Simultaneous determination of intracellular concentrations of tenofovir, emtricitabine, and dolutegravir in human brain microvascular endothelial cells using liquid chromatography-tandem mass spectrometry (LC-MS/MS).

Combination antiretroviral therapy (cART) regimens are recommended for HIV patients to better achieve and maintain plasma viral suppression. Despite adequate plasma viral suppression, HIV persists inside the brain, which is, in part thought to result from poor brain penetration of antiretroviral drugs. In this study, a simple and ultra-sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for simultaneous determination of tenofovir, emtricitabine, and dolutegravir in cell lysates of an immortalized human brain microvascular endothelial cell line (hCMEC/D3) was developed and validated. Analytes were separated on a reverse phase C18 column using water and 0.1% formic acid in acetonitrile as mobile phases. The analytes were detected using positive electrospray ionization mode with multiple reaction monitoring (MRM). The assay was linear in the concentration range of 0.1-100 ng mL-1 for all analytes. Intra- and inter-assay precision and accuracy were within ±13.33% and ±10.53%, respectively. This approach described herein was used to determine the intracellular accumulation of tenofovir, emtricitabine, dolutegravir simultaneously in hCMEC/D3 cells samples.

Simultaneous determination and validation of emtricitabine, rilpivirine and tenofovir from biological samples using LC and CE methods.

A combination of antiretroviral agents is frequently used in effective treatment of the human immunodeficiency virus infection. In this study, two different separation methods are presented for the simultaneous determination of emtricitabine, rilpivirine and tenofovir from raw materials and urine samples. Developed liquid chromatography and capillary electrophoresis methods were thoroughly optimized for high analytical performances. Optimization of multiple variables at the same time by performing a minimum number of experiments was achieved by the Box-Behnken design, which is an experimental design in response surface methodology, in capillary electrophoresis. The results of the experimental design ensure minimum analysis time with well-separated analytes. Separation conditions, such as different stationary phases, pH level, organic modifiers and temperatures in liquid chromatography method, were also optimized. In particular, among stationary phases, the core-shell column especially enhanced the effectiveness of separation in liquid chromatography. Both methods were fully validated and applied to real samples. The main advantage of the developed methods is the separation of the drug combination in a short time with high efficiency and without any time-consuming steps.

Aptamer-based Field-Effect Biosensor for Tenofovir Detection.

During medical treatment it is critical to maintain the circulatory concentration of drugs within their therapeutic range. A novel biosensor is presented in this work to address the lack of a reliable point-of-care drug monitoring system in the market. The biosensor incorporates high selectivity and sensitivity by integrating aptamers as the recognition element and field-effect transistors as the signal transducer. The drug tenofovir was used as a model small molecule. The biointerface of the sensor is a binary self-assembled monolayer of specific thiolated aptamer and 6-mercapto-1-hexanol (MCH), whose ratio was optimized by electrochemical impedance spectroscopy measurements to enhance the sensitivity towards the specific target. Surface plasmon resonance, performed under different buffer conditions, shows optimum specific and little non-specific binding in phosphate buffered saline. The dose-response behavior of the field-effect biosensor presents a linear range between 1 nM and 100 nM of tenofovir and a limit of detection of 1.2 nM. Two non-specific drugs and one non-specific aptamer, tested as stringent control candidates, caused negligible responses. The applications were successfully extended to the detection of the drug in human serum. As demonstrated by impedance measurements, the aptamer-based sensors can be used for real-time drug monitoring.

A novel pretreatment method combining sealing technique with direct injection technique applied for improving biosafety.

AIM: People today have a stronger interest in the risk of biosafety in clinical bioanalysis. A safe, simple, effective method of preparation is needed urgently. METHODOLOGY/RESULTS: To improve biosafety of clinical analysis, we used antiviral drugs of adefovir and tenofovir as model drugs and developed a safe pretreatment method combining sealing technique with direct injection technique. The inter- and intraday precision (RSD %) of the method were <4%, and the extraction recoveries ranged from 99.4 to 100.7%. Meanwhile, the results showed that standard solution could be used to prepare calibration curve instead of spiking plasma, acquiring more accuracy result. CONCLUSION/DISCUSSION: Compared with traditional methods, the novel method not only improved biosecurity of the pretreatment method significantly, but also achieved several advantages including higher precision, favorable sensitivity and satisfactory recovery. With these highly practical and desirable characteristics, the novel method may become a feasible platform in bioanalysis.