Geoffrey Rule (Presenter)
ARUP Laboratories
Bio: After obtaining my BSc in Chemistry at the State University of New York in Syracuse I joined Wendell Roelofs, a noted pheromone chemist, working with isotopically labelled compounds to follow biosynthetic pathways by mass spectrometry. I then joined Jack Henion to begin graduate studies in API LC/MS combined with immunoaffinity purification. After obtaining my MSc I went to work with Richard Durst to specialize in point-of-care type tests for my doctoral research. I have held positions performing LC-MS/MS method development and validation in DMPK, GLP, and clinical chemistry laboratories. I have had the opportunity to contribute to the development of several new pieces of instrumentation and enjoy the challenge of developing greater efficiencies in the analytical lab. Since 2011 I have been working with a number of excellent scientists at ARUP Laboratories including Dr. Alan Rockwood. H
Authorship: Geoffrey S. Rule 1*, Alan L. Rockwood 1, 2
1. ARUP Institute for Clinical and Experimental Pathology 2. Department of Pathology, University of Utah School of Medicine
| Short Abstract We have evaluated the potential for use of a single point calibration over a period of seven months and a total of 340 analytical runs using production laboratory data. The single point calibration data is incorporated into a weighted response factor that provides a more stable estimate of instrument response and therefore better analytical precision. This is shown for three androgen analytes along with several observations that were made over the seven month period. The weighted response factor is shown to behave in a predictable fashion. Several points to consider in adoption of this type of strategy are discussed. |
Long Abstract
Introduction
We have shown in earlier work how use of a weighted average response factor (RF) from a single calibrator can be used to quantify analytical runs with improved precision in comparison to conventional multipoint calibration. Others [1, 2] have shown similar outcomes, both in theory and practice, where use of two calibration levels, and multiple replicates, performs better than use of multiple calibration levels at a single replicate.
Although this type of approach to quantitation is common in other areas of clinical laboratory testing it is somewhat foreign to the MS community. One reason for this may be a lack of familiarity with the implications of using such a strategy aside from the improved efficiency. Here we examine the influence of several factors on quantitative accuracy using weighted single point calibrations with regression forced through a zero intercept. We illustrate, using both simulated and laboratory data, how changes in 1.) internal standard and 2.) calibration standard concentration are predicted to effect quantitative outcomes with use of a weighted RF along with a simple remedy. A third possible contributor to quantitative inaccuracy may be that created by instrumental drift. To better understand potential sources of drift in RF we have evaluated several LC/MS instrument parameters on RF stability and present them here.
Methods and Materials
All laboratory data was collected on production LC-MS instrumentation (AB Sciex triple quadrupole) operated in MS/MS mode with positive ion electrospray ionization. Data was collected in such a way that it could be processed by separate quantitation methods using applications outside of normal processing flows. Data was then processed using either Excel or a separate in-house application created for the purpose of processing by different methods. Simulations were created using PSI-Plot. Weighting was carried out using a factor of 0.75 given to the historical RF value and a factor of 0.25 given to the current, or provisional response factor.
Results and Discussion
We have shown that use of a weighted RF for analyte quantitation can result in improved efficiency as a result of reducing the number of calibrators analyzed with each batch. In addition, the strategy provides better precision than that obtained by conventional multipoint calibration using single replicates at each calibration level. We show that the result of a change in either internal standard concentration (IS) or calibrator concentration behaves in a predictable fashion both with simulation and with data from our dataset of 340 runs. We expect that, in general, use of common strategies to verify analyte stability and “balance” stock solutions can minimize shifts in the analytical accuracy obtained with use of a weighted RF.
We also examine the effect of other instrumental parameters that may influence the stability of an RF when an isotopically labelled internal standard is used. (RF is simply the ratio of an analyte transition to an internal standard transition, divided by the analyte concentration.) Here we assume use of an isotopically labelled internal standard for quantitation.
Ideally the ratio of calibrator to internal standard would be perfectly stable and an identical amount of IS would be added to each calibrator, QC and sample. In reality, this is not the case and we find that some instrumental factors may also influence the ratio of analyte to IS. We consider instrumental factors such as collision energy, collision gas setting, declustering potential, retention time (or K’), peak intensity, and integration parameters, and discuss their influence RF stability. The results of these studies are discussed with regard to their impact on the potential for using weighted average single point calibration.
References & Acknowledgements:
1. Renman, L. and D. Jagner, Asymmetric distribution of results in calibration curve and standard addition evaluations. Analytica Chimica Acta, 1997. 357(1–2): p. 157-166.
2. Tan, A., et al., Comparison of different linear calibration approaches for LC-MS bioanalysis. J Chromatogr B Analyt Technol Biomed Life Sci, 2012. 911: p. 192-202.
| Description | Y/N | Source |
| Grants | no | |
| Salary | no | |
| Board Member | no | |
| Stock | no | |
| Expenses | no |
IP Royalty: no
| Planning to mention or discuss specific products or technology of the company(ies) listed above: | yes |