title: Validation of a rapid and sensitive liquid chromatography-tandem mass spectrometry method for free and total mycophenolic acid.
abstract: BACKGROUND
  Because mycophenolic acid (MPA) is highly protein bound and because the free fraction is the pharmacologically active portion, a rapid, reliable, and sensitive procedure is required to study the relationship between free MPA and treatment efficacy/toxicity. Liquid chromatography-tandem mass spectrometry is ideally suited for such a method.
  
  
  METHODS
  Free MPA was isolated from plasma by ultrafiltration. An online extraction cartridge with a column-switching technique, analytical liquid chromatography over an Aqua Perfect C(18) column, and electrospray tandem mass spectrometry was used to quantify free and total MPA. To investigate ion suppression, a continuous infusion of MPA was introduced into the effluent from the HPLC column, and different ultrafiltrates and extracted plasma samples were injected on the column.
  
  
  RESULTS
  A chromatographic run time of 4 min separated MPA from metabolites and internal standard, thereby avoiding interference from in-source fragmentation. Ion suppression occurred well before elution of MPA and internal standard. The lower limit of quantification for free MPA was 0.5 microg/L, and the method was linear to 1000 microg/L. Interassay imprecision (CV) was <10% for free MPA (0.5-333 microg/L). Agreement was good for free MPA (n = 52) and total MPA (n = 106) between the proposed method and a validated HPLC method with ultraviolet detection. The Passing-Bablok regression line was: y = 0.95x + 0.27 microg/L for free MPA and y = 0.98x + 0.03 mg/L for total MPA.
  
  
  CONCLUSIONS
  The presented method allows the accurate, precise, and rapid determination of free and total MPA in plasma over a wide analytical range covering the concentrations relevant to pharmacokinetic studies and routine monitoring of this drug.
authors.affiliation: Department of Clinical Chemistry, George-August University Gottingen, D-37075 Gottingen, Germany.
authors.forename: Frank
authors.initials: F
authors.lastname: Streit
authors.forename: Maria
authors.initials: M
authors.lastname: Shipkova
authors.forename: Victor William
authors.initials: VW
authors.lastname: Armstrong
authors.forename: Michael
authors.initials: M
authors.lastname: Oellerich
mesh_terms: D001798:Blood Proteins / Q000378:metabolism; D002853:Chromatography, Liquid; D006801:Humans; D009173:Mycophenolic Acid / Q000097:blood* / Q000378:metabolism; D010949:Plasma; D011485:Protein Binding; D012680:Sensitivity and Specificity; D021241:Spectrometry, Mass, Electrospray Ionization; D014462:Ultrafiltration
chemical_list: D001798:Blood Proteins; D009173:Mycophenolic Acid