# Performance Qualification of Automatic System for Antineoplastic Preparation
## 2 Materials and Methods
### Quality Control Procedure  
To perform quantitative assay analysis, reference standards of fluorouracil, paclitaxel and cyclophosphamide were obtained from Sigma Aldrich Merck (Darmstadt, Germany). High-purity water was prepared in-house, using a gradient water purification system. LC-MS grade methanol was purchased from Honeywell-Riedel de Haen.  
Formic acid (LC-MS grade) was bought from Carlo Erba. MS-grade ammonium formate was purchased from Sigma Aldrich.  
Stock solutions of fluorouracil, paclitaxel and cyclophosphamide were prepared in methanol at a concentration of 1000 \(\upmu\)g/mL. Primary dilutions and working standard solutions were prepared from the stock solutions by dilution with water/methanol (90:10, \(v\)/\(v\)).  
These working standard solutions were used to prepare the quality control samples and calibration curve, which are detailed below for each analyte, as shown in Figures 3-5.  
Figure 4: Calibration report of the analyte cyclophosphamide obtained using a UHPLC system with a high-resolution mass spectrometer.  
Figure 3: Calibration report of the analyte 5-fluorouracilç obtained using a UHPLC system with a high-resolution mass spectrometer.  
Calibration samples were prepared at concentrations of 0.1, 0.5 and 1 ug/mL for paclitaxel, at concentrations of 1, 5 and 10 ug/mL for cyclophosphamide and at concentration of 2, 10 and 20 ug/mL for fluorouracil.  
Samples were prepared by dilution in water/methanol (90:10 \(v/v\)). A total of 2 uL of the sample was injected into the UHPLC-HRMS system through the autosampler.  
Chromotographic separation was performed on a Thermo-Dionex ULTIMATE 3000 UHPLC system using a Thermo Scientific Accucore aQ C18 column built by Thermo Fisher Scientific, Waltham, Massachusetts, MAUSA 02451, (100 \(\times\) 2.1 mm i.d., 2.6 um) under multistep gradient conditions. The mobile phase consisted of ammonium formate 5 mM in 0.1% formic acid in water (solvent A) and ammonium formate 5 mM in 0.1% formic acid in methanol (solvent B). The gradient elution program was as follows: 0-2 min 30% B; 2-7 min, 30-70% B; 7-9 min, 70-95% B; 9-12 min, 95% B; 12-12.1 min, 95%- 30% B; 12-120 min, 30% B. The column was maintained at 10 \({}^{\circ}\)C, the autosampler was set at 10 \({}^{\circ}\)C and the injection volume was 2 uL. The HPLC eluent was directly introduced into the heated electrospray ionization source and the total run time for analysis of each sample was 20 min.  
The concentration of each analyte was quantified using a Thermo Scientific Orbitrap Q-Exactive focus equipped with a heated ion spray interface. The ion spray voltage was 4500 V with an auxiliary gas temperature of 250 \({}^{\circ}\)C and a capillary temperature of 320 \({}^{\circ}\)C. Operating conditions were optimized by injecting a mixture of all analytes and were as follows: sheath gas flow 35 arbitrary units (au); auxiliary gas flow 10 au. Quantification was performed in full-scan mode with an operative range of 100 to 900 \(m/z\) in polarity switching. The extracted ions, with 5 ppm tolerance, are shown in Table 4.  
The orbitrap was set to a resolution of 70,000. Automatic data acquisition and analysis were performed with Trace Finder software (version 3.3). For quantification, the peak area of target ions was compared with least-squares calibration curves in which the peak area of calibration standards was plotted against their concentrations.  
\begin{table}
\begin{tabular}{c c c c c} \hline
**Mass (\(m/z\))** & **Formula** & **Species** & **Polarity** & **Analytic** \\ \hline
129.01058 & C4H3 FN2O2 & \(-\)H & Negative & Fluorouracil \\
261.03210 & C7H15 Cl2N2O2P & +H & Positive & Cyclophosphamide \\
286.10709 & C47H51 NO14 & +H-C31H36O10 & Positive & Paclitaxel \\ \hline \end{tabular}
\end{table}
Table 4: Operational methods adopted for quantification of extracted ions.  
Figure 5: Calibration report of the analyte paclitaxel obtained using a UHPLC system with a high-resolution mass spectrometer.