A novel ultrasensitive LC-MS/MS assay for quantification of intracellular raltegravir in human cell extracts.
An assay using ultrahigh pressure liquid chromatography and mass spectrometry detection was developed and validated for measurement of the HIV integrase inhibitor raltegravir (MK-0518) in human cell extracts. The assay is designed to utilize 200 μl of 70% MeOH cell extract derived from human peripheral blood mononuclear cells or human tissue samples. The assay is linear over a range from 0.0023 to 9.2 ng ml(-1). The average %CV (SD/Mean)*100 and %deviation ((observed-target)/target)*100 were less than 20% at the lower limit of quantification and less than 15% over the range of the curve. This assay is an accurate and highly sensitive method for determining raltegravir concentrations in cellular extracts with a lower limit 40 to over 100-fold lower than other methods in the literature. We also present a new processing method where a rapid spin through oil produced a significant increase in apparent intracellular raltegravir concentration compared with conventional processing.
Antiviral Pharmacology Laboratory, University of Nebraska Medical Center, Omaha, NE 68198-6045, USA. blrobbins@unmc.edu
Brian L
BL
Robbins
Sarah R
SR
Nelson
Courtney V
CV
Fletcher
D002138:Calibration; D002457:Cell Extracts; D002498:Centrifugation; D002851:Chromatography, High Pressure Liquid / Q000592:standards; D004355:Drug Stability; D019428:HIV Integrase Inhibitors / Q000097:blood*; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000737:chemistry*; D057230:Limit of Detection; D016014:Linear Models; D000432:Methanol / Q000737:chemistry; D011760:Pyrrolidinones / Q000097:blood*; D000068898:Raltegravir Potassium; D012015:Reference Standards; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D053719:Tandem Mass Spectrometry / Q000592:standards
D002457:Cell Extracts; D019428:HIV Integrase Inhibitors; D011760:Pyrrolidinones; D000068898:Raltegravir Potassium; D000432:Methanol