A multi-detector platform comprising UV/Vis, charged aerosol, and single quadrupole mass spectrometric detection for comprehensive sample analysis

Importance of the Topic

Comprehensive detection and quantification of unknown and unexpected compounds—such as impurities, degradation products, extractables, and leachables—is essential in pharmaceutical and biopharmaceutical analysis to guarantee product safety and regulatory compliance.

Objectives and Study Overview

This application note presents a UHPLC platform combining UV/Vis, charged aerosol detection (CAD), and single quadrupole mass spectrometry (MS) using heated electrospray ionization (HESI) and atmospheric pressure chemical ionization (APCI). The goal was to demonstrate enhanced peak discovery and reliable quantification of both known and unknown analytes, using cell culture bag extracts as a model.

Methodology and Instrumentation

The study employed a Thermo Scientific Vanquish Flex UHPLC system in two setups: a standard quaternary‐pump configuration and an inverse gradient dual‐pump configuration. Reversed‐phase chromatography was performed on a solid core C18 column with a water–isopropanol gradient. Detection included:

• UV/Vis diode array detector (DAD) for chromophore‐containing compounds
• Charged aerosol detector (CAD) for universal detection of semi‐ and nonvolatile analytes
• Single quadrupole MS with dual HESI/APCI source to confirm identity and improve coverage

Dual ionization modes were applied in separate injections to maximize compound coverage. Calibration standards covered 18 reference extractables, with concentrations ranging from 1 to 50 µg/mL (up to 500 µg/mL for semi‐volatiles).

Main Results and Discussion

• Complementary detection: UV/Vis detected 13 standards, CAD detected 11, and MS confirmed 16 compounds. Some analytes ionized better in APCI, others in HESI.
• Inverse gradient CAD response: Calibration curves overlapped more closely when using the inverse gradient, enabling semi‐uniform response and facilitating single‐standard quantification.
• Cell culture bag analysis: Extracts from four bag types revealed erucamide, stearic acid, and oxidation products of Irgafos 168. CAD quantified unknowns with no available standards, while MS provided mass assignments and UV supported peak purity assessment.

Benefits and Practical Applications

• Enhanced peak detection beyond standard UV‐MS setups by combining CAD and dual MS ionization.
• Uniform quantification of unknowns via CAD with a universal calibrant, eliminating the need for individual standards.
• MS-driven identity confirmation allows tentative identification of unknown extractables based on exact mass.

Future Trends and Potential Applications

• Expansion to high‐resolution MS to improve confidence in unknown identification.
• Integration with automated data analysis workflows for rapid extractable and leachable screening.
• Application to other complex matrices, including environmental and food safety testing.

Conclusion

A triple‐detector UHPLC platform with UV/Vis, CAD, and single quadrupole MS (HESI and APCI) offers comprehensive sample characterization. The inverse gradient approach equalizes CAD response, enabling quantification of unknown compounds with a single calibrant. MS confirms identities, and UV/Vis supports quantitation of chromophoric analytes.

Reference

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4. Menz M., Eggart B., Lovejoy K., Acworth I., Gamache P., Steiner F. Thermo Fisher Scientific Technical Note 72806, 2018.