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.).
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.)
Drug Metab Dispos. 2017 Dec;45(12):1215-1224. doi: 10.1124/dmd.117.077073. Epub 2017 Sep 21.
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.
新化学实体的代谢物会影响分子的安全性和疗效，因此在临床前研究中通常需要对其进行定量分析。然而，在早期发现阶段，代谢物的合成标准品非常罕见。需要探索替代方法（如生物合成）来生成这些代谢物。评估这些小量代谢物的数量和纯度变得至关重要。定量 NMR 成为这些样品的首选分析方法。随着低温探头技术在高场 NMR（>500 MHz）中的应用，最近的技术进展有助于提高对这些小微克量样品的分析灵敏度。然而，并非所有实验室都常规配备这种类型的 NMR 仪器。为了在配备宽带氟观测室温探头的常规 400 MHz NMR 仪器上对微克级代谢物进行常规分析，开发了一种新型混合毛细管管装置。为了对样品中的代谢物进行定量分析，在采集 NMR 光谱后，使用人工信号插入（aSICCO）方法引入内部校准的数学信号来计算观察到的浓度（aSICCO）。使用布洛芬作为模型分析物建立了 aSICCO 信号的线性关系。该方法的定量下限为 0.8 mM，扫描次数为 10 K，可以通过增加扫描次数进一步提高。该方法用于定量三种代谢物-苯妥英从磷苯妥英、右美沙芬从右美沙芬和 4-OH-二氯芬酸从二氯芬酸-并适用于体外系统中小分子代谢物的微量生物合成。
