## 2 Results and Discussion
### Method Development  
Method development was undertaken by applying a Plackett-Burman 12-run design. From the data obtained by applying a Plackett-Burman design, it was clear that the conditions needed to perform the sample extraction, allowing the highest sensitivity for both the analytes, were mainly affected by the volume of the washing solution step (x\({}_{2}\)), the composition of the washing solution (x\({}_{4}\)), and the sample loading speed on the cartridge (x\({}_{5}\)), as can be seen in Figure 1. The duration of the sample treatment, instead, depends mainly on the speed of loading and eluting steps and, thus, on the volumes utilized in these steps. The multilinear regression model describing the sensitivity of the internal standard (IS) was not significant, and thus, it is not reported and only briefly mentioned here. None of the model's coefficients were statistically significant, meaning that the selected factors (x\({}_{1}\)-x\({}_{9}\)) have no impact on the IS peak area. Therefore, slight modifications to the extraction procedure do not affect the IS peak area. This observation supports the conclusion that the method is robust as regards the isolation and detection of the IS. So, this model could be neglected and the study can be focused on the one describing the sensitivity of the analytes. The model describing the sensitivity of cyclophosphamide has been validated (confidence interval of 95%) because the experimental response (65,490 \(\pm\) 10,678 counts) was not significantly different from the predicted one (59,339 \(\pm\) 10,678 counts) at the test points. In particular, the cyclophosphamide peak area was greatest when (factors are listed according to the significance of their coefficients):  
* the composition of the washing solution is 90:10 of H\({}_{2}\)O/MeOH (the highest level for factor x\({}_{4}\));
* the volume of basic water utilized to equilibrate the \(\upmu\)SPE cartridge is 0.2 mL (the lowest level for factor x\({}_{2}\));
* the sample loading speed is 25 \(\upmu\)L/s (the highest level for factor x\({}_{5}\)). * the volume of water utilized to equilibrate the \(\upmu\)SPE cartridge is 0.2 mL (the lowest level for factor x\({}_{2}\));
* the sample loading speed is 25 \(\upmu\)L/s (the highest level for factor x\({}_{5}\));
* the dispensing speed4 for the conditioning step is 55 \(\upmu\)L/s (the highest level for factor x\({}_{6}\)). The model describing the duration of the sample extraction is not validated (confidence intervals of 95% probability are: for the experimental response, 716 \(\pm\) 9 s and for the predicted response, 804 \(\pm\) 9 s) but the reason for this is that the experiments at the test points provided results of high precision (RSD 1.3%). So, in our opinion, although the model is not validated, the screening results provide important information that can be used to minimize the time interval for the extraction. The duration of the extraction is the shortest when:  
* the sample loading speed is 25 \(\upmu\)L/s (the highest level for factor x\({}_{5}\));
* the volume of washing solution is 0.2 mL (the lowest level for factor x\({}_{3}\));
* the dispensing speed of the washing solution is 25 \(\upmu\)L/s (the highest level for factor x\({}_{8}\));
* the volume of methanol utilized to condition the \(\upmu\)SPE cartridge is 0.2 mL (the lowest level for factor x\({}_{1}\));
* the volume of water utilized to equilibrate the \(\upmu\)SPE cartridge is 0.2 mL (the lowest level for factor x\({}_{2}\));
* the dispensing speed for the conditioning step is 55 \(\upmu\)L/s (the highest level for factor x\({}_{6}\));
* the dispensing speed for the eluting step is 25 \(\upmu\)L/s (the highest level for factor x\({}_{9}\)). Moreover, the responses for the S/N ratio of cyclophosphamide and iphosphamide were computed, and their models were validated at the confidence interval of 95%.