ABSTRACT
To simultaneously determine the contents of p-coumaric acid, chlorogenic acid, 5-caffeoylquinic acid, 4-caffeoylquinic acid, caffeic acid and ferulic acid in Imperatae Rhizoma concentrated granules, an ultra-high performance liquid chromatography (UPLC) with two internal references method (TIRM) was established and validated. Chromatographic separation was achieved on a ZORBAX RRHD Eclipse Plus C18 column (2.1 mm × 100 mm, 1.8 µm) using 1.7 mmol·L-1 oxalic acid in water and methanol as mobile phase. The flow rate was 0.4 mL·min-1 and the column temperature was set as 35 ℃. The relative correction factors (RCFs) of caffeic acid and ferulic acid using p-coumaric acid as internal reference were calculated and the RCFs of 4-caffeoylquinic acid and 5-caffeoylquinic acid were calculated using chlorogenic acid as the internal reference. The TIRM was fully validated for linearity, accuracy, repeatability, stability and recovery so that it could be compared with the external standard method (ESM). The RCFs of 5-caffeoylquinic acid, 4-caffeoylquinic acid, caffeic acid, and ferulic acid were 1.069, 1.022, 1.368, and 1.493, respectively. The TIRM and ESM were used to determine the contents of six ingredients in Imperatae Rhizoma concentrated granules from different manufacturers and the variation between results was within acceptable limits. In conclusion, the newly established TIRM allowed simultaneous determination of six ingredients (p-coumaric acid, chlorogenic acid, 5-caffeoylquinic acid, 4-caffeoylquinic acid, caffeic acid, ferulic acid) in Imperatae Rhizoma concentrated granules, providing support for the quality control of this traditional Chinese medicine.
ABSTRACT
OBJECTIVE: To identify two different characters of Imperatae Rhizoma on a molecular level. METHODS: A total of 30 samples of Imperatae Rhizoma were analyzed using DNA barcoding method based on ITS1, ITS2, psbA-trnH, matK, and rbcL genes. The DNA sequences were further used for site difference analysis. RESULTS: There is no site difference on psbA-trnH, matK, rbcL of two different characters of Imperatae Rhizoma, three key site differences on ITS1, and one key site difference on ITS2. The key site difference on ITS2 among two different characters of Imperatae Rhizoma can be recognized by the restriction enzyme HpyCH4III. CONCLUSION: ITS2 /ITS1 regions can accurately and efficiently identify two different characters of Imperatae Rhizoma, and PCR-RFLP analysis of the ITS2 region with HpyCH4III is a method to distinguish two different characters of Imperatae Rhizoma.