Ternary Gradient for Tenofovir Disoproxil Fumarate Impurity Profiling

Significance of the Topic

The accurate profiling of organic impurities in tenofovir disoproxil fumarate (TDF) is essential for ensuring the safety, efficacy, and regulatory compliance of antiretroviral therapies.
The simultaneous separation of highly polar and hydrophobic impurities poses analytical challenges, driving the need for robust, sensitive, and rapid methods.

Objectives and Study Overview

This study aimed to adapt a USP pending monograph impurity profiling method for TDF into a mass spectrometry–compatible, faster, and more reproducible UHPLC assay.
The focus was on achieving baseline resolution between early-eluting polar analytes (adenine, tenofovir) and later-eluting nonpolar compounds, while simplifying mobile-phase preparation and reducing run time.

Methodology and Instrumentation

Chromatographic method parameters were optimized using a ternary gradient on a fused-core Accucore aQ column (2.1×100 mm, 2.6 µm).
Mobile phases:

• Solvent A: 25 mM ammonium acetate buffer (pH 3.8)
• Solvent B: Methanol
• Solvent C: Acetonitrile

Gradient program:

• 0–4.0 min: B 0→70%, C 0→15%
• 4.0–4.5 min: B 70%, C 15%
• 4.5–5.0 min: B 70→25%, C 15→70%
• 5.0–6.0 min: B 25%, C 70%
• 6.0–6.1 min: B 25→0%, C 70→0%
• 6.1–15 min: re-equilibration at initial conditions

Other conditions:

• Flow rate: 0.6 mL/min
• Column temperature: 40 °C with active pre-heater
• Injection volume: 1 µL
• Detection: Diode array at 260 nm, 100 Hz data rate

Used Instrumentation

Vanquish Flex UHPLC System consisting of:

• System Base
• Quaternary Pump F
• Split Sampler FT with active pre-heater
• Column Compartment H
• Diode Array Detector HL with LightPipe flow cell

Data acquisition and processing were performed with Chromeleon CDS version 7.2 SR3.

Main Results and Discussion

The ternary gradient enabled a resolution greater than 2.0 between adenine and tenofovir within a total run time under 6 minutes.
This performance surpassed binary gradients and traditional longer methods.

Robustness assessment over 60 consecutive injections (15 h) yielded:

• Retention time RSD: 0.03%
• Peak area RSD: 0.3%

Such precision greatly exceeded the USP pending monograph requirements (≤5–10% RSD) and demonstrated stable performance for high-throughput QC environments.

Benefits and Practical Applications of the Method

• Rapid impurity profiling with run times reduced by over 80% compared to literature methods.
• Simplified mobile-phase preparation using volatile, MS-compatible solvents free of salt precipitation issues.
• High resolution of critical polar impurities and broad applicability to hydrophobic compounds in a single analysis.
• Exceptional method robustness supporting reliable routine operation and data integrity.

Future Trends and Opportunities

Further integration with mass spectrometric detection will enhance sensitivity and structural confirmation of trace impurities.
Advances may include green solvent systems, automated method optimization, and miniaturized columns for even faster separations.
The approach can be extended to other nucleotide analogue drugs and complex biopharmaceutical formulations.

Conclusion

The developed ternary gradient UHPLC method on the Vanquish Flex system with an Accucore aQ column provides a fast, reliable, and MS-compatible approach for comprehensive impurity profiling of tenofovir disoproxil fumarate.
Its exceptional resolution and robustness make it well suited for pharmaceutical QC laboratories and method transfer to regulated environments.

References

1. USP Pending Monograph for Tenofovir Disoproxil Fumarate, Version 1.
2. Thermo Fisher Scientific. Vanquish UHPLC System Overview.
3. Thermo Fisher Scientific. Accucore aQ C18 Polar Endcapped LC Column.