1200 series liquid chromatography system

For LC–MS analysis, the samples were analyzed using an Agilent Technologies 6125B Single Quadrupole LC-MS coupled with an ESI (electron spray ionization) source and interfaced with an Agilent 1200 series liquid chromatography system with a diode-array (UV–Vis) detector. The MS had an identification m/z range of 50–1500, with spectra acquired in positive mode for eugenol and chavicol and in negative mode for hydroxychavicol. The system was equipped with an Agilent ZORBAX Eclipse Plus C18 narrow bore column (2.1 mm internal diameter; 50 mm length; 5 micron particle size; P.N. 959746-902). The LC method used a 12 min gradient ramp from 0.1% formic acid in water to 0.1% formic acid in acetonitrile with an additional 6 minute reset and equilibration time. The injection volume was 10 µL.

Measurements were performed using an HPLC-MS system (Agilent Technologies 1200 series liquid chromatography system). The analytes were separated on a C-18 RP column (5 μm particle size, 250 mm length × 4.6 mm i.d.) (Sigma–Aldrich, Riedel-de Haën, Laborchemikalien, Seelze, Germany) at a flow rate of 1 mL min⁻¹ and an injection volume of 20 μL. The elution gradient followed the method described by the International Olive Council [33 ]. The total biophenol content (natural and oxidized oleuropein and ligstroside derivatives, lignans, flavonoids and phenolic acids), expressed in mg kg⁻¹ of fresh leaves, was calculated by measuring the sum of the areas of the related chromatographic peaks according to the following formula:
where (ΣA) is the sum of the peak areas of the biophenols recorded at 280 nm; Asyr is the area of the syringic acid internal standard recorded at 280 nm; 1000 is the factor used to express the result in mg kg⁻¹; W is the weight of the extract, in grams; RRFsyr/tyr is the multiplication coefficient for expressing the final results as tyrosol equivalents; Wsyr is the weight, in mg, of the syringic acid used as the internal standard in 1 mL of solution added to the sample.

NMR analyses were performed at 25 °C on a Bruker AC 300 spectrometer at 300 MHz and 75 MHz for ¹H and ¹³C NMR, respectively. CDCl₃ and tetramethylsilane were used as the solvent and the internal standard, respectively. HPLC analyses were performed using an Agilent Technologies 1200 series liquid chromatography system equipped with G1379B degasser, G1312A pump, and G1329A autosampler. Mass spectra were obtained in the negative mode using an API 4000 Q-Trap (MSD Sciex Applied Biosystem, Framingham, MA, USA) mass spectrometer equipped with ion max source with ESI probe (ion spray voltage (IS) −4500 V; curtain gas, 20 psi; entry potential (EP), −11 eV; declustering potential (DP), −125 eV; collision energy (CE), −20 eV and collision exit potential (CXP), −16 eV) through direct infusion (10 μL·min⁻¹) of a methanol solution of TyOle (5 μg·mL⁻¹).