2.2. Preparation of the BUO and OMN Samples and Quantification of the Main Secoiridoids

The EVOO extracts were prepared as previously reported [17]: the extracts were prepared starting from 5 mL of oil, obtaining a dry weight of 10 mg and 5.7 mg for BUO and OMN, respectively. Their total phenolic content and total hydroxytyrosol and tyrosol were determined applying to validated methods [17].

To evaluate the concentration of oleacein, oleuropein, oleocanthal, and ligstroside aglycone, the ¹H NMR spectra of BUO and OMN samples were registered by a 400 MHz instrument Advance 400 (Bruker, Bremen, Germany). The extracts were dissolved in 1 mL of CDCl₃, and 1 µL of the solution of the internal standard (maleic acid 30 mg/mL in CH₃CN) was added. According to our previous studies [21], to reference guidelines for quantitation by NMR [22], and to Karkoula et al. [23], the evaluation of oleacein, oleocanthal, oleuropein aglycone, and ligstroside aglycone in their monoaldehyde forms was done applying the following formula:

C(%), concentration of each of the four secoiridoid aldehydes.

I_mal, integral of 2 protons of the ISTD, maleic acid.

I_CHO, integral area of the proton signal of the aldehyde of each secoiridoid.

N_CHO, the number of the aldehyde proton of each secoiridoid.

N_mal, the number of the protons of maleic acid.

MW_X, the molecular weight of each secoiridoid (oleacein 320 g/moL, oleuropein aglycone 378 g/moL, oleocanthal 304 g/moL, and ligstroside aglycone 362 g/moL).

MW_mal, the mw of ISTD maleic acid, 116.1 g/moL.

W_mal, weight in mg of maleic acid.

W_sample, dry weight in mg of BUO or OMN.

P_mal, purity grade of maleic acid.