Improving the specificity and selectivity of your assay

Your LC-MS assay is only as good as its power to discern your target compound from everything else. Standards dissolved in clean solvents can make beautiful assays, but analytes in real-world samples can behave quite differently. Coeluting compounds, background interferences, and other matrix effects can all severely degrade assay quality.  What techniques can be used to improve selectivity when required?

Multiple reaction monitoring (MRM) using a triple quadrupole mass spectrometer monitors the fragment ions of an analyte and is the most common method used for targeted analyte detection and quantitation. MRM experiments substantially reduce interferences versus monitoring the intact analyte alone by adding a second level of specificity caused through analyte fragmentation. But sometimes contaminants still appear even with MRM, particularly with more complex samples where coeluting isobaric compounds can interfere with the MRM detection of the targeted analyte. These interferences can potentially be eliminated through additional sample preparation protocols, but often that can add tedious steps to the assay.

What if your triple quadrupole had a hidden talent to provide a third level of specificity? SCIEX QTRAP^® systems are actually triple quadrupole instruments but with added trapping functionality. The third quadrupole can also operate as a linear ion trap. When challenged with assays that include coeluting compounds, MRM³ can be invoked. MRM³ provides a second level of fragmentation for obscured compounds with the detection and quantitation based upon a-fragment-of-a-fragment of the precursor, i.e, a second-generation fragment ion. MRM³ enables quantitation of analytes when high background and interferences make standard MRM difficult. By removing matrix interferences, MRM³ can significantly improve limits of detection (LOD) and quantitation (LOQ). Besides MRM³ capabilities, QTRAP systems can provide full scan high sensitivity MS/MS data for every MRM peak during the acquisition. By capturing MRM data and MS/MS data in one injection, compounds can be quantified and identified simultaneously, thereby confirming the selection of the analyte versus other possible compounds.

Another way to improve selectivity is by improving the resolution of the MRM experiment. To some extent, resolution can be increased on triple quadrupole and QTRAP instruments. But for truly high resolution, MRM^HR can be used on SCIEX QTOF X500 and TripleTOF^® instruments. Typically, high-resolution instruments must sacrifice sensitivity in order to generate fast enough scan speeds to provide good quantitation. Scan speed is important because the LC peak must be sampled a sufficient number of times across its elution profile to ensure good quantitative statistics. With the SCIEX QTOF X500 and TripleTOF systems, MS/MS spectra are collected at high-resolution and fast scan speeds without sacrificing sensitivity. Fragment ions are extracted post-acquisition to generate MRM-like peaks for integration and quantification. MRM^HR can provide better selectivity by reducing the effects of interferences. Because MS/MS data are always acquired, assay development is simplified since fragment ion selection for quantitation is done post-acquisition. Additionally, the full scan MS/MS data acquired across the entire data set provides confirmation for every analyte.

Timing each MRM acquisition to a specific moment in time is another way to enhance specificity. With MRM scheduling, the instrument is set up to acquire data for each analyte only around its LC elution window. This greatly reduces false positives and improves overall quantitative statistics. The SCIEX Scheduled MRM™ Pro Algorithm simplifies the set-up of scheduled MRM acquisition methods and enables many advanced features that enhance overall specificity, accuracy, and reliability. The Scheduled MRM Pro Algorithm is particularly useful for very complex samples enabling the analysis of thousands of MRM transitions in a single injection and can be used on all SCIEX Triple Quadrupole, QTRAP, QTOF, and TripleTOF platforms.

What about LC conditions? UHPLC systems can provide higher resolution and faster separations than traditional HPLC by using higher pressures. SCIEX ExionLC™ UHPLC systems provide ultra-high pressure separations while also providing very high reproducibility and ultralow carryover. Besides pressure, other factors that can be manipulated to optimize analyte retention times and chromatographic resolution include buffers, solvents, columns, and temperatures. However, for complex samples optimization of conditions for one compound can lead to degradations in performance for others. In these situations wouldn’t it be convenient to simply add another level of separation?

SelexION^® differential mobility separation technology device is a differential mobility device used to separate hard to resolve ions based upon their gas-phase mobility. The SelexION device is simple to install and remove by the user without breaking vacuum and can be used in a transparent mode when not required. Using SelexION, coeluting matrix interferences can be overcome, isobaric compounds can be separated, background can be reduced, and data quality can be greatly improved. Additionally, because the SelexION device provides an additional layer of separation and selectivity, sample preparation can be greatly simplified and tedious LC optimization strategies can be minimized.

A last, but often overlooked option is to use a capillary electrophoresis separation strategy rather than an LC-based strategy. Capillary electrophoresis electrospray ionization MS (CESI-MS) can enable high-resolution separation and enhanced sensitivity while reducing ion suppression bias. CESI-MS has proven to be particularly useful for charged and polar metabolites and degradation products (e.g., anionic and/or hydrophilic analytes), difficult to resolve isobaric metabolites and glycans, proteoforms & peptide PTMs such as those with multiple phosphorylation sites, and intact and native protein conformation and interactions. The SCIEX CESI 8000 Plus, OptiMS cartridge, and adapter kit enable seamless CESI-MS analysis with SCIEX mass spectrometers for those challenging samples, while also consuming minimal sample quantities.

When it comes to specificity and selectivity, SCIEX innovative workflows and technologies provide options for your assay. High specificity and selectivity ensure your target compounds are not contaminated by unexpected interferences. SCIEX solutions arise from both the separation process as well as the MS methodology.

Learn more about SCIEX technologies to enhance selectivity and specificity today at https://sciex.com/products/mass-spectrometers.