Abstract
Objective: The research work unveils the use of nuclear magnetic resonance (NMR) technique for quantitative determination and method validation of obeticholic acid. As standard expository methodology for more up to date medications or formulations may not be available in pharmacopeias, hence it is fundamental need to create novel analytical procedures which should be precise and accurate.
 Methods: Proton (1H) and carbon (13C) NMR analysis were initially performed to confirm the preliminary authenticity of obeticholic acid API. Method validation was accomplished on the basis of standard guidelines for the parameters, in which tetramethylbenzene as an internal standard and deuterated dimethyl sulfoxide as a diluent were used to assess the obeticholic acid.
 Results: For the quantification of the drug, the proton nuclear magnetic resonance signals at 0.602 ppm and 6.86 ppm corresponding to the analyte proton of drug and internal standard respectively were used. The curve equation calculated from the regression method, the relative-standard-deviation and correlation-coefficient were found to be 0.743% and 0.9989 respectively, indicating good linearity. Consequently, the quantitative assay of the drug was found to be 99.91% in linearity with limit of detection and quantification values as 0.0773 mg and 0.2344 mg respectively, making successful the study of method validation for obeticholic acid.
 Conclusion: The advantage of the method was that no reference standard of analyte drug was required for quantification and method validation. The method is non-destructive and can be applied for quantification of drug in commercial pharmaceutical formulation products.
Highlights
nuclear magnetic resonance (NMR) spectroscopy is a well-accepted analytical technique for structure elucidation of simple as well complex molecules and engaged in many branches of biochemistry [1, 2]
The proposed method is relatively fast, simple, precise, selective and sensitive sufficient for all observed related compounds of obeticholic acid compared with previously published studies mainly had used HPLC-MS [12,13,14] to define the concentration of obeticholic acid in plasma, and its methods were not explained whatsoever because they didn’t perform comprehensive method validation, detailed method optimization as per international council for harmonisation (ICH) parameters like Malz et al, El-Sheikh et al and Harahap et al performed [16,17,18]
The proposed method is relatively fast, simple, precise, selective and sensitive sufficient for all observed related compounds of obeticholic acid compared with previously published studies mainly had used HPLC-MS [12,13,14] to define the concentration of obeticholic acid in plasma, and its methods were not explained whatsoever because they cannot perform broad method validation, detailed method optimization as according to ICH parameters
Summary
NMR spectroscopy is a well-accepted analytical technique for structure elucidation of simple as well complex molecules and engaged in many branches of biochemistry [1, 2] It has been deliberated as a rapid, precise, reliable, non-destructive and convenient method for quantitative estimation and is a strategic analytical tool for the unidentified synthetic as well as natural compounds [3, 4]. A major revolution toward the synthetic bile acid compounds was the finding of this potent and selective FXR agonist obeticholic acid which has 100 ~fold greater activity than natural agonist [8, 9]. A numerous analytical method has been reported over time for the determination of obeticholic acid in biological fluids or individually in which mainly includes chromatographic-based HPLC/lC-MS [12,13,14] and other analysis like volumetric with visual end-point detection [15]
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