Determination of the HIV integrase inhibitor, MK-0518 (raltegravir), in human plasma using 96-well liquid–liquid extraction and HPLC-MS/MS

Abstract

An HPLC-MS/MS method was developed for the determination of MK-0518 (raltegravir), an HIV integrase inhibitor, in human plasma over the concentration range of 2–1000 ng/mL. Stable isotope labeled 13C 6-MK-0518 was used as an internal standard. The sample preparation procedure utilized liquid–liquid extraction with hexane:methylene chloride in the 96-well format with a 200 μL plasma sample size. The compounds were chromatographed on an Ace C 18 (50 × 3.0 mm, 3 μm, titanium frits) column with 42.5/57.5 (v/v %) 0.1 mM EDTA in 0.1% formic acid/methanol mobile phase at a flow rate of 0.5 mL/min. Multiple reaction monitoring of the precursor-to-product ion pairs for MK-0518 ( m/ z 445 → 109) and 13C 6-MK-0518 ( m/ z 451 → 367) on an Applied Biosystem API 4000 HPLC-MS/MS was used for quantitation. Intraday precision of standard curve concentrations in five different lots of control plasma was within 3.2%, while accuracy ranged from 94.8 to 106.8%. The mean extraction recovery of spiked plasma samples was 87%. Quality control (QC) samples were stored at −20 °C. Initial within day analysis showed QC accuracy within 7.5% of nominal with precision of 3.1% or less. The plasma QC samples were demonstrated to be stable for up to 23 months at −20 °C. The method described has been used to support over 18 clinical studies during Phase I through III of clinical development.