A validated method for quantification of efavirenz in dried blood spots using high-performance liquid chromatography-mass spectrometry.

BACKGROUND: Efavirenz (EFV) is one of the preferred components of first-line antiretroviral treatment. EFV is characterized by a long plasma half-life (40-55 hours) with large interpatient variability, which raises the potential for individualization of therapy. Analyses of EFV levels in plasma require specialized facilities (cold storage/transport) which, in resource-limited settings, can be problematic; dried blood spots (DBS)-EFV measurements thus provide a cheap easy alternative for therapeutic drug monitoring. Our aim was to develop and validate a liquid chromatography-mass spectrometry method to quantify EFV in DBS collected as part of clinical trials in resource-limited settings. METHODS: DBS for standards, quality control samples, and patient samples were excised and then extracted with ethyl acetate/n-hexane (50/50 vol/vol) after addition of internal standard hexobarbital, and 1 mol/L K2CO3. The extract was evaporated to dryness, the residue reconstituted in mobile phase and analyzed directly by liquid chromatography-mass spectrometry. Gradient elution was on a reverse-phase C18 column using 1 mmol/L ammonium acetate in water and acetonitrile. Quantification was by selected reaction monitoring in negative ionization mode. DBS samples were obtained at several time points over 24 hours from HIV+ patients on either 400 or 600 mg EFV in combination with emtricitabine/tenofovir. RESULTS: The internal standard and EFV eluted at 2.68 and 3.54 minutes, respectively in a 5-minute run time. Matrix effects were minimal (-5.4%). Calibration curves were validated over a concentration range of 25-5000 ng/mL. Intra-assay and interassay variations ranged between 6.7% and 8.7% for imprecision and 100.3% and 104.2% for accuracy. Mean recovery was >64%. The DBS data showed a strong positive correlation with a validated plasma EFV assay (R = 0.9764, P < 0.001). EFV concentrations from DBS were approximately 42% lower than the paired plasma values, and the ratio of blood/plasma did not change over the dosing interval. CONCLUSIONS: The validated assay is now routinely applied to clinical samples measuring DBS EFV for pharmacokinetic analysis. The methodology is robust, accurate, and sensitive.

Pharmacokinetics of lamivudine and lamivudine-triphosphate after administration of 300 milligrams and 150 milligrams once daily to healthy volunteers: results of the ENCORE 2 study.

There is interest in evaluating the efficacy of lower doses of certain antiretrovirals for clinical care. We determined here the bioequivalence of plasma lamivudine (3TC) and intracellular 3TC-triphosphate (3TC-TP) concentrations after the administration of two different doses. ENCORE 2 was a randomized crossover study. Subjects received 3TC at 300 and 150 mg once daily for 10 days (arm 1; n = 13) or vice versa (arm 2; n = 11), separated by a 10-day washout. Pharmacokinetic (PK) profiles (0 to 24 h) were assessed on days 10 and 30. Plasma 3TC and 3TC-TP levels in peripheral blood mononuclear cells were quantified by high-performance liquid chromatography-tandem mass spectrometry. Within-subject changes in PK parameters (the area under the concentration-time curve from 0 to 24 h [AUC(0-24)], the trough concentration of drug in plasma at 24 h [C(24)], and the maximum concentration of drug in plasma [C(max)]) were evaluated by determining the geometric mean ratios (GMRs) adjusted for study arm, period, and intra-individual variation. Regimens were considered bioequivalent if the 90% confidence interval (90% CI) fell within the range of 0.8 to 1.25. A total of 24 subjects completed the study. The GM (90% CI) 3TC AUC(0-24)), expressed as ng·h/ml, for the 300- and 150-mg doses were 8,354 (7,609 to 9,172) and 4,773 (4,408 to 5,169), respectively. Bioequivalence in 3TC PK following the administration of 300 and 150 mg was not demonstrated: the GMRs for AUC(0-24), C(24), and C(max) were 0.57 (0.55 to 0.60), 0.63 (0.59 to 0.67), and 0.56 (0.53 to 0.60), respectively. The GM (90% CI) 3TC-TP AUC(0-24) values (pmol·h/10(6) cells) for the 300- and 150-mg doses were 59.5 (51.8 to 68.3) and 44.0 (38.0 to 51.0), respectively. Bioequivalence in 3TC-TP PK following the administration of 300 and 150 mg was not demonstrated: the GMRs for AUC(0-24), C(24), and C(max) were 0.73 (0.64 to 0.83), 0.82 (0.68 to 0.99), and 0.70 (0.61 to 0.82), respectively. We found that 3TC at 150 mg is not bioequivalent to the standard regimen of 300 mg, indicating that saturation of cytosine phosphorylation pathways is not achieved at a dose of 150 mg.

A rapid and sensitive HPLC-MS method for the detection of plasma and cellular rifampicin.

Rifampicin is active against both intracellular and extracellular Mycobacterium tuberculosis. The ability to measure rifampicin drug concentrations in both plasma and in cells may be useful in evaluating the suitability of dosage regimens for populations and individuals. Here a novel simple, precise and accurate method for the quantification of rifampicin in both cells and plasma is reported. Sample proteins were precipitated with acetonitrile containing the internal standard and then diluted with water. Aliquots of supernatant were then injected into the HPLC-MS system for chromatographic separation and detection. Rifampicin calibration curves encompassed concentrations from 100 to 12,800 ng/mL. Intra- and inter-assay precision and accuracy were determined using low, medium and high concentration quality control samples and was found to be within 10% in all cases. Rifampicin concentrations were found to be unaffected by freeze-thaw cycles, but were significantly affected by heat-inactivation (58 degrees C, 40 min). This assay was successfully utilised to determine the pharmacokinetic profile of rifampicin in plasma and peripheral blood mononuclear cells (PBMC) in 8 tuberculosis patients receiving rifampicin over an 8h period.

Simultaneous determination of HIV protease inhibitors amprenavir, atazanavir, indinavir, lopinavir, nelfinavir, ritonavir and saquinavir in human plasma by high-performance liquid chromatography-tandem mass spectrometry.

We report a precise and accurate method for simultaneous quantification of protease inhibitors (PIs) amprenavir, atazanavir, indinavir, lopinavir, nelfinavir, ritonavir and saquinavir in plasma. An internal standard was added to samples prior to protein precipitation with acetonitrile followed by addition of ammonium formate buffer. Analysis was by HPLC-MS/MS. Calibration curves were validated over concentration ranges encompassing both subtherapeutic and potentially 'toxic' drug concentrations. Inter- and intra-assay variation were below 11% and PI recovery was above 87%. The bioanalytical method described is successfully applied to measure PI concentrations obtained from clinical pharmacokinetic studies and routine therapeutic drug monitoring (TDM).

Intracellular disposition and metabolic effects of zidovudine, stavudine and four protease inhibitors in cultured adipocytes.

OBJECTIVE: The pathogenesis of lipodystrophy caused by the HIV protease inhibitors (PIs) and nucleoside reverse transcriptase inhibitors (NRTIs) is unclear. We have investigated the disposition of these drugs in adipocytes and the consequent effect on adipocyte metabolism and viability. DESIGN: Laboratory study utilizing two murine cell lines, 3T3-L1 and 3T3-F442A. METHODS: Intracellular NRTI phosphate and PI concentrations were determined by HPLC and HPLC-MS/MS, respectively. The cytotoxicity of the drugs was examined on the different adipogenic stages together with their effects on glucose uptake plus or minus insulin, and on glycerol and triglyceride levels. RESULTS: There was rapid intracellular accumulation and phosphorylation of [3H]-zidovudine and -stavudine to their phosphate metabolites in adipocytes. The NRTIs were not cytotoxic, did not affect preadipocyte protein synthesis and did not inhibit adipogenesis or induce lipolysis. PIs accumulated in adipocytes (nelfinavir>saquinavir>ritonavir>indinavir). All PIs, except indinavir, were cytotoxic and inhibited adipogenesis, increased lipolysis and impaired preadipocyte protein synthesis. PIs inhibited glucose uptake in the rank order: indinavir>saquinavir>ritonavir>nelfinavir. CONCLUSION: These data demonstrate that PIs may play a role in the insulin resistance observed in lipodystrophy by affecting glucose uptake, adipogenesis and lipolysis. NRTIs alone do not seem to have any effect on adipocyte metabolism despite undergoing phosphorylation to their triphosphorylated anabolites, although their effects in combination with PIs in perturbing adipocyte metabolism warrants further investigation.

Intracellular indinavir pharmacokinetics in HIV-infected patients: comparison with plasma pharmacokinetics.

OBJECTIVE: To determine intracellular concentrations of indinavir (IDV) and investigate the relationship between plasma and intracellular IDV pharmacokinetics in HIV-infected patients. METHODS: A pharmacokinetic study of 10 patients receiving IDV plus dual nucleoside analogue therapy. Peripheral blood mononuclear cells were isolated by density gradient centrifugation and cell counts estimated. IDV was extracted from cells in the presence of 60% methanol and evaporated to dryness. Both plasma and intracellular IDV samples were assayed by high performance liquid chromatography linked to mass spectrometry. Data were subjected to non-compartmental pharmacokinetic analysis. RESULTS: The mean intracellular IDV area under the curve over 8 h (AUC0-8) was lower than the plasma AUC0-8 (7574 +/- 1003 vs 25060 +/- 4171 ng/ml/h; P<0.004). However, both the elimination half-life (t1/2) and the mean residence time (MRT) of IDV intracellularly were prolonged compared with plasma (t1/2: 2.0 +/- 0.3 vs 1.2 +/- 0.09 h; MRT: 3.6 +/- 0.6 vs 2.1 +/- 0.1 h; P<0.05). All patients were responsive to therapy at the time of the study, as assessed by HIV plasma RNA levels. Individual plasma versus intracellular time course results suggest that, due to the prolonged intracellular half-life, some patients may achieve acceptable intracellular IDV concentrations despite sub-therapeutic plasma levels. Similarly, potentially inadequate intracellular concentrations may occur despite therapeutic plasma concentrations. CONCLUSIONS: There is no significant intracellular accumulation of IDV within the lymphocytes of HIV-1-infected patients relative to plasma. However, intracellular concentrations are compatible with reported IDV-free drug concentrations in plasma. The intracellular elimination half-life and mean residence time of IDV are significantly prolonged compared with plasma. This may in part explain why certain patients maintain adequate viral suppression despite sub-therapeutic plasma IDV levels.

Effect of protein-bound uraemic toxins on the thermodynamic characteristics of human albumin.

The ability of albumin to bind drugs and other lipophilic organic acids is decreased in chronic renal failure by the accumulation of albumin-bound uraemic toxins such as hippuric acid, indoxyl sulphate and 3-carboxy-4-methyl-5-propyl-2-furanpropanoic acid (CMPF). This furan acid is the most highly bound and is not removed by haemodialysis. The inhibitory effects of these three uraemic toxins on the interaction of three marker ligands sodium octanoate (for medium chain fatty acids), salicylic acid and phenol red (bilirubin site/site I) with albumin have been investigated by differential scanning microcalorimetry and flow microcalorimetry. CMPF was the most potent inhibitor and its binding site coincided with that of bilirubin (site I). Indoxyl sulphate binds to the site for medium-chain fatty acids and tryptophan (site II) and hippuric acid, the weakest inhibitor, inhibited binding to the salicylic acid site.

Extraction of uraemic toxins with activated carbon restores the functional properties of albumin.

BACKGROUND: Previous work has demonstrated that a partial normalization of the conformation of albumin from uraemic plasma and a substantial restoration of its binding abilities can be achieved by extraction with activated charcoal. This is best achieved at pH 3, but exposure of whole plasma to this low pH leads to the loss of some essential components. METHODS: The melting curves and ligand-binding abilities of uraemic albumin have been investigated after extraction with a new generation of activated carbon at three pH values (7.2, 3.0 and 5.08). RESULTS: Albumin isolated from uraemic plasma had a characteristically increased melting temperature because of bound ligands. Extraction of uraemic plasma at pH 7.2, 5.08 and 3.0 induced low-temperature shifts of albumin thermo-adsorption maximum T1 of 1.4, 3.8, 2.4 degrees C and T2 of 0.8, 3.9 and 1.2 degrees C, respectively. Flow microcalorimetry data demonstrated a decrease in the ability of uraemic albumin to bind octanoate, phenol red, salicylic acid, warfarin and diazepam. Purification of uraemic plasma at pH 5.08 completely restored the binding affinity of albumin for all the marker ligands. CONCLUSIONS: Highly efficient activated carbons, with clinically feasible acidification of plasma, can remove strongly albumin-bound uraemic toxins. Investigation of the melting curve of the isolated albumin is a new biophysical way to monitor both its molecular condition and the extent of removal of protein-bound toxins by dialysis. The melting curve provides new qualitative and quantitative information about albumin in an analogous way to an electrocardiogram and the heart.

Quantification of rilpivirine in human plasma, cervicovaginal fluid, rectal fluid and genital/rectal mucosal tissues using liquid chromatography-tandem mass spectrometry.

BACKGROUND: A sensitive, specific and robust liquid chromatography-tandem mass spectrometry method has been developed and validated for the quantification of rilpivirine in human plasma, genital/rectal biofluids and mucosal tissues. METHODS: Plasma and tissue samples were extracted using protein precipitation (acetonitrile/water; 5:1 v/v), and genital/rectal biofluids absorbed onto ophthalmic swabs were extracted using liquid-liquid extraction (hexane/ethyl acetate; 80:20 v/v). A stable isotope-labeled internal standard ((13)C-d4-RPV) was used, and the assay was validated over a concentration range of 0.5-400 ng/ml. CONCLUSION: Inter- and intra-assay precision and accuracy met the acceptance as per US FDA bioanalytical guidelines. The validated assay has been used for the determination of rilpivirine concentrations in these matrices as part of an exploratory pharmacokinetic study investigating the suitability of a long-acting formulation of rilpivirine for pre-exposure prophylaxis.

Tenofovir, emtricitabine intracellular and plasma, and efavirenz plasma concentration decay following drug intake cessation: implications for HIV treatment and prevention.

BACKGROUND: In vivo data on tenofovir (TFV), emtricitabine (FTC), and efavirenz (EFV) concentration decay after intake cessation are limited; determinations of "true" elimination half-lives (t½) have often been based on suboptimal sampling windows. Understanding these parameters is important in managing missed doses and planning HIV pre-exposure prophylaxis (PrEP). This study investigated the pharmacokinetics (PK) of plasma TFV/FTC, their intracellular (IC) anabolites, TFV-diphosphate (DP) and FTC-triphosphate (TP), and plasma EFV over 10 days after intake cessation in HIV-negative volunteers. METHODS: Volunteers received an Atripla (TFV/FTV/EFV) tablet daily for 14 days. PK sampling occurred before final dose and up to 228 hours after stopping. Peripheral blood mononuclear cells for [IC](drug) and [plasma](drug) were isolated, with analysis by tandem mass spectrometry. RESULTS: Sixteen participants completed the study. Geometric mean plasma (t½)(228h) of TFV and FTC were 31 and 37 hours. These were longer than the previous reports (TFV 12-18 hours, FTC 10 hours).Geometric mean (t½)(228h) of IC TFV-DP and FTC-TP were 164 and 39 hours, whereas for EFV in plasma was 92 hours. [EFV](plasma) in 5/16 participants were below the suggested MEC of 1000 ng/mL within 48 hours postdose; however, 50% of the participants maintained concentrations above this level after 84 hours. CONCLUSIONS: These data fully characterize the PK of TFV and TFV-DP, FTC and FTC-TP, and EFV after stopping the drug combination. Although decay in concentrations can be related to a target for EFV, this is more difficult for the IC phosphates. Consensus on 'target' triphosphate/diphosphate concentrations will further our understanding of missed/delayed doses in treatment and prevention strategies.

Validation of an electrospray ionisation LC-MS/MS method for quantitative analysis of telaprevir and its R-diastereomer.

A sensitive high-performance reverse phase liquid chromatography-positive ion electrospray tandem mass spectrometry method was developed and validated for the quantification of telaprevir and its inactive R-diastereomer (VRT-127394) in human plasma. The analytes and the internal standard (telaprevir-d11) were extracted from plasma by liquid-liquid extraction using tert-Butyl methyl ether (TBME). Chromatographic separation was achieved on a reversed-phase Accucore C18 column with a gradient programme consisting of water:ammonia (25%), 100:0.01 (v/v) (mobile phase A) and ACN:MeOH:ammonia (25%), 15:85:0.01 (v/v/v) (mobile phase B). The MS acquisition was performed with selective reaction monitoring mode using the respective [M+H](+) ions, m/z 680.59→322.42 for telaprevir and VRT-127394, and 691.15→110.13 for telaprevir-d11. The assay exhibited a linear dynamic range of 5-5000ng/mL for telaprevir and VRT-127394. Acceptable precision (%RSD<6.5%) and accuracy (94-108%) were obtained for concentrations over the range of the standard curve. A procedure was established to stabilise the plasma to prevent ex vivo interconversion of the isomers.

Steady-state pharmacokinetics of lopinavir plus ritonavir when administered under different meal conditions in HIV-infected Ugandan adults.

We investigated the effect of food on the steady-state pharmacokinetics of lopinavir and ritonavir in 12 Ugandan patients receiving lopinavir coformulated with ritonavir (LPV/r) tablets using a crossover design. Intensive pharmacokinetic sampling was performed 7 days apart after LPV/r dosing under moderate fat, high fat, and fasted meal conditions. Lopinavir and ritonavir concentrations were determined by liquid chromatography and tandem mass spectrometry. Compared with the fasted state, a high fat meal reduced lopinavir and ritonavir area under the curve by 14% and 29%, respectively. With a moderate fat meal, area under the curve for both drugs was similar to the fasted state.

Effect of Food on the Steady-State Pharmacokinetics of Tenofovir and Emtricitabine plus Efavirenz in Ugandan Adults.

We investigated the effect of food on the steady-state pharmacokinetics of a proprietary fixed-dose combination (FDC) tablet containing tenofovir disoproxil fumarate (TDF)/emtricitabine/efavirenz. Fifteen Ugandan HIV-1 patients at steady-state dosing with TDF/emtricitabine/efavirenz were admitted for 24-hour intensive pharmacokinetic sampling after dosing in the fasting state. Blood sampling was repeated seven days later with TDF/emtricitabine/efavirenz administered with food (19 g fat). Drug concentrations in plasma were determined by liquid chromatography and tandem mass spectrometry. Geometric mean ratios (GMRs) and confidence intervals (CIs) of parameters were calculated (reference, fasting). For efavirenz, GMRs (90% CIs) for C(max), AUC(0-24), and C(24) were 1.47 (1.24-1.75), 1.13 (1.03-1.23), and 1.01 (0.91-1.11), respectively. Corresponding GMRs were 1.04 (0.84-1.27), 1.19 (1.10-1.29), and 0.99 (0.82-1.19) for tenofovir, 0.83 (0.76-0.92), 0.87 (0.78-0.97), and 0.91 (0.73-1.14) for emtricitabine. Stable patients may take the FDC without meal restrictions. The FDC should be taken without food by patients experiencing central nervous system toxicities.

Validation of a rapid and sensitive high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) assay for the simultaneous determination of existing and new antiretroviral compounds.

Clinical pharmacokinetic studies of antiretrovirals require accurate and precise measurement of plasma drug concentrations. Here we describe a simple, fast and sensitive HPLC-MS/MS method for determination of the commonly used protease inhibitors (PI) amprenavir, atazanavir, darunavir, lopinavir, ritonavir, saquinavir and the non-nucleoside reverse transcriptase inhibitor (NNRTI) nevirapine, as well as the more recent antiretrovirals, the CCR5 antagonist maraviroc and the "second generation" NNRTI etravirine and rilpivirine. An internal standard (quinoxalone; QX) was added to plasma aliquots (100 microl) prior to protein precipitation with acetonitrile (500 microl) followed by centrifugation and addition of 0.05% formic acid (200 microl) to the supernatant. Chromatographic separation was achieved using a gradient (acetonitrile and 0.05% formic acid) mobile phase on a reverse-phase C18 column. Detection was via selective reaction monitoring (SRM) operating in positive ionization mode on a triple-quadrupole mass spectrometer. All compounds eluted within a 5 min run time. Calibration curves were validated over concentration ranges reflecting therapeutic concentrations observed in HIV-infected patients from pharmacokinetic data reported in the literature. Correlation coefficients (r2) exceeded 0.998. Inter- and intra-assay variation ranged between 1% and 10% and % recovery exceeded 90% for all analytes. The method described is being successfully applied to measure plasma antiretroviral concentrations from samples obtained from clinical pharmacokinetic studies.

Effect of boosted fosamprenavir or lopinavir-based combinations on whole-body insulin sensitivity and lipids in treatment-naive HIV-type-1-positive men.

BACKGROUND: Antiretroviral therapy is associated with metabolic complications, including dyslipidaemia, body fat changes and insulin resistance. Healthy volunteer studies have demonstrated a decrease in glucose disposal associated with dosing with specific antiretrovirals. METHODS: HIV-type-1-positive male participants were randomized to receive tenofovir disoproxil fumarate and lamivudine, with either fosamprenavir (FPV)/ritonavir or lopinavir (LPV)/ritonavir twice daily. A hyperinsulinaemic euglycaemic clamp was performed at baseline and at 2 weeks after commencing treatment. The homeostasis model assessment index for insulin resistance (HOMA-IR) was also calculated at these time points. Changes in lipids and lipoprotein subfractions (by nuclear magnetic resonance spectroscopy) were assessed. A pharmacokinetic assessment was undertaken at week 2. RESULTS: A total of 27 participants were enrolled. There was no significant change in whole-body insulin sensitivity or HOMA-IR from baseline or between groups. Total cholesterol increased significantly, by 6.6% with FPV and 10.9% with LPV. The changes in lipids and lipoprotein subfractions were similar between groups with increases in triglycerides, very low-density lipoprotein (VLDL) and chylomicrons, and low-density lipoprotein (LDL) particles. Although the total high-density lipoprotein (HDL) particles were not significantly altered, a decrease in small HDL particles was seen. Changes in VLDL and chylomicron particles in both groups and triglycerides and small HDL particles in the LPV group were statistically significant. CONCLUSIONS: In HIV-type-1-positive men initiating antiretroviral therapy with FPV- or LPV-based regimens, there were no significant changes in whole-body insulin sensitivity after 2 weeks. A proatherogenic lipid profile characterized by increases in triglycerides, VLDL and chylomicron particles and LDL particles, and a decrease in small HDL particles, was observed in both groups.

Intracellular accumulation of human immunodeficiency virus protease inhibitors.

Intracellular accumulation of the protease inhibitors (PIs) saquinavir (SQV), ritonavir (RTV), and indinavir (IDV) was determined in 50 human immunodeficiency virus-positive patients. Following extraction, PIs were quantified by mass spectrometry. Paired plasma and intracellular samples were collected over a full dosing interval from patients (13 on SQV, 6 on RTV, 8 on IDV, 16 on SQV plus RTV, 7 on IDV plus RTV) with a plasma viral load of <400 copies/ml. Data were expressed as intracellular/plasma drug concentration ratios. A hierarchy of intracellular accumulation was demonstrated by the following medians: 9.45 for SQV > 1.00 for RTV > 0.51 for IDV. Coadministration of RTV did not boost ratios of SQV or IDV within the cell or in plasma, although absolute plasma and intracellular SQV concentrations were increased by RTV. Seven individuals receiving SQV in hard-gel capsule form (median, 32 months) had higher intracellular/plasma drug ratios than all other patients receiving SQV (median, 17.62 versus 4.83; P = 0.04), despite consistently low plasma SQV concentrations. How this occurs may provide insight into the mechanisms that limit adequate drug penetration into sanctuary sites.