42 February / March 2019
Figure 2 – Non-ionizing Peak
pH, complicate method development. Utilizing MS detection to identify and track these problematic sample compounds would therefore greatly improve method development effectiveness, resulting in increased performance of, and confidence in, the final method. Though there are a number of MS instruments available to assist with identification of products and impurities, it has long been the desire of the chromatographer to combine the confirmatory power of the MS with familiar chromatography data systems and UV detection typically used in method development. The recent introduction of the Acquity QDa, a miniaturized mass spectrometric detector, coupled with the Empower Chromatography Data Software (CDS) by Waters Corporation (Milford, MA.) has made it both simple and economical to integrate MS data into method development.
To support QbD method development, screening and optimization experiments are conducted that focus primarily on the factors most likely to affect separation – for example factors such as column chemistry, solvent composition, pH, column temperature, and gradient slope in reversed phase chromatography. Statistically designed experiments provide an extremely efficient sampling of a multi-factor experimental
region relative to a brute force all-possible- combinations approach, and also provide much more knowledge-based data than the incremental trial-and-error approach. Designed experiments have the added benefit of enabling the user to study important parameters in combination so that their interactive influences on method performance can be characterized and visualized.
The most valuable and useful knowledge obtainable from method development experimental results is the exact nature of the combined factor effects on the retention and peak shape of each sample molecule as the factor level setting combinations are varied across the experimental region. Extracting this knowledge normally requires the ability to unambiguously locate and track the shape and retention changes of each sample peak across the experiment data set – in other words, peak tracking. Tracking each peak in each experiment chromatogram is a normally a manual and challenging effort, even with the facilitated tools available in chromatography data software. Peak tracking becomes even more complex when two or more peaks co-elute or change elution order between experiment runs.
The limitations of peak tracking using Photodiode Array (PDA) spectral data
are well known [5]. Manually tracking peaks without the benefit of confirmatory mass data requires spiking experiments, or carefully controlled sample mixtures that enable the determination of simple migrations or co-elution to be observed via area or peak height responses. Utilizing mass data from an integrated mass spectrometer greatly facilitates peak identification, but to date has required additional manual manipulation of increased amounts of data.
To alleviate this problem, S-Matrix has developed PeakTracker™ – a powerful new UV/MS peak tracking technology to automate, optimize, and simplify the use of PDA and MS data in LC and LC/MS method development. Fully integrated into S-Matrix’s Fusion QbD software for LC and LC/MS method development, PeakTracker uses 3D PDA spectral data augmented with standard UV peak results data to automatically identify each peak in each experiment chromatogram. PeakTracker also automatically utilizes 3D mass spectral data for experiments run on LC systems configured with the Waters Acquity QDa Mass Detector (QDa). Complex separation and tracking challenges PeakTracker can automatically address include:
• Auto-deconvolution of partially and completely co-eluted peaks.
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