title: High-throughput LC-MS/MS method for monitoring sirolimus and everolimus in the routine clinical laboratory.
abstract: BACKGROUND
  Immunosuppressant therapeutic drug monitoring (TDM) is an important requirement in post-transplant patient care. In recent years, high-performance liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) has become a valid alternative to antibody-based immunoassays in TDM due to its high specificity and sensitivity. Furthermore, this technology allows for the simultaneous measurement of several immunosuppressive drugs. The aim of the present study was to establish a straightforward, robust, and high-throughput LC-MS/MS method for the simultaneous determination of sirolimus and everolimus in whole blood in order to replace immunoassays in our routine practice.
  
  
  METHODS
  Five-level blood calibrators were employed for assay development, while three materials at different concentrations were used for internal quality control. The proposed method uses protein precipitation for sample preparation. Analyses were performed using a triple quadrupole LC-MS/MS, with a C18 held at 60°C. Using an appropriate gradient elution profile and SPE on-line, elution times for all compounds analysed were 2.6 min with a total run-time of 3.5 min.
  
  
  RESULTS
  Calibration curves were linear throughout the selected ranges. The intra- and inter-assay CVs (<7%), the limit of quantification (0.2 μg/L) and accuracy were highly satisfactory. On testing the results using the international proficiency testing scheme (UK-NEQAS), the performance of the proposed method was found to be highly reliable.
  
  
  CONCLUSIONS
  The findings made by us indicate that the proposed method is of value, since it is speedy, straightforward, accurate, and applicable to different LC-MS/MS instruments for the routine TDM of organ transplant recipients.
authors.affiliation: Department of Laboratory Medicine, University-Hospital, Padova, Italy.
authors.forename: Mariela
authors.initials: M
authors.lastname: Ivanova
authors.forename: Carlo
authors.initials: C
authors.lastname: Artusi
authors.forename: Giulia
authors.initials: G
authors.lastname: Polo
authors.forename: Martina
authors.initials: M
authors.lastname: Zaninotto
authors.forename: Mario
authors.initials: M
authors.lastname: Plebani
mesh_terms: D053000:Analytic Sample Preparation Methods; D001774:Blood Chemical Analysis / Q000191:economics / Q000379:methods*; D002853:Chromatography, Liquid / Q000191:economics / Q000379:methods*; D000068338:Everolimus; D006801:Humans; D007166:Immunosuppressive Agents / Q000097:blood*; D015203:Reproducibility of Results; D020123:Sirolimus / Q000031:analogs & derivatives* / Q000097:blood; D053719:Tandem Mass Spectrometry / Q000191:economics / Q000379:methods*; D013997:Time Factors
chemical_list: D007166:Immunosuppressive Agents; D000068338:Everolimus; D020123:Sirolimus