High-performance liquid chromatography with ultraviolet detection for therapeutic drug monitoring of everolimus

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Abstract

We developed and validated a high-performance liquid chromatography–ultraviolet (HPLC–UV) method for determining everolimus concentrations in human whole blood. Sample preparation involved a solid-phase extraction after protein precipitation. The separation of everolimus from internal standard (IS) and endogenous components was achieved using an isocratic elution on an octyl column. The method showed a linear relationship between peak height ratios and blood concentrations in the range of 1–200 ng/mL (r2 = 0.9997). The observed intra- and inter-day assay imprecision had a coefficient of variation (CV) = 12.8%, and inaccuracy was 11.4%. The method was found to be precise, accurate, and sensible making it useful for routine therapeutic monitoring of everolimus.

Introduction

Everolimus (Certican®, Novartis Pharmaceuticals, Basel, Switzerland) is a macrolide bearing a stable 2-hydroxyethyl chain substitution at position 40 on the rapamycin structure, rationally developed to improve the pharmacokinetic characteristics of rapamycin and enhance its bioavailability [1]. Experimental studies [2], [3] and phase II–III clinical trials [4], [5] have shown that everolimus is a potent immunosuppressive agent, and strong correlation between drug trough concentration and clinical outcome has been reported [4], [6], [7]. Everolimus is metabolized by the intestinal and hepatic cytochrome P450 3A4 [8], a system involved in the metabolism of several other agents commonly used to manage transplant recipients [9], underlying the possibility of many drug-to-drug pharmacokinetic interactions. Moreover, everolimus blood levels may be affected by hepatic insufficiency [10] and/or ethnicity [9]. Together, these factors may contribute to the daily exposure of patients to everolimus, ultimately requiring proper drug dose adjustments. Therefore, therapeutic drug monitoring of everolimus concentrations may be crucial to select the optimal dose of everolimus allowing adequate immunosuppression and minimizing potential drug-related toxicity. Different high-performance liquid chromatography (HPLC) assays have been reported so far for the measurement of everolimus concentrations in the whole blood [11], [12], [13], [14], [15], [16]. Although these methods meet the criteria for validated analysis of immunosuppressive drugs, they require mass spectrometric detection, which is not always available in clinical laboratories. Since everolimus is now entering in routine clinical practice medicine, we aimed to set up a simple HPLC method with ultraviolet (UV) detection that was precise and accurate at low concentrations and capable at high throughput. This study describes the chromatographic conditions as well as sample extraction and complete validation of the proposed method.

Section snippets

Materials

Everolimus (purity 97%) was generously provided by Novartis Pharma (Basel, Switzerland) and the internal standard (IS; 32-O-desmethoxyrapamycin) (purity 97%) by Wyeth-Ayerst Research Laboratories (Princeton, NJ) (Fig. 1). Stock solutions containing 50 and 100 μg/mL for everolimus and IS, respectively, were appropriately prepared in methanol. Everolimus working solutions of 100, 500, and 2000 ng/mL were prepared for dilution with methanol/water (50:50) and for IS a working solution of 1000 ng/mL

Results

Under the given conditions, everolimus and IS eluted at retention times of 9 and 10.4 min, respectively, and the total analysis time required was 13 min per sample. Peaks were well resolved, symmetrical and relatively sharp with comparable profiles.

Representative chromatograms of extracts from human drug-free whole-blood samples, a control human whole-blood sample spiked with known amount of everolimus (1 and 2.5 ng/mL), and a sample from a patient receiving everolimus as part of his

Discussion

The assay described herein is a modification of a previous HPLC method with ultraviolet detection we developed for the measurement of whole blood rapamycin levels [20]. The method is currently used in our laboratory for routine drug analysis and its ongoing proficiency is tested by a reference laboratory in UK every month [21]. In that case, the lower limit of quantification was 2.5 ng/mL, a suitable value to detect accurately the expected rapamycin concentration in transplant patients [22].

Acknowledgments

Dr. Dario Cattaneo is a recipient of a “Fondazione Monzino” fellowship. The authors thank the Association for Research on Transplantation (ART) for continuous support. We are also very grateful to Dr. Roberto Fiocchi (Cardiovascular Department, Ospedali Riuniti di Bergamo, Bergamo, Italy) for providing us blood samples from heart transplant patients given everolimus as part of their immunosuppressive therapy.

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