# Clinical Mass Spectrometry in Immunosuppressant Analysis: Toward a Full Automation?
## 3 Results and Discussion
### Method Performance Evaluation  
The lower detection limit (LLOD), the lower quantitation limit (LLOQ), and the linearity range of each analyte for both technologies are reported in Table 3.  
LLOD and LLOQ were found to be higher in Cascadion. Despite this, for both methodologies the values of LLOD and LLOQ are suitable to determine the immunosuppression levels according to the therapeutic range proposed by the recently published consensus recommendations [16], but also to the ranges built on the patient population offering to the \"Maggiore della Carita\" University Hospital of Novara. Moreover, in Tables 4 and 5, intra-assay and inter-assay precision, and accuracy as bias %, measured on three concentration levels for each analyte, are reported. Both platforms \begin{table}
\begin{tabular}{l l l l} \hline \hline  & \multicolumn{1}{c}{**Sample Preparation**} & \multicolumn{1}{c}{**Calibration**} & \multicolumn{1}{c}{**Sample Analysis**} \\ \hline Shimadzu–Recipe & \
\begin{tabular}{l} \(\sim\)1 h, calibrators and controls \\ included (manual operation). \\ 30 min for the first result, then a \\ result at \(\sim\)2.5 min intervals. \\ \end{tabular} & 8 min/day & 100 min/day \\ \cline{2-3} Cascadion &
\begin{tabular}{l} 30 min for the first result, then a \\ result at \(\sim\)2.5 min intervals. \\ \end{tabular} & \(\sim\)18 min/month & 75 min/day \\ \hline \hline \end{tabular}
\end{table} 
Table 2: Times for preparation, calibration, and analysis of 50 samples/day are shown both for Shimadzu–Recipe and for Cascadion. Figure 1: Upper chromatograms: representative chromatograms of level 4 calibrator (tacrolimus = 21.50 ng/mL; CsA = 483.00 ng/mL; everolimus = 22.60 ng/mL; sirolimus = 22.70 ng/mL) used for immunosuppressiveant quantitation obtained by Shimadzu–Recipe analysis. Lower chromatograms: representative chromatograms of level 2 calibrator (tacrolimus = 2.19 ng/mL; CsA = 24.85 ng/mL; everolimus = 1.98 ng/mL; sirolimus = 1.90 ng/mL) used for immunosuppressiveant quantitation obtained by Cascadion. showed low intra-assay CV%: both for Shimadzu-Recipe and Cascadion, the intra-assay CV% is lower than 4.9%. Moreover, with regard to the inter-assay CV%, in Shimadzu-Recipe it was lower than 5.6%, whereas for Cascadion it was lower than 6.3%. Both methodologies provide CV% fully suitable for laboratory and clinical needs whereby the fully automated system shows even lower CV%. \begin{table}
\begin{tabular}{c c c c c c c} \hline \hline \multirow{2}{*}{**Analyte**} & **Control Level** & **Assigned Concentration** & **Experimental Mean** & **Intra-Assay** & **Inter-Assay** & **Bias (\%)** \\  & **Level** & **[ng/mL]** & **Concentration (ng/mL]** & **CV (\%)** & **Inter-Assay** & **Bias (\%)** \\ \hline \multirow{3}{*}{CsA} & I & 58.40 & 59.40 & 2.4 & 5.5 & 1.7 \\  & II & 118.00 & 120.34 & 2.4 & 4.0 & 2.0 \\  & III & 232.00 & 251.10 & 1.4 & 2.1 & 8.2 \\ \hline \multirow{3}{*}{Tacrolimus} & I & 3.67 & 3.81 & 2.8 & 4.7 & 3.9 \\  & II & 7.69 & 7.85 & 3.1 & 4.5 & 2.1 \\  & III & 15.50 & 15.49 & 2.4 & 4.2 & \(-\)0.1 \\ \hline \multirow{3}{*}{Sirolimus} & I & 4.08 & 4.18 & 4.6 & 5.1 & 2.5 \\  & II & 12.70 & 12.69 & 4.1 & 5.6 & \(<\)0.1 \\  & III & 21.70 & 21.32 & 3.3 & 4.7 & \(-\)1.7 \\ \hline \multirow{3}{*}{Everolimus} & I & 3.84 & 3.96 & 4.9 & 4.8 & 3.1 \\  & II & 12.50 & 12.58 & 3.5 & 3.1 & 0.7 \\ \cline{1-1}  & III & 20.50 & 20.32 & 3.5 & 3.7 & \(-\)0.9 \\ \hline \hline \end{tabular}
\end{table} 
Table 4: Intra- and inter-assay precision and accuracy (given as bias %) regarding Shimadzu–Recipe analysis are shown for each control level (I–III).