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

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.