# Clinical Mass Spectrometry in Immunosuppressant Analysis: Toward a Full Automation?
## 3 Results and Discussion  
### Sample Preparation and Analysis  
Two systems based on the gold-standard methodology, LC-MS/MS, for the analysis of immunosuppressants (cyclosporine A, tacrolimus, sirolimus, and everolimus) in whole blood were compared. In particular, the manual sample preparation followed by multiparametric LC-MS/MS analysis (Shimadzu-Recipe) was compared to a recently introduced fully automated technology (Cascadion). The comparison was carried out analyzing in parallel 2022 blood samples for a total of 2119 assays. Few patients, in fact, were subjected to therapy consisting of administration of multiple ISDs, for this reason a small number of samples were tested for multiple analytes.  
\begin{table}
\begin{tabular}{l c c c c c c} \hline \hline \multirow{3}{*}{**Analyte**} & \multirow{3}{*}{\begin{tabular}{c} **RT** \\ **(min)** \\ \end{tabular} } & \multicolumn{3}{c}{**Quantification Ions**} & \multicolumn{3}{c}{**Qualification Ions**} & \multirow{3}{*}{\begin{tabular}{c} **CE** \\ **(V)** \\ \end{tabular} } \\  & & \begin{tabular}{c} **Precursor** \\ **Ion (\(m\)/z)** \\ \end{tabular} & \begin{tabular}{c} **Product** \\ **Ion (\(m\)/z)** \\ \end{tabular} & \begin{tabular}{c} **Precursor** \\ **Ion (\(m\)/z)** \\ \end{tabular} & \
\begin{tabular}{c} **Product** \\ **Ion (\(m\)/z)** \\ \end{tabular} &
\begin{tabular}{c} **(V)** \\ \end{tabular} \\ \hline CsA & 1.42 & 1219.7 & 1202.8 & 1219.7 & 1184.8 & 19 \\ D12-CsA & 1.42 & 1232.0 & 1215.0 & 1232.0 & 1197.1 & 19 \\ Tacrolimus & 1.24 & 821.5 & 768.4 & 821.5 & 576.3 & 23 \\
13C-D2-Tacrolimus & 1.24 & 824.5 & 771.5 & 824.5 & 579.3 & 23 \\ Sirolimus & 1.29 & 931.5 & 864.5 & 931.5 & 882.5 & 18 \\
13C-D3-Sirolimus & 1.29 & 935.6 & 864.7 & 935.6 & 882.2 & 18 \\ Everolimus & 1.31 & 975.6 & 908.4 & 975.6 & 926.7 & 19 \\
13C2D4-Everolimus & 1.31 & 981.6 & 914.7 & 981.6 & 932.6 & 19 \\ \hline \hline \end{tabular}  
*Quantification was achieved by using 4 calibration levels in whole blood and 3 quality controls (Recipe).  
\end{table}
Table 1: MRM transitions adopted for analysis with Shimadzu–Recipe. Monitored analytes are listed together with the corresponding retention time (RT), precursor, and product ions used both for quantification and for qualification, and collision energy (CE).  
With regard to Shimadzu-Recipe, the cumulative chromatogram of tacrolimus, cyclosporine A, sirolimus, and everolimus (Figure 1) shows all of the used mass transitions, and the corresponding retention times for each substance. Representative chromatograms obtained with the Cascadion are also shown for each analyte (Figure 1).  
MS data show good peak shape and separation in both cases, despite the different chromatographic conditions (isocratic conditions vs organic gradient).  
In the Shimadzu-Recipe method, the calibration is performed once a day with the use of four calibration levels. Each calibration point has an analytical run of 2 min, for a total of about 8 min per analytical session, that is, 4 h/month (where 1 month = 30 working days). Cascadion performs calibration every 30 days using six calibration points analyzed in duplicate on each LC channel, then the duration of the calibration is approximately 18 min/month. Hence, the time for calibration provided by the automated method is largely lower with respect to those provided by the open LC-MS/MS. Table 2 shows an estimate of the times required by each of the two methods for both preparation and calibration, and analysis of 50 samples per day.