title: Procedure for determination of immunosuppressive drugs in whole blood with liquid chromatography-isotope dilution mass spectrometry.
abstract: BACKGROUND
  Cyclosporin A, sirolimus, tacrolimus, and everolimus are immunosuppressive drugs used for therapy after organ transplantation. There are several analytical procedures for monitoring the drug level in blood, e.g. immunological methods and high-performance liquid chromatography combined with mass spectrometry (MS). From external quality assessment schemes, it became evident that the analytical results show high dispersion and further standardization is required.
  
  
  METHODS
  Liquid/liquid extraction of the drugs from whole blood samples was performed using ammonium acetate buffer, pH 9.5, and tert-butylmethyl ether/ethyl acetate (1:1 v/v). Separation of the immunosuppressive drugs was achieved by HPLC using a phenyl-hexyl-RP column with a ternary gradient elution profile, consisting of water, methanol, and acetonitrile containing 0.1% v/v formic acid and 0.1 mmol/L Cs+. Quantification of immunosuppressive drugs was performed by isotope-dilution mass spectrometry using [2H12]-Cyclosporin A [13C, 2H3]-Rapamycin, [13C, 2H2]-Tacrolimus, and [13C2, 2H4]-42-O-(2-Hydroxyethyl)rapamycin as internal standards.
  
  
  RESULTS
  The recovery of the new procedure was determined by analysis of spiked blood samples. The recovery in spiked EDTA whole blood samples was 100.8 - 102.5% for cyclosporin A, 101.6 - 103.0% for sirolimus, 100.0 - 101.2% for tacrolimus, and 99.5 - 102.4% for everolimus. The imprecision of the new measurement procedure, expressed as the coefficient of variation (CV), was 1.17 - 2.60% for cyclosporin A in the concentration range between 8.1 and 979 microg/L, 0.92 - 1.72% for sirolimus in the concentration range between 2.1 and 33.2 microg/L, 0.44 - 1.06% for tacrolimus in the concentration range between 2.0 and 30.8 microg/L and 0.82 - 4.34% for everolimus in the concentration range between 2.1 and 31.4 microg/L.
  
  
  CONCLUSIONS
  An isotope dilution LC-MS/MS procedure for determination of four immunosuppressive drugs was developed to provide a basis for further development toward a reference measurement procedure.
authors.affiliation: Department of Biochemistry, Faculty of Medicine, University of Szeged, Szeged, Hungary.
authors.forename: Petra Magdolna
authors.initials: PM
authors.lastname: Molnár
authors.forename: László
authors.initials: L
authors.lastname: Dux
authors.forename: Hans
authors.initials: H
authors.lastname: Reinauer
authors.forename: Michael
authors.initials: M
authors.lastname: Kress
authors.forename: Theodorus
authors.initials: T
authors.lastname: Akerboom
authors.forename: Edit
authors.initials: E
authors.lastname: Szederkényi
authors.forename: Patricia
authors.initials: P
authors.lastname: Kaiser
mesh_terms: D002138:Calibration; D002247:Carbon Isotopes / Q000032:analysis; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D016572:Cyclosporine / Q000097:blood; D003903:Deuterium / Q000032:analysis; D016903:Drug Monitoring / Q000379:methods* / Q000592:standards; D000068338:Everolimus; D006801:Humans; D007166:Immunosuppressive Agents / Q000097:blood*; D007201:Indicator Dilution Techniques; D013058:Mass Spectrometry / Q000379:methods*; D020123:Sirolimus / Q000031:analogs & derivatives / Q000097:blood; D016559:Tacrolimus / Q000097:blood
chemical_list: D002247:Carbon Isotopes; D007166:Immunosuppressive Agents; D016572:Cyclosporine; D000068338:Everolimus; D003903:Deuterium; D020123:Sirolimus; D016559:Tacrolimus