## 2 Materials and Methods
### UHPLC-TQ-MS/MS Analysis  
Liquid chromatographic separations were carried out using an Elute UHPLC system (Bruker Daltonics Inc., Billerica, MA, USA), consisting of an Elute UHPLC HPG 1300 pump with two pairs of individually controlled linear drive pump heads coupled in series, an Elite autosampler maintained at 10 \({}^{\circ}\)C, and an Elute CSV column oven preheater equipped with an Intensity Solo 2 C18 column (100 mm \(\times\) 2.1 mm, 2.0 \(\upmu\)m, Bruker Daltonics Inc., Billerica, MA, USA) maintained at room temperature. Optimum separation conditions were achieved using a solvent mixture as a mobile phase consisting of 0.1% FA in water (solvent A) and 0.1% FA in ACN (solvent B) in a gradient elution mode as follows: 0.00 min 95% solvent A; 0.00-3.00 min 70% solvent A; 3.00-4.00 min 95% solvent A; 4.00-5.00 min 95% solvent A at a flow rate of 0.450 mL/min, and an injection volume of 2 \(\upmu\)L (partial injection), resulting in an analysis time of 5 min, including equilibration time.  
Detection was carried out using a Bruker EvoQ LC-TQ Elite triple quadrupole mass spectrometer (Bruker Daltonics Inc., Billerica, MA, USA) equipped with a heated electrospray ionization interface (HESI). The instrument was operated in positive ionization mode. The mass spectrum is registered with multiple reaction monitoring (MRM) of three transitions per compound (Q1 resolution 2.5; Q3 resolution 2.5; scan time 93.80 ms). Optimization of MRM collision-induced dissociation (CID) energies was performed by direct infusion of individual standard solutions (1 \(\upmu\)g/mL) at a flow rate of 5 \(\upmu\)L/min. HESI source parameters were optimized with multiple runs of UHPLC-MS/MS using gradient and flow rate-optimized conditions. The optimum HESI source parameters were: N2 as drying and nebulizer gas, argon as collision gas, collision pressure at 2.0 mTorr, spray voltage positive at 1535 V, spray amp positive 100.0 \(\upmu\)A, cone pressure 20.0 psi, probe pressure 25.0 PSI, nebulizer pressure 60 psi, cone temperature 280 \({}^{\circ}\)C, and probe temperature 225 \({}^{\circ}\)C. The MS parameters for each analyte are listed in Table 1. Therefore, the most intense production of each analyte in its MS\({}^{2}\) spectrum was used for quantification, while other less intense product ions (at least one of them was mandatory) were controlled for confirmation purposes (Table 1, Figure 2). The software tqControl 2.2.0.1 (Bruker Daltonics, Billerica MA, USA) was used for data processing. Linear ranges were established, from 0.007 to 100 \(\upmu\)g/L for atropine and from 0.14 to 100 \(\upmu\)g/L for scopolamine. LODs and LOQs values ranged from 0.002 to 0.007 \(\upmu\)g/L for atropine and from 0.04 to 0.14 \(\upmu\)g/L for scopolamine.  
\begin{table}
\begin{tabular}{c c c c c c} \hline \multirow{2}{*}{**Compound**} & **R\({}_{\mathbf{t}}\)** & **Precursor** & **Product Ions** & **Collision** & **Scan Time** \\  & **(min)** & **Ion (Q1, _m/z_)** & **(Q3, _m/z_)** & **Energy (eV)** & **(ms)** \\ \hline \multirow{2}{*}{Atropine} & \multirow{2}{*}{2.950} & \multirow{2}{*}{290.5} & \(123.90\) * & 20.5 & \multirow{2}{*}{31.26} \\  & & & 93.60 & 29.0 & & \\  & & & 91.10 & 34.0 & \\ \hline \multirow{2}{*}{Scopolamine} & \multirow{2}{*}{2.465} & \multirow{2}{*}{303.5} & \(137.90\) * & 12.0 & \multirow{2}{*}{23.45} \\  & & & 155.80 & 9.5 & & \\  & & & 120.60 & 16.0 & \\ \hline \end{tabular}
* * Productions used for quantification. Chromatographic conditions with the optimized gradient elution: 0.00 min 95% solvent A; 0.00–3.00 min 70% solvent A; 3.00–4.00 min 95% solvent A; 4.00–5.00 min 95% solvent A. Mobile phase: 0.1% formic acid in water (solvent A) and 0.1% formic acid in acetonitrile (solvent B). Flow rate: 0.450 mL/min. Column at room temperature and total run time analysis of 5 min.  
\end{table}
Table 1: Retention time and optimal parameters to mass spectrometry detection by MRM mode for the analysis of atropine and scopolamine using the UHPLC-TQ-MS/MS method developed in this work.