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Research ArticleArticle

Validation of Isolated Metabolites from Drug Metabolism Studies as Analytical Standards by Quantitative NMR

Gregory S. Walker, Tim F. Ryder, Raman Sharma, Evan B. Smith and Amy Freund
Drug Metabolism and Disposition March 2011, 39 (3) 433-440; DOI: https://doi.org/10.1124/dmd.110.036343
Gregory S. Walker
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Tim F. Ryder
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Raman Sharma
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Evan B. Smith
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Amy Freund
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Abstract

In discovery and development, having a qualified metabolite standard is advantageous. Chemical synthesis of metabolite standards is often difficult and expensive. As an alternative, biological generation and isolation of metabolites in the nanomole range are readily feasible. However, without an accurately defined concentration, these isolates have limited utility as standards. There is a significant history of NMR as both a qualitative and a quantitative technique, and these concepts have been merged recently to provide both structural and quantitative information on biologically generated isolates from drug metabolism studies. Previous methodologies relied on either specialized equipment or the use of an internal standard to the isolate. We have developed a technique in which a mathematically generated signal can be inserted into a spectrum postacquisition and used as a quantitative reference: artificial signal insertion for calculation of concentration observed (aSICCO). This technique has several advantages over previous methodologies. Any region in the analyte spectra, free from interference, can be chosen for the reference signal. In addition, the magnitude of the inserted signal can be modified to appropriately match the intensity of the sample resonances. Because this is postacquisition quantification, no special equipment or pulse sequence is needed. Compared with quantitation via the addition of an internal standard (10 mM maleic acid), the signal insertion method produced similar results. For each method, precision and accuracy were within ±5%, stability of signal response over 8 days was ±5%, and the dynamic range was more than 3 orders of magnitude: 10 to 0.01 mM.

Footnotes

  • Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.

    doi:10.1124/dmd.110.036343.

  • ABBREVIATIONS:

    MIST
    metabolites in safety testing
    LC
    liquid chromatography
    MS
    mass spectrometry
    HPLC
    high-performance liquid chromatography
    NBG
    4-nitrobenzyl glutathione
    DMSO
    dimethyl sulfoxide
    HSQC
    heteronuclear single quantum correlation
    aSICCO
    artificial signal insertion for calculation of concentration observed
    FID
    free induction decay
    AO
    aldehyde oxidase
    qNMR
    quantitative nuclear magnetic resonance
    IStd
    internal standard
    ERETIC
    electronic reference to access in vivo concentrations.

  • Received September 13, 2010.
  • Accepted November 22, 2010.
  • Copyright © 2011 by The American Society for Pharmacology and Experimental Therapeutics
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Drug Metabolism and Disposition: 39 (3)
Drug Metabolism and Disposition
Vol. 39, Issue 3
1 Mar 2011
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Research ArticleArticle

Validation of Isolated Metabolites from Drug Metabolism Studies as Analytical Standards by Quantitative NMR

Gregory S. Walker, Tim F. Ryder, Raman Sharma, Evan B. Smith and Amy Freund
Drug Metabolism and Disposition March 1, 2011, 39 (3) 433-440; DOI: https://doi.org/10.1124/dmd.110.036343

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Research ArticleArticle

Validation of Isolated Metabolites from Drug Metabolism Studies as Analytical Standards by Quantitative NMR

Gregory S. Walker, Tim F. Ryder, Raman Sharma, Evan B. Smith and Amy Freund
Drug Metabolism and Disposition March 1, 2011, 39 (3) 433-440; DOI: https://doi.org/10.1124/dmd.110.036343
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