ABSTRACT
ObjectiveTo develop a quantitative analysis of multi-components by single marker （QAMS） for determination of bufalin， cinobufagin and resibufogenin in Shexiang Baoxin pills， and to provide a method for improving the national standard of the pills. MethodHigh performance liquid chromatography （HPLC） was developed for simultaneous determination of bufalin， cinobufagin and resibufogenin in Shexiang Baoxin pills and the methodology validation was carried out. The chromatographic separation was performed on a Nucleosil 100-5 C18 column （4.6 mm×250 mm， 5 μm） with the mobile phase of acetonitrile -0.1% potassium dihydrogen phosphate aqueous solution （pH adjusted to 3.2 with phosphoric acid） （48∶52）， and the flow rate was 0.6 mL·min-1， the detection wavelength was set at 296 nm and the column temperature was 35 ℃. Taking cinobufagin as the internal standard， the relative correction factors （RCFs） of bufalin and resibufogenin were calculated， and the key influencing factors of RCFs were investigated. Relative retention time was used for the chromatographic peak location of the analyte， combining with the on-line ultraviolet spectroscopy and accurate relative molecular weight obtained by ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry （UPLC-QTOF/MS）. The external standard method was used to verify the contents of three components obtained by QAMS. ResultQAMS was established for the determination of bufalin， cinobufagin and resibufogenin in the samples， and RCFs of cinobufagin to bufalin and resibufogenin were 0.922 and 1.01， respectively. The total content of the three marker compounds in 11 batches of Shexiang Baoxin pills was 33.7-36.0 µg per pill. There was no significant difference between the quantitative results of QAMS and external standard method. ConclusionThe established method can be used for the quality control of bufalin， cinobufagin and resibufogenin in Shexiang Baoxin pills. It is suggested that bufalin should be considered as one of three marker compounds， and the sum of bufalin， cinobufagin and resibufogenin should be used for the content limit of this preparation.
ABSTRACT
As a new strategy capable of uncovering the characteristics of traditional Chinese medicines, the quantitative analysis of multi-components by single-marker(QAMS) has been widely employed for the quality evaluation of Chinese medicinal materials, slices, and extracts. However, its application in the assessment of Chinese patent medicines is yet to be explored. By referring to the determination of three bufogenins in Bufonis Venenum by QAMS described in Chinese Pharmacopoeia(2020 Edition), this paper selected seven representative preparations containing Bufonis Venenum and explored whether the relative correction factors(RCFs) of cinobufagin(CB) to bufalin(BF) and resibufogenin(RB) could be directly used for the quality control of Bufonis Venenum-contained preparations. Based on the qualitative analyses under the same chromatographic conditions as used for toad venom, combing specificity test, five preparations such as Yatong Yili Pills, Houzheng Pills, Xiongdan Jiuxin Pills, Liushen Pills and Niuhuang Xiaoyan Pills, were expected to use validated RCFs for the direct determination of three components. Taking Houzheng Pills as an example, the methodological validation of bufalin, cinobufagin and resibufogenin was carried out, and the recoveries of bufalin, cinobufagin and resibufogenin were 90.64%-106.1%. The obvious difference was not observed between the contents of bufalin and resibufogenin in 24 batches of preparation samples by QAMS and external reference method. In the tested samples, the content of bufalin, cinobufagin and resibufogenin were 1.27-2.61, 2.44-5.66 and 0.988-3.16 mg·g~(-1) in 10 batches of Liushen Pills samples. The contents of bufalin, cinobufagin and resibufogenin were 0.760-1.32, 1.35-2.39 and 0.600-1.55 mg·g~(-1) in 10 batches of Houzheng Pills samples from three manufacturers. The obtained data contribute to improving the quality standard of Bufonis Venenum-contained preparations, and they also provide some ideas for the application of QAMS in the quality evaluation and control of Chinese patent medicines.