Determination of enantiomeric impurity of tenofovir disoproxil fumarate on a cellulose tris(3,5‐dichlorophenyl‐carbamate) chiral stationary phase and the characterization of its related substances

Abstract

A simple and reliable high-performance liquid chromatography method was developed to determine the enantiomeric impurity of tenofovir disoproxil fumarate, an orally bioavailable prodrug of tenofovir, commonly used for the treatment of human immunodeficiency virus and hepatitis B. Tenofovir disoproxil and its enantiomer, were completely separated on a Chiralpak IC column (3 μm, 100 × 4.6 mm, i.d.). The chiral separation was achieved using a mobile phase containing n-hexane, ethanol, methanol, and triethylamine 65/25/10/0.1 (v/v/v/v) at a flow rate of 0.6 mL/min. Ideally, the reversal of enantiomer elution order was achieved on the Chiralpak IC column, to allow the elution of the minor enantiomeric impurity before the major component. Moreover, the proposed method was able to discriminate the active ingredient from the related substances available in the tenofovir disoproxil fumarate raw materials. These compounds were isolated and structurally elucidated by MS and nuclear magnetic resonance. Based on the spectral data, the structures of related substances were confirmed as tenofovir isoproxil monoester and fumaric acid.The high-performance liquid chromatography method was optimized by the design of experiment approach and successfully validated following the International Conference on Harmonization guideline. Proposed method was effectively applied for the quantification of enantiomeric impurity in tenofovir disoproxil fumarate raw materials.

Study on Quality Control of Tenofovir Disoproxil Fumarate Enantiomers by High-Performance Liquid Chromatography–Mass Spectrometry

“…Tenofovir diphosphate competitively binds itself to the natural substrate 5ʹ-deoxyadenosine triphosphate and enters the deoxyribonucleic acid (DNA) to inhibit the activity of the reverse transcriptase of human immunodeficiency virus (HIV) and hepatitis B virus (HBV). Thus, the lengthening of DNA strand and viral replication will be stopped, providing treatment effect against HIV and HBV [1][2][3][4][5]. The hepatoprotective mechanism of TDF is shown in Fig.…”