title: Quantification of the Immunosuppressant Tacrolimus on Dried Blood Spots Using LC-MS/MS.
abstract: The calcineurin inhibitor tacrolimus is the cornerstone of most immunosuppressive treatment protocols after solid organ transplantation in the United States. Tacrolimus is a narrow therapeutic index drug and as such requires therapeutic drug monitoring and dose adjustment based on its whole blood trough concentrations. To facilitate home therapeutic drug and adherence monitoring, the collection of dried blood spots is an attractive concept. After a finger stick, the patient collects a blood drop on filter paper at home. After the blood is dried, it is mailed to the analytical laboratory where tacrolimus is quantified using high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) in combination with a simple manual protein precipitation step and online column extraction. For tacrolimus analysis, a 6-mm disc is punched from the saturated center of the blood spot. The blood spot is homogenized using a bullet blender and then proteins are precipitated with methanol/0.2 M ZnSO4 containing the internal standard D2,(13)C-tacrolimus. After vortexing and centrifugation, 100 µl of supernatant is injected into an online extraction column and washed with 5 ml/min of 0.1 formic acid/acetonitrile (7:3, v:v) for 1 min. Hereafter, the switching valve is activated and the analytes are back-flushed onto the analytical column (and separated using a 0.1% formic acid/acetonitrile gradient). Tacrolimus is quantified in the positive multi reaction mode (MRM) using a tandem mass spectrometer. The assay is linear from 1 to 50 ng/ml. Inter-assay variability (3.6%-6.1%) and accuracy (91.7%-101.6%) as assessed over 20 days meet acceptance criteria. Average extraction recovery is 95.5%. There are no relevant carry-over, matrix interferences and matrix effects. Tacrolimus is stable in dried blood spots at RT and at +4 °C for 1 week. Extracted samples in the autosampler are stable at +4 °C for at least 72 hr.
authors.affiliation: iC42 Clinical Research and Development, University of Colorado, Anschutz Medical Campus.
authors.forename: Touraj
authors.initials: T
authors.lastname: Shokati
authors.affiliation: iC42 Clinical Research and Development, University of Colorado, Anschutz Medical Campus.
authors.forename: Nicholas
authors.initials: N
authors.lastname: Bodenberger
authors.affiliation: iC42 Clinical Research and Development, University of Colorado, Anschutz Medical Campus.
authors.forename: Holly
authors.initials: H
authors.lastname: Gadpaille
authors.affiliation: iC42 Clinical Research and Development, University of Colorado, Anschutz Medical Campus.
authors.forename: Björn
authors.initials: B
authors.lastname: Schniedewind
authors.affiliation: Division of Clinical Pharmacology, Cincinnati Children's Hospital Medical Center.
authors.forename: Alexander A
authors.initials: AA
authors.lastname: Vinks
authors.affiliation: Food and Drug Administration (FDA), Center of Drug Evaluation Research - Office of Generic Drugs.
authors.forename: Wenlei
authors.initials: W
authors.lastname: Jiang
authors.affiliation: Transplant Clinical Research, University of Cincinnati.
authors.forename: Rita R
authors.initials: RR
authors.lastname: Alloway
authors.affiliation: iC42 Clinical Research and Development, University of Colorado, Anschutz Medical Campus; Uwe.Christians@ucdenver.edu.
authors.forename: Uwe
authors.initials: U
authors.lastname: Christians
mesh_terms: D002853:Chromatography, Liquid / Q000379:methods; D059788:Dried Blood Spot Testing / Q000379:methods*; D016903:Drug Monitoring / Q000379:methods; D005260:Female; D006801:Humans; D007166:Immunosuppressive Agents / Q000494:pharmacology*; D008297:Male; D016559:Tacrolimus / Q000494:pharmacology*; D053719:Tandem Mass Spectrometry / Q000379:methods
chemical_list: D007166:Immunosuppressive Agents; D016559:Tacrolimus