ABSTRACT
Therapeutic drug monitoring (TDM) is pivotal to improve the management of HIV infection. Here, a HPLC-UV method has been developed to quantify simultaneously seven HIV protease inhibitors (amprenavir, atazanavir, indinavir, lopinavir, nelfinavir, ritonavir, and saquinavir; PIs), seven nucleoside reverse transcriptase inhibitors (abacavir, didanosine, emtricitabine, lamivudine, stavudine, zalcitabine, and zidovudine; NRTIs), and two non-nucleoside reverse transcriptase inhibitors (efavirenz and nevirapine; NNRTIs) in human plasma. The volume of the plasma sample was 600 microL. This method involved automated solid-phase extraction with Oasis HLB Cartridge 1 cc (divinylbenzene and N-vinylpyrrolidone) and evaporation in a water bath under nitrogen stream. The extracted samples were reconstituted with 100 microL methanol. Twenty microliters of these samples were injected into a HPLC-UV system, the analytes were eluted on an analytical C(18) Symmetry column (250 mm x 4.6mm I.D.) with a particle size of 5 microm. The mobile phase (0.01 M KH(2)PO(4) and acetonitrile) was delivered at 1.0 mL/min with linear gradient elution. The total run time for a single analysis was 35 min, the anti-HIV drugs were detected by UV at 240 and 260 nm. The calibration curves were linear up to 10 microg/mL. The absolute recovery ranged between 88 and 120%. The in vitro stability of anti-HIV drugs (0.005-10 microg/mL) in plasma has been studied at 24.0 degrees C. On these bases, a two to four analyte method has been tailored to the individual needs of the HIV-infected patient. The HPLC-UV method here reported has been validated and is currently applied to monitor PIs, NRTIs, and NNRTIs in plasma of HIV-infected patients. It allows to monitor the largest number of anti-HIV drugs simultaneously, appearing useful in a routine laboratory, and represents an essential step to elucidate the utility of a formal therapeutic drug monitoring for the optimal follow-up of HIV-infected patients.
Subject(s)
Anti-HIV Agents/blood , Chromatography, High Pressure Liquid/methods , Drug Monitoring/methods , Anti-HIV Agents/therapeutic use , Drug Therapy, Combination , HIV Infections/drug therapy , HIV Protease Inhibitors/blood , Humans , Reproducibility of Results , Reverse Transcriptase Inhibitors/blood , Sensitivity and SpecificityABSTRACT
A simple method for the simultaneous determination of zidovudine and nevirapine in human plasma by reversed-phase liquid chromatography with UV detection at 265 nm was developed. A solid-liquid extraction procedure with internal standard was applied to the samples prior to analysis. The system requires a Zorbax SB-C18 column, 250 x 4.6 mm I.D. and a mobile phase composed of potassium dihydrogen phosphate (10 mM; pH 6.5)-acetonitrile (83:17, v/v). Peak-areas are linear; correlation coefficients are better than 0.999; both inter- and intra-day accuracy and precision are lower than 15%. Extraction recoveries are higher than 90% for both zidovudine and nevirapine. The method proposed was employed to determine the levels of the two retroviral drugs in plasma from HIV infected human subjects.
