Development and validation of an LC-MS/MS assay for tenofovir and tenofovir alafenamide in human plasma and cerebrospinal fluid.

A liquid chromatography with triple quadrupole mass spectrometry method was developed and validated for the determination of tenofovir and tenofovir alafenamide concentrations in human plasma and cerebrospinal fluid. Tenofovir and tenofovir alafenamide were extracted from matrix by solid phase extraction. The dried extraction eluents were dissolved in water for LC-MS/MS analysis. Separation was achieved with a Phenomenex Synergi 4 µm Polar-RP 80A column (50 × 2 mm) with a gradient elution of 0.1% formic acid in water and acetonitrile. The total run time was 5 min. Detection of analytes was achieved using electrospray ionization (positive mode) and triple quadrupole selected reaction monitoring. Standard curve concentrations ranged from 0.5 to 500 ng/mL for the plasma assay and 0.1-50 ng/mL for the cerebrospinal fluid assay. The intra- and inter-day accuracy and precision were less than 12% in low, medium, and high quality control samples for both matrices. The validated methods were applied to the analysis of plasma and cerebrospinal fluid samples of a patient undergoing tenofovir therapy which involved the switch from Stribild® (elvitegravir 150 mg/cobicistat 150 mg/emtricitabine 200 mg/tenofovir disoproxil fumarate 300 mg) to Genvoya® (elvitegravir 150 mg/cobicistat 150 mg/emtricitabine 200 mg/tenofovir alafenamide 10 mg).

A Validated Stability Indicating RP-HPLC Method for the Determination of Emtricitabine, Tenofovir Disoproxil Fumarate, Elvitegravir and Cobicistat in Pharmaceutical Dosage Form.

A new simple, rapid stability indicating assay method has been developed and validated for the determination of emtricitabine, tenofovir disoproxil fumarate, elvitegravir and cobicistat using reverse-phase high-performance liquid chromatography in their pharmaceutical dosage form. The chromatographic separation was performed on an ODS column (250 × 4.6 mm, 5 µm) using mobile phase A (potassium dihydrogen orthophosphate, pH adjusted to 2.5) and mobile phase B (acetonitrile) in the ratio of 55:45% v/v at a flow rate of 1 mL/min. The analytes were detected at 250 nm. The method was found to be linear in the concentration range of 2-12 µg/mL for EMT, 3-18 µg/mL for TNDF, 1.5-9 µg/mL for ELV and COB, with the coefficient value (R(2)) of >0.9990. The accuracy was measured via recovery studies and found to be acceptable, and the percentage recoveries were found in the range of 99.93-100.08 ± 0.5%. Forced degradation studies were also conducted, and the drugs were subjected to various stress conditions such as acid hydrolysis, base hydrolysis, oxidative, photolytic and thermal degradation. The proposed method was successfully validated and applied for the quantitative estimation of these drugs in both bulk and tablet dosage forms.