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 2-1000 ng/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 microL plasma sample size. The compounds were chromatographed on an Ace C(18) (50 x 3.0 mm, 3 microm, titanium frits) column with 42.5/57.5 (v/v %) 0.1mM 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 (13)C(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 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.
Merschman
S A
SA
Department of Drug Metabolism, Merck Research Laboratories, WP75B-300, West Point, PA 19486, USA. sheila_merschman@merck.com
Vallano
P T
PT
Wenning
L A
LA
Matuszewski
B K
BK
Woolf
E J
EJ
D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004355:Drug Stability; D005615:Freezing; D019428:HIV Integrase Inhibitors / Q000097:blood*; D006586:Hexanes; D006801:Humans; D008752:Methylene Chloride; D015394:Molecular Structure; D015588:Observer Variation; D009930:Organic Chemicals / Q000097:blood*; D011760:Pyrrolidinones; D000068898:Raltegravir Potassium; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D053719:Tandem Mass Spectrometry / Q000379:methods*
D019428:HIV Integrase Inhibitors; D006586:Hexanes; D009930:Organic Chemicals; D011760:Pyrrolidinones; D000068898:Raltegravir Potassium; D008752:Methylene Chloride