Geoffrey Rule (Presenter)
Bio: Geoff has been doing mass spectrometry for over 30 years in a variety of roles starting with insect pheromone research. He has since worked in FDA regulated drug development laboratories, supporting DMPK studies and clinical trials, and in clinical laboratory testing. He has a special interest in instrumentation and strategies for improving throughput and analytical quality. He is currently a Research Investigator at ARUP Laboratories in Salt Lake City, Utah.
Authorship: Geoffrey Rule 1*, Carmen Gherasim 1, Joely Straseski 1, 2
1. ARUP Institute for Clinical and Experimental Pathology 2. Department of Pathology, University of Utah School of Medicine
Unlike pharmaceutical bioanalysis, clinical mass spec labs use two ions to identify/confirm the identity of each analyte reported. The ratio of the quantitative and qualitative ions is generally required to be within certain limits of an expected value in order to confirm identity and report a result. Oftentimes these limits are arbitrarily set to +/- 35% with values falling outside these limits possibly given as “unreportable due to an interfering substance”. This presentation explores several factors, including tube type, matrix and instrument effects that may influence fragment ion ratios from a given precursor.
The use of ion ratios for analyte confirmation is a step to improve the analytical reliability of a determination. It is based on having reproducible fragmentation of analyte molecules in the mass spectrometer. To be useful it is important to have an understanding of the parameters that may impact ion ratios. For example, it has been shown by others (Kaufmann, 2009) that some compounds have more than one charge site isomers existing, with each isomer giving rise to different fragmentation pathways and product ions. Ordinarily these isomers are not distinguished by the mass spectrometer so the resulting product ion spectrum is a composite of several fragmentation pathways. A shift in the relative abundance of these isomers can therefore cause a change in the ratio of product ions. It has further been shown that sample matrix, solvent composition, and instrument parameters can influence the relative abundance of the charge site isomers.
We describe our studies to establish ion ratios in different collection tube types, at different analyte concentrations, and with different instrument source parameters. In addition to examining fragment ion ratios from one isotopologue we also examine fragmentation of heavy isotopologues (internal standards) and explore their potential impact on analyte calibration stability.
Several analytes and validated methods were utilized for these studies. Collection tube type studies were performed with methods for both estradiol and estrone, as well as for pregnenolone and 17-hydroxypregnenolone. All studies were performed on AB Sciex 5500 or 6500 triple quadrupole mass spectrometers with some experiments utilizing differential mobility spectrometry between the source and high vacuum region (Selexion). Collection tube type studies were collected under an IRB-approved protocol from healthy volunteers donating blood into the tubes of interest. Infusion studies were performed with the analytes ciprofloxacin, repaglinide, and pregenolone. Additional studies with multiple isotopologues of hydroxyindole acetic acid, and tolbutamide were performed with solutions prepared to contain two isotopologues at approximately equivalent concentrations.
We have noticed with some methods that a small percentage of patient samples cannot be reported with quantitative values due to the ion ratio being outside accepted bounds. Samples outside these bounds are often extracted and analyzed a second time with the same result. Bounds are sometimes arbitrarily set and with a broader acceptance range at lower concentrations. The accurate determination of ion ratio variability is essential to minimize reporting of false results. This lead us to ask what actual expected ion ratio variances are and under what conditions might they vary.
With endogenous steroids it is common to utilize calibration standards prepared in a non-matrix solution and to use these as a reference for ion ratios. Caution is warranted with this practice as the ion ratio may differ between matrix collection tubes and calibrators. Care must also be utilized in validation to ensure that ion ratio acceptance criteria are assigned to yield the highest possible specificity.
We have found that ion ratios may differ depending on the sample collection tube type. For example, mean pregnenolone ion ratios generated from six individual donors providing specimens into each of four tube types were compared to matrix matched controls and calibrators prepared in BSA buffer solution. Statistical analysis (Wilcoxon score) indicated a statistically significant difference in ion ratio between EDTA plasma and calibrators.
Others have reported similar findings with regard to matrix effects (Kaufmann, 2009) as well as effects of mobile phase composition, source temperature, and cone voltage on ion ratios. They demonstrated that with some compounds one or more charge site isomers may exist and that these isomers can give rise to different fragmentation pathways. The result is that ion ratios may be skewed by factors favoring one charge site isomer over the other.
Conclusions & Discussion
There are still subtleties to the general understanding of the influences of both matrix and instrumental factors on fragmentation pathways taken by both analytes and their heavy isotopologues. We consider here whether molecular structure and charge location need closer scrutiny in determination of assay specificity as judged by ion ratios. It is important to understand fragmentation pathways for individual transitions selected in assay development, and several simple experiments may be used to evaluate the influence of matrix, source, and instrument conditions on ion ratios. The impact of better understanding these effects on clinical mass spectrometry assays is to provide high specificity quantitative values while minimizing repeat blood draws and sample analysis.
References & Acknowledgements:
Kaufmann A, Butcher P, Maden K, Widmer M, Giles K, Uría D.
Rapid Commun Mass Spectrom. 2009, 23(7):985-98
The authors would like to thank the ARUP Institute for Clinical and Experimental Pathology for making this work possible.
IP Royalty: no
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