PT - JOURNAL ARTICLE AU - Tiwari, Ranjeet AU - Ahire, Deepak AU - Kumar, Hemantha AU - Sinha, Sarmistha AU - Chauthe, Siddheshwar Kisan AU - Subramanian, Murali AU - Iyer, Ramaswamy AU - Sarabu, Ramakanth AU - Bajpai, Lakshmikant TI - Use of Hybrid Capillary Tube Apparatus on 400 MHz NMR for Quantitation of Crucial Low-Quantity Metabolites Using aSICCO Signal AID - 10.1124/dmd.117.077073 DP - 2017 Dec 01 TA - Drug Metabolism and Disposition PG - 1215--1224 VI - 45 IP - 12 4099 - http://dmd.aspetjournals.org/content/45/12/1215.short 4100 - http://dmd.aspetjournals.org/content/45/12/1215.full SO - Drug Metab Dispos2017 Dec 01; 45 AB - 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.