HPLC-DAD-MSn analysis and HPLC quantitation of chemical constituents in the traditional Chinese medicine formula Ya-tong-yi-li-wan

Ya-tong-yi-li-wan (YTYLW) is a well-known traditional Chinese medicine (TCM) formula composed of ChanSu (toad venom), licorice root, realgar, and cinnabaris in the ratio of 24 : 24 : 6 : 5. It is widely used for the treatment of toothache. In this formula, ChanSu accounts for the largest proportion and plays the primary therapeutic role. ChanSu contains multiple bufadienolides as the major effective constituents. Licorice also acts as a key ingredient, with flavonoids and saponins as the major chemical constituents. Realgar and cinnabaris are minerals with As2S2 and HgS as the main constituents, respectively.

YTYLW is recorded in the Chinese Pharmacopoeia, and the total contents of cinobufagin and resibufogenin from ChanSu were selected as markers for its quality control. Contents of these two compounds should be no less than 19.5 mg per gram of YTYLW. The other major bufadienolides in ChanSu like bufalin together with compounds from licorice were not determined in this method. In the present study, a validated and reliable method based on HPLC/DAD/ESI-MSn was established for the comprehensive quality control of YTYLW. A total of 48 compounds were identified or tentatively characterized, including bufadienolides, isoflavones, chalcones, saponins, dihydro flavones, flavonols, flavones, coumarins, and pterocarpan. Simultaneously, of the main compounds in YTYLW were quantitatively determined by HPLC-UV. This method is simple, reliable and sensitive, and could be used for the quality control of YTYLW.

Chemicals and materials

Among the 12 reference standards, liquiritin apioside, liquiritin, isoliquiritin, glycyrrhizic acid and isoangustone A were isolated by the authors from the roots of Glycyrrhiza uralensi Fisch.; gamabufotalin, telocinobufagin, bufotalin, cinobufotalin, bufalin, cinobufagin and resibufogenin were isolated by the authors from the crude drug ChanSu. All these compounds were identified by comparing their <sup>1</sup>H-NMR, <sup>13</sup>C-NMR and MS data with those reported in the literature. The purity of each compound was above 98% as determined by HPLC. A total of nine batches of commercial Ya-tong-yi-li-wan samples were obtained from different drug stores in Guangzhou, China.

Acetonitrile and formic acid were of HPLC grade (J. T. Baker, Phillipsburg, NJ, USA). De-ionized water was purified by a Milli-Q water purification system (Millipore, Bedford, MA, USA). All other chemical solvents were of analytical grade from Beijing Chemical Corporation (Beijing, China).

Preparation of standard stock solutions

Liquiritin apioside (0.63 mg), liquiritin (0.97 mg), gamabufotalin (0.77 mg), isoliquiritin (0.75 mg), telocinobufagin (0.65 mg), bufotalin (1.35 mg), glycyrrhizic acid (3.04 mg), cinobufotalin (1.58 mg), bufalin (1.95 mg), cinobufagin (3.92 mg), resibufogenin (2.95 mg) and isoangustone A (0.15 mg) were accurately weighed, mixed and dissolved in 3.0 mL of 70% (v/v) methanol to produce a stock solution. This stock solution was then accurately diluted to 6/7, 5/7, 4/7, 3/7, 2/7, 1/7 and 1/70 of its original concentration with 70% methanol to obtain eight serial concentrations. All solutions were filtered through membranes (0.22 mm pore size) and stored at 4 oC until use.

Sample preparation

For sample preparation, the Ya-tong-yi-li-wan pills were pulverized into fine powder (40 mesh). The powder was accurately weighed (0.100 g) and extracted with 3 mL of 70% methanol by ultrasonication for 30 min. The sample was then adjusted to the original weight and the supernatant was filtered through 0.45 mm membranes before HPLC analysis.

Quantitative analysis

The quantitative analyses were performed using an Agilent 1100 series HPLC system consisting of a quaternary solvent delivery system, an autosampler, a column compartment and a VWD detector (Agilent Technologies, Waldbronn, Germany). The HPLC conditions were identical to those used for qualitative analysis mentioned above, except that the UV detection wave-length was set at 270 nm.

In this study, a simple, reliable, and sensitive method was developed for qualitative and quantitative analysis of Ya-tong-yi-li-wan. With the aid of HPLC-DAD-MSn, 48 compounds were characterized, including bufadienolides, isoflavones, chalcones, saponins, flavanones, flavonols, flavones, coumarins and 1 pterocarpan. At the same time, 12 major compounds in 9 batches of YTYLW were simultaneously determined by HPLC-UV. This method could be used for quality control of YTYLW.



Ke-di Liu, Xue Qiao, Qing Wang, Wei Song, De-an Guo, Min Ye*. Anal. Methods, 2013, 5, 5241–5247


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