Therapeutic drug monitoring in treatment-experienced HIV-infected patients receiving darunavir-based salvage regimens: A case series.

Therapeutic drug monitoring (TDM) constitutes a compelling approach for the optimization of antiretroviral therapy in treatment-experienced HIV-1 patients. While various inhibitory indices have been proposed to predict virologic outcome, there is a lack of consensus on the clinical value of TDM. Here, we report the comparative results of TDM in 14 HIV-1-infected patients who had previously received at least two different PI-based regimens and who initiated darunavir (DRV)-based salvage therapy. Pharmacokinetic/pharmacodynamics (PK/PD) parameters were calculated for each subject. Seventy-nine percent of subjects had a viral load <50 copies/mL at 48 weeks. The only subject with two consecutive viral loads >50 copies/mL at the end of the study period was the patient with the lowest instantaneous inhibitory potential (IIP). The sample size was insufficient to show an association between any of the PK/PD parameters and virologic response. Based on our observations, we suggest that the utility of IIP for antiretroviral combinations for the prediction of virologic outcome in HIV-1 drug-experienced patients should be studied further.

Negligible effect of tenofovir on atazanavir trough concentrations and genotypic inhibitory quotients in the presence and absence of ritonavir.

BACKGROUND: It is recommended to boost atazanavir with ritonavir (ATV/r) when it is combined with tenofovir disoproxil fumarate (TDF) because of drug interactions. For tolerability, unboosted atazanavir (ATV) is sometimes coadministered with TDF. The objective of this study was to evaluate the impact of this interaction on the proportion of patients achieving target ATV C troughs and genotypic inhibitory quotients (GIQ). MATERIALS AND METHODS: A therapeutic drug monitoring database was screened to evaluate ATV concentrations. Differences in C trough and GIQ values among 4 antiretroviral drug combinations were evaluated. RESULTS: Three hundred eight C troughs, 91 GIQs, and 92 viral loads were evaluated for 238, 68, and 69 patients, respectively. Patients receiving ATV/r and TDF compared with ATV and TDF were more likely to have a therapeutic C trough (odds ratio, 2.27; 95% confidence interval: 1.46-3.52; P < 0.001). Among patients on unboosted ATV, the odds of having a therapeutic ATV C trough did not differ between groups with TDF versus without TDF. Although ritonavir increased the GIQ in patients receiving TDF (odds ratio, 3.38; 95% confidence interval: 1.30-8.81; P = 0.013), a similar proportion of patients on TDF and either ATV/r or ATV achieved a therapeutic GIQ. CONCLUSIONS: In patients receiving TDF, ritonavir increased the ATV C trough and GIQ and patients on ATV/r were more likely to have therapeutic C troughs. However, among subjects without ritonavir boosting, TDF compared with other nucleosides did not influence the odds of achieving a therapeutic ATV C trough. These data suggest that ritonavir boosting of ATV is prudent, particularly in patients with resistance mutations.

Development and optimization of simplified LC-MS/MS quantification of 25-hydroxyvitamin D using protein precipitation combined with on-line solid phase extraction (SPE).

25-Hydroxyvitamin D, the most useful marker of the vitamin D status of an individual, has seen an exponential growth of its routine measurement in recent years. Several methods are currently offered but the most specific is LC-MS/MS. However, the routine use of this technique in the clinical laboratory makes it essential to improve key steps of this method for high throughput delivery. Importantly, the preanalytical steps of this assay and the efficacy of the separation system need to be optimized prior to MS detection. In this report we replaced the standard and time consuming liquid-liquid extraction method of vitamin D metabolites with hexane (LLE) combined with centrifugation (LLE/centrifugation) by a simpler protein precipitation with extraction (PPE) in acetonitrile combined with a fast separation process using a 96-well plate filtration system (PPE/filtration). This rapid extraction was then followed by an on-line solid phase extraction (SPE) using a selective chromatographic separation. We also optimized the operational and consumable costs, by using an inexpensive guard column as a trapping column to significantly enhance the lifespan of the analytical column two to three times as compared to conventional chromatography. The LC-MS/MS technique permits the measurement of both 25-hydroxyvitamin D(2) (25-OH D(2)) and the 25-hydroxyvitamin D(3) (25-OH D(3)) metabolites in electrospray ionization (ESI) mode. The chromatographic system consisted of a 2.1 mm × 50 mm C18 3.5 µM column with a 2.1 mm × 20 mm C18 3.5 µM guard column connected with two 6 ports switching valves. Quantifications were done using the isotopic dilution technique with hexadeutered 25-OH D(3) and 25-OH D(2).The ion suppression problem with phospholipids was also evaluated and optimized to minimize this effect through the chromatography process and the on-line SPE trapping. Calibration curves were prepared by diluting a commercial high calibrator Chromsystems (München, Germany) with either pure triple stripped blank serum or diluted in 6% phosphate buffer saline at pH 7.2. Linearity was tested up to 160 nmol/L for 25-OH D(3) and 75 nmol/L for 25-OH D(2). Low limit of quantification (LLOQ) were established at 3 nmol/L for 25-OH D(2) and 4 nmol/L for 25-OH D(3). Inter-assay and intra-assay precision (CV%) was determined using 3 levels of commercial controls (Utak, CA, USA) for 25-OH D(2) and 25-OH D(3). Results obtained for intra-assay and inter-assay precision (CV%) were 1.1-3.4% and 5-8.9% respectively for the PPE/centrifugation technique and 2.0-3.1% and 4.6-6.6% for the PPE/filtration technique. Accuracy was estimated with the same commercial controls: % bias was -11.2 to 4.9% with PPE/centrifugation and -3.2 to 6.1% with PPE/filtration. 25-OH D(2) and 25-OH D(3) concentrations in human serum with LLE were compared to the new extraction methods using either PPE/centrifugation or PPE/filtration. Correlations comparing the two methods revealed a slope approximately 1.0±0.3 with R≥0.98 with a bias<1 nmol/L. In summary, the new LC-MS/MS method described in this report using an on-line SPE technique with a simple off-line pre-treatment is faster, cost-effective, more reliable and more robust than current and widely used LLE/centrifugation methods coupled with LC-MS/MS.

Isocratic rapid liquid chromatographic method for simultaneous determination of carotenoids, retinol, and tocopherols in human serum.

An improved isocratic and rapid HPLC method was developed for the measurement of carotenoids, retinol and tocopherols in human serum. Vitamins were extracted with hexane. Mobile phase consisted of a mixture acetonitrile:methylene chloride:methanol with 20 mM ammonium acetate. This method used a small bead size (3 microm) Spherisorb ODS2 column with titane frits. Diode array and fluorescence detectors were used respectively for the detection of carotenoids and retinol/tocopherols. Chromatographic separation was complete in 13 min for beta-cryptoxanthin, cis-trans-lycopene, alpha-carotene, beta-carotene, cis-beta-carotene, retinol, delta-tocopherol, gamma-tocopherol and alpha-tocopherol. Echinenone and tocol were employed as internal standards for diode array and fluorescence detection. Accuracy was validated using standard reference material (SRM) 968C. Intra-assay and inter-assay precision were respectively 0.2-7.3% and 3.6-12.6%. Sensitivity was verified using the ICH recommendations and the limit of detection (LOD) obtained was sufficient for routine clinical application.