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

Significance of the Topic

The combination antiretroviral therapy of emtricitabine and tenofovir disoproxil fumarate is widely used in HIV treatment. Accurate impurity profiling of these APIs is essential for patient safety and regulatory compliance. Traditional UV detection struggles with varying chromophores and extinction coefficients, requiring individual standards for each impurity. Charged aerosol detection (CAD) offers a nearly uniform response across nonvolatile analytes, potentially enabling single-calibrant quantitation and simplifying quality control workflows.

Objectives and Study Overview

This work aims to demonstrate the use of a ternary gradient reversed-phase UHPLC method coupled with CAD and inverse gradient compensation for the quantitation of tenofovir and related impurities using a single calibrant. Specific goals include:

• Comparing the quantitative accuracy of CAD with and without inverse gradient compensation.
• Evaluating CAD against UV detection for single-calibrant impurity quantitation.
• Investigating the impact of salt formation between charged analytes and mobile phase additives on CAD response.

Methodology

Sample preparation involved dissolving tenofovir disoproxil fumarate, emtricitabine, tenofovir, and adenine in aqueous solution with acetic acid where needed. Calibration standards ranged from 5 to 2000 µg/mL, with quintuplicate injections.

A ternary mobile phase was employed: eluents were A (water with 0.1% acetic acid, pH 3.5), B (methanol), and C (acetonitrile). A dual-pump gradient approach generated both an analytical gradient for separation and an inverse compensation gradient post-column to maintain constant solvent composition at the CAD interface. The analytical gradient achieved baseline separation of polar and hydrophobic impurities within 10 minutes.

Used Instrumentation

• Thermo Scientific™ Vanquish™ Flex Duo UHPLC system configured for inverse gradient
• Charged Aerosol Detector F (CAD) with evaporator at 35 °C, filter 3.6 s, data rate 20 Hz
• Variable Wavelength Detector at 260 nm for comparative UV detection
• Accucore aQ 2.6 µm, 2.1 × 100 mm reversed-phase column
• Thermo Scientific™ Chromeleon™ CDS 7.2.8 for method control and data processing

Main Results and Discussion

Without inverse gradient compensation, CAD response increased with organic content, producing up to 74% RSD among analytes at 30 ng on column. Implementation of inverse gradient normalization reduced variability to ~8% RSD. Further correction for acetate salt formation—assuming one acetate adduct per positively charged analyte at pH 3.5—improved uniformity to ~7.7% RSD. Flow injection studies confirmed that mobile phase acetate increases apparent analyte mass; correcting for this effect equalized response for all test compounds. Compared to UV detection (57% RSD) and CAD without compensation (99% RSD), the combined CAD/inverse gradient/salt correction workflow provided the most accurate single-calibrant impurity quantitation.

Benefits and Practical Applications

• Eliminates the need for individual impurity standards by using a single API calibrant.
• Provides uniform detector response across a wide polarity range of impurities.
• Reduces baseline drift and peak area variability inherent to gradient elution.
• Enables rapid, robust impurity profiling within pharmaceutical QC settings.

Future Trends and Potential Applications

Wider adoption of CAD with gradient compensation is expected in pharmaceutical analysis for multi-component formulations. Future developments may include:

• Integration with mass spectrometric detection for structural elucidation of unknown impurities.
• Use of low molecular weight mobile phase additives (e.g., formic acid) to control salt formation and simplify quantitative corrections.
• Automated method development within chromatographic data systems to optimize inverse gradient parameters.

Conclusion

The ternary reversed-phase UHPLC method with inverse gradient compensation and CAD enables accurate single-calibrant quantitation of tenofovir and related impurities. Salt correction strategies further enhance response uniformity. This approach overcomes limitations of UV detection and reduces reliance on multiple reference standards, streamlining routine impurity profiling in pharmaceutical quality control.

Reference

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 combined tablet dosage form of emtricitabine and tenofovir disoproxil fumarate by ultraviolet spectroscopy. Int Res J Pharm. 2012;3(12):104–108.
3. Menz M, Eggart B, Lovejoy K, Acworth I, Gamache P, Steiner F. Charged aerosol detection — factors affecting uniform analyte response. Thermo Fisher Scientific Technical Note 72806. 2019.
4. Cohen RD, Liu Y, Gong X. Analysis of volatile bases by high performance liquid chromatography with aerosol-based detection. J Chrom A. 2012;1229:172–179.