Multi-components determination by single reference standard and HPLC fingerprint analysis for Lamiophlomis rotata Pill

Abstract A validated HPLC method was developed to evaluate the quality of Lamiophlomis rotata Pill combining the multi-components analysis by single reference standard with HPLC fingerprint analysis. Five bioactive components (shanzhiside methyl ester, loganin, 8-O-acetylshanzhiside methyl ester, forsythoside B and luteolin-7-O-β-D-glucopyranoside) were selected as markers to control the quality of L. rotata Pill. The results revealed that the chromatographic fingerprint method coupled with multi-components analysis provides an effective and feasible way to determine the components in L. rotata Pill.

Up to now, the lack of chemical reference standards always makes the multi-components determination by external standard method (eSM) impossible in traditional Chinese medicine (TCM). An alternative method named single standard to determine multi-components (SSDMC), which needs only a single reference standard for multi-components determination, can reduce the cost and difficulty of multi-components determination compared with eSM, especially when the component is unstable and hard to purify from the plant. (Hou et al. 2011;Du et al. 2014;Hou et al. 2014). In addition, Chiata et al. (2014) have applied the fingerprint in determination of chemical variability. Sun and Liu (2007), Sun et al. (2013), Liu et al. (2015) have developed the systematically quantified fingerprint method (SQFM), which controls the quality of TCM using the qualitative similarity (S m ), the quantitative similarity (P m ) and the variation coefficient (α).

Validation of the SSDMC method
each analyte had good linear relationship over the concentration range from 6.115 to 114.5 μg . ml −1 . The correlation coefficients of all the calibration curves were more than 0.999. The intra-day RSD values of the five components were less than 2.3%, and the calculated mean inter-day RSD values were less than 2.9%. RSD values in repeatability were less than 2.8%. The recovery was between 98.5-101.2% with the RSD values less than 3.1%.

Calculation of conversion factor and relative retention time
8-O-acetyl shanzhiside methyl ester was the internal reference substance. The conversion factors for shanzhiside methyl ester, loganin, forsythoside B and luteolin-7-O-β-D-glucopyranoside were 1.108, 1.034, 1.791 and 0.452. The values of relative retention time were 0.381, 0.734, 1.145 and 1.332, respectively. Meanwhile, the conversion factor and the relative retention time were in good agreement with each other in varying conditions. After the analysis of paired t-test (p = 0.541 > 0.05) by SPSS 21.0, it was estimated that there was no significant difference between the SSDMC method and the eSM method. Thus, it can be proved that the SSDMC method was suggested as a valid, accurate and feasible method for the quality control of L. rotata Pill.

HPLC fingerprint and similarity analysis
The acceptable values of S m , P m and α were corresponding to grade 4 in Table 1. S m of all 11 samples were more than 0.9 and α were less than 0.2, indicating that the whole sample has high similarity with the reference fingerprint. However, P m of sample S1-S5 and S7 were less than 75%, indicating an unacceptable result of these samples. The result was also consistent with the contents of five components. Therefore, it can be indicated that the SQFM could be applied as an auxiliary method for the quality control of L. rotata Pill.

Conclusion
The SSDMC method coupled with SQFM was firstly developed to quantify the five components in L. rotata Pill. It provides an accurate, feasible and reliable method for the quality control of L. rotata Pill.

Supplementary material
experimental details relating to this article are available online, alongside Figure S1-S2 and Table S1-S4.

Disclosure statement
No potential conflict of interest was reported by the authors.