title: Determination of the acyl glucuronide metabolite of mycophenolic acid in human plasma by HPLC and Emit.
abstract: BACKGROUND
  The acyl glucuronide (AcMPAG) of mycophenolic acid (MPA) has been found to possess pharmacologic and potentially proinflammatory activity in vitro. To establish its pharmacologic and toxicologic relevance in vivo, a reversed-phase HPLC method was modified to simultaneously determine MPA, the phenolic MPA-glucuronide (7-O-MPAG), and AcMPAG. In addition, cross-reactivity of AcMPAG in the Emit assay for MPA was investigated.
  
  
  METHODS
  The procedure used simple sample preparation, separation with a Zorbax Eclipse-XDB-C8 column, and gradient elution. AcMPAG was quantified as 7-O-MPAG-equivalents.
  
  
  RESULTS
  The assay was linear up to 50 mg/L for MPA, 250 mg/L for 7-O-MPAG, and 10 mg/L for AcMPAG (r >0.999). Detection limits were 0.01, 0.03, and 0.04 mg/L for MPA, 7-O-MPAG, and AcMPAG, respectively. The recoveries were 99-103% for MPA, 95-103% for 7-O-MPAG, and 104-107% for AcMPAG. The within-day imprecision was <5.0% for MPA (0.2-25 mg/L), <4.4% for 7-O-MPAG (10-250 mg/L), and < or =14% for AcMPAG (0.1-5 mg/L). The between-day imprecision was <6.2%, <4.5%, and < or =14% for MPA, 7-O-MPAG, and AcMPAG, respectively. When isolated from microsomes, purified AcMPAG (1-10 mg/L) revealed a concentration-dependent cross-reactivity in an Emit assay for the determination of MPA ranging from 135% to 185%. This is in accordance with the bias between HPLC and Emit calculated in 270 samples from kidney transplant recipients receiving mycophenolate mofetil therapy, which was greater (median, 151.2%) than the respective AcMPAG concentrations determined by HPLC. AcMPAG was found to undergo hydrolysis when samples were stored up to 24 h at room temperature or up to 30 days at 4 degrees C or -20 degrees C. Acidified samples (pH 2.5) were stable up to 30 days at -20 degrees C.
  
  
  CONCLUSIONS
  The HPLC and Emit methods for AcMPAG described here may allow investigation of its relevance for the immunosuppression and side effects associated with mycophenolate mofetil therapy.
authors.affiliation: Abteilung Klinische Chemie, Zentrum Innere Medizin, Georg-August-Universität Göttingen, Germany. ewieland@med.uni-goettingen.de
authors.forename: M
authors.initials: M
authors.lastname: Shipkova
authors.forename: E
authors.initials: E
authors.lastname: Schütz
authors.forename: V W
authors.initials: VW
authors.lastname: Armstrong
authors.forename: P D
authors.initials: PD
authors.lastname: Niedmann
authors.forename: M
authors.initials: M
authors.lastname: Oellerich
authors.forename: E
authors.initials: E
authors.lastname: Wieland
mesh_terms: D002851:Chromatography, High Pressure Liquid; D003429:Cross Reactions; D004355:Drug Stability; D017248:Enzyme Multiplied Immunoassay Technique; D005965:Glucuronates / Q000097:blood*; D020719:Glucuronides; D006801:Humans; D007166:Immunosuppressive Agents / Q000097:blood*; D009173:Mycophenolic Acid / Q000031:analogs & derivatives* / Q000097:blood
chemical_list: D005965:Glucuronates; D020719:Glucuronides; D007166:Immunosuppressive Agents; C113145:mycophenolic acid glucuronide; D009173:Mycophenolic Acid