title: Comparison of a stable isotope-labeled and an analog internal standard for the quantification of everolimus by a liquid chromatography-tandem mass spectrometry method.
abstract: BACKGROUND
  Everolimus is an immunosuppressant drug used in solid organ transplantation. Immunoassays and liquid chromatography-mass spectrometry (LC-MS) methods have been used for therapeutic drug monitoring of this drug. In LC-tandem mass spectrometry (MS/MS) methods, both 32-desmethoxyrapamycin and everolimus-d4 have been used as internal standards.
  
  
  OBJECTIVE
  To compare 2 internal standards (32-desmethoxyrapamycin and everolimus-d4) for the quantification of everolimus by an LC-MS/MS method.
  
  
  METHODS
  Both 32-desmethoxyrapamycin and everolimus-d4 were introduced in the method validation process with 2 transitions simultaneously monitored for everolimus (975.6 → 908.7 as the quantifier and 975.6 → 926.9 as the qualifier) by an established LC-MS/MS method. The key performance characteristics were lower limit of quantification, accuracy, precision, and comparison with an LC-MS/MS method offered by another laboratory.
  
  
  RESULTS
  The lower limit of quantification (LLOQ) was 1.0 ng/mL using either internal standard with an analytical recovery of 98.3%-108.1% across the linear range. The total coefficient of variation for everolimus was 4.3%-7.2% with no significant difference between the 2 internal standards. In comparison with an independent LC-MS/MS method, though everolimus-d4 offered a better slope (0.95 versus 0.83), both internal standards showed acceptable results and had a coefficient of correlation r > 0.98 in the tested concentration range of 1.2-12.7 ng/mL.
  
  
  CONCLUSIONS
  Although everolimus-d4 offered a more favorable comparison with an independent LC-MS/MS method, both everolimus-d4 and 32-desmethoxyrapamycin had acceptable performance as the internal standards for everolimus quantification by the LC-MS/MS method.
authors.affiliation: Department of Clinical Pathology, Cleveland Clinic, Cleveland, OH 44195, USA.
authors.forename: Courtney
authors.initials: C
authors.lastname: Heideloff
authors.forename: Drew
authors.initials: D
authors.lastname: Payto
authors.forename: Sihe
authors.initials: S
authors.lastname: Wang
mesh_terms: D002853:Chromatography, Liquid / Q000379:methods / Q000592:standards; D000068338:Everolimus; D006801:Humans; D007166:Immunosuppressive Agents / Q000097:blood*; D007553:Isotope Labeling / Q000379:methods / Q000592:standards; D020123:Sirolimus / Q000031:analogs & derivatives* / Q000097:blood; D053719:Tandem Mass Spectrometry / Q000379:methods / Q000592:standards*
chemical_list: D007166:Immunosuppressive Agents; D000068338:Everolimus; D020123:Sirolimus