## 2 Materials and Methods
### Development and Validation of LC-MS/MS-Based Methodology  
CHG standard was initially used to set up and validate a detection method for CHG. EMA guidelines for the validation of bioanalytical methods were followed. The LC-MS/MS analysis system consisted of a UHPLC (Ultrahigh-Performance Liquid Chromatography) UtiMate 3000 Dioneek(r) chromatograph from Thermo Fisher Scientific(tm) (Waltham, MA, USA) equipped with a binary pump, a refrigerated autosampler and a thermostated chromatographic column coupled to the TSQ Endura(tm) mass spectrometer (MS) from Thermo Fisher Scientific(tm) (Waltham, MA, USA), equipped with electrospray ionization (ESI) source and a triple quadrupole analyzer (QqQ). Chromatographic separation was performed using a Kinetex C18 column (50 \(\times\) 2.1 mm--1.73 um particle size) (Phenomenex, Torrance, CA, USA). Compound elution was obtained using a two-component mobile phase: 0.1% formic acid in water (eluent A) and methanol (eluent B). The following multi-step gradient was optimized: 30% B at time 0, from 0 to 2.5 min 95% B, 2.5 to 3.5 min at \begin{table}
\begin{tabular}{c c c c} \hline \hline
**Active Principles** & **Formulation 1** & **Formulation 2** & **Formulation 3** \\ \hline CHG & 0.20\% & 0.20\% & 0.20\% \\ CPC & – & + & – \\ _Aloe barbadensis_ (Aloe Vera) & – & + & + \\ Leaf Extract & – & + & + \\ _Salvia officinalis_ (Sage) & – & + & + \\ Leaf Extract & + & – & – \\ Ethyl alcohol & + & – & – \\ \hline \hline \end{tabular}
\end{table} 
Table 1: Some of the active principles contained in the three commercial CHG mouthwash formulations (according to the manufacturer’s descriptions). 95% B, at 3.5 return to initial conditions and equilibration of the column at 30% B up to 4.5 min. The flow rate was 0.4 mL/min, and the column temperature was maintained at 20 \({}^{\circ}\)C. The injection volume was 5 \(\mu\)L, and all samples were analyzed in triplicate. Mass spectrometric analyses were carried out in selected reaction monitoring (SRM) and electrospray positive mode. A CHG mono-charged precursor ion and a double-charged precursor ion were identified with direct infusion of pure standard (\(m/z\) 506 and 253, respectively). For each analyte, a precursor and two products (quantifier and qualifier) were selected and used for the detection and quantification: CHG mono-charged, 506 \(\rightarrow\) 336 and 202; CHG double-charged, 253 \(\rightarrow\) 170 and 153. Selectivity, specificity, absence of carryover, the lower limit of detection (LLOD) and quantification (LLOQ) were also determined. Selectivity was assessed by analyzing the diluent solvent (MeOH 20%, TCA 1%). The absence of interfering components was verified. Specificity was evaluated based on the consistency of retention times across runs and the presence of both products (quantifier and qualifier) in a constant ratio. The LLOD was defined as the lowest concentration at which the analytical test can detect the analyte signal (S), discriminating it from the background noise (N) (LLOD: S/N \(\geq\) 3). The LLOQ is the lowest concentration at which precision and trueness were within 20%, with a peak intensity at least 5 times higher than baseline noise (LLOQ: S/N \(\geq\) 5). Linearity was evaluated using a 6-point calibration curve, built by using the CHG standard, in a range of concentrations from 0.25 to 2 \(\mu\)g/mL, including a zero value. Peak areas obtained were plotted against analyte concentrations, and a linear regression analysis was performed. The slope and the correlation coefficient were calculated for each calibration curve. Quality controls (QCs) of quantifications were used at 0.6 and 1.2 \(\mu\)g/mL. Precision and trueness were evaluated by analyzing LLOQ and the two QCs samples in five replicates to assess the intraday accuracy. Precision was expressed as the percentage coefficient of variation (%CV), and the trueness was evaluated by measuring the relative error, expressed as %bias, between experimental measurements and theoretical concentrations.