Revealing Fine Isotope Structure with Resolution Enhancement Mode on the SELECT SERIES™ MRT

This study demonstrates the benefit of confirmation of putative empirical formulae by interrogation of the fine isotope structure that is provided by high resolving power obtained at chromatographic timescales.

High resolution mass spectrometers (HRMS), are prevalent as screening tools for metabolite identification in drug discovery and development, where the constituents of interest are present in complex biological matrices, including urine and blood. Confident identification of both xenobiotic and endogenous analytes is key to the drug discovery and development pipeline, while accurate mass facilitates putative empirical formulae generation. Confidence in an identification is increased when the constituents of the empirical formula for the compound of interest, are confirmed by their presence in the fine isotope structure, which is observed at high resolving power.

Here we present data obtained using a novel acquisition mode on the SELECT SERIES MRT that enables the flight path to be extended such that a second pass of the multi-reflecting time of flight (MRT) analyzer is enabled, which increases the resolving power by >50% and enables >300,000 FWHM. A non-targeted LC-MS screening experiment using the Resolution Enhancement Mode (REM) was undertaken for the analysis of pharmaceutical drug xenobiotics in the urine of a healthy volunteer. This study demonstrates the benefit of confirmation of putative empirical formulae by interrogation of the fine isotope structure that is provided by high resolving power obtained at chromatographic timescales.

The SELECT SERIES MRT is a hybrid Q-Tof mass spectrometer that employs a multi-reflecting time of flight analyzer.¹ Figure 1 shows the ion path of the MRT operating in a standard single pass mode, the ions enter the analyzer through a orthogonal accelerator and follow a trajectory between lenses P1 and P23 shown in blue, once the ions reach lens P23 they are deflected back in a trajectory towards P1, shown in red, and the detector, resulting in a pathlength of ~47m and a resolving power of >200,000 FWHM, this mode of operation is called multi-reflecting time of flight (MRT) mode. In the MRT mass analyzer ions travel a fixed pathlength from the orthogonal accelerator to the detector, with the observed resolving power, R (being defined as R=pathlength/2x time spread), increasing proportionately with pathlength, this can be achieved by making the analyzer physically longer or by deflecting the ions within the analyzer and enabling them to make multiple passes of the device. The former has practical physical limitations whereas the latter can be achieved by applying a timed pulse to an electrode deflecting the ions within the device.

The analyzer can also be operated with a timed pulse applied to lens P1, deflecting a restricted mass range so as to undergo two passes, increasing the pathlength and hence observed resolving power to >300,000 FWHM. This is shown in Figure 2, the ions enter the analyzer through a orthogonal accelerator and follow a trajectory between lenses P1 and P23 shown in blue (Figure 2A), once the ions reach lens P23 they are deflected back in a trajectory towards P1, shown in red (Figure 2B), a timed pulse is then applied to lens P1 (shown in orange) deflecting the ions back towards P23 (Figure 2C) shown in green, once the ions reach lens P23 they are deflected back again in a trajectory, shown in yellow, towards P1 and finally the detector (Figure 2D), resulting in a pathlength of ~92m and a resolving power of >300,000 FWHM.

The transmittable mass range is a function of the selected low m/z, with the high m/z limit being approximately four times that of the lowest m/z, so for a start mass of m/z 300 the upper m/z would be ~1100 m/z.

Another consideration of the described approach to increase pathlength is duty cycle, this can be maintained through a synchronization of the time-of-flight of an ion packet release between the upstream gas cell and the orthogonal accelerator for the m/z range of interest. This novel mode of operation is called Resolution Enhancement Mode (REM).

Data were acquired in Resolution Enhancement Mode (REM) and the base peak intensity chromatogram for the urine four-hour time point sample is shown in Figure 3A, this is a complex chromatogram with many overlapping features. Extracted mass chromatograms for two co-eluting metabolites at 4.8 minutes, desmethyl naproxen sulfate (m/z 295.03, C13H10O6S) and carbamazepine hydroxy sulfate (m/z 331.04, C15H10N2O5S), are shown in Figures 3B and 3C respectively.

The corresponding mass spectrum for the chromatographic peak at retention time 4.8 minutes is shown in Figure 4, featuring two metabolites at m/z 295.03, corresponding to desmethyl naproxen sulfate and m/z 331.04, corresponding to carbamazepine hydroxy sulfate with an example of the representative resolving power observed at m/z 331.04 of >300.000 FWHM shown in the inset.

The observed mass accuracy for the two metabolites was 542 ppb for desmethyl naproxen sulfate and -423 ppb for carbamazepine hydroxy sulfate. Although ppb levels of mass accuracy give confidence in putative empirical formulae, the observation of fine isotope structure significantly increases the confidence in an identification.

The fine isotope structure for desmethyl naproxen sulfate is shown in Figure 5. Confidence in the identification is increased through the confirmation of the presence of oxygen and sulfur in the formula by the presence of the 16O, 18O, 32S, 33S, and 34S isotopes.

The fine isotope structure for the second metabolite, carbamazepine hydroxy sulfate, is shown in Figure 6. Confidence in the identification is also increased through the confirmation of the presence of nitrogen, oxygen and sulfur in the formula by the presence of the 14N, 15N, 16O, 18O, 32S, 33S, and 34S isotopes.

A novel approach to increasing mass resolution on the SELECT SERIES MRT allows an increased confidence in analyte identification. Multiple examples of fine isotope structure being used to corroborate putative empirical formulae as a result of improved resolving power obtained using Resolution Enhancement Mode (REM) at chromatographic timescales.