Xiaolei Xie (Presenter)
Thermo Fisher Scientific
Bio: Dr. Xiaolei Xie is a scientist at Thermo Fisher Scientific. He focuses on the application development for clinical research and forensic toxicology laboratories using LC-MS technology. Prior to Thermo, he has been working for Caprion Proteomcis and specialized on proteomics based biomarker discovery and verification using mass spectrometry. Dr. Xie got his Ph.D. degree on Biochemistry from Peking University, Beijing followed by postdoctoral research at the University of Michigan, Ann Arbor.
Authorship: Xiaolei Xie, Mindy Gao, Marta Kozak
Thermo Fisher Scientific, San Jose, CA
| Short Abstract Selected reaction monitoring (SRM) has emerged as the MS “gold-standard” for targeted quantification. An alternative quantitative method is parallel reaction monitoring (PRM). In PRM, the third quadruple of a triple quadrupole is substituted with a high resolution mass analyzer to permit parallel detection of all product ions in one high resolution mass analysis. Here, we evaluate the analytical performance of the PRM method and draw a comparison to SRM. We demonstrated comparable quantitative performance between PRM and SRM in terms of run-to-run reproducibility, matrix effects, and measurement accuracy using epi-vitamin D quantitation in plasma and barbiturate measurement in urine. |
Long Abstract
Introduction:
Selected reaction monitoring (SRM), primarily performed on triple quadrupole (QqQ) mass spectrometers (MS), has emerged as the MS “gold-standard” for targeted quantification in clinical research and forensic toxicology applications. With the development of the hybrid quadrupole-Orbitrap MS instruments, an alternative quantitative method is parallel reaction monitoring (PRM). In PRM, the third quadruple of a triple quadrupole is essentially substituted with a high resolution accurate mass Orbitrap mass analyzer to permit the parallel detection of all target product ions in one, concerted high resolution mass analysis.
Here, we evaluate the analytical performance of the PRM method and draw a performance comparison to SRM in terms of run-to-run reproducibility, matrix effects, and measurement accuracy. We assess the two methods performance in the context of epi-vitamin D quantitation in plasma and barbiturate measurement in urine.
Methods:
After preparation, the same samples were divided equally to two aliquots and were run on a hybrid quadrupole-Orbitrap MS (PRM) and on a triple quadrupole MS (SRM) respectively. The two instruments had identical front ends (autosampler, LC pump, and column heater). In PRM, a single precursor ion was selected in the quadrupole with an isolation width of 2.0 m/z and fragmented in the HCD cell using an optimized, compound-specific collision energy. The resulting MS/MS product ion spectrum was detected in the Orbitrap detector at a resolution of 35,000 (FWHM at m/z of 200). In SRM, the cycle time was 0.5 second. Q1 and Q3 resolution was set as 0.7 Da (FWHM).
In the first clinical research application of epi-vitamin D quantitation, plasma samples were processed by protein precipitation followed by solid phase extraction. 25-hydroxy vitamin D3, epi-25-hydroxy vitamin D3, and 25-hydroxy vitamin D2 were separated chromatographically and quantified by the two MS methods in positive polarity with APCI probes.
For the second method regarding measurement of five barbiturates (amobarbital, butalbital, phenobarbital, pentobarbital, secobarbital) used in forensic toxicology laboratories, urine samples were diluted 20 fold and directly analyzed by LC-MS in negative polarity with heated ESI probes.
Results:
For the PRM analysis of barbiturates, for each analyte and internal standard (IS), the precursor extract mass in the MS/MS spectrum was used for quantification and the most abundant fragment was used for confirmation. For epi-vitamin D analysis, a specific fragment from the MS/MS spectrum was selected as the quantifying ion. In both cases, the chromatograms were reconstructed with a mass accuracy of 5 ppm for quantification.
In SRM analysis of both sets of compound, two products ions were collected. One was for quantification and another one was for ion ratio confirmation. To ensure confident quantification, at least 15 scans were collected across each peak for both PRM and SRM analysis.
The linearity range in PRM was 1-100 ng/mL for 25-hydroxy vitamin D3, epi-25-hydroxy vitamin D3, and 25-hydroxy vitamin D2. The linearity range in SRM was 2-100 ng/mL for each analyte. The inter-assay precision in PRM was 2.5-6.4% RSD. The inter-assay precision in SRM was 3.4-9.4% RSD. The bias in PRM was 3-14%. The bias in SRM was 0-7%.
The linearity range in PRM was 5-2000 ng/mL for amobarbital, butalbital, pentobarbital, secobarbital and 25-2000 ng/mL for phenobarbital. The linearity range in SRM was 5-2000 ng/mL for butalbital and secobarbital, 10-2000 ng/mL for amobarbital and pentobarbital, 25-2000 ng/mL for phenobarbital. The inter-assay precision in PRM was 4.1-9.7% RSD. The inter-assay precision in SRM was 0.9-8.4% RSD. The recovery rate of internal standards (matrix effect) in 48 donor urine samples in PRM ranged from 76% to 108%. The IS recovery rate in SRM ranged from 74% to 112%.
Conclusions:
We demonstrated comparable quantitative performance between PRM and SRM in terms of run-to-run reproducibility, matrix effect, and measurement accuracy in the context of clinical research and forensic toxicology applications. Similar quantitative performance was observed using both methods across multiple differing parameters including matrix (plasma and urine), polarity (positive and negative) and ionization mode (APCI and heated ESI).
In addition to comparable quantitative performance, the PRM technique has several potential advantages over the traditional SRM approach. First, PRM spectra would be highly specific because all potential product ions are available to confirm the identity of the analyte. Second, PRM could provide a higher tolerance for co-eluting interferences. And third, PRM could eliminate much of the effort required to develop and optimize the traditional SRM assay because PRM monitors all transitions.
References & Acknowledgements:
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