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 ZnSO 4 containing the internal standard D 2, 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. Video LinkThe video component of this article can be found at

A Steady-State Head-to-Head Pharmacokinetic Comparison of All FK-506 (Tacrolimus) Formulations (ASTCOFF): An Open-Label, Prospective, Randomized, Two-Arm, Three-Period Crossover Study

“…Dried blood spot analysis was performed using previously validated and described technique . Following extraction of tacrolimus from the DBS card, concentrations were evaluated by LC‐MS/MS on a platform consisting of Agilent components (Agilent, Santa Clara, CA) in combination with AB Sciex (AB Sciex, Foster City, CA) mass spectrometers at iC42 Clinical Research and Development (University of Colorado, CO).…”

Assessment of tacrolimus intrapatient variability in stable adherent transplant recipients: Establishing baseline values

“…DBS monitoring has been shown to be a reliable method of determining tacrolimus levels. [47][48][49][50][51][52][53][54] Subjects were instructed to obtain capillary blood by piercing the finger with a lancet, immediately prior to the morning tacrolimus dose. The resulting blood drop was placed on a filter paper card (Whatman 903 protein saver cards, GE Whatman, Marlborough, MA).…”

Application of dried blood spot for analysis of low molecular weight fraction (metabolome) of blood

“…Solid-phase extraction conjugated to LC-MS/MS is designed to automatically facilitate desorption of samples and is effective both in time and in cost by a method for analyzing DBSs [74,[93][94][95][96]. Compared with protein precipitation, solid phase extraction is an improved purification of the sample [97]. There are specific problems with the use of automated extraction in comparison with the methods of autonomous extraction, which can also be a significant source of analysis errors: an inhomogeneous mixture of the internal standard with the analyte in the extract; dilution of the sample, and as a result, broadening the band in chromatographic separation and/or difficulty separating the extract on the analytical column [44].…”

“…The 2D chromatography process reduced matrix and transport effects for DBSs, and related inaccuracies were reduced [112,113]. With the further addition of automated extraction coupled to the 2D-C system, sensitivity and specificity become maximum when combined with a triple quadrupole tandem mass spectrometer or high-resolution quadrupole mass spectrometer (QTOF) [97,114].…”