Simultaneous determination of the HIV protease inhibitors indinavir, amprenavir, saquinavir, ritonavir, nelfinavir and the non-nucleoside reverse transcriptase inhibitor efavirenz by high-performance liquid chromatography after solid-phase extraction

As part of an on-going study on the suitability of a formal therapeutic drug monitoring (TDM) of antiviral drugs for improving the management of HIV infection, a high-performance liquid chromatography method has been developed to quantify simultaneously in plasma five HIV protease inhibitors (PIs) (i.e., indinavir, amprenavir, saquinavir, ritonavir, nelfinavir) and the novel non-nucleoside reverse transcriptase inhibitor efavirenz. After viral inactivation by heat (60 degrees C for 60 min), plasma (600 microl), with clozapine added as internal standard, is diluted 1:1 with phosphate buffer, pH 7 and subjected to a solid-phase extraction on a C18 cartridge. Matrix components are eliminated with 2 x 500 microl of a solution of 0.1% H3PO4 neutralised with NaOH to pH 7. PIs and efavirenz are eluted with 3 x 500 microl MeOH. The resulting eluate is evaporated under nitrogen at room temperature and is reconstituted in 100 microl 50% MeOH. A 40-microl volume is subjected to HPLC analysis onto a Nucleosil 100, 5 microm C18 AB column, using a gradient elution of MeCN and phosphate buffer adjusted to pH 5.15 and containing 0.02% sodium heptanesulfonate: 15:85 at 0 min-->30:70 at 2 min-->32:68 at 8 min-->42:58 at 18 min-->46:54 at 34 min, followed by column cleaning with MeCN-buffer, pH 5.15 (90:10), onto which 0.3% AcOH is added. Clozapine, indinavir, amprenavir, saquinavir, ritonavir, efavirenz and nelfinavir are detected by UV at 201 nm at a retention time of 8.2, 13.0, 16.3, 21.5, 26.5, 28.7 and 31.9 min, respectively. The total run time for a single analysis is 47 min, including the washing-out and reequilibration steps. The calibration curves are linear over the range 100-10,000 ng/ml. The absolute recovery of PIs/efavirenz is always higher than 88%. The method is precise with mean inter-day relative standard deviations within 2.5-9.8% and accurate (range of inter-day deviations -4.6 to +4.3%). The in vitro stability of plasma spiked with PIs/efavirenz at 750, 3000 and 9000 ng/ml has been studied at room temperature, -20 degrees C and +60 degrees C. The method has been validated and is currently applied to the monitoring of PIs and efavirenz in HIV patients. This HPLC assay may help clinicians confronted to questionable compliance, side effects or treatment failure in elucidating whether patients are exposed to adequate circulating drug levels. The availability of such an assay represents an essential step in elucidating the utility of a formal TDM for the optimal follow-up of HIV patients.