Abstract

HIGH THROUGHPUT LC-MS/MS METHOD FOR THE QUANTITATION OF DIDANOSINE IN HUMAN PLASMA BY SOLID PHASE EXTRACTION USING 96 WELL PLATE FORMAT

Tangudu Nagabhusana Rao*, Guntuku Girija Sankar, Lade Jyothi Rani

ABSTRACT

High-throughput Liquid chromatography–mass spectrometry method has been developed and validated for the quantification of Didanosine in human plasma using Zidovudine as an internal standard (ISTD). Following solid phase extraction (SPE) in 96 well plate format, the analyte and ISTD were run on Inertsil ODS-3, 4.6 X 100 mm, 5 μm column using an isocratic mobile phase consisting of 10mM Ammonium formate buffer (pH 6.4 ± 0.5): Methanol 10:90 v/v. The precursor and product ions of the drugs were monitored on a triple quadrupole instrument operated in the positive ionization mode. The method was validated over a concentration range of 1.01 to 2002.18 ng/mL with relative recoveries ranging from 92.4 to 96.4%. The inter batch precision (%CV) across three validation runs was ≤ 8.9%. The Inter batch percent relative errors (%RE) determined at four QC levels (LLOQ, LQC, MQC and HQC) was between -7.7 to 4.9%. According to the validated results, the proposed method was found to be specific, accurate, precise and high throughput method and could be used for the estimation of Didanosine in human plasma and can be applied for the routine analysis.

Keywords: Didanosine, Zidovudine, Solid Phase Extraction and 96 well plates.