A Multi-Detector Set-up Comprising UV/Vis Detection, Charged Aerosol Detection and Single Quadrupole Mass Spectrometric Detection for Comprehensive Quantitative Sample Analysis

Importance of the Topic

Comprehensive sample analysis is critical in analytical chemistry for detecting and quantifying both known and unknown contaminants, degradation products, or extractables from laboratory consumables. Combining complementary detection techniques enhances confidence in assigning compound identities and improves quantitation accuracy, especially when reference standards are unavailable.

Aims and Study Overview

This work evaluates a multi-detector platform integrating UV/Vis, charged aerosol detection (CAD), and single quadrupole mass spectrometry (MS) to achieve comprehensive quantitative analysis of extractables from single-use cell culture bag linings. The study compares a standard gradient set-up with an inverse gradient configuration, assesses detector complementarity, and demonstrates quantification strategies for unknowns using a universal calibrant.

Methodology and Instrumentation

• Chromatography: Thermo Scientific Vanquish Flex UHPLC system in two configurations—single-pump analytical gradient and dual-pump inverse gradient.
• Column: Thermo Scientific Accucore C18, 100 × 2.1 mm, 2.6 µm, operated at 45 °C.
• Detectors: Vanquish Diode Array Detector (UV/Vis) set at multiple wavelengths; Charged Aerosol Detector with 35 °C evaporator; ISQ EC/EM single quadrupole MS with alternating HESI and APCI sources, performing full scans and SIM.
• Gradient Programs: Standard gradient from 5 % to 100 % organic; inverse gradient for CAD and MS to maintain constant post-column composition, improving CAD uniformity.
• Samples: 18 reference standards (1–50 µg/mL, adjusted for semivolatiles) and extracts of ethylene-vinyl acetate/polyethylene bag lining prepared in 50:50 isopropanol/water.

Main Results and Discussion

• Detection: UV/Vis detected 13 of 18 standards; CAD detected 11; MS confirmed identities for 16 via HESI and APCI. Combining detectors ensured all standards were observed.
• Inverse Gradient Benefits: CAD calibration curves overlapped more closely, enabling use of a single universal calibrant (bisphenol A) to quantify unknown and known compounds, except for low-response semivolatiles.
• Cell Culture Bag Extracts: 18 known extractables and 19 unknowns quantified. High levels of UV-invisible slip agent erucamide and derivatives of Irgafos 168 were identified. CAD provided quantitation for compounds without chromophores; MS delivered identity confirmation and tentative assignments for unknowns.

Benefits and Practical Applications

• Enhanced Coverage: Combined detection maximizes analyte coverage across a range of volatilities and chemistries.
• Quantification of Unknowns: Universal calibration with CAD and inverse gradient allows quantification without individual standards.
• Identity Confirmation: Single quadrupole MS with both ionization modes supports robust confirmation of both known and unknown compounds.

Future Trends and Opportunities

• Advanced Detectors: Integration of high-resolution MS or additional universal detectors to improve identification of trace unknowns.
• Automated Workflows: Implementation of real-time data processing and AI-driven identification for faster screening in QA/QC.
• Broader Applications: Extension of inverse gradient multi-detector strategies to other complex matrices such as biopharmaceutical formulations and environmental samples.

Conclusion

This study demonstrates that a multi-detector HPLC platform combining UV/Vis, CAD, and single quadrupole MS, particularly with an inverse gradient configuration, delivers comprehensive quantitative analysis of extractables from single-use cell culture bags. The approach enhances detectability, enables quantitation of unknowns using a universal calibrant, and leverages MS for identity confirmation, offering a robust workflow for complex sample characterization.

References

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4. Menz M, Eggart B, Lovejoy K, Acworth I, Gamache P, Steiner F. Charged aerosol detection—factors affecting uniform analyte response. Thermo Fisher Technical Note 72806; Oct 2018.
5. Meding S, Lovejoy K, Swart R, Steiner F, Ruehl M. A multi-detector set-up comprising UV/Vis, CAD and single quadrupole MS for comprehensive quantitative sample analysis. Thermo Fisher Application Note 72869; Dec 2018.