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

Importance of Comprehensive Multi-Detector Analysis

Ensuring the reliable detection, identification, and quantification of trace impurities, degradation products, and extractables/leachables is critical in pharmaceutical, bioprocessing, and quality control laboratories. Single detectors often overlook analytes lacking chromophores or ionization propensity. A combined platform integrating UV/Vis, charged aerosol (CAD), and single‐quadrupole mass spectrometry (MS) with dual ionization modes (HESI and APCI) provides deeper insight into complex samples and unknown contaminants.

Aims and Overview of the Study

This application study evaluated a Thermo Scientific Vanquish Flex UHPLC coupled to a Vanquish Charged Aerosol Detector and ISQ EC/EM single quadrupole MS. Goals included:

• Demonstrate complementary detection of known and unknown extractables.
• Compare standard versus inverse gradient setups for CAD response uniformity.
• Assess HESI and APCI ionization for broad compound coverage.
• Apply the platform to analyze cell culture bag leachables.

Methodology and Instrumentation

Reversed‐phase chromatography used an Accucore C18 core–shell column (100×2.1 mm, 2.6 µm) with water/formic acid (4 mM, pH 3.1) and isopropanol gradient at 0.5 mL/min. Two pump configurations were tested:

• Standard quaternary pump delivering only analytical gradient.
• Inverse gradient dual‐pump setup delivering a counter‐gradient to normalize solvent composition reaching CAD/MS.

Detectors:

• Diode array UV/Vis (210–345 nm, multi‐wavelength acquisition).
• Charged aerosol detector (35 °C evaporator, 10 Hz).
• ISQ EC/EM single quadrupole MS with HESI and APCI sources (positive/negative full–scan and SIM acquisition).

Samples included 18 reference extractables (phthalides, phenols, parabens, acids, antioxidants) calibrated from 1–50 µg/mL and extracts from four single‐use cell culture bags. Cell bag linings (ethylene vinyl acetate + polyethylene) were extracted with 50:50 isopropanol/water, concentrated, and reconstituted for injection.

Main Results and Discussion

• UV/Vis and CAD detected complementary sets: some analytes lacked chromophores but were well‐detected by CAD; others ionized poorly yet showed strong UV response.
• Combining HESI and APCI improved MS coverage: 16/18 standards detected by both ionization modes; volatile analytes favored HESI or APCI based on protonation/gas‐phase ionization.
• Inverse gradient significantly improved CAD response uniformity across compounds eluting in different solvent strengths, enabling quantification with a single universal calibrant (bisphenol A) for non‐volatile analytes.
• Application to cell bag extracts revealed known leachables (stearic acid, erucamide) plus eleven unknowns. MS provided mass assignments for two additional compounds (oxidized Irgafos 168 and bis(2,4-di-tert-butylphenyl)phosphate).

Benefits and Practical Applications

• Enhanced detection of UV‐silent, non‐ionic, or poorly ionizable impurities.
• Single calibrant quantification via CAD reduces need for reference standards.
• Dual ionization delivers robust ID confirmation of unknowns.
• Inverse gradient minimizes response bias, improving QA/QC throughput and accuracy.

Future Trends and Potential Uses

• Integration with high‐resolution MS for definitive structural elucidation of unknowns.
• Automated workflows combining inverse gradients with machine‐learning-guided peak classification.
• Expansion into lipidomics and polymer additive profiling where diverse chemical properties challenge single detectors.
• Wider adoption in biopharma extractables/leachables risk assessment and regulatory submissions.

Conclusion

The multi‐detector platform coupling UV/Vis, CAD, and single‐quadrupole MS with HESI and APCI delivers comprehensive analysis of complex samples. The inverse gradient approach equalizes CAD response, enabling universal calibrant quantification of unknown impurities. This configuration is highly suited for extractables/leachables studies, impurity profiling, and routine QC when standards are unavailable.

Reference

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