An Offline SPE-LC-MS/MS Method for Simultaneous Quantification of Tacrolimus, Cyclosporine A, Kynurenine, Tryptophan,...

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.

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.

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.

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.