Quantification of the HIV-integrase inhibitor raltegravir (MK-0518) in human plasma by high-performance liquid chromatography with fluorescence detection

A simple and sensitive HLPC method with fluorescence detection was developed for the accurate determination of the first licensed HIV integrase inhibitor raltegravir in human plasma. A 500-μL plasma sample was spiked with delavirdine as internal standard and subjected to liquid–liquid extraction based on a previously described assay i.e. using hexane/methylene chloride (1:1, v/v%) at pH 4.0. HPLC was performed using a Symmetry Shield RP18 column (150 mm × 4.6 mm), a gradient elution of acetonitrile −0.01% (v/v) triethylamine in water adjusted to pH 3.0 at a flow rate of 1 mL/min and a fluorimetric detector set at 299 and 396 nm as excitation and emission wavelengths, respectively. The retention time was 5.0 min for internal standard and 6.4 min for raltegravir. Calibration curves were linear in the range 5–1000 ng/mL and the accuracy of quality control samples in the range 10–750 ng/mL varied from 98.3 to 99.1% and 98.3 to 101.0% of the nominal concentrations for intra-day and day-to-day analysis, respectively with a precision of 6.3% or less. Among the other antiretroviral drugs which can be given in association to HIV-infected patients, none was found to interfere with internal standard or raltegravir. The described assay was developed for the purpose of therapeutic drug of this HIV integrase inhibitor.