Selecting a Structural Analog as an Internal Standard for the Quantification of 6-Methylmercaptopurine by LC-MS/MS

Author:

Smith Kathryn A1,Merrigan Stephen D1,Johnson-Davis Kamisha L12

Affiliation:

1. ARUP Institute for Clinical and Experimental Pathology, Salt Lake City, UT

2. Department of Pathology, University of Utah Health Sciences Center, Salt Lake City, UT

Abstract

Abstract Background When choosing an analog internal standard (IS) in a quantitative LC-MS/MS assay, careful selection and thorough verification are important for developing an accurate quantitative assay. The IS is a critical component in quantitative mass spectrometry because it is used to normalize results by compensating for variations in sample preparation and instrument performance. Here we present the results of our investigation in the selection process for a structural analog IS (SA-IS) to be used in the quantification of 6-methylmercaptopurine (6-MMP) in cytolysed red blood cell (RBC). Methods A cocktail solution of 9 SA-ISs including the isotopically labeled structural isomer and the 6-MMP stable isotope-labeled IS (SIL-IS) was spiked into cytolysed RBC controls and patient samples. Linearity, accuracy, sensitivity, precision, run stability, method comparison, and reinjection reproducibility experiments were performed. Ion suppression was also assessed by T-infusing the cocktail solution. Results All analogs were linear from 100 to 1200 ng/mL 6-MMP with acceptable precision and sensitivity by use of a spiked blank lysate. Method comparison plots of 6-MMP concentrations in patient samples had excellent agreement for 2 of the SA-ISs (i.e., the isotopically labeled structural isomer and an SA-IS with an added methyl group) when compared to the SIL-IS. Halogen-substituted analogs (i.e., Cl and Br) also met the criteria as an acceptable IS. However, 2 of the selected SA-ISs having substituted amine moieties showed unacceptable performance, with ≥15% bias when compared to the SIL-IS. Conclusion There are many parameters to consider when determining if an analog will be a good IS choice, and the approaches highlighted in this article can be applied to the selection of SA-IS in the development of other LC-MS/MS assays.

Funder

ARUP Institute for Clinical and Experimental Pathology

Publisher

Oxford University Press (OUP)

Subject

General Medicine

Reference40 articles.

1. Detection sensitivity and selectivity;Snyder;Practical HPLC method development,1997

2. Liquid chromatography-mass spectrometry methods; approved guideline;CLSI,2014

3. The role of liquid chromatography-tandem mass spectrometry in the clinical laboratory;van den Ouweland;J Chromatogr B Analyt Technol Biomed Life Sci,2012

4. Strategies for the assessment of matrix effect in quantitative bioanalytical methods based on HPLC-MS/MS;Matuszewski;Anal Chem,2003

5. A retrospective analysis of urine drugs of abuse immunoassay true positive rates at a national reference laboratory;Johnson-Davis;J Anal Toxicol,2016

同舟云学术

1.学者识别学者识别

2.学术分析学术分析

3.人才评估人才评估

"同舟云学术"是以全球学者为主线,采集、加工和组织学术论文而形成的新型学术文献查询和分析系统,可以对全球学者进行文献检索和人才价值评估。用户可以通过关注某些学科领域的顶尖人物而持续追踪该领域的学科进展和研究前沿。经过近期的数据扩容,当前同舟云学术共收录了国内外主流学术期刊6万余种,收集的期刊论文及会议论文总量共计约1.5亿篇,并以每天添加12000余篇中外论文的速度递增。我们也可以为用户提供个性化、定制化的学者数据。欢迎来电咨询!咨询电话:010-8811{复制后删除}0370

www.globalauthorid.com

TOP

Copyright © 2019-2024 北京同舟云网络信息技术有限公司
京公网安备11010802033243号  京ICP备18003416号-3