A HPLC–mass spectrometric method suitable for the therapeutic drug monitoring of everolimus

Abstract

We report here the validation of an HPLC–electrospray–tandem mass spectrometry method for the quantification of everolimus, an immunosuppressant drug. Whole blood samples (100 μl) were extracted by protein precipitation which involved sample pre-treatment with zinc sulphate followed by acetonitrile (containing internal standard, 40- O-(3′-hydroxy)propyl-rapamycin). HPLC was performed using a step-gradient at a flow rate of 0.6 ml/min on a Waters TDM C 18 column (10 mm × 2.1 mm I.D.) with a resultant chromatographic analysis time of 2 min. Mass spectrometric detection by selected reaction monitoring (everolimus m/ z 975.5 → 908.3; internal standard m/ z 989.5 → 922.3). The assay was linear from 0.5 to 40 μg/l ( r 2 > 0.994, n = 11). The inter- and intra-day analytical recovery and imprecision for quality control samples (1.25, 12.5 and 30 μg/l) were 93.4–98.2% and <10.7%, respectively ( n = 10). At the lower limit of quantification (0.5 μg/l) the inter- and intra-day analytical recovery was 94.4–95.8% with imprecision of <14.1% ( n = 10). The absolute recovery of everolimus (6.5 μg/l) and internal standard (12.5 μg/l) was 96.5 and 88.3%, respectively ( n = 3). A comparison of our method against the mean of all HPLC methods for a series of samples from an external proficiency testing scheme revealed good correlation as shown by the regression analysis: y = 0.973 x + 0.301 ( r 2 = 0.986, n = 71). In conclusion, the method described is suited to the current requirements for therapeutic drug monitoring of everolimus.