An Offline SPE-LC-MS/MS Method for Simultaneous Quantification of Tacrolimus, Cyclosporine A, Kynurenine, Tryptophan, and Creatinine Using Volumetric Absorptive Microsampling Device Mitra

Kajetan Nierychlewski; Katharina Habler; Stephan Kemmner; Tobias Seibt; Michael Fischereder; Markus Schwarz

doi:10.1097/FTD.0000000000001341

An Offline SPE-LC-MS/MS Method for Simultaneous Quantification of Tacrolimus, Cyclosporine A, Kynurenine, Tryptophan, and Creatinine Using Volumetric Absorptive Microsampling Device Mitra

Ther Drug Monit. 2025 May 22. doi: 10.1097/FTD.0000000000001341. Online ahead of print.

Authors

Kajetan Nierychlewski¹, Katharina Habler¹, Stephan Kemmner², Tobias Seibt², Michael Fischereder³, Markus Schwarz¹

Affiliations

¹ Institute of Laboratory Medicine, LMU University Hospital, LMU Munich, Germany.
² Transplant Center, LMU University Hospital, LMU Munich, Germany; and.
³ Renal Division, Department of Internal Medicine IV, LMU University Hospital, LMU Munich, Germany.

PMID: 40401811
DOI: 10.1097/FTD.0000000000001341

Abstract

Background: Therapeutic drug monitoring of immunosuppressants is critical in balancing insufficient immunosuppression due to underdosing, and severe adverse effects due to overdosage. For a more comprehensive therapeutic drug monitoring and follow-up of transplant patients, the aim was to develop a liquid chromatography-tandem mass spectrometry method for the simultaneous quantification of tacrolimus, cyclosporine A, tryptophan, kynurenine, and creatinine using a volumetric absorptive microsampling device.

Methods: Venous and capillary blood samples were simultaneously collected using a volumetric absorptive microsampling device called Mitra. The method involved protein precipitation followed by offline solid-phase extraction using a positive pressure manifold. Chromatographic separation was achieved by a formic acid-ammonium formate-methanol gradient on a Synergi Polar reversed-phase column. Multiple reaction monitoring in the positive ion mode and stable isotope-labeled internal standards were used for quantification. Validation was performed according to the European Medicines Agency and US Food and Drug Administration (FDA) guidelines.

Results: Validation was successful, meeting European Medicines Agency and FDA guidelines. Investigation of selectivity, accuracy, and precision met the required criteria of a deviation <15%. Internal standards successfully compensated potential matrix effects. A comparison of 26 anonymized samples from transplant patients on Mitra with venous blood controls demonstrated the method's suitability.

Conclusions: For the first time, we herein describe a liquid chromatography-tandem mass spectrometry method for the simultaneous quantification of tacrolimus, cyclosporine A, tryptophan, kynurenine, and creatinine on Mitra. Self-collection of samples may facilitate therapeutic monitoring. Simultaneous determination of creatinine may help monitor kidney function, while tryptophan and kynurenine may serve as a biomarker for early detection of transplant rejection.

Keywords: high-performance liquid chromatography–tandem mass spectrometry; immunosuppressants; kynurenine pathway; therapeutic drug monitoring; volumetric absorptive microsampling device.