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Use of Hybrid Capillary Tube Apparatus on 400 MHz NMR for Quantitation of Crucial Low-Quantity Metabolites Using aSICCO Signal.

Authors
  • Tiwari, Ranjeet1
  • Ahire, Deepak1
  • Kumar, Hemantha1
  • Sinha, Sarmistha1
  • Chauthe, Siddheshwar Kisan1
  • Subramanian, Murali1
  • Iyer, Ramaswamy1
  • Sarabu, Ramakanth1
  • Bajpai, Lakshmikant2
  • 1 Discovery Analytical Sciences (R.T., H.K., S.K.C., R.S., L.B.) and Pharmaceutical Candidate Optimization (D.A., S.S., M.S.), Bristol-Myers Squibb-Biocon Research Center, Bangalore, India; and Pharmaceutical Candidate Optimization, Bristol-Myers Squibb, Princeton, New Jersey (R.I.). , (India)
  • 2 Discovery Analytical Sciences (R.T., H.K., S.K.C., R.S., L.B.) and Pharmaceutical Candidate Optimization (D.A., S.S., M.S.), Bristol-Myers Squibb-Biocon Research Center, Bangalore, India; and Pharmaceutical Candidate Optimization, Bristol-Myers Squibb, Princeton, New Jersey (R.I.) [email protected] , (India)
Type
Published Article
Journal
Drug metabolism and disposition: the biological fate of chemicals
Publication Date
Dec 01, 2017
Volume
45
Issue
12
Pages
1215–1224
Identifiers
DOI: 10.1124/dmd.117.077073
PMID: 28935657
Source
Medline
License
Unknown

Abstract

Metabolites of new chemical entities can influence safety and efficacy of a molecule and often times need to be quantified in preclinical studies. However, synthetic standards of metabolites are very rarely available in early discovery. Alternate approaches such as biosynthesis need to be explored to generate these metabolites. Assessing the quantity and purity of these small amounts of metabolites with a nondestructive analytical procedure becomes crucial. Quantitative NMR becomes the method of choice for these samples. Recent advances in high-field NMR (>500 MHz) with the use of cryoprobe technology have helped to improve sensitivity for analysis of small microgram quantity of such samples. However, this type of NMR instrumentation is not routinely available in all laboratories. To analyze microgram quantities of metabolites on a routine basis with lower-resolution 400 MHz NMR instrument fitted with a broad band fluorine observe room temperature probe, a novel hybrid capillary tube setup was developed. To quantitate the metabolite in the sample, an artificial signal insertion for calculation of concentration observed (aSICCO) method that introduces an internally calibrated mathematical signal was used after acquiring the NMR spectrum. The linearity of aSICCO signal was established using ibuprofen as a model analyte. The limit of quantification of this procedure was 0.8 mM with 10 K scans that could be improved further with the increase in the number of scans. This procedure was used to quantify three metabolites-phenytoin from fosphenytoin, dextrophan from dextromethorphan, and 4-OH-diclofenac from diclofenac-and is suitable for minibiosynthesis of metabolites from in vitro systems.

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