title: A direct method for the measurement of everolimus and sirolimus in whole blood by LC-MS/MS using an isotopic everolimus internal standard.
abstract: BACKGROUND
  Whole-blood concentrations of the immunosuppressant drugs everolimus and sirolimus should be monitored. A sensitive and selective method offering the detection of both analytes in small sample volumes would optimize the throughput of samples for sirolimus or everolimus analysis. This study reports the validation of a liquid chromatography tandem mass spectrometry method, including a stable isotope internal standard, for the simultaneous measurement of everolimus and sirolimus.
  
  
  METHODS
  Whole-blood samples (20 μL) were treated with ammonium bicarbonate (20 μL), zinc sulfate (20 μL), and internal standard solution ((13)C(2)D(4)-everolimus in acetonitrile, 100 μL). After centrifugation, 20 μL of the supernatant was injected onto a Waters Symmetry C18 high-performance liquid chromatography column. The aqueous and organic mobile phases were 2 mmole/L of ammonium acetate containing 0.1% (vol/vol) formic acid, in water and methanol, respectively. Analytes were detected using tandem mass spectrometry (Waters Acquity TQD).
  
  
  RESULTS
  Analytes were eluted at around 2 minutes within a 6-minute analytical run time. Detector response was linear for both analytes across the ranges studied (1-49 μg/L for sirolimus, 1-41 μg/L for everolimus), and a lower limit of quantitation of 1 μg/L was reliably attained. Intraassay and interassay imprecision and inaccuracy were <15% (coefficient of variation) in all cases. Analyte recovery was in the range of 72%-117%. The analytes were stable in blood after freezing and thawing, and for at least 12 hours after preparation while waiting to be injected. Ion suppression and interference from phospholipids were not significant.
  
  
  CONCLUSIONS
  A straightforward, robust liquid chromatography tandem mass spectrometry assay has been developed and validated for the simultaneous measurement of everolimus and sirolimus in small volumes of whole blood.
authors.affiliation: *Immunosuppressive Drug Monitoring Service, Institute of Liver Studies, King's College Hospital, London, United Kingdom; †Department of Clinical Chemistry, Wansbeck General Hospital, Ashington, United Kingdom; and ‡King's College London, London, United Kingdom.
authors.forename: Phillip E
authors.initials: PE
authors.lastname: Morgan
authors.forename: Nigel W
authors.initials: NW
authors.lastname: Brown
authors.forename: J Michael
authors.initials: JM
authors.lastname: Tredger
mesh_terms: D002138:Calibration; D002851:Chromatography, High Pressure Liquid; D016903:Drug Monitoring / 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
chemical_list: D007166:Immunosuppressive Agents; D000068338:Everolimus; D020123:Sirolimus