# Clinical Mass Spectrometry in Immunosuppressant Analysis: Toward a Full Automation?
## 3 Results and Discussion
### Method Performance Evaluation
\begin{table}
\begin{tabular}{c l l c c} \hline \hline  & **Analyte** & **LLOQ [ng/mL]** & **LLOQ [ng/mL]** & **Linearity [ng/mL]** \\ \hline \multirow{3}{*}{Shimadzu–Recipe} & CsA & 1.75 & 5.26 & 5.6–1713.0 \\  & Tacrolimus & 0.04 & 0.13 & 0.130–60.9 \\  & Sirolimus & 0.05 & 0.15 & 0.150–67.6 \\  & Everolimus & 0.05 & 0.15 & 0.150–67.7 \\ \hline \multirow{3}{*}{Cascadion} & CsA & 2.83 & 8.5 & 8.5–920.0 \\  & Tacrolimus & 0.28 & 0.85 & 0.85–34.5 \\  & Sirolimus & 0.28 & 0.85 & 0.85–34.5 \\  & Everolimus & 0.28 & 0.85 & 0.85–34.5 \\ \hline \hline \end{tabular}
\end{table} 
Table 3: LLOD (Lower Limit of Detection), LLOQ (Lower Limit of Quantitation), and linearity are shown both for Shimadzu–Recipe and for Cascadion, together with the corresponding times for calibration and analysis of 50 samples per day for a total of 30 days. \begin{table}
\begin{tabular}{c l 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 (\%)** & **CV (\%)** & **Bias (\%)** \\ \hline \multirow{3}{*}{CsA} & I & 15.43 & 15.95 & 1.5 & 2.9 & 3.4 \\  & II & 374.64 & 375.94 & 1.0 & 2.9 & 0.3 \\  & III & 644.44 & 644.94 & 1.1 & 3.0 & 2.2 \\ \hline \multirow{3}{*}{Tacrolimus} & I & 2.03 & 2.10 & 4.9 & 5.6 & 3.7 \\  & II & 12.50 & 12.80 & 3.2 & 4.5 & 2.4 \\  & III & 25.22 & 25.82 & 2.9 & 4.3 & 2.4 \\ \hline \multirow{3}{*}{Sirolimus} & I & 2.06 & 2.08 & 4.8 & 6.3 & 0.8 \\  & II & 12.34 & 12.59 & 4.0 & 5.8 & 2.0 \\ \cline{1-1}  & III & 24.22 & 25.62 & 3.4 & 5.1 & 5.8 \\ \hline \multirow{3}{*}{Everolimus} & I & 1.93 & 2.05 & 4.6 & 5.5 & 6.4 \\ \cline{1-1}  & II & 12.13 & 12.40 & 4.4 & 5.4 & 2.2 \\ \cline{1-1}  & III & 24.55 & 25.21 & 4.5 & 5.7 & 2.7 \\ \hline \hline \end{tabular}
\end{table} 
Table 5: Intra- and inter-assay precision and accuracy (given as bias %) regarding Cascadion analysis are shown for each control level (I–III). Furthermore, maximum bias observed in Shimadzu-Recipe methodology was 8.2% for CsA whereas that observed in Cascadion was 6.4% for everolimus analysis. In both cases, all observed values are suitable for a clinical setting. In Figure 2 and Table 6, the correlation plots for each analyte are shown, together with the number of samples analyzed, the equations of the fitting plots, and the confidence intervals (CI). A good correlation between the two methods was observed. Moreover, it can be noted that for all four analytes there are strong correlation values without significant variations from linearity as the correlation coefficients for cyclosporine A (0.997), tacrolimus (0.979), \begin{table}
\begin{tabular}{l c c c c c} \hline \hline \multirow{2}{*}{**Analyte**} & \multirow{2}{*}{**N**} & **Shimadzu–Recipe Mean** & **Cascadion Mean** & **Intercept (95\% CI)** & **Slope (95\% CI)** \\  & & **(SD) [ng/mL]** & **(SD) [ng/mL]** & **[ng/mL]** & **Slope (95\% CI)** \\ \hline CsA & 203 & 165.62 (158.68) & 158.27 (152.18) & \(-\)2.03 (\(-\)3.07 to \(-\)1.13) & 0.96 (0.95 to 0.98) \\ Tacrolimus & 1757 & 7.09 (2.85) & 6.65 (2.67) & \(-\)0.04 (\(-\)0.09 to 0.02) & 0.94 (0.93 to 0.95) \\ Sirolimus & 42 & 6.85 (2.22) & 6.68 (2.04) & 0.37 (\(-\)0.30 to 1.14) & 0.90 (0.78 to 1.04) \\ Everolimus & 117 & 5.03 (2.00) & 5.44 (1.97) & 0.30 (\(-\)0.10 to 0.89) & 1.00 (0.90 to 1.09) \\ \hline \hline \end{tabular}
\end{table} 
Table 6: For each analyte, mean (standard deviation) and Passing–Bablok regression parameters are shown both for the reference and test method. Figure 2: Passing–Bablok fit (red line) for cyclosporine A (**A**), tacrolimus (**B**), sirolimus (**C**), and everolimus (**D**). Grey line indicates the perfect correlation (slope = 1, intercept = 0). Each plot is coupled to regression results. sirolimus (0.939), and everolimus (0.929) show. A good correlation, however, was expected as both the methodologies are based on LC-MS/MS analysis which is the gold standard for immunosuppressive analysis.