatives (b) of both compounds at the concentration of 20 mg L<sup>−</sup>1. The application of a second derivative treatment allows highlighting minor or subtle spectral features, and resolves spectral overlapping [23–25]. Valleys in the second derivative spectra correspond to peaks in direct absorption spectra, and vice versa.

**Figure 1.** Mean spectra (**a**), and second derivatives (**b**) of OHTyr and Tyr solutions in natural deep eutectic solvent (NADES) (20 mg L−1, *n* = 3). The red dotted reference lines correspond to the wavelengths selected for the calibration curves.

As regards OHTyr, the spectrum showed a large peak at 280 nm (λmax = 280 nm, ελmax = 2793 M−<sup>1</sup> cm−1). The molar extinction (e) determined for OHTyr in NADES was lower than that reported for OHTyr in water [26]. The second derivative showed more spectral features, with two negative peaks at 281 nm and 287 nm, and a positive peak at 294 nm, with a slight shoulder on the right. Spectra of NADES solutions at different concentrations of OHTyr (3, 5, 10, 20, 30 mg L−1) were acquired. Then, a rapid screening via analysis of correlation (data not shown) indicated that the wavelengths that showed the best correlation of the second derivative of the spectrum with the concentrations of

OHTyr were 294, 299, 286, and 280 nm (*r* = 0.99878, *r* = 0.9986, *r* = 0.99853, and *r* = 0.99839, respectively, *n =* 15).

As regards Tyr, the spectrum showed a peak at 276 nm and a shoulder at about 283 nm (λmax = 276 nm, ελmax = 1676 M−<sup>1</sup> cm<sup>−</sup>1). Therefore, hypsochromic and hypochromic shifts of the absorbance were observed for the monophenolic group of Tyr compared to the *o*-diphenolic structure of OHTyr. Spectra of NADES solutions at different concentrations of Tyr (5, 10, 20, 30, 40 mg L−1) were acquired. The analysis of correlation (data not shown) indicated that the wavelengths that showed the best correlation of the second derivative of the spectrum with the concentrations of Tyr were 290, 274, and 282 nm (*r* = 0.99911, *r* = 0.99897, *r* = 0.99883 respectively, *n =* 15).
