# Study of the Phenolic Compound Profile of _Arbutus unedo_ L.
## 2 Materials and Methods
### Chromatographic Analysis by HPLC-TQ-MS/MS and Analytical Parameters  
The HPLC system consisted of a Varian 1200/1200L LC/MS-MS containing two solvent delivery modules ProStar 210/215, a ProStar 410 autosampler (equipped with a 20 \(\upmu\)L loop), a thermostatic column compartment, and a 1200L TQ triple quadrupole mass spectrometry detector with an electrospray ionization (ESI) ion source (data acquisition system MS Workstation version 6.3). The chromatographic separation of the analytes was achieved on a Kromaphase 100 C18 column (150 \(\times\) 2.0 mm I.D., 3.5 \(\upmu\)m particle size) from Scharlau (Barcelona, Spain). The mobile phase consisted of ACN (A) and Milli Q water (B), both containing 2 mM ammonium acetate and 0.1% formic acid in a gradient elution program: t = 0 min 95% B, t =18 min 0% B, and t =20 min 95% B (2 min). The total run-time  
Figure 2: Scheme of the sample preparation of _A. unedo_ L. berries analyzed by UV-Vis and HPLC-TQ-MS/MS at three ripening stages.  
of the method was 22 min. The flow rate was 0.2 mL/min, the column temperature 30 \({}^{\circ}\)C, and the injection volume 10 \(\mu\)L. The TQ was operated using the ESI ion source in negative ion mode. Nitrogen was used as both drying and nebulizer gas, whereas argon was used as collision gas under the following conditions: the drying and nebulizer gas was set at 300 \({}^{\circ}\)C with 21 and 63 psi, respectively; the capillary voltage was held at \(-4500\) V, and shield at 600 V. Collision gas was set at 2.20 mTorr, and the detector operated at 1395 V. The ESI source parameters were initially optimized by direct infusion of standard solutions of each analyte (5 mg/L) at a flow rate of 10 \(\mu\)L/min selecting the molecular ion at \(-40\) V cone voltage and optimizing the collision cell voltage to obtain the product ions. Multiple reaction monitoring (MRM) mode was employed for all analytes (mass peak width Q1 2.5 uma; mass peak width Q3 1.5 uma; scan width in MRM 0.70 uma).  
Analytical parameters of the HPLC-TQ-MS/MS method were determined, including linearity, detection (LOD) and quantification (LOQ) limits, and precision. Linearity was evaluated for each analyte according to the sensitivity of the HPLC-TQ-MS/MS system. Calibration curves were constructed by linear regression analysis, plotting peak area versus analyte concentration. Regression coefficients (R\({}^{2}\)) were calculated, and the criterion for good linearity was considered R\({}^{2}\) > 0.99. The LOD and LOQ of each analyte were calculated considering the concentration that produces a signal-to-noise ratio (S/N) of 3 and 10, respectively, for the chromatographic response obtained from the lowest concentration of the calibration curve. Precision (expressed as relative standard deviation, %RSD) was evaluated in terms of intra-day (repeatability, six replicates on the same day) and inter-day (reproducibility, three replicates analyzed three different days) precision for an intermediate fortification level.  
The PCs found in the strawberry tree berry samples were identified according to their retention times and mass spectra (precursor and product ions), which were compared with the ones obtained with standard solutions. For the quantification of each analyte, the peak area was interpolated in the corresponding calibration curve, considering the matrix effect produced in the analysis. The matrix effect was studied through the comparison of the peak areas of the standards in solvent with the peak areas of the spiked sample extracts at the same concentration of each standard (subtracting the analyte peak area when it was presented in the sample).