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

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.

High-throughput and sensitive screening of compounds with deoxyribonucleic acid-binding activity by a high-performance liquid chromatography-tandem mass spectrometry-fluorescence detection technique using palmatine as a fluorescence probe.

A high-throughput biochemical detection method based on the combination of high-performance liquid chromatography (HPLC), multiple-stage mass spectrometry (MS(n)) and DNA-binding activity assay was developed and validated for the simultaneous screening and identification of DNA-binding compounds in complex samples. Palmatine was used as a sensitive, nontoxic and environmentally friendly DNA fluorescence probe. HPLC fingerprints, ultraviolet absorption spectra, MS(n) fragments of components, and DNA-binding activity profiles could be simultaneously recorded during real-time analysis. Using the proposed method, 25 compounds were identified from Lophatherum gracile Brongn extracts, of which 18 were novel compounds first identified in these extracts. Nineteen compounds showed DNA-binding activity, most of which were flavone glycosides, with distinct dose-effect and structure-activity relationships. The method was validated and was proven to have a good linearity in the range of concentrations used in the study. The limit of detection was 0.2020nmol. Our study indicated that the proposed method was sensitive, accurate, precise and reliable to be used for simultaneous screening and identification of DNA-binding compounds in complex samples.

Simultaneous determination of 19 flavonoids in commercial trollflowers by using high-performance liquid chromatography and classification of samples by hierarchical clustering analysis.

The flowers of Trollius species, named Jin Lianhua in Chinese, are widely used traditional Chinese herbs with vital biological activity that has been used for several decades in China to treat upper respiratory infections, pharyngitis, tonsillitis, and bronchitis. We developed a rapid and reliable method for simultaneous quantitative analysis of 19 flavonoids in trollflowers by using high-performance liquid chromatography (HPLC). Chromatography was performed on Inertsil ODS-3 C18 column, with gradient elution methanol-acetonitrile-water with 0.02% (v/v) formic acid. Content determination was used to evaluate the quality of commercial trollflowers from different regions in China, while three Trollius species (Trollius chinensis Bunge, Trollius ledebouri Reichb, Trollius buddae Schipcz) were explicitly distinguished by using hierarchical clustering analysis. The linearity, precision, accuracy, limit of detection, and limit of quantification were validated for the quantification method, which proved sensitive, accurate and reproducible indicating that the proposed approach was applicable for the routine analysis and quality control of trollflowers.

Simultaneous qualitative and quantitative analysis of phenolic acids and flavonoids for the quality control of Apocynum venetum L. leaves by HPLC-DAD-ESI-IT-TOF-MS and HPLC-DAD.

A reliable method based on high performance liquid chromatography-diode array detector-electrospray ionization-ion trap-time of flight-mass spectrometry (HPLC-DAD-ESI-IT-TOF-MS) was developed for the identification of phenolic acids and flavonoids in Apocynum venetum L. leaves and its adulterant, Pocynum hendersonii (Hook. f.) Woodson leaves. A total of 21 compounds were identified or tentatively identified, including 4 phenolic acids and 17 flavonoids. 3-O-caffeoylquinic acid (3-CQA) and caffeic acid were detected for the first time in A. venetum leaves; 4-O-caffeoylquinic acid (4-CQA), 3-CQA, caffeic acid, quercetin-3-O-(6"-O-malonyl)-galactoside, quercetin-3-O-(6"-O-malonyl)-glucoside, kaempferol-3-O-(6"-O-malonyl)-glucoside, kaempferol-3-O-(6"-O-acetyl)-glucoside, and kaempferol-3-O-dihexoside were detected for the first time in P. hendersonii leaves. Cluster analysis was employed to analyze 24 batches of A. venetum leaves and 5 batches of P. hendersonii leaves collected from various regions in China. The analysis, which was based on the 21 compounds, indicated that profiles of these compounds were distinct between the two species, and among A. venetum leaf samples from different origins. 18 of these 21 compounds were selected as the markers and simultaneously analyzed by HPLC-DAD for the first time. The quantitative analytical method was validated and subsequently applied to the comprehensive quality evaluation of 24 batches of A. venetum leaves.