MSACL 2024 Abstract

Development and Validation of a LC-MS/MS Method for Concentrated Tacrolimus & Cyclosporine from Pharmaceutical Products to Assess Adsorption by Feeding Tubes

Yi Xiao (1,2), Mari Gabra (1), Edward Leung (1,2)
(1) Department of Pathology and Laboratory Medicine, Children’s Hospital of Los Angeles, Los Angeles (2) Department of Pathology, Keck School of Medicine, University of Southern California, Los Angeles, CA

Yi Xiao, Ph.D. (Presenter) Children’s Hospital Los Angeles

Presenter Bio: Yi Xiao, Ph.D., DABCC, CLS is currently an assistant professor of Clinical Pathology at Keck School of Medicine of USC, and assistant director of the core laboratory and the special chemistry/biochemical genetics laboratory at Children’s Hospital Los Angeles. He received his PhD in Biochemistry from Vanderbilt University, and completed clinical chemistry fellowship from a ComACC-accredited fellowship program from Children’s Hospital Los Angeles.

Relevant Financial Disclosures (within past 24 months, reported on Jul 17, 2025) No relevant financial relationship(s) to disclose. Conflict mitigation NOT REQUIRED.

Background: Tacrolimus and cyclosporine are common immunosuppressive drugs used in the prevention and treatment of solid-organ transplant rejection (e.g., kidney, liver, heart). Other indications include several autoimmune diseases such as Crohn’s disease, autoimmune hepatitis, and rheumatoid arthritis. To prevent toxicity while achieving therapeutic efficacy, tacrolimus and cyclosporine in whole blood specimens are commonly measured in medical centers so that their dosages can be precisely adjusted. Different from adult patients, pediatric patients often require smaller doses and the use of feeding tubes for drug administration. In order to assess nonspecific adsorption by feeding tubes, we modified our in-house clinical method to measure highly concentrated tacrolimus and cyclosporine specimens prepared from clinical pharmaceutical products.

Method: Highly concentrated tacrolimus and cyclosporine specimens were serially diluted with dimethyl sulfoxide (2,000 times for tacrolimus and 20,000 times for cyclosporine) before measurements by our clinical method using liquid chromatography mass spectrometry. The method for highly concentrated tacrolimus and cyclosporine was validated, with analytical measurement range determined with in-house prepared concentrated specimens at varying concentrations, precision (repeatability and reproducibility) assessed by multiple measurements of concentrated specimens at three different concentrations, accuracy assessed by recovery studies, and matrix effect assessed by post-column infusion and by matrix dilution with dimethyl sulfoxide.

Results: The method was linear from 10 ug/mL to 80 ug/mL for tacrolimus, and 1 mg/mL to 8 mg/mL for cyclosporine with r2 > 0.99. Repeatability and reproducibility were <10% CV. Minimal matrix effect was observed. Recovery studies using commercial calibrators to measure specimens prepared from reference materials and dimethyl sulfoxide identified no significant bias for tacrolimus but an average positive bias of 20% for cyclosporine, and a correction factor was used to finalize cyclosporine results for specimens prepared from clinical pharmaceutical products. The positive bias was eliminated when calibrators were prepared in dimethyl sulfoxide.

Conclusions: We have developed and validated a LC-MS/MS method for the measurement of highly concentrated tacrolimus and cyclosporine in specimens prepared from clinical pharmaceutical products. We hypothesize that one or more components in the commercial calibrators have contributed to the positive bias of cyclosporine in those specimens. Our method can be used to support studies that assess nonspecific adsorption by feeding tubes.