14
Confirming Peak Identification in Bioanalytical Studies Utilising Xevo TQ MS Product Ion Confirmation
Paul D. Rainville, Joanne Mather, and Robert S. Plumb, Waters Corporation, Milford, MA, U.S. INTRODUCTION
The accurate quantification of candidate pharmaceuticals in bioanalysis relies on the use of sensitive, specific methodology. The advent of atmospheric pressure ionisation 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.
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
is acquired after the apex of the MRM peak and before the return to baseline, Figure 2.
ADVERTORIAL
This MS or MS/MS spectrum
Figure 2. Schematic illustrating PIC data acquisition.
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.
RESULTS
During the development of an LC/MS/MS method for fluticasone propionate, two peaks with the same MRM transitions were detected, Figure 3.
CONCLUSION
Figure 1. The ACQUITY UPLC System with the Xevo TQ MS. EXPERIMENTAL
Rat plasma was spiked with fluticasone propionate, a corticosteroid used for the treatment of asthma. The sample was then prepared by solid phase extraction utilising an Oasis® HLB µElution plate. The sample was analyzed using UltraPerformance LC® mass spectrometry.
(UPLC® ) coupled with tandem
LC conditions LC system: Column:
Column temp.: Flow rate:
Waters ACQUITY UPLC® System
ACQUITY UPLC BEH C18 Column 2.1 x 50 mm, 1.7 µm 45 °C
500 µL/min
Mobile phase A: 0.1% NH4OH Mobile phase B: Gradient:
Methanol 15 to 95 %B/1 min
MS conditions MS system:
Ionization mode:
Waters Xevo TQ MS ESI positive
Capillary voltage: 3000 V Cone voltage:
30 V
Desolvation temp.: 600 °C Source temp.:
150 °C
Desolvation flow: 1000 L/Hr Collision energy: 17 V MRM transition: m/z 501 > 293
Figure 3. Analysis of rat plasma spiked with 100 ng/mL fluticasone propionate.
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.
Figure 4. (A) Product scan comparison of fluticasone propionate standard and (B) Product scan acquired by PIC of analyte peak eluting at 0.85 min.
REFERENCE
1. Twohig M, Fujimoto G, Mather J, Plumb RS. Simultaneous Confirmation and Quantification using Xevo TQ MS: Product Ion Confirmation (PIC). Waters Application Note. 2008; 720002829en.
Circle no. 33
INTERNATIONAL LABMATE - APRIL/MAY 2010 - ADVERTORIAL
Page 1 |
Page 2 |
Page 3 |
Page 4 |
Page 5 |
Page 6 |
Page 7 |
Page 8 |
Page 9 |
Page 10 |
Page 11 |
Page 12 |
Page 13 |
Page 14 |
Page 15 |
Page 16 |
Page 17 |
Page 18 |
Page 19 |
Page 20 |
Page 21 |
Page 22 |
Page 23 |
Page 24 |
Page 25 |
Page 26 |
Page 27 |
Page 28 |
Page 29 |
Page 30 |
Page 31 |
Page 32 |
Page 33 |
Page 34 |
Page 35 |
Page 36 |
Page 37 |
Page 38 |
Page 39 |
Page 40 |
Page 41 |
Page 42 |
Page 43 |
Page 44 |
Page 45 |
Page 46 |
Page 47 |
Page 48 |
Page 49 |
Page 50 |
Page 51 |
Page 52 |
Page 53 |
Page 54 |
Page 55 |
Page 56 |
Page 57 |
Page 58 |
Page 59 |
Page 60 |
Page 61 |
Page 62 |
Page 63 |
Page 64 |
Page 65 |
Page 66 |
Page 67 |
Page 68
