Zlatuse Clark (Presenter)
Bio: B.S. in analytical chemistry from Masaryk University, Brno, Czech Republic Ph.D. in bioanalytical chemistry from Brigham Young University, Provo, Utah Currently an R&D scientist at ARUP Laboratories, Salt Lake City, Utah
Authorship: Zlatuse D Clark
ARUP Laboratories, Salt Lake City, UT
This session segment will discuss: • The use of internal standard in mitigating interference • How do we monitor for interference?
The popularity of LC-MS/MS-based methods for clinical testing continues to rise. However, despite their superior analytical specificity, these methods may still suffer from interference affecting method accuracy and precision, and hence negatively impacting patient care. The aim of this Practical Training Track presentation is to introduce the participant to what analytical interference is, where it may come from, how we test for it in LC-MS/MS, ways to mitigate it, and how to monitor for it (1). Practical examples of interference issues and how they were resolved will be shown.
The use of internal standard in mitigating interference
While the above tactics help identify and/or reduce interferences, no method is completely immune to them. Many labs use stable isotope-labeled internal standards to increase LC-MS/MS method robustness and mitigate interference such as matrix effects (6, 11). When analytes elute in a signal suppression region, compensating with an internal standard is often adequate. Problems arise, however, when the suppression severely decreases the signal-to-noise ratio of the analyte and internal standard peaks. This compromises assay performance, especially near the assay’s lower limit of quantitation (7). Another issue may stem from the internal standard not exactly co-eluting with the analyte, which sometimes happens with deuterium-labeled analogs when the deuterium atoms are in positions that impact chromatographic retention. For example, if the internal standard elutes outside of a suppression zone while the analyte peak is partially in it, the two compounds experience a different degree of suppression, which results in inaccurate quantitation. The strategies for resolving these issues include using internal standards having stable labels that do not impact chromatographic retention time (e.g., 13C, 15N, 18O) and thus better compensate for matrix effects, or other approaches aimed toward reducing interferences, such as LC gradient profile modifications and more selective sample preparation.
How do we monitor for interference?
Even the best method development strategies rarely are able to prevent interference completely. Consequently, the need to monitor for interference in routine testing in order to avoid reporting compromised results is undisputable. The most relevant data quality metrics are ion ratios, absolute internal standard areas, and retention times. Deviations in these metrics can signal the presence of interference in either the analyte or internal standard mass chromatograms (12, 13).
Conclusions & Discussion
After this segment attendees should be able to:
1. Discuss the role of internal standard in mitigating interference
2. Name parameters used for interference monitoring
References & Acknowledgements:
1. Clark ZD, Balloch S, Calton L, Mason D. Interference Testing and Mitigation in LC-MS/MS Assays. Clinical Laboratory News 2017;43(8):22-5.
2. CLSI. Evaluation of matrix effects; Approved guideline – second edition. CLSI document EP14-A2.Wayne (PA): CLSI; 2005.
3. CLSI. Interference testing in clinical chemistry; Approved guideline – second edition. CLSI document EP7-A2.Wayne (PA): CLSI; 2005.
4. Clark ZD, Cutler JM, Pavlov IY, et al. Simple dilute-and-shoot method for urinary vanillylmandelic acid and homovanillic acid by liquid chromatography tandem mass spectrometry. Clin Chim Acta 2017;468:201–8.
5. Clark ZD, Cutler JM, Frank EL. Practical LC-MS/MS method for 5-hydroxyindoleacetic acid in urine. J Appl Lab Med 2017;1:387–99.
6. Matuszewski BK, Constanzer ML, Chavez-Eng CM. Strategies for the assessment of matrix effect in quantitative bioanalytical methods based on HPLC-MS/MS. Anal Chem 2003;75:3019–30.
7. Annesley TM. Ion suppression in mass spectrometry. Clin Chem 2003;49:1041–4.
8. Bonfiglio R, King RC, Olah TV, et al. The effects of sample preparation methods on the variability of the electrospray ionization response for model drug compounds. Rapid Commun Mass Spectrom 1999;13:1175–85.
9. King R, Bonfiglio R, Fernandez-Metzler C, et al. Mechanistic investigation of ionization suppression in electrospray ionization. J Am Soc Mass Spectrom 2000;11:942–50.
10. Clark ZD, Strathmann FG, McMillin GA. Diluting and shooting yourself in the foot: Complications with sample-to-sample variations in signal suppression. MSACL 2013 podium presentation.
11. CLSI. Liquid chromatography-mass spectrometry methods; Approved guideline. CLSI document C62-A. Wayne (PA): CLSI; 2014.
12. Lynch KL. LC-MS/MS quality assurance in production: The real work begins after validation. Clinical Laboratory News 2017;43(5):28–9.
13. Zabell APR, Stone J, Julian RK. Using big data for LC-MS/MS quality analysis. Clinical Laboratory News 2017;43(5):30–1.
Much gratitude to Dr. Frederick Strathmann for collaboration on several projects addressing the various aspects of interference testing and for facilitating exciting opportunities to present and publish on these topics. Many thanks as well to Donald Mason, Lisa Calton, and Stephen Balloch for their contributions as coauthors of our recent Clinical Laboratory News article “Interference Testing and Mitigation in LC-MS/MS Assays,” which was used in preparing this presentation.
The work presented here was supported in part by ARUP Institute for Clinical and Experimental Pathology®.
IP Royalty: no
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