DOSY-Assisted Multicomponent Characterization Enables Classification of Precious Chinese Materia Medica and Identification of Adulteration: A Case Study of Calculus Bovis.

Introduction: Despite significant advances in analytical methods, the quality evaluation and adulteration detection of chemically complex traditional Chinese medicines continues to be a major challenge.

Objectives: We developed a custom diffusion-ordered spectroscopy (DOSY) approach for the qualitative analysis of precious Calculus Bovis (CB) and detection of its adulterants.

Methods: Using Artificial Calculus Bovis as an example, we focused on the effects of NMR spectrometers, solvent viscosity, matrices, matrix concentration, and temperature on separation efficiency of DOSY. The optimal conditions also successfully applied for the identification of Natural Calculus Bovis, Calculus Bovis Sativus, and adulterated samples. The accuracy of DOSY was verified by ¹³C NMR.

Results: Via inspection of NMR spectrometers, solvents, matrices, matrix concentration, and temperature, the optimal conditions for DOSY experiments of CB were found to be using methanol-d₄ as the solvent, adding 4 mg of polyvinyl pyrrolidone (PVP) as the matrix, and a temperature of 298 K. Under these conditions, five types of compounds, cholic acid (CA), deoxycholic acid (DCA), hyodeoxycholic acid (HDCA), taurine-conjugated bile acids (tBAs), and glycine-conjugated bile acids (gBAs), were unequivocally identified in three types of CB. ¹³C NMR validated the accuracy of the DOSY results. As for the sugar-adulterated CB, the signals of sugar could be clearly identified on the DOSY spectrum.

Conclusions: This study verified the feasibility of DOSY for the quality evaluation of CB and provided a reference point for future qualitative and quantitative analysis of other complex traditional Chinese medicines, as well as the characterization of adulterated samples.