title: An UPLC-MS/MS method coupled with automated on-line SPE for quantification of tacrolimus in peripheral blood mononuclear cells.
abstract: BACKGROUND
  Tacrolimus is an immunosuppressor used to treat patients undergoing liver transplantation. TDM of hematic tacrolimus by liquid chromatography became standard practice, but it does not necessarily reflect its concentration at its active site. Our aim was to validate a new method for tacrolimus quantification into target cells (peripheral blood mononuclear cells, PBMCs) and testing it on 100 real samples from 37 pediatric patients.
  
  
  METHODS
  PBMCs were collected using cell-preparation-tubes; cells number and MCV were evaluated. Tacrolimus was quantified using UPLC-MS/MS coupled with a new automated on-line SPE platform. Chromatographic run was performed on an Acquity UPLC(®) BEH C18 1.7 μm (2.1 mm × 50 mm) column for 5 min, with a gradient of water and methanol (both with 2 mM/L ammonium acetate and 1 mL/L formic acid). XBridge(®) C8 10 μm (1 mm × 10 mm) SPE cartridges were used. The internal standard was 6,7-dimethyl-2,3-di(2-pyridyl)quinoxaline.
  
  
  RESULTS
  Full validation following FDA guidelines was performed: the method showed high sensitivity and specificity (LLOQ of 0.010 ng; LLOD of 0.005 ng). Intra- and inter-day imprecision and inaccuracy were <15%. A positive and stable matrix effect was observed, with a good recovery for tacrolimus. All drug amounts in real samples resulted within the calibration range and calibration curves were linear (r(2)=0.998). Concentrations from each patient were standardized using their evaluated MCV: intra-PBMCs concentration was meanly 12.7 times higher than the hematic one.
  
  
  CONCLUSIONS
  This method might be eligible and useful for a clinical routine use, giving more reliable data on drug concentration at the active site.
authors.affiliation: Laboratory of Clinical Pharmacology and Pharmacogenetic(2), Unit of Infectious Diseases, University of Turin, Department of Medical Sciences, Amedeo di Savoia Hospital, Turin, Italy. Electronic address: debora.pensi@unito.it.
authors.forename: Debora
authors.initials: D
authors.lastname: Pensi
authors.affiliation: Laboratory of Clinical Pharmacology and Pharmacogenetic(2), Unit of Infectious Diseases, University of Turin, Department of Medical Sciences, Amedeo di Savoia Hospital, Turin, Italy.
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 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: Unit of Pediatric Gastroenterology and Hepatology, University of Turin, "Città della Salute e della Scienza" Hospital, Turin, Italy.
authors.forename: Andrea
authors.initials: A
authors.lastname: Brunati
authors.affiliation: Laboratory of Clinical Pharmacology and Pharmacogenetic(2), Unit of Infectious Diseases, University of Turin, Department of Medical Sciences, Amedeo di Savoia Hospital, Turin, Italy.
authors.forename: Giovanni
authors.initials: G
authors.lastname: Di Perri
authors.affiliation: Laboratory of Clinical Pharmacology and Pharmacogenetic(2), Unit of Infectious Diseases, University of Turin, Department of Medical Sciences, Amedeo di Savoia Hospital, Turin, Italy.
authors.forename: Antonio
authors.initials: A
authors.lastname: D'Avolio
mesh_terms: D000293:Adolescent; D002138:Calibration; D002648:Child; D002675:Child, Preschool; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D006801:Humans; D007166:Immunosuppressive Agents / Q000097:blood* / Q000737:chemistry*; D007963:Leukocytes, Mononuclear / Q000737:chemistry*; D012015:Reference Standards; D012680:Sensitivity and Specificity; D016559:Tacrolimus / Q000097:blood* / Q000737:chemistry*; D053719:Tandem Mass Spectrometry / Q000379:methods
chemical_list: D007166:Immunosuppressive Agents; D016559:Tacrolimus
keywords: FK-506; Intracellular; OSM; PBMC; Tandem-mass