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

• Published in: Biomedical chromatography 2010-05, Vol.24 (5), p.497-505
• Main Authors: Gurule, Sanjay, Goswami, Dipanjan, Khuroo, Arshad H., Monif, Tausif
• Format: Article
• Language: English
• Subjects: Anti-Infective Agents - blood; atovaquone; Atovaquone - blood; bioanalytical; Chromatography, Liquid - methods; Humans; LC-MS/MS; method-development/validation; Naphthoquinones; Reference Standards; Sensitivity and Specificity; Tandem Mass Spectrometry - methods
• ISSN: 0269-3879; 1099-0801
• DOI: 10.1002/bmc.1317

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 μ Polar‐RP 80A (150 × 2.0 mm) column, using 100 μL of plasma extraction volume along with 10 μL 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. Copyright © 2009 John Wiley & Sons, Ltd.