MSACL 2024 Abstract

Evaluating Analytical Performance of Tacrolimus LC-MS/MS Assay Using Ascomycin Versus Tacrolimus-C13D2 Internal Standards. Kwaku Twum, PhD, Philip Bates, Nichole Korpi-Steiner, PhD, DABCC, FADLM. Department of Pathology and Lab Medicine, School of Medicine, University of North Carolina-Chapel Hill, USA. Kwaku Twum, Ph.D (Presenter) University of North Carolina - Chapel Hill >> POSTER (PDF)

Presenter Bio: Dr. Kwaku Twum is a postdoctoral fellow in clinical chemistry at the University of North Carolina at Chapel Hill. He received his Ph.D. in Biomedical Science with a focus on health and environmental chemistry from Oakland University, Michigan. Before immigrating to the United States, he completed his bachelor and masters’ education in pharmaceutical chemistry, became a fellow of the Pharmaceutical Society of Ghana, and a licensed pharmacist. Currently, he is a member of the American Chemical Society (ACS), the Association of Diagnostic and Laboratory Medicine (ADLM), and an active member of the Society of Young and Clinical Laboratorians (SYCL).

Objective: To investigate and compare imprecision, accuracy, linearity, and potential matrix interference in tacrolimus LC-MS/MS assay using ascomycin or tacrolimus-C13D2 as internal standards.

Introduction: Therapeutic monitoring of whole blood tacrolimus concentration via liquid chromatography-tandem mass spectrometry (LC-MS/MS) remains essential to reduce the risk of rejection of a transplant organ. Conventional LC-MS/MS approaches for tacrolimus measurement have utilized the internal standard ascomycin, a structural analog. Recently, deuterated isotopically labeled tacrolimus internal standards have become increasingly commercially available, which may provide improved performance over ascomycin. This study evaluated the method performance for measurement of tacrolimus in whole blood using ascomycin versus deuterated tacrolimus (tacrolimus-C13D2) internal standards.

Methods: Whole blood tacrolimus concentrations are measured by LC-MS/MS as part of routine patient care at UNC Health McLendon Clinical Laboratories (Chapel Hill, NC). In brief, EDTA whole blood tacrolimus is extracted using an automated TECAN auto-pipetting system, which involves pipetting (in the following order) 100 µL of zinc sulfate, 100 µL of calibrator/control/sample, 250 µL lysing solution containing acetonitrile and internal standard (ascomycin or tacrolimus-C13D2) into microcentrifuge tubes. Samples are vortexed and centrifuged at 13000g for 3 minutes. A 100 µL aliquot is transferred to a 96-well microtiter plate and analyzed using a Waters Xevo TQD Mass Spectrometer equipped with an Acquity UPLC BEH C18 Column (2.1x50 mm) with an Acquity UPLC BEH C18 guard column (2.1 x 5mm). An electrospray ionization interface is operated in positive ion [ES(+)] mode and MS/MS detection in multiple reaction monitoring mode. The performance in the linearity of calibrators, precision, accuracy, and interference of the matrix using the two internal standards were investigated. To assess imprecision, three levels of UTAK quality control materials [L1(3.65 ng/mL), L2(13.26 ng/mL), and L3(20.32 ng/mL)] spanning the analytical measuring range (AMR) were tested in over 20 analytical runs spanning ten days to calculate percent coefficient of variation (CV). Accuracy was assessed by comparing a) measured results in residual external quality assessment materials with peer results and calculating standard deviation index (SDI) and b) patient result comparisons using tacrolimus-C13D2 to a validated assay that uses ascomycin. Linearity and % recovery of six-point calibration results prepared using tacrolimus-C13D2 or ascomycin for each analytical run were compared. Statistical analysis of agreement between results was all performed using Microsoft Excel.

Results: Between-day imprecision was measured at the CV of 8.40% (L1), 2.66% (L2), 3.67% (L3), and 3.63% (L1), 3.87% (L2), 9.27% (L3) for QC measured with tacrolimus-C13D2 and ascomycin respectively. Using previously reported patient samples (n=17) with ascomycin as the internal standard, tacrolimus-C13D2 showed a mean bias of +0.55 ng/ml or 3.00%. Testing of residual CAP survey samples showed a better CAP survey challenge closer to the peer group mean for tacrolimus-C13D2 (SDI -1.48, -1.17, and -1.35) compared to ascomycin (SDI -2.1, -2.2, -2.1). Six calibrator levels measured each day of testing demonstrated linear response and showed a slightly better recovery of manufacturer-assigned concentrations using tacrolimus-c13d2 (mean bias -0.02%, R2 of 0.9997) compared to ascomycin (mean bias -0.58%, R2 of 0.9984).

Conclusion: Whole blood tacrolimus measurement via LC-MS/MS demonstrated comparable analytical performance using tacrolimus-C13D2 or ascomycin as an internal standard. Linearity, imprecision, and accuracy in tacrolimus measurements showed acceptable performance for both internal standards, though a negative bias in measured tacrolimus compared with peers was observed. In evaluating the choice of internal standard, other factors such as cost and ease of obtaining pure isotopically labeled analog must also be considered.