(W1230-01-06) A Multi-detector Platform Combining Liquid Chromatography with High Resolution Mass Spectrometry, UV and Charged Aerosol Detection for Comprehensive Extractables and Leachables Analysis

Purpose: LC/MS, often hyphenated with UV detection, is commonly used to detect and annotate the non-volatile fraction of compounds in Extractables & Leachables (E&L) studies. Industry guidance and regulatory requirements indicate an analytical evaluation threshold (AET), above which the identification of the compounds in question is necessary for toxicological risk assessment. The use of AET requires quantitation of compounds during the identification process, which is commonly carried out with surrogate standards. However, MS signal intensity can vary significantly between different compounds. UV detection is often used as an alternative, however it requires that the analyte contains a chromophore. To overcome these limitations and lower the uncertainty of the concentration estimate of unknowns, charged aerosol detection (CAD) has been proposed as an orthogonal detector of non-volatile compounds. One benefit of this detector is that when constant mobile phase composition is maintained, the signal response is more universal, thus enabling the surrogate quantitation of unknown compounds based on an internal standard with lower uncertainty than using MS or UV response. Here, we demonstrate the combination of the three detection techniques in one system to allow the concurrent measurement of UV absorption, CAD signal and high resolution-accurate mass (HRAM) mass spectra from one injection. The performance of this system for extractables analysis is evaluated in the analysis of an Extractable & Leachable (E&L) standard mixture. Additionally, the application to the analysis of a sample extract and the workflow for identification of compounds prioritized based on CAD response is highlighted.

Methods: The LC/UV/CAD/HRAM-MS analysis was carried out using a Thermo Scientific Vanquish Duo UHPLC system for inverse gradient with Diode Array and Charged Aerosol detectors coupled to a Thermo Scientific Orbitrap Exploris 120 MS. The mass spectrometry analysis was carried out using Full Scan experiments employing polarity switching at a Resolution setting of 60,000 (@ m/z 200) on the TraceCERT Extractables and Leachables Screening Standard for LC (MilliporeSigma) Untargeted screening experiments on a representative extract prepared from commercially available rubber stoppers for pharmaceutical applications were carried out using single polarity data-dependent MS² (ddMS²) experiments and the Thermo Scientific AcquireX intelligent data acquisition workflow (Iterative precursor exclusion workflow). For qualitative data processing, data were imported into Thermo Scientific™ Compound Discoverer™ 3.3 SP1 software for spectral deconvolution and compound identification.

Results: The UHPLC system used in this study effectively uses two pumps to provide the analytical gradient and  inverse gradient which were combined after the UV detector and then split using a variable flow splitter to allow optimization of the mass-dependent CAD signal without reducing the concentration-dependent ESI signal. An E&L screening standard mixture of 21 compounds was used to demonstrate the performance of the system. With the fast polarity switching HRAM MS data from the Orbitrap Exploris 120 MS, 17 of the 21 compounds could be detected with electrospray ionization (ESI) in either positive or negative mode at 1 µg/mL (with two additional compounds detected at higher concentration). Two compounds, namely 1,3-di-tert-butylbenzene and 2,6-di-tert-butyl-4-methylphenol, were not detected in this analysis, however both are expected to show better response in GC/MS experiments. 9 compounds carrying chromophores such as aromatic or conjugated double bonds were detectable by UV at this concentration, while 11 compounds were detectable by CAD. Those compounds not detectable at 1 µg/mL with CAD had lower molecular weights, indicating their higher volatility. Notably, of the 19 compounds detected in this analysis, 15 were detectable by at least two detectors. MS sensitivity was generally higher than UV or CAD, with the latter having detection limits in the range of 0.1-1 µg/mL. Comparing the relative response of the compounds, MS signal showed the largest variance in response across the different compounds, while response from CAD was significantly more uniform, as indicated by lower standard deviations. To demonstrate the application of the multidetector system to the analysis of an extractables solution, it was used to analyze the isopropanol extract of pharmaceutical-grade rubber stoppers. Compounds that showed detectable CAD peaks above the background level in the rubber stopper extract could be prioritized for annotation. In the example of a CAD peak at RT 19.38 min, the MS data revealed two co-eluting compounds with monoisotopic MW 338.3344 Da and 227.1885 Da. Based on the Full Scan and fragmentation data match to the mzCloud spectral library, the former compound could be identified as Erucamide with high confidence.

Conclusion: UV, CAD and HRAM MS provide orthogonal detection based on different properties, which increases compound coverage and annotation confidence. The universal response of the CAD detector, when used with the Vanquish Duo for inverse gradient system, enables more confident semi-quantitation of unknown compounds against surrogate standards, aiding in determining which compounds need to be identified in the context of Extractables studies.

Multi-detector platform combining liquid chromatography with high resolution mass spectrometry, UV and charged aerosol detection for comprehensive extractables and leachables analysis