title: Validation of an LC-MS/MS method for the quantification of mycophenolic acid in human kidney transplant biopsies.
abstract: Mycophenolic acid (MPA) has a low therapeutic index and large inter-individual pharmacokinetic variability necessitating therapeutic drug monitoring to individualise dosing after transplantation. There is an ongoing discrepancy as to whether plasma MPA concentrations sufficiently predict kidney rejection or toxicity and whether immunosuppressant concentrations within the graft tissue may better predict transplant outcomes. The aim of the study was to develop an LC-MS/MS method for the quantification of MPA concentrations in human kidney biopsies taken as part of routine clinical procedures. A total of 4 surplus human kidney biopsies obtained from 4 different kidney transplant recipients were available to use for this study. MPA was also quantified in 2 kidney samples from rats administered MPA to assess tissue extraction reproducibility. Human kidney biopsies and rat kidneys were homogenised mechanically and underwent liquid-liquid extraction before analysis by LC-MS/MS. MPA-free human kidney tissue was used in calibrators and quality control samples. Analyte detection was achieved from multiple reaction monitoring of the ammonium adducts of both MPA (m/z 321.1→207.3) and N-phthaloyl-l-phenylalanine (PPA, internal standard, m/z 296.2→250.2) using positive electrospray ionisation. The method was linear (calibration curves R(2)>0.99, n=10), precise, and accurate with coefficients of variation and bias less than 15%. Extraction efficiencies for MPA and PPA were approximately 97% and 86%, respectively, and matrix effects were minimal. In 4 kidney transplant recipients, tissue MPA concentrations ranged from 1.3 to 7.7ng/mg of tissue, however, the correlation between blood (C0) and tissue MPA concentrations could not be established. The method was successfully applied to the quantification of MPA in human kidney biopsies without the need to alter current clinical protocols.
authors.affiliation: Department of Clinical Pharmacology, The Queen Elizabeth Hospital, Woodville, SA 5011, Australia; Discipline of Pharmacology, School of Medical Sciences, The University of Adelaide, Adelaide, SA 5005, Australia.
authors.forename: Zaipul I
authors.initials: ZI
authors.lastname: Md Dom
authors.affiliation: Department of Clinical Pharmacology, The Queen Elizabeth Hospital, Woodville, SA 5011, Australia.
authors.forename: Benjamin D
authors.initials: BD
authors.lastname: Noll
authors.affiliation: Discipline of Pharmacology, School of Medical Sciences, The University of Adelaide, Adelaide, SA 5005, Australia.
authors.forename: Janet K
authors.initials: JK
authors.lastname: Coller
authors.affiliation: Discipline of Pharmacology, School of Medical Sciences, The University of Adelaide, Adelaide, SA 5005, Australia.
authors.forename: Andrew A
authors.initials: AA
authors.lastname: Somogyi
authors.affiliation: Central Northern Adelaide Renal Transplantation Services, The Royal Adelaide Hospital, Adelaide, SA 5000, Australia.
authors.forename: Graeme R
authors.initials: GR
authors.lastname: Russ
authors.affiliation: Department of Internal Medicine, Renal Transplant Unit, Erasmus MC, University Medical Centre Rotterdam, Rotterdam, The Netherlands.
authors.forename: Dennis A
authors.initials: DA
authors.lastname: Hesselink
authors.affiliation: Department of Internal Medicine, Renal Transplant Unit, Erasmus MC, University Medical Centre Rotterdam, Rotterdam, The Netherlands.
authors.forename: Teun
authors.initials: T
authors.lastname: van Gelder
authors.affiliation: Department of Clinical Pharmacology, The Queen Elizabeth Hospital, Woodville, SA 5011, Australia; Discipline of Pharmacology, School of Medical Sciences, The University of Adelaide, Adelaide, SA 5005, Australia. Electronic address: benedetta.sallustio@health.sa.gov.au.
authors.forename: Benedetta C
authors.initials: BC
authors.lastname: Sallustio
mesh_terms: D000818:Animals; D000903:Antibiotics, Antineoplastic / Q000032:analysis / Q000493:pharmacokinetics*; D001706:Biopsy; D002853:Chromatography, Liquid / Q000379:methods; D016903:Drug Monitoring / Q000379:methods; D006801:Humans; D007668:Kidney / Q000737:chemistry / Q000473:pathology*; D016030:Kidney Transplantation; D059625:Liquid-Liquid Extraction; D008297:Male; D009173:Mycophenolic Acid / Q000032:analysis / Q000493:pharmacokinetics*; D051381:Rats; D017208:Rats, Wistar; D053719:Tandem Mass Spectrometry / Q000379:methods
chemical_list: D000903:Antibiotics, Antineoplastic; D009173:Mycophenolic Acid
keywords: LC–MS/MS; Mycophenolic acid; Tissue MPA concentrations; Transplantation