Improved performance of comprehensive two-dimensional HPLC separation of traditional Chinese medicines by using a silica monolithic column and normalization of peak heights

Performance of comprehensive two-dimensional liquid chromatography system is greatly improved than we reported previously by using a silica monolithic column as for the second dimensional separation. Due to the increase of the elution speed on the second dimensional monolithic column, the first dimensional column efficiency and analysis rate can be greatly improved as comparing with conventionally second dimensional column. The developed system was applied to analysis of methanol extraction of two umbelliferae herbs Ligusticum chuanxiong Hort. and Angelica sinensis (Oliv.) Diels by using CN column as for the first dimensional separation and a silica monolithic ODS column for the second dimensional separation, and the obtained three-dimensional chromatograms were treated by normalization of peak heights with the value of the highest peak or setting a certain value using a software written in-house. It was observed that much more peaks for low-abundant components in TCM extract can clearly be detected here than we reported before, due to the large difference for the amount of components in TCMs' extract. With the above improvements in separation performance and data treatment, totally about 120 components in methanol extraction of Rhizoma chuanxiong and 100 in A. sinensis were separated with UV detection within 130 min. This result meant that both the number of peaks detected increase twice but the analysis time decease twice if comparing with the previously reported result. (c) 2005 Published by Elsevier B.V.

Spatial and temporal variations of microcystins in hepatopancreas of a freshwater snail from Lake Taihu

In this paper, spatial and temporal variations of three common microcystins (MC-RR, MC-YR, and MC-LR) in the hepatopancreas of a freshwater snail (Bellamya aeruginosa) were studied monthly in two bays of Lake Taihu. Microcystins (MCs) concentration in hepatopancreas was quantified by liquid chromatography-mass spectrometry (LC-MS). The MCs concentrations in hepatopancreas were higher at Site 1 than those at other sites, which was in agreement with the changes of intracellular MCs concentrations in the water column. There was a significant correlation between MCs concentrations in the hepatopancreas and that in the seston, suggesting that spatial variances of MCs; concentrations in hepatopancreas among the five sites were due to spatial changes of toxic Microcystis cells in the water column. PCCA indicates that in addition to Microcystis, other factors (e.g., water temperature) also substantially affected the accumulation of MCs in hepatopancreas of the snail. (C) 2008 Published by Elsevier Inc.

Cytotoxicity evaluation of three pairs of hexabromocyclododecane (HBCD) enantiomers on Hep G2 cell

Hexabromocyclododecanes (HBCDs) are additive brominated flame retardants mainly used in plastics and textiles. At the present time, these compounds are found in almost all environmental and human samples. In order to evaluate the environmental safety and health risk of HBCDs, the enantiomerically pure alpha-, beta-, and gamma-HBCD were prepared using high performance liquid chromatography (HPLC) on a PM-P-CD column and the cytotoxicities of their enantiomers were evaluated in Hep G2 cells. Results from the 3-(4,5-dimethylthioazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), resazurin reduction and lactate dehydrogenase (LDH) release assays showed a good agreement that the order of cytotoxicity was gamma-HBCD >= beta-HBCD > alpha-HBCD, and that significantly lower cell viability and higher LDH release were observed in all (+)-enantiomers ((+) alpha-, (+) beta- and (+) gamma-HBCD) than the corresponding (-)-forms ((-) alpha-, (-) beta- and (-) gamma-HBCD). Additionally, the formation of reactive oxygen species (ROS) induced by these HBCD enantiomers were detected. The positive correlation between the LDH release and ROS formation demonstrated that the toxic mechanism might be mediated by oxidative damage. These results suggest that environmental and human health risks of HBCDs must be evaluated at the level of individual enantiomers. (C) 2008 Published by Elsevier Ltd.

Optimization of RP-HPLC analysis of low molecular weight organic acids in soil

RP-HPLC analysis for low molecular weight organic acids in soil solution has been optimized. An Atlantis (TM) C-18 column was used for the analyses. An optimal determination for eleven organic acids in soil solution was found at room temperature (25 degrees C) and 220 nm detection wavelength, with a mobile phase of 10 mM KH2PO4 -CH3OH (955, pH 2.7), a flow rate of 0.8 mL/min and 10 mu L sample size. The detection limits ranged 3.2-619 ng/mL, the coefficients of variation ranged 1.3-4.6%, and the recoveries ranged 95.6-106.3% for soil solution with standard addition on the optimal conditions proposed.

The optimization of high performance liquid chromatographic method for analysis of trace microcystins in natural water bodies

The ratio of methanol., water and trifluoroacetic acid ( TFA) was regulated to change the polarity and the pH of the rinse solution and the eluent, so as to improve the high performance liquid chromatography HPLC) detection method for trace microcystines (MCs) in natural water bodies. The results showed that 40 % similar to 45 % methanol-water solution containing 0. 1 % TFA could get good effects on the rinse of impurity, and 70% methanol-water solution containing 0. 1% TFA could elute all the MCs in solid phase extraction ( SPE) cartridge ( C-18), In this way. it is suggested that, in analysis of environmental samples with high concentration of impurity, impurity should be washed with 40% similar to 45% methanol-water solution containing 0. 1% TFA, and MCs should be eluted with 70% similar to 100% methanol-water solution containing 0. 1% TFA.

千金子和暖地大叶藓的化学成分研究

本论文由三部分共4 章组成。第一部分阐述了大戟科大戟属传统中药千金子（Euphorbia lathyris L.）化学成分、生物学活性以及千金子化学成分的HPLC、UPLC-MS、GC-MS 分析成果。第二部分介绍了民族药材暖地大叶藓（Rhodobryum giganteum (Schwaegr.) Par.）的化学成分研究和结构鉴定。第三部分概述了大戟属 植物中大环二萜酯的研究进展。 第一章包括1－3 节。在第1, 2 节中报道了千金子（Euphorbia lathyris L.）95% 乙醇提取物的化学成分分离鉴定。我们采用正、反相硅胶柱层析、重结晶等各种分离方法，凭借MS、IR、NMR、X-ray 等现代仪器手段,从中共分离鉴定22 个化合物。其中8 个是高活性化合物前体-续随子烷型大环二萜及3 个巨大戟烷型二萜，还有香豆素、生物碱、甾体等类型，其中完成对5 个大环二萜酯构型的确认，对2 个二萜酯构型进行了修正。第3 节中介绍对千金子化学成分的细胞毒性、α-葡萄糖苷酶抑制活性、P-gp 表达抑制活性的模型筛选结果。 第二章包括3 节，第1 节报道不同产地千金子高效液相色谱定量分析结果。第2 节介绍了各大环二萜酯的HPLC-MS/MS 的分析结果，并且对其质谱裂解规律、UPLC-MS 快速鉴定方法做了进一步讨论。第3 节介绍了千金子挥发油成分分析。采用传统水蒸气蒸馏方法提取千金子中的挥发油，并经气相色谱－质谱联用（GC-MS）技术共分离鉴定出 49 个化合物，占挥发油总量的90.48％。 第三章包括1, 2 两节，第1 节报道了暖地大叶藓化学成分。采用正、反相硅胶，凝胶柱层析等各种分离方法和MS、IR、NMR 等解析手段，共分离鉴定10个化合物，其中一个环肽化合物为新化合物。第2 节介绍了暖地大叶藓挥发油成分分析，共分离鉴定出 52 个化合物，占其挥发油总量的85.67％。 第四章概述了大戟科大戟属植物中大环二萜酯的研究进展。 This dissertation consists of three parts. In the first part, it is elaborated that the phytochemical investigation from the traditional Chinese medicine: seeds of Euphorbia lathyris L.. Biological activity and constituents analysis by HPLC、UPLC-MS、GC-MS were reported. In the second part, it is discussed that the chemical constituents were isolated and identificated from minority nationalitical herb－Rhodobryum giganteum (Schwaegr.) Par.. The third part is a review about the progress of studies on macrocyclic diterpenes from Euphorbia. The first part is composed of 1－3 sections. The section 1and 2 is focused on the isolation and identification of chemical constituents from seeds of E. lathyris. 22 compounds were isolated from the seeds of E. lathyris. by isolation methods of column chromatography (silica gel, including reversed phase) and recrystallisation on the basis of spectroscopic methods including IR, MS, NMR and X-ray. In 8 macrocyclic and 3 ingenane diterpenes, the relative configuration of 5 macrocyclic diterpenes were confirmed, in which 2 were amended. In the third section, cell cytotoxic activity, restraining activity of α-Glucosidase and multidrug resistance (MDR) reversing activity about P-gp were tested. 5 potential revsering reagents were found. The second part is composed of 1－3 sections. In first section it is described that the quality of the chemical constituents of E. lathyris from 5 sources , which were analyzed by high-performance liquid chromatography. In addition, the fractionation rules of some macrocyclic diterpenes were discussed and Ultra Performance Liquid Chromatography/ electrospray ionization mass spectrometry (UPLC-MS) was applied for quick determination of compounds in the second section. In the third section, chemical analysis of the essential oil from seeds of E. lathyris by GC-MS were reported. The essential oil from the seeds of E. lathyris L. in Sichuan was extracted by steam distillation and 49 compounds were isolated and identified from the essential oil by gas chromatography-mass spectrometer (GC-MS). These compounds are accounted for 90.46% of the total essential oil. The second part, including section 4 and 5, is about the phytochemical investigation of R. giganteum. In the former section, ten compounds were isolated and identified. Among them, a new peptide was characterized by spectroscopic analysis including IR, MS and NMR. In the other section, 52 compounds were isolated and identified from the essential oil by gas chromatography-mass spectrometer (GC-MS). These compounds are accounted for 85.67% of the total essential oil. The third part is a review about the progress of studies on macrocyclic diterpenes from Euphorbia.

Using HPLC retention parameters to estimate fish bioconcentration factors of organic compounds

Reversed-phase high performance liquid chromatography (RP-HPLC) was employed to develop predictive models for fish bioconcentration factors (BCF) of organic compounds. Estimation of BCF from RP-HPLC retention parameters on octadecyl-bonded silica gel (ODS), cyanopropyl-bonded silica gel (CN), and phenyl-bonded silica gel (Ph) columns were investigated. The results show that, for a set of compounds belonging to different chemical classes, the CN stationary phase is the best one among the three columns and better than n-octanol/water model for BCF estimation. A multi-column RP-HPLC model, using the retention parameters on the CN and Ph columns as the variables of multiple linear regression equations, was further evaluated to estimate BCF of organic compounds belonging to different chemical classes, and the results show that the multi-column RP-HPLC model is better than that of any single RP-HPLC column for BCF estimation.

Separation of enantiomers by nanoliquid chromatography and capillary electrochromatography using a bonded cellulose trisphenylcarbamate stationary phase

A cellulose trisphenylcarbamate-bonded chiral stationary phase was applied to nano-liquid chromatography (nano-LC) and capillary electrochromatography (CEC) with nonaqueous and aqueous solutions as the mobile phases. Several chiral compounds were successfully resolved on the prepared phase by nano-LC. The applicability of nonaqueous CEC on a cellulose derivative stationary phase was investigated with the organic solvents methanol, hexane, 2-propanol, and tetrahydrofuran (THF) containing acetic acid, as well as triethylamine as the mobile phases. Enantiomers of warfarin and praziquantel were baseline-resolved with plate numbers of 82 300 and 38 800 plates/m, respectively, for the first eluting enantiomer. The influence of applied voltage, concentration of nonpolar solvent, apparent pH, and buffer concentration in the mobile phase on the electroosmotic flow (EOF) and the mobility of the enantiomers was evaluated. Enantioseparations of traps-stilbene oxide and praziquantel were also achieved in aqueous CEC with plate numbers of 111 100 and 107 400 plates/m, respectively, for the first eluting enantiomer. A comparison between nonaqueous CEC and aqueous CEC based on a cellulose trisphenylcarbamate stationary phase was discussed. Pressure-assisted CEC was examined for the chiral separation of praziquantel and faster analysis with high enantioselectivity was acquired with the proper pressurization of the inlet vial.

Multivariate optimization in micellar electrokinetic capillary chromatography

The optimization of the organic modifier concentration in micellar electrokinetic capillary chromatography (MECC) has been achieved by a uniform design and iterative optimization method, which has been developed for the optimization of composition of the mobile phase in high performance liquid chromatography. According to the proposed method, the uniform design technique has been applied to design the starting experiments, which can reduce the number of experiments compared with traditional simultaneous methods, such as the orthano design. The hierarchical chromatographic response function has been modified to evaluate the separation quality of a chromatogram in MECC. An iterative procedure has been adopted to search the optimal concentration of organic modifiers for improving the accuracy of retention predicted and the quality of the chromatogram. Validity of the optimization method has been proved by the separation of 31 aromatic compounds in MECC. (C) 2000 John Wiley & Sons, Inc.

Separation of ternary systems of hydrophilic ionic liquid with miscible organic compounds by RPLC with refractive index detection

A rapid and simple analytical method was developed for the simultaneous and quantitative determination and separation of hydrophilic imidazolium ionic liquids (ILs) (1-butyl-3-methylimidazolium chloride, [C(4)mim]Cl; 1-hexyl-3-methylimidazolium chloride, [C(6)mim]Cl; 1-octyl-3-methylimidazolium chloride, [C(8)mim]Cl; 1-allyl-3-methylimidazolium chloride, [Amim]Cl; or 1-allyl-3-methylimidazolium bromide, [Amim]Br) with miscible ethyl acetate and EtOH and their mixtures using reverse phase liquid chromatography coupled with refractive index detection (RPLC-RI). The influence of 60 to 100% (volume percentage) methanol in the mobile phase on the IL systems ([C(4)mim]Cl, [C(6)mim]Cl, [C(8)mim]Cl, [Amim]Br, or [Amim]Cl)-ethyl acetate-EtOH was investigated.

Facile separation and determination of Aconitine alkaloids in traditional Chinese medicines by CE with tris(2,2 '-bipyridyl) ruthenium(II)-based electrochemi-luminescence detection

A facile CE method coupled with tris(2,2'-bipyridyl) ruthenium(ll)-based electrochem iluminescence [Ru(bpy)(3)(2+)] detection was developed for simultaneous determination of Aconitum alkaloids, i.e., hypaconitine (HA), aconitine (AC), and mesaconitine (MA) in baseline separation. The optimal separation of these Aconitum alkaloids was achieved in a fused-silica capillary column (50 cm x 25 mu m id) with 30 mM phosphate solution (pH 8.40) as running buffer at 12 kV applied voltage. The three alkaloids can be determined within 10 min by a single run. The calibration curves showed a linear range from 2.0 x 10(-7) to 2.0 x 10(-5) M for HA, 3.4 x 10(-7) to 1.7 x 10(-5) M for AC, and 3.8 x 10(-7) to 1.9 x 10(-5) M for MA. The RSDs; for all analytes were below 3.01%. Good linear relationships were found with correlation coefficients for all analytes exceeding 0.993. The detection limits were 2.0 x 10(-8) M for HA, 1.7 x 10(-7) M for AC, and 1.9 x 10(-7) M for MA under optimal conditions. This method was successfully applied to determine the three alkaloids in Aconitum plants.

Direct tris(2,2 '-bipyridyl)ruthenium(II) electrochemiluminescence detection of polyamines separated by capillary electrophoresis

Capillary electrophoresis (CE) with tris(2,2'-bipyridyl) ruthenium (II) (Ru(bpy)(3)(2+)) electrochemiluminescence (ECL) detection technique was developed for the analysis of four polyamines (putrescine (Put), cadaverine (Cad), spermidine (Spd), and spermine (Spm)) analysis. The four polyamines contain different amine groups, which have different ECL activity. There are several parameters which influence the resolution and ECL peak intensities, including the buffer pH and concentrations, separation voltage, sample injection, electrode materials, and Ru(bpy)(3)(2+) concentrations. Polyamines are separated by capillary zone electrophoresis in an uncoated fused-silica capillary (50 cm x 25 mum (ID) filled with acidic phosphate buffer (200 mmol/L phosphate, pH 2.0) - 1 mol/L phosphoric acid (9:1 v/v) and a separation voltage of 5 kV (25 muA), with end-column Ru(bpy)(3)(2+) ECL detection. A 5 mmol/L Ru(bpy)(3)(2+) solution plus 200 mmol/L phosphate buffer (pH 11.0) is added into the reagent reservoir. The calibration curve is linear over a concentration range of two or three orders of magnitude for the polyamines. The analysis time is less than 25 min. Detection limits for Put and Cad are 1.9 x 10(-7) mol/L and 7.6 x 10(-9) mol/L for Spd and Spm, respectively.

Capillary electrophoresis with electrochemiluminescence detection of procyclidine in human urine pretreated by ion-exchange cartridge

This paper describe a Ru(bpy)(3)(2+) based electrochemiluminescence (ECL) method to detect procyclidine in human urine following separation by capillary electrophoresis (CE). An ECL detection cell was designed for post-column addition of Ru(bpy)(3)(2+). Parameters affecting separation and detection were optimized, leading to a detection limit of 1 x 10(-9) mol/l in an on-capillary stacking mode. For application in urine, a cartridge packed with slightly acidic cation-exchange resin was used to eliminate the matrix effects of urine and improve the detection sensitivity. Extraction recovery was nearly 90%.

Correlation analysis of the structures and gas-liquid chromatographic retention indices of amines

Quantitative structure-retention relationship(QSRR) was studied for amines to gas-liquid chromatography on three stationary phases of different polarities with the topological indices A(m) (A(m1), A(m2), A(m3)) and gravitational index GI. The algorithm of "Leaps and Bounds" was performed for selection of the variables. And the multi-regression and the quasi-Newton neural networks were employed for the calculation with better results.