An on-line analytical approach for detecting haptens in Shuang-huang-lian powder injection

- An HPLC-DAD-ESI-IT-TOF-MS-BSA-FLD system was established for the first time.
- Fourteen of 35 identified major compounds in SHLIP were BSA-binding active.
- The structure-activity relationships of different types of compounds were studied.
- The hapten properties of 10 BSA-binding active compounds was validated by ELISA.

Shuang-huang-lian powder injection (SHLPI) is a traditional Chinese medicine injection (TCMI) frequently used in the clinical treatment of faucitis, bronchitis, and other viral and bacterial infections of upper respiratory tract. However, its allergenic reactions, being the main adverse effects (AEs) of SHLPI, have been a serious problem of its clinical safety. This problem has not been solved due to short of methods for detecting haptens in complex TCMIs. In this study, an on-line high-performance liquid chromatography-diode-array detector-mass spectrometry combined with bovine serum albumin-fluorescence detector (HPLC-DAD-MS-BSA-FLD) system was established for the first time, validated and applied for identification of haptens in SHLPI. Fourteen of 35 identified compounds showed BSA binding activity, and they were six flavonoids, six caffeoylquinic acids (CQAs), and two phenylethanoid glycosides. The structure-activity relationships of 10 active components were studied, and their ability of sensitization together with that of two CQAs were further verified by ELISA assay. It was found that 10 compounds had sensitization, and flavonoids showed stronger sensitizability than CQAs while the diCQAs were slightly stronger than caffeoylquinic acids. The system was validated using 3-CQA as a positive control, and was proved to have good reproducibility, stability, precision (RSD < 0.1%) and linearity (R2 > 0.9993). This online system is fast, sensitive and efficient for screening haptens in traditional Chinese medicine injection (TCMI), provides a new approach to reveal the chemical basis of haptens in TCMIs.