Quantitation using high-resolution mass spectrometry (HRMS)

Abstract

Objective

To provide an overview of the technical differences between triple quadrupole (QQQ), quadrupole-time of flight (QTOF) and orbitrap mass spectrometers, then to provide a commentary on the quantitative performance of each instrument type, with examples from the author's own laboratory.

Discussion

QQQ instruments are primarily used in multiple reaction monitoring (MRM) mode in a targeted fashion, meaning compounds are only detected if pre-programmed in the acquisition method. QTOF and orbitrap instruments are commonly used in full scan mode and therefore capture a far larger amount of information, with all ionisable compounds capable of being detected, whether they are targeted or not.

QTOF spectra are typically composed of hundreds to thousands of individual ion packets, termed transients. The sensitivity of a QTOF method can be increased by summing a greater number of transients at the expense of scan speed. Orbitrap instruments are not affected by this but the resolution increases as a transient spends a longer time in the orbitrap, which reduces the scan speed.

In an orbitrap instrument, ions are first accumulated in the C-trap before being injected into the orbitrap analyser. To prevent overfilling of the orbitrap, the number of ions injected is controlled at this step. The resulting spectrum is then mathematically adjusted using automatic gain control (AGC) to maintain the quantitative accuracy of the data. AGC can improve the overall dynamic range across different spectra, but it may restrict the simultaneous measurement of very abundant and trace compounds within a single spectrum compared to QTOF instruments.

All three instrument designs commonly offer excellent precision, but modern QQQ instruments may provide superior sensitivity. The aspects of selectivity most relevant to quantitation are the amount of noise, and the frequency and abundance of interfering peaks from the matrix. It has been the experience of Forensic Science SA that extracted ion chromatograms from LC-QTOF data using appropriately narrow mass extraction windows usually show less noise and fewer interfering matrix peaks than MRM data from LC-QQQ methods.

Ease of use is subjective and can often be influenced by the experience of a person or the laboratory. Although HRMS instruments still have a reputation as being difficult to set up and use, at Forensic Science SA, most if not all scientists would consider it easier to set up a quantitative method using an LC-QTOF instrument, than determine and optimise MRMs on an LC-QQQ. Troubleshooting mass spectral issues that can affect quantitative results including co-eluting analyte suppression and formation of adducts, dimers and multiply charged ions is much simpler using full scan HRMS data.

Conclusion

High-resolution mass spectrometers demonstrate excellent quantitative capabilities in addition to their established screening advantages. While modern high-end QQQ instruments generally offer superior sensitivity and potentially wider linear dynamic ranges than current HRMS instruments, HRMS instruments provide comparable precision and often superior selectivity. Although some specific analyses may benefit from QQQ analysis, HRMS instruments prove suitable for most quantitative applications in forensic toxicology.