Evaluation of performance of new, isotopically labeled internal standard ([13c2d4]RAD001) for everolimus using a novel high-performance liquid chromatography tandem mass spectrometry method.

INTRODUCTION

Everolimus was approved for the prevention of organ rejection of kidney transplants in adult patients in April 2010 by the US Food and Drug Administration. Therapeutic drug monitoring of everolimus is recommended for all patients receiving this immunosuppressant. The goal of this study was to improve and revalidate our previously published high-performance liquid chromatography tandem mass spectrometry method for the measurement of everolimus and to assess the performance of the recently introduced isotopically labeled internal standard (IS).

METHOD

For this method, the following innovative changes were incorporated: (1) sample preparation includes the addition of water to hemolyze red blood cells; (2) a separate extraction column is added to the system; (3) different settings of the switching valve are used to minimize dead volume; (4) a more sensitive mass spectrometer is used (API 5000) for drug detection; (5) isotopically labeled everolimus is used as the IS.

RESULTS

The addition of water to blood before adding ZnSO4 with methanol improves absolute recovery of everolimus from 77.3% ± 6.18% to 82.3% ± 6.3%. The use of the more sensitive mass spectrometer permitted the use of a smaller sample volume and lowered the lower limit of quantification from 1.0 to 0.5 ng/mL. Between-day precision for quality control samples is below 9%, and the accuracy ranged from 94.8% to 106.4%. Neither carryover nor matrix effects were observed. A comparison study showed very good agreement between the results obtained by our laboratory and those obtained by a reference laboratory (r = 0.93). The performance of the new IS, [13c2d4] RAD001, was compared with that of SDZ RAD 223-756. Everolimus concentration results obtained using the isotopically labeled IS agreed more closely with the results from the reference laboratory (using 13c2d4) as compared with everolimus concentration results obtained using the analog (SDZ RAD 223-756).

CONCLUSIONS

This revised methodology together with the comparison study data proved that our novel method for the measurement of everolimus is sensitive, precise, reliable, and robust.