Confirming Peak Identification in Bioanalytical Studies Utilizing Xevo TQ MS Product Ion Confirmation

The accurate quantification of candidate pharmaceuticals in bioanalysis relies on the use of sensitive, specific methodology. The advent of atmospheric pressure ionization sources, in the late 1980s, allowed the simple interfacing of liquid chromatography with tandem quadrupole mass spectrometry. The selectivity and sensitivity provided by LC/MS/MS has made it the technique of choice for bioanalytical studies.

Bioanalytical scientists have relied on the specificity and sensitivity of the multiple reaction monitoring (MRM) MS acquisition mode to ensure that the correct analyte is quantified. However, even with the specificity of MRM analysis and modern sample preparation techniques, such as solid phase extraction, extraneous peaks can interfere during sample analysis. The appearance of an extra peak during MRM analysis could be due to chromatographic peak  splitting, associated drug metabolites, or matrix interferences.

The occurrence of extra peaks during analysis would require additional experiments to correctly identify the analyte of interest. With a conventional tandem quadrupole mass spectrometer, this would require separate analysis to acquire full scan MS or full scan MS/MS spectra to confirm the identity of the peaks in question. With the introduction of the Waters® Xevo™ TQ MS, both MRM and full scan MS or product scan MS/MS can now be acquired during a single injection.

Rat plasma was spiked with fluticasone propionate, a corticosteroid used for the treatment of asthma. The sample was then prepared by solid phase extraction utilizing an Oasis® HLB µElution plate. The sample was analyzed using UltraPerformance LC® (UPLC®) coupled with tandem mass spectrometry.

Product Ion Confirmation (PIC)

The novel collision cell design of the Xevo TQ MS allows for simultaneous acquisition of MRM and full scan MS data. Product Ion Confirmation (PIC) takes advantage of this capability to collect a high-quality full scan MS or MS/MS spectrum during MRM acquisition.¹ This MS or MS/MS spectrum is acquired after the apex of the MRM peak and before the return to baseline, Figure 2.

Furthermore, the fast data capture rate of the Xevo TQ MS allows for multiple full scan MS or MS/MS spectra to be acquired across the narrow, 2- to 3-second-wide chromatographic peaks that are typically generated during analysis when coupled with UPLC.

During the development of an LC/MS/MS method for fluticasone propionate, two peaks with the same MRM transitions were detected, Figure 3.

The product ion scan spectra simultaneously generated by the PIC function during analysis is displayed in Figure 3. Spectra (A) is that obtained from the peak eluting with a retention time of 0.69 minutes. Spectra (B) is that obtained from the peak eluting with a retention time of 0.85 minutes.

Each of the product ion scan spectra generated from the analysis were then compared to the product ion scan spectra obtained during sample tuning of the fluticasone propionate standard, Figure 4.

Here we can see that comparison of the two spectra show good agreement, confirming that the peak with the retention time of 0.85 minutes is the analyte of interest, fluticasone propionate. Thus, in one analytical run, we were able to identify the peak at 0.85 minutes as the analyte of interest without the need for further analysis.

Multiple peaks with the same MRM transition can be observed during method development or routine analysis in bioanalytical studies. These spurious peaks can be generated by chromatographic peak splitting, metabolites, or matrix interferences. Product Ion Confirmation (PIC) functionality enables the confirmation of peak identity during MRM quantitative analysis by obtaining a quality product scan spectra during MRM MS analysis. The ability of the Xevo TQ MS to perform this function results in the reduction of further confirmatory experiments, saving time and money, which ultimately increases laboratory productivity. Furthermore, this function can be carried out with the narrow, 2- to 3-second-wide chromatographic peaks generated by UPLC.

1. Twohig M, Fujimoto G, Mather J, Plumb RS. Simultaneous Confirmation and Quantification using Xevo TQ MS: Product Ion Confirmation (PIC). Waters Application Note. 720002829, 2008.