## 3 Materials and Methods
### Chemometric Evaluation  
The application of a Plackett-Burman design allowed us to evaluate the effect of 9 factors on sample extraction by performing 12 experiments.  
The responses selected to develop the method were four in number: the peak areas of the analytes (cyclophosphamide,ophosphamide, and internal standard) and the duration of the sample extraction. The aim of the experimental design was to maximize the peak areas, both for the analytes and the internal standard, which means maximizing their sensitivity and, at the same time, minimizing the sample extraction duration.  
The factors evaluated to study the sample extraction on the ePrep ONE workstation were the volume of methanol utilized to condition the \(\upmu\)SPE cartridge (x\({}_{1}\)), the volume of basic water utilized to equilibrate the \(\upmu\)SPE cartridge (x\({}_{2}\)), the volume of the washing solution (x\({}_{3}\)), the composition of the washing solution (x\({}_{4}\)), the sample loading speed (x\({}_{5}\)), the dispensing speed for the conditioning step (x\({}_{6}\)), the dispensing speed for the equilibration step (x\({}_{7}\)), the dispensing speed for the washing step (x\({}_{8}\)), and the dispensing speed for the elution step (x\({}_{9}\)). Two dummy factors were added to the experimental plan (Table 2, factors x\({}_{10}\) and x\({}_{11}\)) to estimate the random error; additionally, three validation points (experiments 1, 6, and 7) were randomly chosen to assess the concordance between the experimental observations and the model predictions.  
The data were collected using Microsoft Excel and processed using Rstudio Version 1.2.1335 (c2009-2019 Rstudio, Inc., as GUI for R version 3.6.1 (5 July 2019) \"Action of the Toes\", c2019 (Rstudio PBC, Boston, MA, USA). The R Foundation for Statistical Computing.  
\begin{table}
\begin{tabular}{c c c c c c c c c c c c c} \hline \hline \multirow{2}{*}{\#} & \(\mathbf{x_{1}}\) & \(\mathbf{x_{2}}\) & \(\mathbf{x_{3}}\) & \(\mathbf{x_{4}}\) & \(\mathbf{x_{5}}\) & \(\mathbf{x_{6}}\) & \(\mathbf{x_{7}}\) & \(\mathbf{x_{8}}\) & \(\mathbf{x_{9}}\) & \(\mathbf{x_{10}}\) & \(\mathbf{x_{11}}\) \\  & **(mL)** & **(mL)** & **(mL)** & **(\%MoOH, \%er)** & **(mL/s)** & **(mL/s)** & **(mL/s)** & **(mL/s)** & **(mL/s)** & **(mL/s)** & **(Dummy)** & **(Dummy)** \\ \hline
1 & 0.35 & 0.35 & 0.35 & 5 & 15 & 55 & 55 & 15 & 15 & 0 & 0 \\
2 & 0.5 & 0.2 & 0.2 & 0 & 25 & 15 & 55 & 25 & 5 & 1 & 1 \\
3 & 0.5 & 0.2 & 0.5 & 10 & 5 & 55 & 55 & 25 & 5 & \(-1\) & \(-1\) \\
4 & 0.2 & 0.2 & 0.5 & 0 & 25 & 55 & 15 & 25 & 25 & 1 & \(-1\) \\
5 & 0.2 & 0.5 & 0.2 & 10 & 25 & 15 & 55 & 25 & 25 & \(-1\) & \(-1\) \\
6 & 0.35 & 0.35 & 0.35 & 5 & 15 & 55 & 55 & 15 & 15 & 0 & 0 \\
7 & 0.35 & 0.35 & 0.35 & 5 & 15 & 55 & 55 & 15 & 15 & 0 & 0 \\
8 & 0.5 & 0.5 & 0.2 & 10 & 25 & 55 & 15 & 5 & 5 & 1 & \(-1\) \\
9 & 0.2 & 0.2 & 0.2 & 0 & 5 & 15 & 15 & 5 & 5 & \(-1\) & \(-1\) \\
10 & 0.2 & 0.2 & 0.2 & 10 & 5 & 55 & 55 & 5 & 25 & 1 & 1 \\
11 & 0.5 & 0.2 & 0.5 & 10 & 25 & 15 & 15 & 5 & 25 & \(-1\) & 1 \\
12 & 0.2 & 0.5 & 0.5 & 0 & 25 & 55 & 55 & 5 & 5 & \(-1\) & 1 \\
13 & 0.2 & 0.5 & 0.5 & 10 & 5 & 15 & 15 & 25 & 5 & 1 & 1 \\
14 & 0.5 & 0.5 & 0.2 & 0 & 5 & 55 & 15 & 25 & 25 & \(-1\) & 1 \\
15 & 0.5 & 0.5 & 0.5 & 0 & 5 & 15 & 55 & 5 & 25 & 1 & \(-1\) \\ \hline \hline \end{tabular}
\end{table}
Table 2: Experimental plan applied in the study. The experiments are listed in the random order applied during the working session.