Development of an HPLC-UV assay method for the simultaneous quantification of nine antiretroviral agents in the plasma of HIV-infected patients

Abstract

A new method using high-performance liquid chromatography coupled with ultra violet detection (HPLC-UV) was developed and validated for the simultaneous quantification of atazanavir, dolutegravir, darunavir, efavirenz, etravirine lopinavir, raltegravir, rilpivirine and tipranavir in human plasma. For the first time we reported here the development and validation of an HPLC-UV assay to quantify the frequently administered 9 antiretroviral compounds including dolutegravir and rilpivirine. A simple solid phase extraction procedure was applied to 500 µL aliquots of plasma. The chromatographic separation of the drugs and internal standard (quinoxaline) was achieved with a gradient of acetonitrile and sodium acetate buffer on a C₁₈ reverse-phase analytical column with a 25 min analytical run time. Calibration curves were optimised according to the therapeutic range of drug concentrations in patients, and the coefficient of determination (r²) was higher than 0.99 for all analytes. Mean intraday and interday precisions (RSD) for all compounds were less than 15.0%, and the mean accuracy (% deviation from nominal concentration) was also found to be less than 15.0%. Extraction recovery range was between 80% and 120% for all drugs analysed. The solid phase extraction and HPLC-UV method enable a specific, sensitive, and reliable simultaneous determination of nine antiretroviral agents in plasma. Good extraction efficiency and low limit of HPLC-UV quantification make this method suitable for use in clinical trials and therapeutic drug monitoring.

Keywords: Antiretrovirals; Bioanalytical method validation; HPLC–UV.

Fig. 1

Representative chromatograms of standard point 1 and blank plasma of (A) calibration curves A and (B) calibration curves B at 260 nm and (C) RPV and ETV at 305 nm.

Fig. 2

Comparative chromatograms of standard point 6 and standard point 1 of (A) calibration curves A and (B) calibration curves B at 260 nm and (C) RPV and ETV at 305 nm.

Fig. 3

Box-plot of (A) DRV (n=342), (B) ATV (n=885), (C) ETV (n=68), (D) LPV (n=174), (E) EFV (n=181), (F) RGV (n=344), (G) TPV (n=5), (H) RPV (n=48) and (I) DTG (n=3), where n=number of samples above LOQ.