Ternary Gradient for Tenofovir Disoproxil Fumarate Impurity Profiling

Significance of the Topic

The analysis of tenofovir disoproxil fumarate and its organic impurities is critical in pharmaceutical quality control to ensure drug purity and patient safety. Rapid and robust separation of both polar and nonpolar compounds supports regulatory compliance and enhances throughput in research and production laboratories.

Study Objectives and Overview

This study establishes a fast impurity profiling method for tenofovir disoproxil fumarate (TDF) using a ternary gradient on a Vanquish Flex UHPLC system with an Accucore aQ column. Objectives include achieving clear separation of early eluting polar analytes adenine and tenofovir, reducing analysis time, and ensuring mass spectrometry compatibility of eluents.

Methodology and Instrumentation

Chromatographic Method

• Column: Accucore aQ 2.6 μm, 2.1 × 100 mm
• Mobile Phases: A 25 mM ammonium acetate buffer pH 3.8, B methanol, C acetonitrile
• Ternary Gradient: 0–4.0 min from 100% A to 70% B and 15% C; 4.5–5 min to 25% B and 70% C; reequilibration to initial conditions by 6.1–15 min
• Flow Rate: 0.6 mL/min; Column Temperature: 40 °C; Injection Volume: 1 μL; Detection at 260 nm

Instrumentation

• Vanquish Flex UHPLC with quaternary pump and active pre-heater
• Split Sampler FT, Column Compartment H, Diode Array Detector HL with LightPipe flow cell
• Data acquired and processed with Chromeleon CDS software version 7.2 SR3

Results and Discussion

The optimized ternary gradient achieved resolution greater than 2 between adenine and tenofovir, surpassing the USP requirement of 1.5. Hydrophobic compounds including emtricitabine and TDF impurities eluted within a 6-minute window. Over 60 consecutive injections spanning 15 hours, tenofovir disoproxil fumarate exhibited retention time RSD of 0.03% and peak area RSD of 0.3%, demonstrating excellent method robustness and precision.

Benefits and Practical Applications

This method simplifies eluent preparation by using an MS-compatible buffer and two organic solvents mixed in-line, eliminating salt precipitation and handling issues of tertiary butyl alcohol. The shortened run time and consistent retention behavior improve sample throughput and reliability in routine quality control and impurity screening.

Future Trends and Opportunities

Ongoing developments in fused-core column technology and UHPLC instrumentation will further reduce analysis times and increase sensitivity. Coupling with high-resolution mass spectrometry may allow deeper impurity characterization. Automation of buffer preparation and closed-loop monitoring of system performance will drive efficiency and reproducibility in pharmaceutical analytics.

Conclusion

A ternary gradient method on the Vanquish Flex UHPLC system paired with an Accucore aQ column delivers a rapid, robust, and MS-compatible impurity profiling approach for tenofovir disoproxil fumarate. It meets USP resolution criteria, shows outstanding precision over extended operation, and supports high-throughput pharmaceutical testing.

Reference

1 Authorized USP Pending Monograph for Tenofovir Disoproxil Fumarate Version 1 accessed May 2015
2 Thermo Fisher Scientific Vanquish UHPLC System Overview accessed May 2015
3 Thermo Fisher Scientific Accucore aQ C18 Polar Endcapped LC Column Details accessed May 2015