title: A high performance liquid chromatography tandem mass spectrometry for the quantification of tacrolimus in human bile in liver transplant recipients.
abstract: Tacrolimus whole-blood concentrations imperfectly reflect concentrations at the effect site. Tacrolimus concentrations in the transplanted organ could be more relevant to predict rejection events. Because liver biopsy cannot be repeatedly performed after liver transplantation, we suggested measuring tacrolimus in the bile to have a cost-effective and clinically implementable surrogate marker of intra-hepatic tacrolimus concentration. We developed and fully validated a liquid chromatography-tandem mass spectrometry method for the determination of tacrolimus in human bile. Sample purification was achieved using protein precipitation and liquid-liquid extraction with ethyl-acetate. Gradient elution was performed using a C18 analytical column with a 5min run-time. The method was linear from 0.5ng/mL to 20ng/mL. In this concentration range, within-day and between-day precisions as well as overall bias were within ±15%. Matrix effect was fully corrected by the internal standard (ascomycin). The assay was optimized to achieve good selectivity in this complex biological matrix. Tacrolimus was found to be stable in bile stored 6 months at -80°C, after 3 freeze and thaw cycles, 20h at room temperature and 24h in extracts kept at 15°C in the auto-sampler. The method was applied to quantify tacrolimus in bile from liver transplant recipients. It allowed getting preliminary data about tacrolimus excretion profile in bile and showed the lack of correlation between tacrolimus whole blood concentration and tacrolimus liver exposition. This alternative and innovative analytical approach of tacrolimus bio-analysis appears suitable for further studies evaluating relevance of biliary tacrolimus concentration as a new pharmacological marker of immunosuppressive activity.
authors.affiliation: Rennes University Hospital, Department of Clinical and Biological Pharmacology and Pharmacovigilance, Pharmacoepidemiology and Drug Information Center, Rennes, France; Rennes 1 University, Faculty of Medicine, Laboratory of Experimental and Clinical Pharmacology, Rennes, France; Inserm, CIC-P 1414 Clinical Investigation Center, Rennes, France. Electronic address: camille.tron@chu-rennes.fr.
authors.forename: Camille
authors.initials: C
authors.lastname: Tron
authors.affiliation: Inserm, CIC-P 1414 Clinical Investigation Center, Rennes, France; Rennes University Hospital, Hepato-Biliary and Digestive Surgery Unit, Rennes, France.
authors.forename: Michel
authors.initials: M
authors.lastname: Rayar
authors.affiliation: Rennes University Hospital, Department of Clinical and Biological Pharmacology and Pharmacovigilance, Pharmacoepidemiology and Drug Information Center, Rennes, France; Rennes 1 University, Faculty of Medicine, Laboratory of Experimental and Clinical Pharmacology, Rennes, France; Inserm, CIC-P 1414 Clinical Investigation Center, Rennes, France.
authors.forename: Antoine
authors.initials: A
authors.lastname: Petitcollin
authors.affiliation: Rennes University Hospital, Hepato-Biliary and Digestive Surgery Unit, Rennes, France.
authors.forename: Jean-Marie
authors.initials: JM
authors.lastname: Beaurepaire
authors.affiliation: Rennes University Hospital, Hepato-Biliary and Digestive Surgery Unit, Rennes, France.
authors.forename: Caterina
authors.initials: C
authors.lastname: Cusumano
authors.affiliation: Rennes University Hospital, Department of Clinical and Biological Pharmacology and Pharmacovigilance, Pharmacoepidemiology and Drug Information Center, Rennes, France; Rennes 1 University, Faculty of Medicine, Laboratory of Experimental and Clinical Pharmacology, Rennes, France; Inserm, CIC-P 1414 Clinical Investigation Center, Rennes, France.
authors.forename: Marie-Clémence
authors.initials: MC
authors.lastname: Verdier
authors.affiliation: Inserm, CIC-P 1414 Clinical Investigation Center, Rennes, France; Rennes University Hospital, Hepato-Biliary and Digestive Surgery Unit, Rennes, France.
authors.forename: Pauline
authors.initials: P
authors.lastname: Houssel-Debry
authors.affiliation: Inserm, CIC-P 1414 Clinical Investigation Center, Rennes, France; Rennes University Hospital, Medical Intensive Care Unit, Rennes, France.
authors.forename: Christophe
authors.initials: C
authors.lastname: Camus
authors.affiliation: Inserm, CIC-P 1414 Clinical Investigation Center, Rennes, France; Rennes University Hospital, Hepato-Biliary and Digestive Surgery Unit, Rennes, France.
authors.forename: Karim
authors.initials: K
authors.lastname: Boudjema
authors.affiliation: Rennes University Hospital, Department of Clinical and Biological Pharmacology and Pharmacovigilance, Pharmacoepidemiology and Drug Information Center, Rennes, France; Rennes 1 University, Faculty of Medicine, Laboratory of Experimental and Clinical Pharmacology, Rennes, France; Inserm, CIC-P 1414 Clinical Investigation Center, Rennes, France.
authors.forename: Eric
authors.initials: E
authors.lastname: Bellissant
authors.affiliation: Rennes University Hospital, Department of Clinical and Biological Pharmacology and Pharmacovigilance, Pharmacoepidemiology and Drug Information Center, Rennes, France; Rennes 1 University, Faculty of Medicine, Laboratory of Experimental and Clinical Pharmacology, Rennes, France; Inserm, CIC-P 1414 Clinical Investigation Center, Rennes, France.
authors.forename: Florian
authors.initials: F
authors.lastname: Lemaitre
mesh_terms: D001646:Bile / Q000737:chemistry*; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D006801:Humans; D007166:Immunosuppressive Agents / Q000032:analysis* / Q000302:isolation & purification; D059625:Liquid-Liquid Extraction; D016031:Liver Transplantation; D016559:Tacrolimus / Q000032:analysis* / Q000302:isolation & purification; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D007166:Immunosuppressive Agents; D016559:Tacrolimus
keywords: Alternative matrix; Bile; High performance liquid chromatography; Mass spectrometry; Tacrolimus; Therapeutic drug monitoring