Thomas Laha (Presenter)
University of Washington
Bio: Tom is a scientist in the developmental chemistry division of the department of Laboratory Medicine, University of Washington. His focus is developing quantitative assays for endogenous and exogenous small molecules and peptides in biological samples using liquid chromatography tandem mass spectrometry.
Authorship: Thomas J. Laha, MT(ASCP) (1), Clark M. Henderson, PhD (1), Andrew N. Hoofnagle, MD PhD (1)
(1)Department of Laboratory Medicine, University of Washington
| Short Abstract To aid in the selection of a new mass spectrometer, we developed an experimental model which allowed the concurrent evaluation of both small molecule and peptide performance across top-tier quadrupole mass spectrometers from four LC-MS/MS vendors. Each Vendor was supplied a seven-sample test set, composed of linear admixtures of two trypsin-digested serum samples. One sample contained the human vitamin D binding globulin variant GC2 (tryptic peptide LPDATPK) and the other sample, deficient for this variant, was spiked with amphetamine to a final concentration of 150pg/mL. Vendors were asked to analyze each test mix in quadruplicate, concurrently monitoring multiple transitions for amphetamine, GC2 and GC2 internal standard. Key parameters that were compared included the y-intercept of the linear regressions, %CV at each of the concentrations, and %CV of transition ion ratios. |
Long Abstract
Introduction
When a clinical laboratory finds itself in the envious position of acquiring a new quadrupole mass spectrometer, the difficult task of evaluating instruments and vendors begins. To narrow the ever growing list of instrument vendors and models, we often consult with colleagues who have experience with the vendors under consideration as well as reviewing our unique experimental requirements such as:
• Intended use
• Instrument functionality and robustness
• Laboratory space and resources
• Timely vendor support
• Costs to purchase and maintain the instrument
While this list contributes valuable information, it does not provide critical empirical data on which to base a final decision. Experimental sample sets could be constructed and provided to the vendors to properly evaluate the analytical performance of the instrument under consideration. Recently we have developed an experimental model to evaluate the performance of top-tier quadrupole mass spectrometers from four LC-MS/MS vendors (A-D). The goal was to examine the performance of each instrument for specific applications in small molecule and peptide analysis.
Methods
A set of seven serum samples was prepared and consisted of admixtures of two serum trypsin digests. This test set was developed to evaluate the quantitative performance of each instrument while simultaneously measuring small molecules and peptides. One sample contained the human vitamin D binding globulin variant GC2 (LPDATPK) and the other was deficient for this variant. The samples were digested by a previously published method after which amphetamine was added to the GC2-deficient sample at a final concentration of 150 pg/mL(1). 40µL of each sample was injected onto a Waters Acquity HSS T3 1.8µm, 2.1x50mm column and a linear gradient was employed using 0.1% formic acid in water and 0.1% formic acid in methanol as mobile phase A and B respectively. The gradient started at 98%:2% and ended in 5.5 minutes at 45%:55% mobile phase A and B respectively. Amphetamine and GC2 eluted at approximately 3.3 minutes.
Each vendor was supplied a blinded sample set consisting of one 210 µL aliquot of Test mix samples 1-7 as well as solutions for optimization of their mass spectrometers. Additionally each vendor was provided a Waters Acquity HSS T3; 1.8µm, 2.1 mm x 50 mm column of the same lot, along with detailed gradient and injector conditions. Each Vendor was asked to analyze the samples in quadruplicate, concurrently monitoring transitions for amphetamine (1-2 transitions), GC2 (at least 4 transitions) and GC2 internal standard (at least four transitions). It was requested that they report peak areas and signal-to-noise (peak-to-peak) for all transitions of GC2, GC2-IS and amphetamine as well as chromatographic images for all test samples/transitions.
Results
Linear regression was performed based on the peak area response of GC2 or amphetamine peak area versus concentration on column. Linear regressions were then evaluated for linearity as well as injector carryover. The amphetamine y-intercept expressed as a percent of the most abundant peak area (0% – 20%) became a surrogate for detector noise and endogenous peak area CV (amphetamine, 2.3%-6.0%; GC2, 1.8%-3.3%) and peak area response CV (GC2, 1.9%-3.5%) were evaluated to assess instrument precision. Ion ratio CV (amphetamine, 5.2%-9.8%; GC2, 5.9%-22.8%) was used as an indicator of detector sensitivity(2). Lastly, integrated peak shape was used as a qualitative measure of instrument performance.
Conclusions
The data revealed that the mass spectrometer from Vendor A best fit our required performance criteria. It produced acceptable CVs across all measures and had a y‐intercept that demonstrated reasonably low detector noise compared to vendors B and D. While vendor C also demonstrated low detector noise, the CV of the GC2 ion ratios was unacceptable as was also the case for vendor B; this metric can be used to evaluate overall mass spectrometer sensitivity(2).
References & Acknowledgements:
References
1. Henderson CM, Lutsey PL, Misialek JR, et al. Measurement by a Novel LC-MS/MS Methodology Reveals Similar Serum Concentrations of Vitamin D-Binding Protein in Blacks and Whites. Clin Chem. 2015;000:1-9. doi:10.1373/clinchem.2015.244541.
2. MacCoss MJ, Toth MJ, Matthews DE. Evaluation and optimization of ion-current ratio measurements by selected-ion-monitoring mass spectrometry. Anal Chem. 2001;73(13):2976-2984. http://www.ncbi.nlm.nih.gov/pubmed/11467543.
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