Preparative isolation of cyclolanostane-type saponins from Astragalus membranaceus bge. var. mongholicus (Bge.) Hsiao by TLC-MS/MS guided high-speed counter-current chromatography

Two cyclolanostane-type saponins, astragalosides I and II, were first identified by TLC-MS/MS in the ethyl acetate extract of the roots of Astragalus membranaceus Bge var. mongholicus (Bge.) Hsiao without chemical reference substances. They were then isolated by high-speed counter-current chromatography with a two-step two-phase solvent system of ethyl acetate-2-propanol-water (5:1:5, 50:1:50, v/v/v). The quantities of astragalosides I and II isolated from 1 g of the crude extract were 30.2 mg and 16.5 mg, respectively. Their purities were found to be over 95% by HPLC-ELSD analysis. Their chemical structures were confirmed by 1H and 13C nuclear magnetic resonance analysis.     

Two new saponins from the aerial part of Astragalus membranaceus var. mongholicus

Two new saponins named mongholicoside A (1) and mongholicoside B (2) were isolated from the aerial part of Astragalus membranaceus var. mongholicus. Their structures were determined by 1D and 2D NMR, ESI-MS techniques and chemical methods.     

The Different Resistance of Two Astragalus Plants to UV‐B Stress is Tightly Associated with the Organ‐specific Isoflavone Metabolism

In this work, the changes in isoflavone levels and the expression of genes involved in their biosynthesis were studied in two Astragalus by UPLC ‐MS and real‐time PCR after 10 days of UV ‐B treatment (λ _max = 313 nm, 804 J m⁻²). Isoflavones were significantly induced by UV ‐B irradiation. The influence might be activated by the regulation of these target genes. Our results indicate that (1) the resistance of Astragalus membranaceus might not be as good as Astragalus mongholicus in the enhanced UV ‐B radiation environment; (2) the enhanced accumulation of calycosin and calycosin‐7‐glucoside with UV ‐B treatment in roots of A. mongholicus might be derived from formononetin which is synthesized in the leaves; (3) the glycosylation process could be stimulated and activated by the enhanced UV ‐B radiation in both A. mongholicus and A. membranaceus . In other words, glycosylation of isoflavones might play a crucial role for two Astragalus plants in response to UV ‐B stress. Overall, this study offered a feasible elicitation strategy to understand the accumulation pattern of isoflavone in A. mongholicus and A. membranaceus , and also provided a reference for the changes in isoflavone levels of Astragalus in UV ‐B enhanced environment in the future.     

Preparative isolation and purification of two isoflavones from Astragalus membranaceus Bge. var. mongholicus (Bge.) Hsiao by high-speed counter-current chromatography

Two isoflavones, calycosin-7-O-beta-D-glycoside and formononetin-7-O-beta-D-glycoside, were separated from n-butanol extract of the root of Astragalus membranaceus Bge. var. mongholicus (Bge.) Hsiao by high-speed counter-current chromatography in two steps using two different solvent systems composed of ethyl acetate-ethanol-n-butanol-water (30:10:6:50, v/v) and ethyl acetate-ethanol-water (5:1:5, v/v). From 200 mg of crude extract, calycosin-7-O-beta-D-glycoside (12 mg) and formononetin-7-O-beta-D-glycoside (10 mg) were isolated at over 95% purity by HPLC analyses, and their structures were identified by MS, 1H NMR and 13C NMR.     

Preparative isolation and purification of isoflavan and pterocarpan glycosides from Astragalus membranaceus Bge. var. mongholicus (Bge.) Hsiao by high-speed counter-current chromatography

(3R)-(-)-7,2'-Dihydroxy-3',4'-dimethyl isoflavan-7-O-beta-D-glucopyranoside and (6aR, 11aR) 9,10-di-methoxypterocarpan-3-O-beta-D-glucopyranoside were separated from the ethyl acetate extract of the root of Astragalus membranaceus Bge. var. mongholicus (Bge.) Hsiao by high-speed counter-current chromatography (HSCCC). A two-phase system composed of ethyl acetate-ethanol-acetic acid-water (4:1:0.25:5, v/v) was selected by analytical HSCCC. Preparative HSCCC yielded, from 100 mg of the partially purified extract, 50 mg of isoflavan glycoside and 10 mg of pterocarpan glycoside each at over 95% purity by high-performance liquid chromatography (HPLC) analysis. Their structures were identified by MS, 1H NMR and 13C NMR.     

Determination of five toxic alkaloids in two common herbal medicines with capillary electrophoresis

Calycosin was purified from an ethyl acetate extract of the root of Astragalus membranaceus Bge. var. mongholicus (Bge.) Hsiao by high-speed counter-current chromatography. The separation was performed in two steps with a two-phase solvent system composed of n-hexane-chloroform-methanol-water (1:3:3:2, v/v). From 200 mg of the crude extract, 14.8 mg of calycosin was obtained at over 99% purity as determined by HPLC analysis, and its chemical structure was confirmed by MS, 1H and 13C nuclear magnetic resonance.     

高速逆流色谱法分离纯化黄芪中的芒柄花素和毛蕊异黄酮

采用高速逆流色谱法(HSCCC),以正己烷-氯仿-甲醇-水组成二相系统作为固定相与流动相,对黄芪的乙酸乙酯粗提 物进行了分离纯化。 结果发现:以正己烷-氯仿-甲醇-水(体积比为1.5∶3∶3∶2)组成的系统可以从黄芪的乙酸乙酯粗 提物中分离出毛蕊异黄酮,纯度可达95％以上,并可以初步纯化芒柄花素;接着用正己烷-氯仿-甲醇-水(体积比为4∶4∶5 ∶4)组成的系统进一步纯化芒柄花素,其纯度达95％以上。利用该方法,可以对中药黄芪中的异黄酮进行快速的分离和纯 化。     

Simultaneous analysis of seven astragalosides in Radix Astragali and related preparations by liquid chromatography coupled with electrospray ionization time-of-flight mass spectrometry

A method has been developed for the qualitative and quantitative analysis of pharmacologically active astragalosides isolated from several species of the genus Astragalus by high performance liquid chromatography coupled with electrospray ionization time-of-flight mass spectrometry. Seven astragalosides in Radix Astragali and their commercial pharmaceutical preparations were analyzed using the developed method. The extracted ion current chromatograms were obtained from the total ion current chromatogram using the m/z of [M+Na]+ ions produced by target compounds for peak determination. The limits of detection and limits of quantification were in the range of 0.10-0.22 ng and 0.22-0.52 ng in full scan mode, respectively. All calibration curves showed good linear regression (r2 > or = 0.9965) within the test range. The overall intra- and inter-day precision was less than 2.86% for peak area and the accuracy was higher than 92.9% on using ginsenoside I as internal standard. The assay was successfully utilized to analyze the major biologically active astragalosides in six samples of Astragalus membranaceus (Fisch.) Bge var. mongholicus (Bge.) Hsiao. and eight commercial preparations. The overall results demonstrate that this method is simple, selective, and suitable for the quality control of Chinese medicine and their preparation in the low nanogram range.     

Liquid chromatography-electrospray ionization mass spectrometry study of the flavonoids of the roots of Astragalus mongholicus and A. membranaceus

High-performance liquid chromatography-electrospray ionization mass spectrometry has been applied to analyze the flavonoids of Huangqi, the roots of Astragalus mongholicus and A. membranaceus. Eight flavonoids were identified as calycosin-7-O-beta-D-glucoside, calycosin-7-O-beta-D-glucoside-6"-O-malonate (2), ononin, (6aR,11aR)-3-hydroxy-9,10-dimethoxypterocarpan-3-O-bet a-D-glucoside, calycosin, (3R)-7,2'-dihydroxy-3',4'-dimethoxyisoflavan-7-O-beta-D-glucoside, formononetin-7-O-beta-D-glucoside-6"-O-malonate and formononetin by direct comparison with the isolated standards from Huangqi. The existence of (6aR,11aR)-3-hydroxy-9,10-dimethoxypterocarpan, (3R)-7,2'-dihydroxy-3',4'-dimethoxyisoflavan, astrapterocarpanglucoside-6'-O-malonate and astraisoflavanglucoside-6'-O-malonate was detected. This is the first report of flavonoid glycoside malonates in these two Astragalus species, and malonate 2 is a structurally completely identified new compound.     

Identification and determination of glycosides in tobacco leaves by liquid chromatography with atmospheric pressure chemical ionization tandem mass spectrometry

HPLC-atmospheric pressure chemical ionization MS (HPLC-APCI-MS) was used to screen and identify glycosides in tobacco leaf. MS/MS and MS3 and photodiode array (PDA) detection were also used in the characterization. A total of 12 glycosides were found and four of them were identified based on their abundant [M + H]+ ions, UV spectra, and MS/MS analysis and they are scopolin, rutin, quercetin-3-glycoside, and kaempferol-3-rutinoside. Analytical characteristics of the method were investigated. The contents of these glycosides were obtained and compared based on the relative peak area to the internal standard in seven kinds of tobacco leaf.     

混合型离子交换液相色谱-串联质谱法检测鸡蛋中10种氨基糖苷类药物残留

该研究系统地优化了样品前处理过程及仪器分析中影响氨基糖苷残留分析准确度与灵敏度的各主要因素,建立了鸡蛋中10种氨基糖苷类药物(链霉素、双氢链霉素、潮霉素B、卡那霉素、阿米卡星、妥布霉素、安普霉素、大观霉素、新霉素、庆大霉素)残留量的混合型离子交换液相色谱-串联质谱分析方法。样品经10 mmol/L乙酸铵缓冲溶液(含0.4 mmol/L EDTA和50 g/L三氯乙酸)超声提取,调节pH至6~7后,经PRiME HLB固相萃取柱富集净化,采用SIELC Obelisc R色谱柱分离,以乙腈和1.0%(v/v)甲酸水溶液(含1 mmol/L甲酸铵)为流动相进行梯度洗脱,在正离子、多反应监测模式下经串联质谱仪测定,外标法定量。该方法在5~200 μg/L质量浓度范围内线性关系良好,相关系数(r²)均大于0.99;方法的检出限(LOD, S/N≥3)为2~5 μg/kg,定量限(LOQ, S/N≥10)为5~10 μg/kg。在空白鸡蛋中进行LOQ、20 μg/kg、100 μg/kg 3个水平的加标回收实验,方法的平均回收率(n=6)为68.1%~111.3%,相对标准偏差为1.2%~12.3%。利用该方法对市售的20批次鸡蛋样品进行测定,均未检出目标物。本方法简单、灵敏、准确,可实现鸡蛋中10种氨基糖苷类药物残留的批量检测。     

Analysis of the Aconitine Alkaloids in Chuanwu by Electrospray Ionization ／ Tandem Mass Spectrometry

An electrospray ionization / tandem mass spectrometric (ESUMS/MS) method was developed for the simultaneous identification and analysis of three aconitine alkaloids [mesacontine (MA), hypaconitine (HA), and aconitine (A)] as intact molecules at low nanogram level in Chinese traditional medicine Chuanwu decoction as well as in human whole blood extract without chromatographic separation.     

Solvent effect in 1H NMR spectra of 3'-hydroxy-4'-methoxy isoflavonoids from Astragalus membranaceus var. mongholicus

Four 3'-hydroxy-4'-methoxy-isoflavonoids: calycosin-7-O-beta-D-glucopyranoside (1), calycosin (2), pratensein-7-O-beta-D-glucopyranoside (3), and pratensein (4) were isolated from Astragalus membranaceus var. mongholicus, among which compound 4 was obtained from this plant for the first time. The solvent effect obscuring (1)H signal patterns of B ring of compounds 1-4 was reported to avoid mis-assignments. Previously reported NMR data of compounds 1 and 2 were corrected based on 1D and 2D NMR experiments.     

Identification of certain chemical agents in complex organic solutions by gas chromatography/tandem mass spectrometry

Analytical methods that are used for the detection and identification of chemicals related to the Chemical Weapons Convention in complex organic solutions were developed. A matrix modified by the addition of complex diesel oil background compounds and interferences was used for the development of a gas chromatography-mass spectrometry or gas chromatography-tandem mass spectrometry (GC-MS or GC-MS/MS) method for unambiguous identification of the scheduled chemicals. Co-elution of diethyl N,N-dimethylphosphoramidate and ethyl N,N-dimethylphosphoramidocyanidate, which are not separated by GC, was identified by GC-MS. Although GC-MS can identify the compounds, GC-MS/MS identification has greater accuracy and sensitivity in the case of complex matrixes. Four scheduled chemicals were accurately and selectively identified against numerous interfering substances by GC-MS/MS. The fragmentation chemistry of these compounds using MS/MS analysis was investigated.     

Improvements in protein identification confidence and proteome coverage for human liver proteome study by coupling a parallel mass spectrometry/mass spectrometry analysis with multi-dimensional chromatography separation

Recently, matrix-assisted laser desorption ionization (MALDI) technique has been shown to be complementary to electrospray ionization (ESI) with respect to the population of peptides and proteins that can be detected. In this study, we tried to hyphenate MALDI-TOF-TOF-MS and ESI-QUADRUPOLE-TOF-MS with a single 2D liquid chromatography for complicated protein sample analysis. The effluents of RPLC were split into two parts for the parallel MS/MS detection. After optimizing the operation conditions in LC separation and MS identification, a total of 1149 proteins were identified from the global lysate of normal human liver (NHL) tissue. Compared to the single MS/MS detection, the combined analysis increased the number of proteins identified (more than 25%) and enhanced the protein identification confidence. Proteins identified were categorized and analyzed based upon their cellular location, biological process and molecular function. The identification results demonstrated the application potential of a parallel MS/MS analysis coupled with multi-dimensional LC separation for complicated protein sample identification, especially for proteome analysis, such as human tissues or cells extracts.     

MSPD-RRLC-UV/MS法同时检测果蔬中9种三嗪类除草剂残留量

基于基质固相分散萃取(MSPD)-快速高分离液相色谱紫外和质谱检测法, 测定了果蔬中9种三嗪类除草剂的残留量. 以浓度与色谱峰面积之间的线性关系定量, 保留时间结合质谱信息进行定性. 9种待测物在0.02～4.0 mg/kg范围内峰面积与浓度具有良好的线性关系, 相关系数(r2)在0.9996～0.9998之间; 方法的最低检出限(LOD)为0.002 mg/kg; 低、中和高3个浓度点的加标回收率介于75.2%～95.6%之间, 相对标准偏差(RSD)小于11%.     

不同品种的黄芪微量元素比较

采用原子吸收光谱测定了商品黄芪、梭果黄芪、金翼黄芪及云南栽培黄芪中Ｃｕ、Ｆｅ、Ｚｎ、Ｍｎ、Ｃａ、Ｍｇ、Ｃｏ、Ａｌ等元素,不同品种的黄芪中微量元素含量均有差异。     

Elucidation of the composition of bovine milk fat triacylglycerols using high-performance liquid chromatography-atmospheric pressure chemical ionisation mass spectrometry

Bovine milk fat triacylglycerols (TAGs) have been characterised using high-performance liquid chromatography-atmospheric pressure chemical ionisation mass spectrometry (HPLC-APCI-MS) and high-temperature gas chromatography-mass spectrometry (GC-MS). The complex nature of the fat meant that prefractionation was necessary to provide simpler fractions for more detailed molecular analyses. Silica thin-layer chromatography gave rise to two fractions, one of which contained predominantly butyric acid containing TAGs. Gel permeation chromatography (GPC) gave rise to 16 fractions, which were subsequently analysed using HPLC-APCI-MS. Twelve of the GPC fractions were also analysed by high-temperature GC-MS using a capillary column coated with a polarisable stationary phase. TAGs present in the fractions were correlated with those in the chromatogram of the whole milk fat through retention time comparison and the use of mass chromatograms. In total, 120 TAGs were identified.     

Identification of Ginsenosides in Panax quinquefolium by LC-MS

An HPLC-APCI-MS method for the identification of ginsenosides in Panax quinquefolium has been developed. HPLC-APCI-MS could effectively identify ocotillol, protopanaxadiol, protopanaxatriol and oleanane-type ginsenosides in a single MS experiment since [M-H]⁻ ions and characteristic thermal degradation ions of ginsenosides could be simultaneously observed under negative and positive ionization conditions. Nine ocotillol-type ginsenosides including 24(R)-pseudoginsenoside F₁₁ were firstly identified and a total of 30 ginsenosides were identified in Panax quinquefolium. The ginsenoside profile differences between Chinese and American P. quinquefolium were investigated by HPLC-APCI-MS.     

Identification and determination of the major constituents in Kai‐Xin‐San by UPLC‐Q/TOF MS and UFLC‐MS/MS method

In order to have overall chemical material information of Kai‐Xin‐San (KXS), the reliable ultra‐high‐performance liquid chromatography quadrupole time‐of‐flight mass spectrometer (UHPLC–Q‐TOF‐MS) and ultra‐fast liquid chromatography mass spectrometer (UFLC‐MS/MS) methods were developed for the identification and determination of the major constituents in KXS. Moreover, the UHPLC–Q‐TOF‐MS method was also applied to screen for multiple absorbed components in rat plasma after oral administration of KXS. The UHPLC–Q‐TOF‐MS method was achieved on Agilent 6520 Q‐TOF mass and operated in the negative ion mode. Good separation was performed on a ZORBAX Eclipse Plus C18 column with a gradient elution at a flow rate of 0.2 ml/min. A total of 92 compounds in KXS were identified or tentatively characterized based on their exact molecular weights, fragmentation patterns, and literature data. A total of 26 compounds including 23 prototype components and three metabolites were identified in rat plasma after oral administration of KXS. Then, 16 major bioactive constituents were chosen as the benchmark substances to evaluate the quality of KXS. Their quantitative analyses were performed by a triple quadrupole tandem mass spectrometer (MS/MS) operating in multiple‐reaction monitoring mode(MRM). The analysis was completed with a gradient elution at a flow rate of 0.4 ml/min within 35 min. The simple and fast method was validated and showed good linearity, precision, and recovery. Furthermore, the method was successful applied for the determination of 16 compounds in KXS. All results would provide essential data for identification and quality control of active chemical constituents in KXS. Copyright © 2016 John Wiley & Sons, Ltd.