Quantitation of tenofovir and impurities in multi-component drug products by ternary gradient reversed-phase chromatography with charged aerosol detection

Significance of the Topic

Reversed-phase impurity profiling of multi-component antiretroviral drugs such as tenofovir disoproxil and emtricitabine is challenged by diverse polar and hydrophobic impurities and differing chromophores. Charged aerosol detection (CAD) offers near-uniform response across nonvolatile analytes, enabling single-calibrant quantitation without individual impurity standards. However, gradient elution and salt formation with mobile phase additives can distort CAD response.

Goals and Overview of the Study

This study demonstrates the application of a ternary reversed-phase gradient combined with an inverse compensatory gradient and CAD to quantify tenofovir, emtricitabine, and related impurities. Key objectives include comparing CAD versus UV detection, evaluating inverse gradient compensation for response uniformity, and proposing a simple salt-formation correction for single-calibrant quantitation.

Methodology

• Sample Preparation: API and impurity standards at 1.0 mg/mL in water; adenine at 0.1 mg/mL in 0.1% acetic acid. Calibration standards for tenofovir disoproxil from 2000 to 5 µg/mL; other analytes from 50 to 5 µg/mL. Quintuplicate injections.
• Mobile Phases: A = water + 0.1% acetic acid (pH 3.5), B = methanol, C = acetonitrile; weekly solvent refresh to minimize background.
• Chromatographic Conditions: Accucore aQ column (2.1×100 mm, 2.6 µm) at 40 °C; flow rate 0.6 mL/min; ternary gradient over 15 min to separate adenine, tenofovir, emtricitabine, and TD impurities.

Used Instrumentation

• UHPLC System: Thermo Scientific Vanquish Flex Duo with dual pumps for analytical and inverse gradients.
• Detectors: Vanquish Charged Aerosol Detector (CAD) and UV detector at 260 nm.
• Data System: Chromeleon CDS v7.2.8.

Main Results and Discussion

CAD provided uniform response enabling single-calibrant quantitation of all nonvolatile analytes. Without inverse gradient compensation, late-eluting analytes showed up to 74% RSD in response. Inverse gradient compensation normalized solvent composition at the detector, reducing RSD to 8.1%. A simple mass correction accounting for one acetate adduct per analyte further improved uniformity to 7.7% RSD. UV detection and uncompensated CAD showed up to 57% and 99% RSD, respectively. Flow injection experiments confirmed that acetate in mobile phase increases analyte response proportionally to its molar mass, supporting the salt-formation correction strategy.

Benefits and Practical Applications

• Enables accurate impurity quantitation in pharmaceutical QA/QC and R&D without individual impurity standards.
• Streamlines multi-component drug analysis through single-calibrant methodology.
• Improves robustness and reduces baseline drift via inverse gradient compensation.

Future Trends and Opportunities

Further work may extend this approach to other charged analyte mixtures, evaluate alternative counterions (e.g., formate, TFA) for optimized salt-formation control, and integrate fully automated inverse gradient workflows in routine pharmaceutical analysis.

Conclusion

Combining CAD with a dual-pump ternary/inverse gradient and simple salt-formation correction offers a robust, accurate single-calibrant method for impurity profiling of multi-component drug products, outperforming traditional UV detection.

References

1. Fabel S. Ternary gradient for tenofovir disoproxil fumarate impurity profiling. Thermo Fisher Scientific Application Note 1129.
2. Iavanya B. et al. Method development and validation of emtricitabine and tenofovir disoproxil fumarate by UV spectroscopy. Int. Res. J. Pharm. 2012;3(12):104–108.
3. Menz M., Eggart B., Lovejoy K. et al. Charged aerosol detection—factors affecting uniform analyte response. Thermo Fisher Scientific Technical Note 72806.
4. Cohen RD., Liu Y., Gong X. Analysis of volatile bases by HPLC with aerosol-based detection. J. Chrom. A. 2012;1229:172–179.