Validation of a practical liquid chomatography with ultraviolet detection method for quantification of whole-blood everolimus in a clinical TDM laboratory.

Abstract

Until now, only LC/MS methods for quantification of everolimus have been published. The authors validated an LC/UV method for quantification of everolimus from whole blood. The authors sought to improve on the protocol for sirolimus determination previously reported by French et al. Everolimus and the internal standard 32-desmethoxy-rapamycin were extracted from whole blood with n-butyl chloride after precipitation of proteins and then reconstituted in mobile phase and washed with hexane to remove lipids. Everolimus was quantified by reverse-phase chromatography of the extraction product at 60 degrees C, using an isocratic 60% acetonitrile/water mobile phase at a flow rate of 1.0 mL/min. Everolimus eluted at approximately 9.6 minutes, and internal standard at approximately 11.6 minutes. A series of 32 calibration curves were linear over the concentration range of 2-100 ng/mL using 0.5 mL of whole blood per sample with r > 0.990 and slope displaying an 8.8 interassay %CV. At the lower limit of quantification, 2 ng/mL, the percentage bias and %CV were -5.0% and 14.7%, respectively. Intraassay precision at weighed-in levels of 6, 12, and 32 ng/mL were 2.4% to 6.4%, and biases were -10.7% to -8.5%. These same quality control materials yielded -6.3% to -0.8% biases from the expected values and 2.4% to 10.9% interday precision, respectively. This method for everolimus determination, validated according to FDA guidelines, provides longer column life and better sensitivity than that of French et al for sirolimus determination. This protocol also provides acceptable accuracy and precision over the expected therapeutic range and allows 1 technologist using 1 LC/UV system to run up to 5000 samples per year with confidence.