# Clinical Mass Spectrometry in Immunosuppressant Analysis: Toward a Full Automation?
###### Abstract  
The analysis of immunosuppressive drugs allows the physician to monitor, and eventually correct, immunosuppressive therapy. The panel of molecules under evaluation includes cyclosporine A (CsA), tacrolimus, sirolimus, and everolimus. Initially, assays were performed by immunometric methods, but in the past few years this methodology has been largely superseded by a more accurate and specific technique, liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS), which is now considered the \"gold standard\" for immunosuppressiveant analysis. Both LC-MS/MS and often also immunoassays require a preanalytical manual sample preparation, which involves time-consuming sequential operations whose traceability is often hampered and adds up to the probability of gross errors. The aim of this work was to compare an \"open\" LC-MS/MS with a fully automated system, consisting of LC instrumentation combined with a triple quadrupole MS, named Thermo ScientificTM CascadionTM SM Clinical Analyzer (Cascadion). Such automated systems suit the requirements of the reference method and are designed to completely eliminate all of the manual procedures. More than 2000 immunosuppressant samples were analyzed both with the open LC-MS/MS and with Cascadion. Statistics allowed the evaluation of linearity, intra- and inter-assay CV%, bias %, limit of detection and of quantitation, and Passing-Bablok and Bland-Altman plots. Results indicated a good correlation between the two methods. In both cases, methods confirmed their suitability for diagnostic settings. Cascadion could provide support when the presence of specialized personnel is lacking, and/or when great productivity and continuous workflow are required. 24 February 20224 September 5 April 2  
The goal of the treatment, in fact, is to reduce the risk of graft rejection while avoiding the negative effects related to toxicity (nephrotoxicity, neurotoxicity, diabetes, gastralgia, hyperlipidemia) [2]. From an analytical point of view, initially ISD assays were performed by using immunometric methods which offer high throughput, satisfy the need for automation, and are easy to use. However, it is well known that such techniques are susceptible to interferences [3; 4], and are affected by antibody cross-reactivity with drug metabolites, causing an overestimation of ISD concentration, which limits analytical specificity [3; 5; 6]. In the last 10 years, a growing role was taken by liquid chromatography coupled to mass spectrometry (LC-MS), a more accurate, precise, and specific technique, as it is not affected by interferents and by the presence of inactive ISD metabolites. The features of LC-MS, and in particular of tandem mass spectrometry (LC-MS/MS), allow the simultaneous quantification of a large number of molecules, rapid execution times, and confident quantitative analyses [7; 8], which led this methodology to be the gold standard for ISD analysis. On the other side, LC-MS/MS is more complex with regard to instrumental management and usually requires highly specialized personnel, as the main challenge connected to this methodology consists of LC-MS/MS method development and time requirements for proper validation. However, regardless of the technique used, the ISD assays require a pre-analytical preparation of the sample, consisting of the lysis and precipitation of the red blood cells and proteins, in order to collect the ISDs containing supernatant to analyze. ISDs, in fact, are mainly bound to erythrocytes, so this purification step is mandatory [9; 10]. Often, these steps are manually performed as well as operations such as numbering/transcription, programming of the work list on the instrument, and the subsequent insertion of the results in the laboratory information system. Beyond being time-consuming, these steps also amplify the probability of gross errors. In ISD analysis, the need for timely, 24/7 diagnostics often collides with the parallel need for \"LC-MS grade\" analytical precision.