2.2. Extraction and Phenolic Quantification by HPLC-ESI-TOF/MS

L. citriodora leaves, purchased from Monteloeder (Alicante, Spain), were milled by an ultra-centrifugal mill ZM200 (Retsch GmbH, Haan, Germany) to get an average particle size of 500 µm. The powder was stored in a dry and dark place until extraction. Microwave-assisted extraction (MAE) was performed in a Multiwave 3000SOLV (Anton Paar, Graz, Austria). An extract rich in polar compounds was obtained using 42% ethanol at 113 °C for 22 min [17]. Analytical assays were carried out by reverse-phase HPLC. The chromatographic separation was performed by using a RRLC 1200 series (Agilent Technologies, Palo Alto, CA, USA) with a Zorbax Eclipse Plus C18 analytical column (150 × 4.6 mm id, 1.8 µm particle diameter. Agilent Technologies, Palo Alto, CA, USA) and a multistep gradient elution with a mobile phase of water: acetonitrile 90:10 (v:v) with 0.1% formic acid (A) and acetonitrile (B) for 35 min.

The HPLC was coupled to a mass spectrometer equipped with an orthogonal electrospray interface (ESI; model G1607 from Agilent Technologies, Palo Alto, CA, USA) operating in negative ionization mode considering a mass range of 50–1000 m/z. An external calibration with sodium formate cluster was done in a quadratic high-precision calibration (HPC) regression mode. Source and transfer parameters were set according to a previously reported method [8].

The data provided by the analytical platform were processed by using DataAnalysis 4.0 software (Bruker Daltonics, Bremen, Germany), which allows the identification and quantification of the polar components of extracts. Hence, the qualitative characterization of each compound was made upon an interpretation of their mass spectra data provided by TOF-MS (m/z calculated and experimental, mSigma and molecular formula) and the information available in the literature [19]. For the quantitative characterization, four different calibration curves were built with verbascoside, loganic acid, quercetin and kaempferol-3-glucoside. The calibration curves were built by plotting the standard concentration as a function of the peak area given by HPLC-ESI-TOF analyses (area standard/area internal standard). Apigenin was used as internal standard (25 µg/mL). In all cases, the linearity of calibration curves was higher than 0.99. Detection and quantification limits, as well as repeatability of the method (intraday and interday precision) was previously reported [8].

The extract was dissolved in water:ethanol (50:50 v:v) to reach a concentration of 5 mg/mL. The concentration of the phytochemicals was calculated by interpolation of the area analyte/area internal standard ratio in the corresponding calibration curve. Iridoid glycosides were quantified using loganic acid calibration curve. According to their structures, luteolin-7-diglucuronide, chrysoeriol-7-diglucuronide, apigenin-7-diglucuronide and acacetin-7-diglucuronide were quantified by using the kaempferol-3-glucoside curve. The amounts of the rest of flavonoids were calculated by using the quercetin calibration curve. Lastly, verbascoside calibration curve was used to quantify the phenylpropanoids group. The results were expressed as mg of analyte per g of L. citriodora extract.