Achieve confident impurity detection with the Thermo Scientific ISQ EC single quadrupole mass spectrometer

Importance of the Topic

Impurity profiling of small molecule pharmaceuticals is crucial to ensure safety and efficacy during drug development and quality control. Regulatory guidelines from the International Council for Harmonisation (ICH Q3B) require characterization and monitoring of impurities above defined thresholds, typically between 0.1 % and 1.0 % of the active dose. Conventional UV detection is widely used in routine QC but often lacks specificity and sensitivity when compared with mass spectrometric methods.

Study Objectives and Overview

This study demonstrates the capability of the Thermo Scientific ISQ EC single quadrupole mass spectrometer for quantitative impurity analysis in pharmaceutical workflows. Tenofovir disoproxil fumarate, an anti-retroviral API, and its two known impurities (adenine and tenofovir) were selected to showcase targeted quantification and peak purity assessment. Key goals included comparing UV and MS detection limits, validating method suitability against USP requirements, and detecting unexpected degradation products.

Methodology

Sample preparation involved dilution of standard solutions of adenine, tenofovir, emtricitabine, and tenofovir disoproxil in aqueous methanol. Calibration standards ranged from 10 ppb to 10 ppm. A ternary gradient UHPLC separation on an Accucore aQ column (2.1 × 100 mm, 2.6 μm) employed water, methanol, and acetonitrile each with 0.1 % acetic acid. Gradient conditions were optimized for baseline separation of impurities and APIs within a 6-minute run time. Quantification used Serial Ion Monitoring (SIM) windows for individual compounds, while full scan (120–600 m/z) data supported peak purity and impurity screening.

Instrumentation Used

• Thermo Scientific Vanquish Flex Quaternary UHPLC system (pump, autosampler, column compartment, variable wavelength detector)
• Thermo Scientific ISQ EC single quadrupole mass spectrometer with orthogonal heated electrospray source
• Chromeleon 7.2 chromatography data system for instrument control and data analysis

Main Results and Discussion

System suitability testing achieved USP criteria: tenofovir disoproxil peak tailing factor ≤ 2.0 (observed 1.7, 4 % RSD) and resolution between adenine and tenofovir ≥ 1.5 (observed 4.0). MS detection limits outperformed UV by up to three orders of magnitude. In SIM mode, adenine at 1 pg on column yielded S/N ~10 and tenofovir at 10 pg yielded S/N ~7, implying practical detection limits in the low picogram range. Calibration curves showed excellent linearity (R2 > 0.999) over the relevant range. Quantification of impurities at levels down to 0.01 % delivered recovery within 90–115 % and precision better than 10 % RSD. Full scan data confirmed peak purity of tenofovir disoproxil, revealing only [M+H]+ and [M+Na]+ with no co-eluting species. Untargeted full scan screening detected three additional unknown impurities, which were identified by accurate mass as tenofovir isoproxil monoester, tenofovir methyl isoproxil, and tenofovir isopropyl isoproxil, each within 0.1 amu of theoretical mass.

Benefits and Practical Applications

• Enhanced sensitivity for trace-level impurity quantification compared with UV detection
• Rapid targeted analysis via timed SIM for routine QC assays
• Simultaneous peak purity assessment and untargeted impurity screening using full scan data
• Robust, integrated workflow with Chromeleon CDS for streamlined method setup and automated data processing

Future Trends and Opportunities

Advances in single quadrupole MS sensitivity and software-driven workflows will further simplify impurity profiling in regulated environments. Future developments may include higher multiplexing of SIM windows, integration of AI-based data review for automated impurity flagging, miniaturized LC-MS platforms for on-site QC, and cloud-enabled collaboration for global quality monitoring.

Conclusion

The Thermo Scientific ISQ EC single quadrupole mass spectrometer demonstrates reliable, sensitive, and specific quantitative impurity analysis of pharmaceutical APIs. Combining SIM-based targeted quantification with full scan peak purity and screening provides robust compliance with regulatory requirements and supports efficient QC workflows.

References

• ICH Harmonised Tripartite Guideline Q3B(R2): Impurities in New Drug Products, June 2006
• United States Pharmacopeia Pending Monograph 1 for Tenofovir Disoproxil Fumarate
• Thermo Scientific Application Note 1129: Ternary Gradient for Tenofovir Disoproxil Fumarate Impurity Profiling, 2016