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Abstract BACKGROUND
Sirolimus and its derivative everolimus are widely used today as immunosuppressive agents in transplantation medicine. Therapeutic drug monitoring is recommended for maintenance of immunosuppression due to the large inter- and intra-individual variability in their pharmacokinetic characteristics, as well as the narrow therapeutic windows of these drugs. Therefore, a fast, simple, and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay was developed and validated for quantification of these two immunosuppressants.
METHODS
Sirolimus and everolimus, along with their deuterated internal standards sirolimus-d3 and everolimus-d4, were extracted by protein precipitation. To prepare the samples, 100 µL of calibrator (6 levels of ClinCal® Whole Blood Calibrator set for immunosuppresants, RECIPE Chemicals + Instruments GmbH, Germany), control (UTAK, Valencia, CA), or sample (EDTA whole blood) were mixed with 500 µL of precipitation solution (aqueous 0.1M ZnSO4 in acetonitrile, 1:4 v/v) containing the internal standards (sirolimus-d3 and everolimus-d4 at 8 ng/mL). After a 10 minute equilibration period, the mixture was centrifuged for 10 minutes and the supernatant was transferred to an injection vial. The sample was analyzed on a Transcend II LX-2 LC coupled to a TSQ QuantisTM triple quadrupole mass spectrometer (ThermoFisher). The injection volume was 25 μL. The analytes were separated with a Raptor Biphenyl column (Restek, 50 mm × 2.1 mm, 1.8µm) heated to 70 °C. Multiple reaction monitoring (MRM) was used to monitor sirolimus (quantifier 931.6>864.6 and qualifier 931.6>882.6), sirolimus-d3 (quantifier 935.6>865.6 and qualifier 935.6>883.6), everolimus (quantifier 975.7>908.6 and qualifier 975.7>926.6), and everolimus-d4 (quantifier 979.7>912.6 and qualifier 979.7>930.6). Ion suppression or enhancement was assessed by qualitative and quantitative matrix effect studies. Linearity, precision (inter- and intra-assay), accuracy, sensitivity, and specificity were validated. The total analytical runtime was 3.5 min.
RESULTS
Ion enhancement was observed for everolimus and sirolimus, but was compensated for by their internal standards. After the quantitative matrix effect study, the response ratio of post-spiked patient samples to neat solution samples was determined to range from 91.3% to 114.5%. Quantitation of sirolimus and everolimus was linear from 1.0 to 50.0 ng/mL. For sirolimus, the analytical recoveries of the calibration verification standards ranged from 97.1% to 101.8 %, and the %CV at the LOQ was <10%. For everolimus, the analytical recoveries of the calibration verification standards ranged from 93.9% to 101.6 %, and the %CV at the LOQ was <15%. Dilution with commercial RECIPE matrix ClinCal Level 0 up to 10-fold recovered within 87.5% - 112.5%. The total imprecision of sirolimus was 4.2% at 4.3 ng/mL, 4.7% at 9.5 ng/mL, and 4.3% at 14.4 ng/mL. The total imprecision of everolimus was 5.0% at 4.3 ng/mL, 4.3% at 9.8 ng/mL, and 2.9% at 15.4 ng/mL. Comparison with an independent LC-MS/MS method showed an average bias of 3.1% for sirolimus and 6.8% for everolimus. Deming regression analysis of the results indicated a slope of 1.018 and intercept of -0.996 (R=0.9849) for sirolimus, and a slope of 0.933 and intercept of -0.019 (R=0.9988) for everolimus. No carryover was observed at twice the ULOQ (50 ng/mL) for both analytes. Whole blood specimens were stable up to 7 days, 14 days and 8 weeks at ambient, refrigerated and frozen temperatures, respectively. Extracted samples were stable for 7 days at refrigerated temperature. No interferences were observed from hemolysis, icterus, lipemia, and over 100 exogenous compounds.
CONCLUSIONS
This LC-MS/MS method for sirolimus and everolimus in whole blood is accurate and efficient. It requires a simple one-step protein precipitation for sample preparation, thus enabling a short turnaround time.
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= Discovery stage. (19.79%, 2022)
= Translation stage. (37.97%, 2022)
= Clinically available. (42.25%, 2022)
