title: First UHPLC-MS/MS method coupled with automated online SPE for quantification both of tacrolimus and everolimus in peripheral blood mononuclear cells and its application on samples from co-treated pediatric patients.
abstract: Tacrolimus (TAC, FK-506) and everolimus (EVE, RAD001) are immunosuppressors used to treat pediatric patients undergoing liver transplantation. Their hematic TDM by liquid chromatography became standard practice. However, it does not always reflect concentrations at their active site. Our aim was to develop and validate a new method for the simultaneous TAC and EVE quantification into target cells: peripheral blood mononuclear cells (PBMCs). Peripheral blood mononuclear cells were collected using cell preparation tubes; cells number and mean cell volume were evaluated by an automatic cell counter. TAC and EVE were quantified using UHPLC-MS/MS coupled with an automated online solid-phase extraction platform. Chromatographic run was performed on an Acquity UPLC® BEH C18 1.7 μm (2.1 × 50 mm) column at 45 °C, for 6 min at 0.5 ml/min. Mobile phases were water and methanol, both with 2 mm ammonium acetate and 1 ml/l formic acid). XBridge® C8 10 μm (1 × 10 mm) SPE cartridges were used, and the internal standard was ascomycin. Following Food and Drug Administration guidelines, method validation resulted in high sensitivity and specificity. Calibration curves were linear (r2= 0.998) and intra-day and inter-day imprecision and inaccuracy were <15%. A reproducible matrix effect was observed, with a good recovery for all compounds. Drug amounts in 15 'real' PBMCs samples from five pediatric patients in co-treatment resulted within the calibration range (0.039-5 ng). Concentrations from each patient were standardized using their evaluated mean cell volume: intra-PBMCs concentration was meanly 19.23 and 218.61 times higher than the hematic one for TAC and EVE, respectively. This method might be useful in clinical routine, giving reliable data on drugs concentration at the active site. Copyright © 2017 John Wiley & Sons, Ltd.
authors.affiliation: Unit of Infectious Diseases, Department of Medical Sciences, University of Turin, Amedeo di Savoia Hospital (Laboratory of Clinical Pharmacology and Pharmacogenetics#), Turin, Italy.
authors.forename: Debora
authors.identifier: 0000-0003-4095-8312
authors.initials: D
authors.lastname: Pensi
authors.forename: Amedeo
authors.initials: A
authors.lastname: De Nicolò
authors.affiliation: Unit of Pediatric Gastroenterology and Hepatology, University of Turin, Città della Salute e della Scienza Hospital, Turin, Italy.
authors.forename: Michele
authors.initials: M
authors.lastname: Pinon
authors.affiliation: Unit of Infectious Diseases, Department of Medical Sciences, University of Turin, Amedeo di Savoia Hospital (Laboratory of Clinical Pharmacology and Pharmacogenetics#), Turin, Italy.
authors.forename: Clarissa
authors.initials: C
authors.lastname: Pisciotta
authors.affiliation: Unit of Pediatric Gastroenterology and Hepatology, University of Turin, Città della Salute e della Scienza Hospital, Turin, Italy.
authors.forename: Pier Luigi
authors.initials: PL
authors.lastname: Calvo
authors.affiliation: Clinical Biochemistry Unit, Department of Diagnostic Laboratory, A.O.U. Città della Salute e della Scienza Hospital, Turin, Italy.
authors.forename: Antonello
authors.initials: A
authors.lastname: Nonnato
authors.affiliation: Liver Transplantation Center, General Surgery 2U, A.O.U. Cittàdella Salute e della Scienza Hospital, University of Turin, Turin, Italy.
authors.forename: Renato
authors.initials: R
authors.lastname: Romagnoli
authors.affiliation: Liver Transplantation Center, General Surgery 2U, A.O.U. Cittàdella Salute e della Scienza Hospital, University of Turin, Turin, Italy.
authors.forename: Francesco
authors.initials: F
authors.lastname: Tandoi
authors.affiliation: Unit of Infectious Diseases, Department of Medical Sciences, University of Turin, Amedeo di Savoia Hospital (Laboratory of Clinical Pharmacology and Pharmacogenetics#), Turin, Italy.
authors.forename: Giovanni
authors.initials: G
authors.lastname: Di Perri
authors.affiliation: Unit of Infectious Diseases, Department of Medical Sciences, University of Turin, Amedeo di Savoia Hospital (Laboratory of Clinical Pharmacology and Pharmacogenetics#), Turin, Italy.
authors.forename: Antonio
authors.initials: A
authors.lastname: D'Avolio
mesh_terms: D000293:Adolescent; D002648:Child; D002675:Child, Preschool; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D000068338:Everolimus / Q000097:blood*; D006801:Humans; D007166:Immunosuppressive Agents / Q000097:blood*; D007963:Leukocytes, Mononuclear / Q000737:chemistry*; D016031:Liver Transplantation; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D052616:Solid Phase Extraction; D016559:Tacrolimus / Q000097:blood*; D053719:Tandem Mass Spectrometry / Q000379:methods
chemical_list: D007166:Immunosuppressive Agents; D000068338:Everolimus; D016559:Tacrolimus
keywords: OSM; PBMCs; everolimus; intracellular; tacrolimus; tandem mass