HPTLC METHOD FOR DETERMINATION OF DARUNAVIR IN RAT PLASMA AND ITS APPLICATION IN PHARMACOKINETIC STUDIES

Abstract

A new, rapid, and sensitive high-performance thin-layer chromatography coupled with electrospray ionization mass spectrometry (HPTLC-ESI/MS) has been developed and validated for the quantification of darunavir in rat plasma and its application in pharmacokinetic studies. Detection and quantification were performed without using an internal standard. Protein precipitation method was followed for extracting darunavir from plasma. A saturated mixture of toluene: acetone: methanol (6:2:2 v/v) was used as mobile phase. Densitometric quantification was performed at λ = 262 nm by reflectance scanning. The method was linear over a concentration range of 5–150 ng/µL. The intra-day and inter-day precisions, expressed as RSD, were in the range of 2.15–2.77 (n = 3) and 2.13–2.47 (n = 3) respectively. The LOD was 1.24 ng/µL and LOQ was 3.85 ng/µL. The method proved to be accurate with a recovery between 94.77–98.44 and it was selective for the active principle tested. Additionally, selectivity of the method was confirmed by mass spectrometry. The mass spectra showed darunavir ion at m/z 569.80 [M +Na]⁺ being acquired directly from the rat plasma sample bands spiked with darunavir by an elution-based interface. The method was applied for the determination of plasma levels as well as pharmacokinetic study of darunavir administered orally to rats. Supplemental materials are available for this article. Go to the publisher's online edition of Journal of Liquid Chromatography & Related Technologies to view the free supplemental file.

Keywords:

• darunavir
• HPTLC-ESI/MS
• interface
• pharmacokinetics
• protein precipitation
• rat plasma

ACKNOWLEDGMENT

The authors are grateful to Dr. J. S. Yadav, Director, IICT and Dr. J. Madhusudana Rao, Head of Natural Products Lab, IICT for providing facilities to perform this work. Bokka Ramesh¹ thanks^the university grants commission for his junior research fellowship.

Notes

^(a) n = 3; analyzed on the same day (three solutions of each concentration).

^(b) n = 9; analyzed on three different days (three solutions of each concentration prepared for 3 days).

^(a)Each value is the mean ± standard deviation.

^(b)(Found concentration/added concentration) × 100.