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

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 2-1000ng/mL. Stable isotope labeled 13 C-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 mu L plasma sample size. The compounds were chromatographed on an Ace C-18 (50 x 3.0 mm, 3 mu m, 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 (mlz 445 -> 109) and C-13(6)-MK-0518 (inlz 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 degrees 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 degrees C. The method described has been used to support over 18 clinical studies during Phase I through III of clinical development. (c) 2007 Elsevier B.V. All rights reserved.