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.

Paired plasma and intracellular samples were obtained from 12 HIV-infected adults taking raltegravir twice daily (b.i.d.), and after switching to once daily. With b.i.d. dosing, no plasma trough concentrations were below the IC95, in contrast to 33% for once daily dosing. Fifty percent of the once daily group had intracellular trough concentrations below the inhibitory concentration 95 (IC95), 25% in the b.i.d. group. Lower plasma and intracellular concentrations may contribute to inferior virologic suppression rates observed with once daily raltegravir dosing.

Current procedures for obtaining and measuring plasma concentrations of HIV protease inhibitors (PIs) are technically challenging. Dried blood spot (DBS) assays offer a way to overcome many of the obstacles. We sought to develop a DBS assay for quantitation of the PI atazanavir (ATV) and to compare this method with a previously validated plasma assay. We prospectively enrolled 48 patients with well-controlled HIV disease who had been on ATV for at least 7 days. ATV was quantified from plasma by use of high-performance liquid chromatography (HPLC). A reversed-phase ultrahigh-performance liquid chromatography (UPLC) assay was utilized for DBS samples. The concentrations of ATV quantified in a DBS matrix showed very strong agreement with those measured in plasma (r 2 ‫؍‬ 0.988). The mean difference in ATV concentration between the two methods was ؊10.8% (95% confidence interval [95% CI], ؊7.65% to ؊13.95%), indicating that the DBS method has a slight negative bias. A majority (97.8%) of the differences in concentration between the two assays fell within ؎2 standard deviations. ATV concentrations were lower in subjects who had detectable HIV RNA in plasma (mean, 543 ng/ml) than in those with HIV RNA of <50 copies/ml (mean, 1,582 ng/ml) (P ‫؍‬ 0.03, Wilcoxon rank-sum test). In conclusion, our study demonstrated that ATV quantitation in a DBS matrix is feasible and accurate. DBS use offers a convenient alternative for measuring plasma concentrations of ATV and may have utility in monitoring of drug concentrations in clinical practice and in future studies.Presently, approximately 33 million persons are living with human immunodeficiency virus (HIV) disease (35). Treatment options for HIV disease have expanded over the last 15 years, particularly with the introduction of protease inhibitors (PIs) as a component of combination antiretroviral therapy (ART). Use of these agents has been associated with significant decreases in morbidity and mortality (13,16,29). Despite the efficacy of PIs, a substantial number of patients still experience virological failure (23). PIs show significant interindividual pharmacokinetic variability for identical dosing regimens (7,14,31,33). High PI concentrations have been associated with toxicity, while subtherapeutic concentrations have been associated with virologic failure (2,3,9,11,12,28,30,31,32). These findings have led to interest in the use of therapeutic drug monitoring (TDM), which individualizes therapy to maximize outcomes and minimize toxicity (1, 7, 10). Currently, the literature does not support and guidelines do not recommend routine use of TDM in HIV-infected adults (8, 21).Atazanavir (ATV) is an azapeptide PI approved for use in both treatment-naïve and treatment-experienced patients (18). It has the advantage of being dosed once a day and can be used with or without ritonavir (RTV), although coadministration of RTV is preferred (8). The current techniques for quantitation of ATV (as well as all other PIs) are plasma-or serum-based analytical procedures. These procedures ...

The COBAS AmpliPrep/COBAS AMPLICOR HIV-1 MONITOR Test, version 1.5 (CAP/CA), and the COBAS AMPLICOR HIV-1 MONITOR Test, version 1.5, were compared. CAP/CA reduced and consolidated labor while modestly increasing assay throughput without increased failure rates or direct costs, regardless of batch size and assay format.