LC‐APCI mass spectrometric method development and validation for the determination of atovaquone in human plasma

Abstract

A newly developed LC-APCI mass spectrometric method is described for human plasma determination of atovaquone using lapachol internal standard. A single-step protein precipitation technique for plasma extraction of atovaquone achieving mean recovery of 94.17% (CV 8%) without compromising sensitivity (limit of quantitation 50.3 ng/mL) or linearity (50.3 ng/mL-23924.6 ng/mL) is delineated in this paper. Heated nebulizer in negative multiple reaction monitoring mode was employed with transitions m/z 365.2 --> m/z 337.1 and m/z 240.9 --> m/z 185.7 for atovaquone and lapachol respectively in this liquid chromatographic-tandem mass spectrometric method. Excellent chromatographic separation on a Synergi 4 micro Polar-RP 80A (150 x 2.0 mm) column, using 100 microL of plasma extraction volume along with 10 microL of injection load, completing analysis run-time within 2.5 min, highlights this simple yet unique bioanalytical method. The developed method can be successfully applied to pharmacokinetic studies on atovaquone suspension administered in healthy volunteers or HIV-infected patients. Moreover full method validation results not published before are presented and discussed in detail for the first time in this article.

The anti-malarial atovaquone increases radiosensitivity by alleviating tumour hypoxia

“…Peak areas are presented relative to the DMSO control and normalized to the cell number obtained by a Sceptre cell counter (Millipore). Plasma and tumour atovaquone concentrations were determined using lapachol as an internal standard 45 by negative electrospray ionization on a Waters EMD1000. HPLC was performed on a Cortecs C18 column, 100 × 3 mm (Waters), with eluents of 5 mm ammonium formate in 50% acetonitrile (A) and acetonitrile (B) using a gradient of 10–100% B in 5 min.…”

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

“…The assay requires only 10 µL of plasma, which is suitable for pediatric populations. This volume requirement is considerably lower than previously developed mass spectrometry-based assays (25 – 500 µL) [14] , [15] , [16] . The total run-time for each sample analysis in our assay was 7.3 min, which is longer than what has been reported in previous studies (1.3 – 2.5 min), but fulfills the throughput requirements of our laboratory.…”

An Ultraperformance LC-MS/MS Method for the Quantification of the Antimalarial Atovaquone in Plasma

“…Notably, the method has a per-sample runtime of 1.3 min and requires only 25 μL of plasma, offering significant improvement from 2 previously published methods (11,12). Interestingly, while a previously published method validation study stated superior performance of a liquid-liquid-extraction approach for ATQ quantification (11), the protein precipitation method described herein was dynamic, showing good recovery from plasma (85.2%-96.4%) and circumventing any sample dry down and reconstitution.…”

“…Therefore, analytical methods to quantify ATQ should be sufficiently dynamic. Previously described assays are limited by large sample volumes of up to 0.5 mL, complex sample preparation, and/or narrow analytical measuring ranges, with previous methods reaching an upper limit of quantification (ULOQ) of 24000 ng/mL (11,12). Here, we describe the development and validation of an ultraperformance (UP) LC-MS/MS assay for the quantification of ATQ in human plasma.…”