Efavirenz In Plasma Research Articles

A validated method for simultaneous quantification of four antiretrovirals in dried blood spot and plasma using LC-MS/MS: Application to efavirenz therapeutic drug monitoring in pregnant patients

IntroductionThe specific physiological background induced by pregnancy leads to significant changes in maternal pharmacokinetics, suggesting potential variability in plasma concentrations of antiretrovirals. Pregnant HIV patients exposed to subtherapeutic doses, particularly in the last trimester of the pregnancy, have higher chances to transmit the infection to their children. Therefore, the therapeutic drug monitoring of antiretrovirals in HIV pregnant patients would be of great value. ObjectivesThis study aimed to develop and validate a sensitive liquid chromatograph tandem mass spectrometry (LC-MS/MS) method for simultaneous quantification of efavirenz, raltegravir, atazanavir, and ritonavir in dried blood spots (DBS) and plasma. Design and MethodsThe analytes were extracted from the DBS punch and plasma with a mixture of methanol:zinc sulfate 200 mM (50:50, v/v) and 100 % methanol, respectively. For the chromatographic separation a Shim-pack® C18, 4.6 mm × 150 mm, 5 μm column was used. Detection was performed in a 3200-QTRAP® mass spectrometer, with a run time of 6 min. ResultsThe assay was linear in the range of 15–1,000 ng/mL for raltegravir, 50–10,000 ng/mL for both atazanavir and ritonavir, 50–5,000 ng/mL for efavirenz. Precision and accuracy at these concentrations were less than 15 % for all analytes. Raltegravir, atazanavir, and ritonavir were stable for seven days at 23 °C and 40 °C, whereas efavirenz was stable for twenty-four hours at the same conditions. ConclusionsThe method was successfully applied to quantify efavirenz in DBS samples obtained from HIV-1 infected pregnant volunteers under antiretroviral therapy. The concentrations of efavirenz in DBS and plasma were comparable according to Passing-Bablok regression and Bland-Altman analysis.

A simple high-performance liquid chromatographic assay for concurrent quantification of lumefantrine and efavirenz in human plasma from malaria–HIV co-infected individuals

BackgroundAs per current treatment guidelines, artemether-lumefantrine and efavirenz-based antiretroviral therapy are recommended drugs for falciparum malaria and human immunodeficiency virus (HIV) infections, respectively. A liquid chromatography-ultraviolet detection method for simultaneous quantification of lumefantrine and efavirenz was developed and validated for efficacy and pharmacokinetic clinical studies. Lumefantrine and efavirenz were separated using a 100 × 4.6 mm × 3 µm Fortis C18 chromatographic column, and a multistep gradient mobile phase. Calibration curves were obtained with a series of standard solutions containing known concentrations of the chemical reference of both analytes prepared concomitantly in drug-free plasma. The assay was validated within the calibration ranges of 78.125–20,000 ng/mL for lumefantrine and 187.15–24,000 ng/mL for efavirenz. Stability assessment was carried out with or without heating the quality control sample to 58 °C for 45 min. The method was employed to measure the plasma concentrations of lumefantrine and efavirenz in a study conducted among malaria-HIV co-infected patients.ResultLumefantrine and efavirenz were well separated from each other and from the biological matrix. The method demonstrated a good recovery of 72.64% for lumefantrine and 117.17% for efavirenz. The intra- and inter-day accuracy presented as 95.36–105.14% for lumefantrine and 104.11–115% for efavirenz and precision ranged from 1.15 to 6.45% for lumefantrine and 0.43 to 13.12 for efavirenz, were within ± 15% at the three quality control levels. The analytes from both quality control lots and samples collected from HIV-malaria co-infected individuals were found to be stable post-deactivation of infectious virus by heating to 58 °C for 45 min.ConclusionThe assay is accurate, precise and shown to simultaneously measure the lumefantrine and EFV in human plasma.

Instability of Efavirenz Metabolites Identified During Method Development and Validation.

Accurate quantification of efavirenz metabolites in patient samples is required to investigate their potential contribution to efavirenz adverse events. This study aimed to validate a LC-MS/MS method to quantify and investigate the stability of efavirenz and metabolites in human plasma. Compounds were extracted from plasma by supported liquid extraction and resolved on a C18 column. Validation was performed following FDA bioanalytical method validation guidelines. Stability under common conditions of sample pre-treatment and storage were assessed. Efavirenz and 8-hydroxyefavirenz were stable for all conditions tested. 7-Hydroxyefavirenz and 8,14-dihydroxyefavirenz were not stable in plasma at room temperature for 24 h (46%-69% loss), -20°C for 90 days (17%-50% loss), or 60°C for 1 h (90%-95% loss). Efavirenz and 8-hydroxyefavirenz concentrations in HIV/AIDS patient (n=5) plasma prepared from pre-treated (60°C for 1 h) whole blood varied from 517-8564 ng/mL and 131-813 ng/mL, respectively. 7-Hydroxyefavirenz and 8,14-dihydroxyefavirenz concentrations were below validated lower limits of quantification (0.25 and 0.5 ng/mL, respectively), most likely due to sample pre-treatment. This is the first report of 7-hydroxyefavirenz and 8,14-dihydroxyefavirenz instability under conditions commonly used in preparation of samples from HIV/AIDS patients. Alternative biosafety measures to heat pre-treatment must therefore be used for accurate quantification of plasma 7-hydroxyefavirenz and 8,14-dihydroxyefavirenz.

A fast-screening dispersive liquid-liquid microextraction-gas chromatography-mass spectrometry method applied to the determination of efavirenz in human plasma samples.

We demonstrate the suitability of a fast, green, easy-to-perform, and modified sample extraction procedure, i.e., dispersive liquid-liquid microextraction (DLLME) for the determination of efavirenz (EFV) in human plasma. Data acquisition was done by gas chromatography-mass spectrometry (GC-MS) in the selected ion monitoring (SIM) mode. The simplicity of the method lies in, among others, the avoidance of the use of large organic solvent volumes as mobile phases and non-volatile buffers that tend to block the plumbing in high-performance liquid chromatography (HPLC). Chromatographic and mass spectral parameters were optimized using bovine whole blood for matrix matching due to insufficient human plasma. Method validation was accomplished using the United States Food and Drug Administration (USFDA) 2018 guidelines. The calibration curve was linear with a dynamic range of 0.10-2.0μg/mL and an R2 value of 0.9998. The within-run accuracy and precision were both less than 20% at the lower limit of quantification (LLOQ) spike level. The LLOQ was 0.027μg/mL which compared well with some values but was also orders of magnitude better than others reported in the literature. The percent recovery was 91.5% at the LLOQ spike level. The DLLME technique was applied in human plasma samples from patients who were on treatment with EFV. The human plasma samples gave concentrations of EFV ranging between 0.14-1.00μg/mL with three samples out of seven showing concentrations that fell within or close to the recommended therapeutic range.

Mechanistic Modeling of Maternal Lymphoid and Fetal Plasma Antiretroviral Exposure During the Third Trimester.

Pregnancy-induced changes in plasma pharmacokinetics of many antiretrovirals (ARV) are well-established. Current knowledge about the extent of ARV exposure in lymphoid tissues of pregnant women and within the fetal compartment is limited due to their inaccessibility. Subtherapeutic ARV concentrations in HIV reservoirs like lymphoid tissues during pregnancy may constitute a barrier to adequate virological suppression and increase the risk of mother-to-child transmission (MTCT). The present study describes the pharmacokinetics of three ARVs (efavirenz, dolutegravir, and rilpivirine) in lymphoid tissues and fetal plasma during pregnancy using materno-fetal physiologically-based pharmacokinetic models (m-f-PBPK). Lymphatic and fetal compartments were integrated into our previously validated adult PBPK model. Physiological and drug disposition processes were described using ordinary differential equations. For each drug, virtual pregnant women (n = 50 per simulation) received the standard dose during the third trimester. Essential pharmacokinetic parameters, including Cmax, Cmin, and AUC (0–24), were computed from the concentration-time data at steady state for lymph and fetal plasma. Models were qualified by comparison of predictions with published clinical data, the acceptance threshold being an absolute average fold-error (AAFE) within 2.0. AAFE for all model predictions was within 1.08–1.99 for all three drugs. Maternal lymph concentration 24 h after dose exceeded the reported minimum effective concentration (MEC) for efavirenz (11,514 vs. 800 ng/ml) and rilpivirine (118.8 vs. 50 ng/ml), but was substantially lower for dolutegravir (16.96 vs. 300 ng/ml). In addition, predicted maternal lymph-to-plasma AUC ratios vary considerably (6.431—efavirenz, 0.016—dolutegravir, 1.717—rilpivirine). Furthermore, fetal plasma-to-maternal plasma AUC ratios were 0.59 for efavirenz, 0.78 for dolutegravir, and 0.57 for rilpivirine. Compared with rilpivirine (0 h), longer dose forgiveness was observed for dolutegravir in fetal plasma (42 h), and for efavirenz in maternal lymph (12 h). The predicted low lymphoid tissue penetration of dolutegravir appears to be significantly offset by its extended dose forgiveness and adequate fetal compartment exposure. Hence, it is unlikely to be a predictor of maternal virological failure or MTCT risks. Predictions from our m-f-PBPK models align with recommendations of no dose adjustment despite moderate changes in exposure during pregnancy for these drugs. This is an important new application of PBPK modeling to evaluate the adequacy of drug exposure in otherwise inaccessible compartments.

Multiresponse Optimization of HPLC Method: Simultaneous Estimation of Protease Inhibitors and NNRTI in Human Plasma.

Multiresponse optimization approach to develop a simple isocratic, highly sensitive and accurate HPLC method for the simultaneous determination of Efavirenz, Atazanavir, Lopinavir and Ritonavir in human blood plasma along with carvedilol as an internal standard. Optimized the factors (ACN, buffer concentration and flow rate) effecting and interacting with the responses (k1, Rs2,1, Rs3,2 and tR5) applying Central Composite Design a chemometric tool. All the mathematical models as well as response surfaces were defined and derived for the separation using this strategy. Chromatography was performed on Thermo Hypersil C18 column using mobile phase comprising of ACN: 10mM KH2PO4 (51.2:48.8) with 1mLmin-1 flow rate and detection wavelength was fixed at 210nm. The analysis time was within 9min. The method developed was validated by following "Bioanalytical method validation" [USFDA-CDER, 2001]. The developed method can be applied for bioavailability and pharmacokinetic studies.

Simple and Rapid Method by Ultra High-Performance Liquid Chromatography (UHPLC) with Ultraviolet Detection for Determination of Efavirenz in Plasma: Application in a Preclinical Pharmacokinetic Study.

A simple and rapid ultra-high-performance liquid chromatography (UHPLC) method for determination of efavirenz (EFV) in plasma was developed and applied in a preclinical pharmacokinetic study. The method involves only addition of acetonitrile to precipitation of plasma proteins followed by solvent evaporation. The mobile phase consisted of methanol, acetonitrile and 0.1M formic acid (20:50:30) at a flow rate of 0.3mL/min with run time of 5min. A CSH C18 column and a UHPLC-UV system operating at 245nm were used. There was a linear response in the range of 0.078 to 10μg/mL, and the equation was obtained by weighting (1/x2) with r2=0.9965. The pharmacokinetic disposition of EFV was investigated in rabbits (two groups, n=7) following a single intravenous administration (IV group) at a dose of 2.7mg/kg and a single oral administration (oral group) of EFV co-administered with lamivudine (3TC) and tenofovir (TNF) at a dose of 50, 25 and 25mg, respectively. The study demonstrated the applicability of the method for determination of EFV in plasma without interference from other co-administered drugs, and the pharmacokinetic parameters were calculated. The method showed advantages over other methods in the literature, such as simplicity of sample processing and fast results.

No Relevant Pharmacokinetic (PK) Interaction between Rezafungin and Nine Probe Drugs: Results from a Drug-Drug Interaction (DDI) Study

Introduction Rezafungin (RZF) is a new echinocandin being developed to treat patients with systemic fungal infections and to prevent invasive fungal infections in allogeneic hematopoietic stem cell transplant (HSCT) recipients and other patients at high risk of infection. RZF is differentiated by stable and efficacy-driving pharmacokinetics (PK) that allow for once-weekly dosing. Several in vitro studies have been conducted to assess the potential for rezafungin to alter the PK of other drugs. Based on the results, possible interactions were identified in which the risk of drug-drug interactions (DDIs) could not be ruled out relative to the assumed Ph3 dose of 400 mg once weekly and known PK exposure in healthy subjects. Clinical in vivo evaluations of drug interaction potential were performed proactively. Methods This was an open-label, inpatient study of 26 healthy subjects to assess DDIs between RZF and probe drugs selected for known interactions with CYP enzymes and transporters (and consequently commonly used in evaluations of PK interaction), as well as drugs likely to be coadministered with RZF. A loading dose of RZF 600 mg was administered on the first dosing day, to approximate a steady state plasma concentration of multiple once-weekly 400-mg doses, followed by 400-mg doses on days 10 and 15. Probe drugs were administered as part of a cocktail containing ≥2 drugs, once prior to and once after receiving RZF on a schedule designed to allow for washout between doses and to limit interactions with other drugs (Figure 1). Samples were analyzed to determine RZF, tacrolimus, repaglinide, metformin, rosuvastatin, pitavastatin, caffeine, efavirenz, midazolam and digoxin concentrations in plasma (except for tacrolimus which was in whole blood) to characterize the PK profile of each analyte. PK exposure parameters (area under curve [AUC] and maximum concentration [Cmax]) were calculated from the plasma concentration-time profiles by noncompartmental analysis. Results When rezafungin was dosed concomitantly with metformin, pitavastatin, caffeine, efavirenz, midazolam, digoxin, tacrolimus, repaglinide, and rosuvastatin, no relevant change in systemic concentrations of these probe drugs was observed (Figure 2). PK exposure of all drugs were similar with or without RZF. Maximum changes in mean Cmax or AUC were Conclusion No meaningful PK interactions occurred between RZF and the 9 probe drug substrates tested, providing evidence that no RZF dose adjustment is expected when co-administered with these commonly used drugs.

Solid-phase extraction combined with dispersive liquid-liquid microextraction/HPLC-UV as a sensitive and efficient method for extraction, pre-concentration and simultaneous determination of antiretroviral drugs nevirapine, efavirenz and nelfinavir in pharmaceutical formulations and biological samples

Therapeutic drug monitoring (TDM) of antiretroviral drugs requires accurate and precise analysis of their trace amounts in plasma samples. Solid-phase extraction (SPE) coupled with dispersive liquid-liquid microextraction based on solidification of floating organic drop (DLLME-SFO) was introduced as a simple and sensitive method for extraction, pre-concentration and simultaneous determination of efavirenz (EFV), nelfinavir (NFV) and nevirapine (NVP) in human plasma and pharmaceutical formulations by using high performance liquid chromatography (HPLC) with UV detection. A response surface methodology (RSM) based on central composite design (CCD) was used for optimizing the main variables in the extraction procedure. Under the optimum experimental conditions, the calibration curves were linear in the range of 0.1–400 ng mL−1 with correlation coefficients from 0.9982 to 0.9997 and limits of detection (at S/N = 3) of 0.03 to 0.07 ng mL−1. Moreover the intra-day and inter-day precision (RSD%) were in the range of 2.2–4.2% and 3.1–5.2%, respectively. The enrichment factors and relative recoveries of the anti-HIV drugs were 459–1507 and 93.7–105.4%. The method was successfully applied to the simultaneous determination of the trace amounts of the antiretroviral drugs in blood plasma and pharmaceutical formulations.

Quantification of efavirenz, emtricitabine and tenofovir in plasma from HIV-positive pregnant women. Analytical development and clinical application

Quantification of efavirenz, emtricitabine and tenofovir in plasma from HIV-positive pregnant women. Analytical development and clinical application

Comparison of efficiencies of selected sample extraction techniques for the analysis of selected antiretroviral drugs in human plasma using LC-MS

IntroductionSample preparation in bio analytical chemistry poses a challenge because it can be compound dependent. We compared six sample extraction techniques i.e. QuEChERS (Q), liquid extraction (LE), protein precipitation (PPT), Q-PPT, Q-LE and LE-PPT for the extraction of antiretroviral drugs emtricitabine, tenofovir, efavirenz, lopinavir and rotinavir in human blood plasma. MethodA multiple reaction monitoring liquid chromatography- tandem mass spectrometry method for the determination of the same antiretroviral drugs developed and validated in this laboratory was used. Comparisons were based on the efficiencies of extraction, the precisions and accuracies. Using United States Food and Drug Administration guidelines, analytical performance characteristics i.e. limits of detection, lower limits of quantification and upper limits of quantification were also compared. ResultsThe percent mean recoveries ranged between 68.8 and 81.2% for single modes and 52.4–70.5% for mixed mode techniques. The precisions of all the extraction techniques were within the Using United States Food and Drug Administration guidelines acceptable range of <15% at all concentration levels for all analytes. Accuracy ranged between 8.73 and 65.94% for single mode techniques and between 21.73 and 51.59% for mixed mode techniques. DiscussionThe mixed modes gave slightly lower recoveries but Q-LE compared well with the single modes at slightly higher spike levels. Limits of detection for all the six sample preparation techniques fell below the clinically relevant therapeutic range of approximately 3–8ppm. Therefore all techniques can be employed for routine therapeutic drug monitoring studies.

Population Pharmacokinetic Model Linking Plasma and Peripheral Blood Mononuclear Cell Concentrations of Efavirenz and Its Metabolite, 8-Hydroxy-Efavirenz, in HIV Patients.

The objectives of this study were to characterize the population pharmacokinetics (PK) of efavirenz (EFV) and 8-hydroxy-efavirenz (8OHEFV) in plasma and peripheral blood mononuclear cells (PBMCs) and to explore covariates affecting the PK parameters. Fifty-one patients had steady-state 0-to-24-h concentrations of EFV and 8OHEFV in plasma with corresponding concentrations in PBMCs, while 261 patients had one or two sparse concentrations at 16 ± 1 h postdose at weeks 4 and/or 16. The pharmacogenetic markers CYP2B6*6, CYP3A5*3, CYP3A5*6, UGT2B7*2, ABCB1 (3435C→T, 3842A→G), OATP1B1*1B, and OATP1B1*5, the presence of a rifampin-based antituberculosis (anti-TB) regimen, baseline body weight and organ function values, and demographic factors were explored as covariates. EFV concentration data were well described by a two-compartment model with first-order absorption (Ka ) and absorption lag time (Alag) (Ka = 0.2 h-1; Alag = 0.83 h; central compartment clearance [CLc/F] for CYP2B6*1/*1 = 18 liters/h, for CYP2B6*1/*6 = 14 liters/h, and for CYP2B6*6/*6 = 8.6 liters/h) and PBMCs as a peripheral compartment. EFV transfer from plasma to PBMCs was first order (CLp/F = 32 liters/h), followed by capacity-limited return (Vmax = 4,400 ng/ml/h; Km = 710 ng/ml). Similarly, 8OHEFV displayed a first-order elimination and distribution to PBMCs, with a capacity-limited return to plasma. No covariate relationships resulted in a significant explanation of interindividual variability (IIV) on the estimated PK parameters of EFV and 8OHEFV, except for CYP2B6*6 genotypes, which were consistent with prior evidence. Both EFV and 8OHEFV accumulated to higher concentrations in PBMCs than in plasma and were well described by first-order input and Michaelis-Menten kinetics removal from PBMCs. CYP2B6*6 genotype polymorphisms were associated with decreased EFV and 8OHEFV clearance.

Development and validation of reversed-phase HPLC gradient method for the estimation of efavirenz in plasma.

Efavirenz is an anti-viral agent of non-nucleoside reverse transcriptase inhibitor category used as a part of highly active retroviral therapy for the treatment of infections of human immune deficiency virus type-1. A simple, sensitive and rapid reversed—phase high performance liquid chromatographic gradient method was developed and validated for the determination of efavirenz in plasma. The method was developed with high performance liquid chromatography using Waters X-Terra Shield, RP18 50 x 4.6 mm, 3.5 μm column and a mobile phase consisting of phosphate buffer pH 3.5 and Acetonitrile. The elute was monitored with the UV-Visible detector at 260 nm with a flow rate of 1.5 mL/min. Tenofovir disoproxil fumarate was used as internal standard. The method was validated for linearity, precision, accuracy, specificity, robustness and data obtained were statistically analyzed. Calibration curve was found to be linear over the concentration range of 1–300 μg/mL. The retention times of efavirenz and tenofovir disoproxil fumarate (internal standard) were 5.941 min and 4.356 min respectively. The regression coefficient value was found to be 0.999. The limit of detection and the limit of quantification obtained were 0.03 and 0.1 μg/mL respectively. The developed HPLC method can be useful for quantitative pharmacokinetic parameters determination of efavirenz in plasma.

Bio Analytical Method Development Of an Anti-HIV-1 Drug Efavirenz Using High Performance Liquid Chromatography In Human Plasma

A highly sensitive, selective and validated high performance liquid chromatography (HPLC) method has been developed for the quantitative determination of efavirenz in human CPDA plasma. The analyte and internal standard (IS) duloxetin (DXT) were extracted from human plasma using liquid-liquid extraction technique, then separated on a BDS hypersil C18 (15cm, 4.6mm x 5μm), reverse phase column using a mobile phase comprising of acetonitrile and 50 mM sodium dihydrogen orthophosphate solution (pH 5.5) (48:52, v/v) and column oven temperature was maintained at 30 °C. It was pumped at a flow rate of 0.8 mL/min with an injection volume of 15 µL, with a total run time of 15 min. The detection was performed on a tandem mass spectrometer equipped with electro spray ionization (ESI) source. Calibration curve for EFZ was linear within the selected range of 0.03–4.57 µg/mL in human plasma with mean correlation coefficient of 0.999. The LLOQ for efavirenz was found to be 0.03 µg/mL. The intra- and inter-assay precision and accuracy of the quality control samples, expressed as the percent relative standard deviation (% RSD) was less than 2%. The mean recovery was 96.63% to 112.71%, for EFZ and DXT, respectively. This method has been demonstrated to be an improvement over existing methods due to its greater sensitivity and specificity. The data obtained show that the sensitivity and selectivity of this method are adequate for drug monitoring in clinical research and pharmacokinetic investigation of EFZ

Development of an HPLC–UV assay method for the simultaneous quantification of nine antiretroviral agents in the plasma of HIV-infected patients

A new method using high-performance liquid chromatography coupled with ultra violet detection (HPLC–UV) was developed and validated for the simultaneous quantification of atazanavir, dolutegravir, darunavir, efavirenz, etravirine lopinavir, raltegravir, rilpivirine and tipranavir in human plasma. For the first time we reported here the development and validation of an HPLC–UV assay to quantify the frequently administered 9 antiretroviral compounds including dolutegravir and rilpivirine. A simple solid phase extraction procedure was applied to 500µL aliquots of plasma. The chromatographic separation of the drugs and internal standard (quinoxaline) was achieved with a gradient of acetonitrile and sodium acetate buffer on a C18 reverse-phase analytical column with a 25min analytical run time. Calibration curves were optimised according to the therapeutic range of drug concentrations in patients, and the coefficient of determination (r2) was higher than 0.99 for all analytes. Mean intraday and interday precisions (RSD) for all compounds were less than 15.0%, and the mean accuracy (% deviation from nominal concentration) was also found to be less than 15.0%. Extraction recovery range was between 80% and 120% for all drugs analysed. The solid phase extraction and HPLC–UV method enable a specific, sensitive, and reliable simultaneous determination of nine antiretroviral agents in plasma. Good extraction efficiency and low limit of HPLC–UV quantification make this method suitable for use in clinical trials and therapeutic drug monitoring.

In Vivo Profiling and Distribution of Known and Novel Phase I and Phase II Metabolites of Efavirenz in Plasma, Urine, and Cerebrospinal Fluid.

Efavirenz (EFV) is principally metabolized by CYP2B6 to 8-hydroxy-efavirenz (8OH-EFV) and to a lesser extent by CYP2A6 to 7-hydroxy-efavirenz (7OH-EFV). So far, most metabolite profile analyses have been restricted to 8OH-EFV, 7OH-EFV, and EFV-N-glucuronide, even though these metabolites represent a minor percentage of EFV metabolites present in vivo. We have performed a quantitative phase I and II metabolite profile analysis by tandem mass spectrometry of plasma, cerebrospinal fluid (CSF), and urine samples in 71 human immunodeficiency virus patients taking efavirenz, prior to and after enzymatic (glucuronidase and sulfatase) hydrolysis. We have shown that phase II metabolites constitute the major part of the known circulating efavirenz species in humans. The 8OH-EFV-glucuronide (gln) and 8OH-EFV-sulfate (identified for the first time) in humans were found to be 64- and 7-fold higher than the parent 8OH-EFV, respectively. In individuals (n = 67) genotyped for CYP2B6, 2A6, and CYP3A metabolic pathways, 8OH-EFV/EFV ratios in plasma were an index of CYP2B6 phenotypic activity (P < 0.0001), which was also reflected by phase II metabolites 8OH-EFV-glucuronide/EFV and 8OH-EFV-sulfate/EFV ratios. Neither EFV nor 8OH-EFV, nor any other considered metabolites in plasma were associated with an increased risk of central nervous system (CNS) toxicity. In CSF, 8OH-EFV levels were not influenced by CYP2B6 genotypes and did not predict CNS toxicity. The phase II metabolites 8OH-EFV-gln, 8OH-EFV-sulfate, and 7OH-EFV-gln were present in CSF at 2- to 9-fold higher concentrations than 8OH-EFV. The potential contribution of known and previously unreported EFV metabolites in CSF to the neuropsychological effects of efavirenz needs to be further examined in larger cohort studies.

An Investigation of Liquid Chromatography-Mass Spectral Attributes and Analytical Performance Characteristics of Tenofovir, Emtricitabine and Efavirenz in Human Plasma.

Liquid chromatography (LC) and mass spectral behavior and analytical performance characteristics of efavirenz (EFV), emtricitabine (EMT) and tenofovir (TFV), i.e., individual components of Atripla(®), were probed. This was followed by estimation of their analytical performance characteristics employing LC and a parallel direct infusion sample introduction procedure. Performance characteristics using both types of sample introduction procedures were compared. Using liquid chromatography-mass spectrometry (LC-MS), linearities, i.e., correlation coefficients of the calibration curves of EFV, EMT and TFV, ranged between 0.9300 and 0.9990 in the full scan, selected ion monitoring and mass spectrometry/mass spectrometry (MS-MS) modes. The limits of detection (LODs) ranged between 0.5 and 11.6 µg/L. The lower limits of quantification (LLOQs) and the upper limits of quantification (ULOQs) were in the ranges of 0.9-23.2 and 1.6-38.7 µg/L, respectively. The LODs ranged between 0.8 and 114.7 µg/L. The LLOQs and the ULOQs were in the ranges of 1.6-29.4 and 2.7-49.0 µg/L, respectively. In the case of EMT, sodiated molecular ion at m/z 270 was used to adduce analytical performance characteristics from which lower detection limits were obtained compared with those in the literature where [M+H](+) at m/z 248 was used.

Cloud point extraction for analysis of antiretrovirals in human plasma by UFLC-ESI-MS/MS

An analytical methodology based on cloud point extraction (CPE) coupled to Ultra-Fast Liquid Chromatography and electrospray tandem mass spectrometry (UFLC-MS/MS) was developed for analysis of Abacavir (ABC), Efavirenz (EFV), Lamivudine (3 TC) and Nelfinavir (NFV) in human plasma. It is the first time that CPE was used for extraction of antiretrovirals (ARV) from plasma. The effects of relevant physic-chemical variables on analytical response of each ARV, including pH, surfactant concentration, equilibration time and temperature, were study and optimized; as well as its coupling to UFLC-ESI-MS/MS. Under optimized conditions, the resulting methodology was as follows: a 500 μL aliquot of human plasma was diluted with 2 mL deionized water in a 10 mL centrifuge tube. A 500 μL aliquot Triton X-114 5% w/v was added and homogenized using a vortex stirrer. The resulting cloudy solution was kept at 65 °C for 20 min for promoting the condensation of surfactant micelles. Then it was centrifuged at 3000 × g for 5 min for separation of the surfactant-rich phase. After discarding the aqueous supernatant, 400 μL ACN were added to the remaining surfactant rich phase and centrifuged in order to precipitate proteins and separate them. A 150 μL aliquot of the supernatant was transferred to 2 mL vial and further diluted with 400 μL deionized water. A 30 μL aliquot of the so-prepared solution was injected and analyzed into the UFLC-MS/MS. The method detection limits for ABC, EFV, 3 TC and NFV under optimized conditions were 31, 77, 57 and 21 ng mL−1, respectively. The RSD% for the studied analytes were <15%, except at the LOQ, which were <19%. Recovery values ranged from 81 to 107%. The proposed methodology was successfully applied for the analysis of ABC, EFV, 3 TC and NFV in human plasma within the concentration range of 43–6816, 125–4992, 81–3248 and 49–7904 ng mL−1, respectively. Under optimized working conditions the proposed analytical methodology meets standard requirements of international guidelines, which makes it suitable for pharmacokinetic studies of the four ARV, as well as for therapeutic monitoring of HIV patients.

Determination of Efavirenz in Plasma by Dispersive Liquid-Liquid Microextraction Coupled to High-Performance Liquid Chromatography

Dispersive liquid–liquid microextraction (DLLME) coupled with high-performance liquid chromatography (HPLC) equipped with UV detector was used for the extraction and determination of efavirenz in plasma sample. Acetonitrile and chloroform were used as disperser and extraction solvents, respectively. Several parameters, including extraction solvent, the type of dispersion solvent, volume of extraction and dispersion solvents, pH, ionic strength of the media, as well as centrifuging time were optimized. Samples were injected on a reversed-phase C18 analytical column. The mobile phase consisted of 20 mM phosphate buffer (pH=3.0) and acetonitrile (40:60, v/v). The calibration curve of the proposed method was linear in the range of 0.1–100 μg/L. The relative standard deviations (RSD, %) were 3.4–7.5% (n=6) and the limit of detection (LOD) was 0.01 µg/L. The proposed method was applied to the analysis of plasma sample and spiked recoveries in the range of 98.5–102.5% were obtained. The obtained results show that DLLME is a very simple, rapid, sensitive, and efficient analytical method for the determination of efavirenz in plasma. Keywords: Efavirenz, DLLME, plasma, HPLC, HIV, UV detection.

Rapid Quantification of Efavirenz in Human Plasma by Electrospray Ionization Tandem Mass Spectrometry

AbstractWe report a rapid and sensitive method for detecting efavirenz in human plasma by electrospray ionization tandem mass spectrometry (ESI‐MS/MS). Detection of efavirenz in human plasma samples was confirmed by acquiring MS/MS of protonated efavirenz at m/z 316.5 to yield the characteristic fragment ions at m/z 298, 274, 272, 251, 246, 237, 232, 203 and 168, respectively. This approach provided good linearity for the quantification of efavirenz (R2 = ∼0.9989), with good precision (RSD &gt;10%) and limit of detection (0.20 nM). This method can be widely applied for precise quantitative analysis of a variety of drugs in clinical samples.