An improved cell membrane chromatography method for the simultaneous screening of two epidermal growth factor receptor antagonists from radix scutellariae

A high‐expression epidermal growth factor receptor cell membrane chromatography using the silica gel with the average particle size of 3 μm as the stationary phase carrier coupled with high‐performance liquid chromatography and mass spectrometry was established for the online screening of epidermal growth factor receptor antagonists from Radix Scutellariae (Huang Qin in Chinese), a traditional Chinese medicine. In this study, the growth factor receptor cell membrane chromatography model using the smaller particle size carrier showed a higher efficiency for simultaneous screening baicalein, another one of the potential epidermal growth factor receptor antagonists from Radix Scutellariae extract besides wogonin, which was found in our previous work. The molecular docking result showed the occupancy site and binding mode of baicalein and wogonin with epidermal growth factor receptor tyrosine kinase were similar to gefitinib. The result of the assay for the in vitro inhibitory activity showed that baicalein and wogonin inhibited the growth of the high‐expression epidermal growth factor receptor cell in a dose‐dependent manner and even achieved a better inhibition effect than gifitinib in the low‐dosage range.     

Sodiation‐based in‐source collision for profiling of pyranocoumarins in Radix Peucedani (Qianhu): utility of sodium adducts' stability with in‐source collision

Full scan mode of liquid chromatography‐mass spectrometry equipped with an electrospray ionization source offers a chance on global detection of complicated components; however, the scan mode carries significant challenges in rapidly capturing information of analysts. Sodiation‐based in‐source collision was proposed here, as a technique for rapid detecting untargeted analytes in full scan analysis, which was based on the stability of sodium adducts and the nonselectivity of in‐source collision. Then the technique was applied to profile of angular‐type pyranocoumarins (APs) in Radix Peucedani, with full scan analysis performed at two specific in‐source collision energy: a high energy 50 V that is tolerated by the sodium adducts of APs, and a low energy 10 V, at which abundant adducts were offered. The spectra list of two average mass spectra was exported, and stable ions were selected based on the intensity ratio of standards at the two collision energy. Then 27 plausible [M + Na]⁺ m/z values of APs were acquired after filtering the fragment ion and isotope ions and validating with [M + NH₄]⁺. Eighty‐two APs finally were tentatively identified based on their accurate spectral data of MSⁿ, fragmentation rules, and elution order regardless of their absolute configuration, which included 25 reported APs from Peucedanum praeruptorum Dunn. The technique provided a novel application of sodium adduct in qualitative analysis. And it was valuable for rapidly capturing information of analytes in full scan analysis, not only for APs but also for other compounds that could form sodium adducts. Copyright © 2017 John Wiley & Sons, Ltd.     

Establishment of A431 cell membrane chromatography-RPLC method for screening target components from Radix Caulophylli

We describe here an analytical method of A431 cell membrane chromatography (A431/CMC) (CMC, cell membrane chromatography) combined with RPLC for recognition, separation, and identification of target components from traditional Chinese medicines (TCMs) Radix Caulophylli. The A431 cells with high expressed epidermal growth factor receptor (EGFR) were used to prepare the stationary phase in the CMC model. Retention fractions on the A431-CMC model were collected using an automated fraction collection and injection module (FC/I). Each fraction was analyzed by RPLC under the optimized conditions. Gefitinib and erlotinib were used as standard compounds to investigate the suitability and reliability of the A431 cell membrane chromatography-RPLC method prior to screening target component from Radix Caulophylli total alkaloids. The results indicated that caulophine and taspine were the target component acting on the epidermal growth factor receptor. This method could be an efficient way in drug discovery using natural medicinal herbs as a source of novel compounds.     

Rapid simultaneous determination of isoflavones in Radix puerariae using high-performance liquid chromatography-triple quadrupole mass spectrometry with novel shell-type column

A high-performance liquid chromatography (HPLC) coupled with triple quadrupole mass spectrometry (MS/MS) method was developed for rapid determination of 13 isoflavones in Radix puerariae. A novel shell-type column, namely Kinetex core-shell C(18) column (50 mm×2.1 mm id, 2.6 μm), and gradient elution were used during the analysis. The chromatographic peaks of 13 investigated compounds were identified by comparing their retention time and MS data with the related reference compounds. Multiple-reaction monitoring (MRM) was employed for the quantitative analysis with negative ionization mode. All calibration curves showed good linearity (r(2)>0.9990) within test ranges. The LOD and LOQ were lower than 0.017 and 0.873 μg/mL on column, respectively. The intra- and inter-day precisions for 13 analytes were <1.17 and 2.17%, respectively, and the recoveries were 93.1-104.4%. The validated method was applied for quantitative analysis of 13 isoflavones in 7 species of Radix puerariae. The result demonstrated that HPLC-MS/MS system with Kinetex column could be a promising analytical tool for the determination of isoflavones in traditional Chinese medicines, which is helpful for comprehensive evaluation of quality of R. puerariae.     

Liquid‐Chromatography Quantitative Analysis of 20 Amino Acids after Derivatization with FMOC‐Cl and Its Application to Different Origin Radix isatidis

We developed a simple, rapid and reliable method for determination of 20 common amino acids based on derivatization with 9‐fluorenylmethyl chloroformate (FMOC‐Cl) and RP‐LC/UV, this method was first introduced into quantitative analysis of amino acids. The amino groups of amino acids were trapped with FMOC‐Cl to form amino acid‐FMOC‐Cl adducts which can be suitable for LC‐UV. Chromatographic separation was performed on a C₁₈ column with a mobile phase gradient consisting of acetonitrile and sodium acetate solution. This method was shown to be sensitive for 20 common amino acids. In the intra‐day precisions assay, the range of RSDs was 3.21‐7.67% with accuracies of 92.34‐102.51%; for the inter‐day precisions assay, the range of RSDs was 5.82‐9.19% with accuracies of 90.25‐100.63%. The results also indicated that solutions of amino acids‐FMOC‐Cl can be kept at room temperature for at least 24 h without showing significant losses in the quantified values. The validated method was successfully applied to the determination of major four kinds of amino acids in R. isatidis samples (Arg, Pro, Met and Val). The total content of amino acids in different origin R. isatidis was 13.32‐19.16 mg/g. The differences between R. isatidis samples were large using HCA.     

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.     

活血逐瘀颗粒剂中芍药甙的含量测定

本文采用了薄层色谱法将复方制剂活血逐瘀颗粒中的芍药甙与其他成分分离，用紫外分光光度法测定芍药甙的含量，其平均回收率为９８．７４％，变异系数为１．４３％，本文方法抗干扰能力强，适合于中药复方制剂中芍药甙的定量分析。     

超临界CO_2萃取板蓝根靛玉红的工艺优化研究

采用正交试验法,对板蓝根靛玉红超临界CO2萃取工艺进行优化。选用萃取温度、萃取压力、萃取时间作为正交试验的3个因素,每个因素选3个水平,以提取物中靛玉红的含量来确定最佳提取条件,并且在最佳条件下确定所加夹带剂的最佳剂量。结果表明最佳工艺为:萃取温度45℃、萃取压力35MPa、萃取时间3h,夹带剂为75%的乙醇200mL。最佳工艺条件下提取靛玉红的得率为4.95μg/g,为传统提取方法的1.6倍。     

丹参超微粉碎前后丹参酮ⅡA的溶出行为比较

采用高效液相色谱法测定丹参超微粉体和常规粉体中丹参酮ⅡA体外溶出量和溶出速率.丹参超微粉体和常规粉体中丹参酮ⅡA体外溶出量无显著性差异,超微粉体的溶出速率较常规粉体明显增加.实验表明超微粉碎可明显提高丹参酮ⅡA溶出速率.