Sihe Wang1, Jenny E Magill, Faye B Vicente. 1. Department of Pathology and Laboratory Medicine, The Feinberg School of Medicine, Northwestern University, Chicago, Ill, USA. swang@childrensmemorial.org
Abstract
CONTEXT: Combined immunosuppressant therapy using tacrolimus and sirolimus has demonstrable benefits. Simultaneous chromatographic monitoring of whole blood tacrolimus and sirolimus is useful for reducing reagent consumption and turnaround time. We report here a simple and rapid method using high-performance liquid chromatography/mass spectrometry for simultaneous measurement of whole blood tacrolimus and sirolimus. OBJECTIVE: To develop and validate a high-performance liquid chromatography/mass spectrometry method that is suitable for clinical laboratories and that is simple, rapid, and cost-effective. DESIGN: Whole blood (80 microL) was mixed with zinc sulfate solution, followed by protein precipitation with acetonitrile containing the internal standards. After brief centrifugation, the supernatant (20 microL) was injected onto a C18 guard column. The drug and the internal standard ammonium adducts were monitored by multiple reaction monitoring. One-point calibration at levels of 200 ng/mL (249 nM) tacrolimus and 100 ng/mL (109 nM) sirolimus was prepared by adding tacrolimus and sirolimus to immunosuppressant-free whole blood. RESULTS: The assay took 2.5 minutes per sample injection. The total imprecision was between 2.46% and 7.04% for tacrolimus and between 5.22% and 8.30% for sirolimus across the concentrations tested. No carryover was observed, and recoveries were 92% to 98% for tacrolimus and 100% for sirolimus at all levels tested. The tacrolimus was linear from 0.52 to 155.5 ng/mL (0.65-193.4 nM), and sirolimus was linear from 0.47 to 94.8 ng/mL (0.51-103.7 nM). Biases of correlations with commercial methods were within 7%. CONCLUSIONS: This improved method is simple, fast, cost-effective, and suitable for clinical laboratories. It has been implemented for routine clinical monitoring of posttransplantation immunosuppressant therapy.
CONTEXT: Combined immunosuppressant therapy using tacrolimus and sirolimus has demonstrable benefits. Simultaneous chromatographic monitoring of whole blood tacrolimus and sirolimus is useful for reducing reagent consumption and turnaround time. We report here a simple and rapid method using high-performance liquid chromatography/mass spectrometry for simultaneous measurement of whole blood tacrolimus and sirolimus. OBJECTIVE: To develop and validate a high-performance liquid chromatography/mass spectrometry method that is suitable for clinical laboratories and that is simple, rapid, and cost-effective. DESIGN: Whole blood (80 microL) was mixed with zinc sulfate solution, followed by protein precipitation with acetonitrile containing the internal standards. After brief centrifugation, the supernatant (20 microL) was injected onto a C18 guard column. The drug and the internal standard ammonium adducts were monitored by multiple reaction monitoring. One-point calibration at levels of 200 ng/mL (249 nM) tacrolimus and 100 ng/mL (109 nM) sirolimus was prepared by adding tacrolimus and sirolimus to immunosuppressant-free whole blood. RESULTS: The assay took 2.5 minutes per sample injection. The total imprecision was between 2.46% and 7.04% for tacrolimus and between 5.22% and 8.30% for sirolimus across the concentrations tested. No carryover was observed, and recoveries were 92% to 98% for tacrolimus and 100% for sirolimus at all levels tested. The tacrolimus was linear from 0.52 to 155.5 ng/mL (0.65-193.4 nM), and sirolimus was linear from 0.47 to 94.8 ng/mL (0.51-103.7 nM). Biases of correlations with commercial methods were within 7%. CONCLUSIONS: This improved method is simple, fast, cost-effective, and suitable for clinical laboratories. It has been implemented for routine clinical monitoring of posttransplantation immunosuppressant therapy.