Over the last decade mass spectrometry (MS) has been increasingly implemented in QC laboratories to support e.g. research, quantification, release analysis or the control of LC methods.
Hereby, coupling of LC-UV and MS analysis has been proven particularly useful as it offers an orthogonal way of detection by linking UV responses with their associated molecular masses. Thus, co-elution of non-isobaric compounds can be discovered rapidly by LC-MS and, since the underlying principle is discrimination by mass, MS/MS may be applied for supplementary structural investigations. In turn, because MS is constantly developing into a more widely used standard QC method, software and instrument compliance is another crucial aspect of MS-related analyses that needs the attention of users and vendors.
We developed a semi-automated LC-MS data analysis method to facilitate identification of impurities, that are co-eluting with UV-Peaks of interest and are thus not detectable by UHPLC analysis alone. Additionally, identification and absolute quantification of identified impurities has been accomplished using tandem mass spectrometry and spiking experiments, respectively.
LC-MS data acquisition was performed applying standard parameters and a MS acquisition frequency of 2 Hz. After integration of the UV Peak of interest a custom made DataAnalysis script automatically reads out the extracted ion chromatograms (EIC’s) of all m/z values over a previously defined intensity threshold. All generated EIC’s are subsequently listed by the software and evaluated for congruency. EIC’s that show congruent trends with the main compound are discarded as “artefacts” generated due to e.g. in-source CID, while EIC’s diverging from the main compounds EIC are further analyzed to confirm or rule out the presence of an impurity. We also show that the utilization of a 2D map (m/z vs. retention time) is a very useful tool to facilitate detection of co-eluting compounds, especially when corresponding EIC’s are inconclusive or not precisely generated.
Furthermore, we also want to highlight from a routine QC lab point of view, the needs regarding MS-software usability in a regulated environment. It seems that while the technical advancement of mass spectrometers proceeded rapidly, corresponding software development often lags behind, particularly in terms of usability, and compliance-related functionalities are merely applied “on-top” of existing core software rather than being an integral part of the software development process right from the start. Consequently, we want MS vendors to understand the current bottle necks when performing MS in a GxP setting and where small changes or add-ons would greatly facilitate routine lab processes.