Validation of atovaquone plasma levels by liquid chromatography-tandem mass spectrometry for therapeutic drug monitoring in pediatric patients

J Mass Spectrom Adv Clin Lab. 2022 Sep 14:26:23-27. doi: 10.1016/j.jmsacl.2022.09.004. eCollection 2022 Nov.

Abstract

Background: Atovaquone has traditionally been used as an antiparasitic and antifungal agent, but recent studies have shown its potential as an anticancer agent. The high variability in atovaquone bioavailability highlights the need for therapeutic drug monitoring, especially in pediatric patients. The goal of our study was to develop and validate the performance of an assay to quantify atovaquone plasma concentrations collected from pediatric cancer patients using LC-MS/MS.

Methods: Atovaquone was extracted from a 10 µL volume of K2-EDTA human plasma using a solution consisting of ACN: EtOH: DMF (8:1:1 v:v:v), separated using reverse-phase chromatography, and detected using a SCIEX 5500 QTrap MS system. LC-MS/MS assay performance was evaluated for precision, accuracy, carryover, sensitivity, specificity, linearity, and interferences.

Results: Atovaquone and its deuterated internal standard were analyzed using a gradient chromatographic method that had an overall cycle-time of 7.4 min per injection, and retention times of 4.3 min. Atovaquone was measured over a dynamic concentration range of 0.63 - 80 µM with a deviation within ≤ ± 5.1 % of the target value. Intra- and inter-assay precision were ≤ 2.7 % and ≤ 8.4 %, respectively. Dilutional, carryover, and interference studies were also within acceptable limits.

Conclusions: Our studies have shown that our LC-MS/MS-based method is both reliable and robust for the quantification of plasma atovaquone concentrations and can be used to determine the effective dose of atovaquone for pediatric patients treated for AML.

Keywords: (CSEH) plasma, Charcoal Stripped K2-EDTA Human; ACN, Acetonitrile; AML, Acute Myeloid Leukemia; ATF4, Activating Transcription Factor 4; Acute myeloid leukemia; Atovaquone; CRR, Clinical Reportable Range; DMF, Di-Methyl Formamide; EtOH, Ethanol; FDA, Food and Drug Administration; HPLC, High-Performance Liquid Chromatography; IPA, Isopropanol; LC-MS/MS; MeOH, Methanol; PJP, Pneumocystis jirovecii pneumonia; REDD1, REgulated in Development and DNA damage responses 1; STAT3, Signal Transducer and Activator of Transcription 3; TDM, Therapeutic Drug Monitoring, LC-MS/MS, Liquid Chromatography-tandem Mass Spectrometry; Therapeutic drug monitoring; XIC, eXtracted Ion Chromatogram; eIF2α, Eukaryotic Initiation Factor 2α; mTOR, mammalian Target Of Rapamycin.