Study On HPLC Fingerprints Of Several Animal Gelatins In Chinese Traditional Medicine

As a kind of Chinese traditional medicine, animal gelatin is obtained from animal skin, bone, nail, horn, and so on. The large similarity in structure and properties of gelatins from different origins makes their differentiation difficult. Now the quality control of animal gelatin is mainly depending on pharmacognosia methods which need more experiences and have a lower accuracy. The objective of this research was to develop the fingerprints of four animal gelatins including Dong E donkey-hide glue, Dong E-zhen donkey-hide glue, Tortoiseshell glue, Deerhorn glue with modern analytical technique, according to the liposoluble constituents. Then the fingerprints were evaluated by semblance analysis with the similarity evaluation system for chromatographic fingerprint of CTM and cluster analysis with the SPSS 13.0 program. In addition, a further study was carried out for donkey hide-glue to identify the spurious breed Dong E donkey hide-glue.An reversed phase high performance liquid chromatography (RP-HPLC) method has been developed and validated for the quality evaluation of TCM Dong E donkey-hide glue, Dong E-zhen donkey-hide glue, Tortoiseshell glue, Deerhorn glue by fingerprinting analysis. Meanwhile a novel sample preparation procedure has been designed which named liquid-liquid-liquid state extraction could simultaneously extract both the hydrophilic and lipophilic components for a single run HPLC system. Separations were achieved using a 250mm×4.6 mm, 5μm, reversed-phase column. The eluent system consisted of two components: water and acetonitrile with gradient elute. Column temperature was kept at 30℃, injection 50μL. Blank tests and gelatins were detected at 205 run as well as 200～400nm to gain the three dimensional pictures with the DAD. The results revealed that there were 19 common chromatographic peaks in Dong E donkey-hide glue, 18 common chromatographic peaks in Dong E-zhen donkey-hide glue, 20 common chromatographic peaks in Tortoiseshell glue and 17 common chromatographic peaks in Deerhorn glue. Semblances among 10 batches were 0.95, 0.93, 0.99, 0.95 of four kind of glues respectively. The developed method proved practicable and reliable for quality control.In this study, the identification of 10 batches of Dong E donkey-hide glue and 10 batches of spurious breed Dong E donkey hide-glue was studied based on the developed HPLC fingerprints of lipophilic components in donkey-hide glue. Semblance results showed that the semblances of 10 batches of spurious breed Dong E donkey hide-glue were 0.48～0.82 compared with reference chromatograph obtained by 10 batches of Dong E donkey hide-glue. And cluster analysis revealed the 10 batches of Dong E donkey hide-glue were gathered as a species; however, 10 batches of spurious breed Dong E donkey hide-glue were separated into two groups. Meanwhile determined the molecular mass of common peaks and special peaks in Donkey-hide glue, Tortoiseshell glue, Deerhorn glue by HPLC-MS. The identification can be based on the molecular mass of special peaks.The ratio of mobile phase, flow, column temperature, chromatographic columns from different companies or different batches were taken into consideration. The objective of this study was to make sure the practicable of the HPLC fingerprints. And the considered items followed to Chinese pharmacopeia. Our results revealed that the chromatographic fingerprints obtained by different ratio of mobile phase, flow, column temperature showed fine similarity (> 0.99). The R.S.D. of relative retention time and relative peak area of all common peaks in samples were estimated to be smaller than 2.0% and 6.0% respectively. In addition, this study evaluated five chromatographic columns from four companies. And the five chromatographs had good similarity (>0.99). From all of these, we gained the conclusion that the HPLC fingerprints had good tolerance and could be used for routine control.The developed method proved practicable and reliable for quality control of medicative glues with multivalent components in a complicated matrix. Our results revealed that the chromatographic fingerprint combining similarity evaluation could efficiently identify and distinguish medicative glues from different sources.