Qualitative and quantitative determination of polyacetylenes in different Bupleurum species by high performance liquid chromatography with diode array detector and mass spectrometry.

Polyacetylenes are main toxic ingredients in Bupleurum longiradiatum, a poisonous plant that has ever been misused as substitutes for Chaihu (Bupleuri Radix). For the first time, a high-performance liquid chromatography method coupled with diode array detector and mass spectrometry (HPLC-DAD-MS) was developed for qualitative and quantitative analysis of nine polyacetylenes in Bupleurum species. All references, including two new polyacetylenes, were isolated from B. longiradiatum and purified using a semi-preparation HPLC chromatography. The analysis was performed on a TSKgel ODS-100V C18 column (3 µm, 150 mm x 4.6 mm i.d.) using a gradient system of acetonitrile and water, with diode array detection (254 nm). The method was validated for linearity, precision, accuracy, limit of detection and quantification. A total of 27 Bupleurum samples were examined with this method, which showed a great variety in the distribution and contents of the polyacetylenes. It was found that polyacetylenes (1-8) were the main ingredients in B. longiradiatum, while a few kinds of polyacetylenes (5-8) were also identified in B. smithii, B. smithii var. parvifolium, B. bicaule and B. angustissimum. However, no polyacetylenes (1-9) were detected in the authentic Chaihu samples and the other Bupleurum species. The results indicated that the toxic B. longiradiatum could readily be distinguished from other medicinal Bupleurum species based on the polyacetylene profiles, and the guidelines and quality control of polyacetylenes for Chaihu are useful. The acute toxicity of the ethanol extract of B. longiradiatum and its fractions was also investigated.

Identification and determination of the major constituents in traditional Chinese medicine Longdan Xiegan Pill by HPLC-DAD-ESI-MS.

A novel and sensitive HPLC-UV method has been developed for the simultaneous determination of twelve major compounds in Longdan Xiegan Pill. The chemical profile of the twelve compounds, including geniposidic acid (1), geniposide(2), gentiopicroside(3), liquiritin(4), crocin(5), baicalin(6), wogonoside(7), baicalein(8), glycyrrhizic acid (9), wogonin (10), oroxylin A (11) and aristolochic acid A (12), was acquired using high-performance liquid chromatography-diode array detector coupled with an electrospray tandem mass spectrometer (HPLC-DAD-ESI-MS). The analysis was performed on a Dikma Platisil ODS C18 column (250 mm × 4. 6 mm, 5 µm) with a gradient solvent system of acetonitrile-0. 1% aqueous formic acid. The validation was carried out and the linearities (r > 0. 9996), repeatability (RSD<1. 8%), intra- and inter-day precision (RSD< 1. 3%), and recoveries (ranging from 96. 6% to 103. 4%) were acceptable. The limits of detection (LOD) of these compounds ranged from 0.29 to 4. 17 ng. Aristolochic acid A, which is the toxic ingredient, was not detected in all the batches of Longdan Xiegan Pill. Furthermore, hierarchical cluster analysis was used to evaluate the variation of the herbal prescription. The proposed method is simple, effective and suitable for the quality control of this traditional Chinese medicine (TCM).

Polyacetylenes from Bupleurum longiradiatum.

Eight new polyacetylenes (1-8) and six known polyacetylenes were isolated from the entire parts of Bupleurum longiradiatum, a poisonous plant. The structures of the new compounds were determined by spectroscopic data interpretation. The absolute configuration of the known compound bupleurotoxin (9) was established by the modified Mosher's method. All isolates were also tested for their cytotoxicity against a human leukemia cell line (HL-60).

Fast determination of saikosaponins in Bupleurum by rapid resolution liquid chromatography with evaporative light scattering detection.

A rapid resolution liquid chromatography coupled with evaporative light scattering detection method was established for simultaneous determination of six saikosaponins, namely saikosaponin a, saikosaponin c, saikosaponin d, 6''-O-acetylsaikosaponin a, 3''-O-acetylsaikosaponin d and 6''-O-acetylsaikosaponin d in Bupleurum. The analysis was performed by using an Agilent Zorbax SB-C18 column (1.8 microm, 3.0 mm x 50 mm i.d.) at gradient elution of water and acetonitrile, and the saikosaponins were well separated within 12 min, which provided about a fourfold reduction in analysis time by comparing a conventional high performance liquid chromatography method. Owing to their low ultraviolet absorption, the saikosaponins were detected by evaporative light scattering. The standard curves to quantify the saikosaponins were constructed by the log-log plot, which showed good linearity with the correlation coefficients exceeding 0.9954. The detection limits and quantification limits ranged in 8.38-25.00 microg/mL and 25.13-45.00 microg/mL, respectively. Satisfactory intra-day and inter-day precisions were achieved with the relative standard deviation (R.S.D.) less than 6.58%, and the average recoveries obtained were in the range of 96.9-100.4%. In addition, MeOH-1.0% (v/v) pyridine was found to be the best the extraction solvent when compared to MeOH and MeOH-1.0% (v/v) ammonia water. A total of 23 samples of roots of Bupleurum from different species or locations were examined with this analytical method, and their chemical profiles provided information for the chemotaxonomic investigation. The results demonstrated that the analytical method is highly effective for the quality evaluation of Bupleurum species.

Characterization and identification of saikosaponins in crude extracts from three Bupleurum species using LC-ESI-MS.

A LC-diode array detection (DAD)-ESI-MS/MS method was established for the online characterization and identification of saikosaponins (SSs) from extracts of roots of Bupleurum scorzonerifolium Willd, B. marginatum var. stenophyllum and B. komarovianum. In ESI-MS/MS spectra of SSs, [M-H](- )ions were subjected to the cleavage of glycosidic bonds and produced Y type ions, which can be used to elucidate the structures of saccharide chains and aglycones. Fragmentation of aglycones provided mass information about their major substitutions. For three structural types of SSs, type III can be easily identified by their fragmentation behaviors; while type I and II often occur as isomers and they can be discriminated by their typical UV absorption data. The only sugar ring-cross cleavage corresponding to 76 Da took place at a furanose sugar moiety. As a result, more than 75 SSs, including eight novel compounds, were identified or tentatively characterized based on their UV and mass spectra and retention times. The approach established here allows a comprehensive analysis of the SSs in the genus of Bupleurum and will be helpful for quality control of the crude materials and their related preparations.

Characterization and identification of steroidal alkaloids in the Chinese herb Veratrum nigrum L. by high-performance liquid chromatography/electrospray ionization with multi-stage mass spectrometry.

Electrospray ionization multi-stage mass spectrometry (ESI-MS(n)) was performed to study the fragmentation behaviour of seventeen steroidal alkaloids (4 protoverine-type alkaloids, 10 germine-type alkaloids and 3 zygadenin-type alkaloids) from the Chinese herb Veratrum nigrum L. The MS(n) spectra of the [M+H](+) ions for steroidal alkaloids provided a wealth of structural information on the substituted groups. In positive ion mode, the three types of alkaloids showed very different characteristic ions: m/z 436 or 418 for protoverine-type alkaloids; m/z 438, 420 or 402 for germine-type alkaloids; m/z 440 or 422 for zygadenin-type alkaloids. These fragments were used to deduce their mass spectral fragmentation mechanisms. Furthermore, the primary compounds in methanolic extracts of the herb of Veratrum nigrum L. were investigated by using liquid chromatography (LC)/ESI-MS(n). As a result, 21 steroidal alkaloids (5 protoverine-type alkaloids, 14 germine-type alkaloids and 2 zygadenin-type alkaloids) were selectively identified from 27 determined peaks. Eleven compounds were unambiguously identified by comparing with standard compounds and ten compounds were tentatively identified or deduced according to their MS(n) data. Two of these compounds (xingangermine and deacetyl xinganveratrine) were found to be novel steroidal alkaloids. In addition, the chemical structures of two pairs of steroidal alkaloid isomers were deduced by comparing their fragment ions. Given the important structural information of known and unknown steroidal alkaloids in crude herbal extracts, this study is useful for identifying these types of steroidal alkaloids in crude materials rapidly and selectively.