A simple method for HPLC retention time prediction: linear calibration using two reference substances

作者：Sun Lei; Jin Hong yu; Tian Run tao; Wang Ming juan; Liu Li na; Ye Liu ping; Zuo Tian tian; Ma Shuang cheng

来源：Chinese Medicine, 2017, 12(1): 16.

DOI：10.1186/s13020-017-0137-x

• Linear calibration using two reference substances
• Multi-component analysis
• RP-HPLC
• Relative retention
• Retention time
• Traditional Chinese medicines

BACKGROUND(#br)Analysis of related substances in pharmaceutical chemicals and multi-components in traditional Chinese medicines needs bulk of reference substances to identify the chromatographic peaks accurately. But the reference substances are costly. Thus, the relative retention (RR) method has been widely adopted in pharmacopoeias and literatures for characterizing HPLC behaviors of those reference substances unavailable. The problem is it is difficult to reproduce the RR on different columns due to the error between measured retention time (t\u003csub\u003eR\u003c/sub\u003e) and predicted t\u003csub\u003eR\u003c/sub\u003e in some cases. Therefore, it is useful to develop an alternative and simple method for prediction of t\u003csub\u003eR\u003c/sub\u003e accurately.(#br)METHODS(#br)In the present study, based on the thermodynamic theory of HPLC, a method named linear calibration using two reference substances (LCTRS) was proposed. The method includes three steps, procedure of two points prediction, procedure of validation by multiple points regression and sequential matching. The t\u003csub\u003eR\u003c/sub\u003e of compounds on a HPLC column can be calculated by standard retention time and linear relationship.(#br)RESULTS(#br)The method was validated in two medicines on 30 columns.(#br)CONCLUSION(#br)It was demonstrated that, LCTRS method is simple, but more accurate and more robust on different HPLC columns than RR method. Hence quality standards using LCTRS method are easy to reproduce in different laboratories with lower cost of reference substances.

• 出版日期2017-6-19