Determination of saponins and alkaloids in Caulophyllum thalictroides (blue cohosh) by high-performance liquid chromatography and evaporative light scattering detection

The roots of Caulophyllum thalictroides, traditionally used for the treatment of menstrual difficulties and as an aid in childbirth, contain saponins, which are considered to be responsible for the uterine stimulant effects, together with teratogenic alkaloids. An HPLC method has been developed which permits the determination of the triterpene saponins in the plant and also the separation of four alkaloids. The best results were obtained with a C-12 stationary phase using ammonium acetate buffer (pH 8.0) and acetonitrile as mobile phase. Owing to their low UV absorbance, the saponins were detected by evaporative light scattering, whereas the alkaloids were monitored by UV at 310 nm. The identities of the compounds were confirmed in an LC-MS experiment. Different plant samples and commercial products have been analysed using the described method, and remarkable qualitative and quantitative variations were revealed. Comparing the daily uptake of total saponins, a difference of greater than 100-fold was observed within the various products; the alkaloid content on the other hand was more uniform.     

Preparative separation of four triterpene saponins from radix astragali by high-speed counter-current chromatography coupled with evaporative light scattering detection

Four triterpene saponins, including astragaloside IV, astragaloside II, astragaloside I and acetylastragaloside I, were successfully isolated and separated by high-speed counter-current chromatography coupled with evaporative light scattering detection from Radix Astragali using stepwise elution with a pair of solvent systems composed of n-hexane:ethyl acetate:ethanol:water in volume ratios of 1:0.6:0.6:1 and 1:1:1:1 (by volume). The isolation produced 26.5 mg astragaloside IV, 28.2 mg astragaloside II, 48.7 mg astragaloside I and 17.6 mg acetylastragaloside I with purities of 97.6, 96.4, 98.8 and 96.8%, respectively, determined by high-performance liquid chromatography from 250 mg crude extract. The chemical structures of the isolated compounds were identified by UV, NMR and MS, and confirmed by authentic standards.     

Molar mass distribution of hydroxypropyl cellulose by size exclusion chromatography with dual light scattering and refractometric detection

Physico-chemical properties of cellulose derivatives are of considerable interest in many technical applications, for example, in the food and drug industry. Efficient and careful characterisation of these properties is thus highly desirable. In this study, two different size exclusion chromatography (SEC) systems, connected on-line either to a low-angle laser light scattering detector (LALLS) or to a multi-angle laser light scattering detector (MALLS), are employed for size characterisation of three batches of hydroxypropyl cellulose (HPC) from different manufacturers. All three samples turned out to have a weight average molar mass around 100,000 g/mol, but considerable differences concerning conformational properties were found. Two of the samples contained compact components, presumably aggregates of HPC, which were clearly detected by both SEC-systems. The third sample, obtained from another manufacturer, did not show any indication of aggregation. Both SEC-MALLS and SEC-LALLS are proven to be efficient techniques for characterisation of complex polysaccharides like HPC containing mixtures of solvated polymer chains, as well as micelle-like aggregates.     

A preparative method for isolation of fucoxanthin from Eisenia bicyclis by centrifugal partition chromatography

Introduction – Eisenia bicyclis (Kjellman) Setchell (Laminariaceae) is a common brown alga that inhabits around the coast of Korea, Japan and China. It contains fucoxanthin, a major carotenoid of brown algae which shows a variety of pharmaceutical functions. Objective – The aim of this investigation was the quantification and preparative isolation of fucoxanthin from fresh E. bicyclis using a new separation scheme, centrifugal partition chromatography (CPC). Methodology – The fucoxanthin fraction (Fuco fraction) was prepared by solvent partition method from the acetone extract of fresh E. bicyclis. Fuco fraction was used for CPC using a two‐phase solvent system of n‐hexane–ethyl acetate–ethanol–water (5:5:7:3, v/v/v/v). The flow rate of mobile phase was 2 mL/min with descending mode while rotating at 1000 rpm. The eluate was monitored at 410 nm. The content and structure of fucoxanthin in the CPC fraction were confirmed with HPLC, UV, APCI/MS and NMR spectra. Results – A preparative CPC yielded 20 mg of fucoxanthin (87% recovery from Fuco fraction) in a two‐step separation from 516 mg of Fuco fraction containing 4.59% fucoxanthin. The purity of the isolated fucoxanthin was about 81% in the first CPC step and over 98% in the second CPC step based on the calibration curve. The isolated fucoxanthin was identified as all‐trans‐fucoxanthin with APCI/MS (parent ion at m/z 641 [M + H ? H₂O]⁺) and ¹H, ¹³C and 2‐D NMR spectra. Conclusion – High‐purity fucoxanthin was successfully isolated from fresh E. bicyclis, suggesting further potential applications in the industrial use of this valuable carotenoid. Copyright © 2011 John Wiley & Sons, Ltd.     

Preparative isolation and purification of triterpene saponins from Clematis mandshurica by high-speed counter-current chromatography coupled with evaporative light scattering detection

Preparative high-speed counter-current chromatography (HSCCC) coupled with evaporative light scattering detection (ELSD) was successfully applied to the isolation and purification of four triterpene saponins from Clematis mandshurica by one-step separation. The solvent system was composed of ethyl acetate-n-butanol-ethanol-0.05% TFA (5:10:2:20, v/v). The purities of all the saponins obtained were better than 97%. The structures of the compounds were elucidated by the means of spectroscopic methods.     

Simultaneous quantification of five triterpenoid saponins in Clematis L. spp. by high-performance liquid chromatography with evaporative light scattering detection

A simple and accurate method involving high-performance liquid chromatography with evaporative light scattering detection was developed for the simultaneous determination of five triterpenoid saponin components in Clematis L. spp. for the first time. The analysis was performed on a Zorbax SB-C(18) column and gradient elution with acetonitrile and water with 0.1% formic acid was utilised. All the calibration curves exhibited good linear characteristics with correlation coefficients in the range from 0.9979 to 0.9997. The limits of detection and limits of quantification were less than 0.152 and 0.506 microg, respectively. The overall recoveries for the five analytes were between 91.3 and 99.5%. A total of 10 samples from Clematis L. spp. were analysed under optimised conditions and the chemical profiles provided information for the identification of botanical origin, the development of new medicinal resources and chemotaxonomic investigation.     

Preparative isolation and purification of chemical components from Aconitum coreanum by high-speed counter-current chromatography coupled with evaporative light scattering detection

Aconitum coreanum (Lèvl.) Rapaics (Guanbaifu in Chinese) is a widely used, centuries-old Chinese herb. A preparative high-speed counter-current chromatography (HSCCC) coupled with evaporative light scattering detection (ELSD) method was employed for isolation and purification of alkaloids from the crude extract of Aconitum coreanum (Lèvl.) Rapaics using ethyl acetate-n-butanol-methanol-0.2 m HCl (7:2:2:7, v/v) as a two-phase solvent system. Six alkaloids, including GFO, GFQ, GFZ, hetisinone, hetisine and GFAA, were obtained in one-step separation. The purity of these compounds was 97.6, 93.8, 91.8, 91.9, 96.2 and 91.1%, respectively.     

Identification and quantification of C21 steroidal saponins from Radix Cynanchi Atrati by high-performance liquid chromatography with evaporative light scattering detection and electrospray mass spectrometric detection

High-performance liquid chromatography coupled with electrospray tandem mass spectrometry (HPLC-ESI/MS) and evaporative light scattering detection (HPLC-ELSD), respectively, has been performed for the simultaneous identification and quantification of six C(21) steroid saponins, including cynanversicoside A, B, D, G, glaucoside C and glaucogenin C-3-O-beta-d-thevetopyranoside in Radix Cynanchi Atrati. The extraction of the C(21) steroidal saponins was performed using a B-811 Buchi Universal Extraction System in Warm Solvent Mode, and the analyte was concentrated by column chromatography before HPLC analysis. The chromatographic separation was performed on an Agilent Zorbax Extend C(18) analytical column efficiently using gradient elution with acetonitrile and water. The method was validated with acceptable linearities (r > 0.9991) and recoveries (98.2-101.3%). The limits of detection of the C(21) steroid saponins were from 0.2 microg for glaucogenin C-3-O-beta-d-thevetopyranoside to 0.5 microg for cynanversicoside B. The intra- and inter-day precisions of the method were evaluated and were less than 5.0%. The method was successfully used to analyse 20 batches of Radix Cynanchi Atrati. The content of C(21) steroid saponins in the plant material varied significantly from habitat to habitat, confirming the necessity to control the quality of Radix Cynanchi Atrati during its preparation and application in the clinic.     

Preparative separation of chlorogenic acid by centrifugal partition chromatography from highbush blueberry leaves (Vaccinium corymbosum L.)

Introduction – Blueberries (genus Vaccinium) have gained worldwide focus because of the high anthocyanin content of their fruits. In contrast, the leaves of blueberry have not attracted any attention, even though they contain large quantities of chlorogenic acid, a strong antioxidant compound. Objective – The aim of this investigation was the quantification and preparative isolation of chlorogenic acid (5‐caffeoylquinic acid, 5‐CQA) from blueberry leaves using a new separation scheme, centrifugal partition chromatography (CPC). Methodology – A water fraction containing a high concentration of 5‐CQA (14.5% of dry weight extract) was obtained by defatting a crude methanol extract from blueberry leaves. CPC was applied to isolate 5‐CQA from this water fraction using a two‐phase solvent system of ethyl acetate–ethanol–water at a volume ratio 4:1:5 (v/v/v). The flow‐rate of mobile phase was 2 mL/min with the ascending mode while rotating at 1200 rpm. The eluate was monitored at 330 nm. The structure of chlorogenic acid in the CPC fraction was confirmed with HPLC, UV, ESI/MS and NMR spectra. Results – The HPLC chromatogram showed that the fractions collected by CPC contained chlorogenic acid with 96% purity based on peak area percentage. The total amount of chlorogenic acid isolated from 0.5 g of a water fraction was 52.9 mg, corresponding to 10.6% of the water fraction. The isolated compound was identified successively as 5‐CQA with MS (parent ion at m/z 355.1 [M + H]⁺) and ¹H NMR spectra [caffeoyl moiety in the down field (δ 6.0–8.0 ppm) and quinic acid moiety in the up field (δ 2.0–5.5 ppm)]. Conclusion – 5‐CQA was successfully isolated from blueberry leaves by the CPC method in a one‐step procedure, indicating a further potential use for blueberry leaves. Copyright © 2010 John Wiley & Sons, Ltd.     

HSCCC-ELSD法分离纯化青葙子中的皂苷

连接有蒸发光散射检测器的高速逆流色谱仪首次成功的应用于制备和分离青葙子中的皂苷celosins A和B.二氯甲烷∶正丁醇∶甲醇∶水(4∶0.3∶3∶2)+0.5％冰醋酸作为洗脱溶剂系统.从半制备型HSCCC收集到的组分进行HPLC分析,可以得到:celosin A纯度为98.9％,celosin B的纯度为98.1％.这是高速逆流色谱仪首次被用于纯化青葙子中的皂苷,两个化合物的结构通过碳谱和质谱来确定.     

Determination of propafenone hydrochloride by flow‐injection analysis coupled with resonance light scattering detection

A simple, sensitive and rapid flow injection analysis (FIA) method with resonance light scattering (RLS) was described for the determination of propafenone (PPF). The method was based on the ion‐association reaction of 12‐tungstophosphoric acid (TP) with propafenone. In pH 1.0 acidic medium, TP reacted with PPF to form an ion‐associate complex, which resulted in a significant enhancement of RLS intensity. The maximum scattering peak was located at 340 nm, the RLS intensity was proportional to the concentration of PPF in the range 0.003–9.0 µg/mL, and the detection limit (3σ) of 1.0 ng/mL was obtained at a sampling rate of 60 samples/h. The feasible reaction conditions and FIA parameters for the system were optimized. The method proposed in this paper shows satisfactory reproducibility with a relative standard deviation (RSD) of 2.1% for 10 successive determinations of 2.0 µg/mL PPF. The present method had been successfully applied to the determination of PPF in serum samples and pharmaceutical samples. The results obtained were in agreement with the method used in the Chinese Pharmacopoeia. Copyright © 2008 John Wiley & Sons, Ltd.     

高效液相尺寸排阻色谱-多角度激光散射定量检测灭活禽流感病毒抗原中完整病毒颗粒

本研究旨在建立一种灭活禽流感病毒原料液中完整病毒颗粒准确定量的方法。针对直接采用高效液相尺寸排阻色谱法（high performance size exclusion chromatography,HPSEC）检测灭活禽流感病毒原液存在杂质干扰的问题,首先以H5N8型抗原为对象,分别考察了聚乙二醇（polyethylene glycol,PEG）沉淀和离子交换色谱法（ion exchange chromatography,IEC）进行预处理。在优化条件下,经DEAE FF阴离子交换层析纯化预处理,杂蛋白去除率为86.87%,病毒血凝回收率为100%。HPSEC分析预处理后的样品,8.5–10.0 min处色谱峰样品电泳检测显示主要为H5N8病毒蛋白,动态光散射分析平均粒径为127.7 nm,推测为完整病毒特征峰;在IEC预处理后的样品中加入抗体进行HPSEC检测,8.5-10.0 min处特征峰消失,显示IEC预处理有效去除了杂质干扰。通过将HPSEC与多角度激光散射技术（multi-angle laser scattering technique,MALLS）联用,可以准确获得样品中完整病毒颗粒的数量,且病毒颗粒数与色谱峰面积具有良好线性关系（R²=0.997）。建立的IEC预处理-HPSEC-MALLS检测方法应用于其他亚型（H7N9）、批次和浓度的病毒原液中完整病毒颗粒数的准确检测,均具有良好适用性,且重复性好,相对标准偏差（relative standard deviation,RSD）<5%,n=3。     

Speciation of Al in human serum by convective-interaction media fast-monolithic chromatography with inductively coupled plasma mass spectrometric detection

A new analytical procedure using anion-exchange separation support based on convective-interaction media (CIM) was developed for the speciation of Al in human serum. The separation of proteins was performed on a weak anion-exchange CIM diethylamine (DEAE) fast-monolithic disk. To prevent co-elution of low molecular mass (LMM) Al species with high molecular mass (HMM) Al compounds on CIM disk serum proteins were first separated from LMM-Al species by the use of size exclusion chromatography (SEC). For this purpose 1 mL of serum was injected onto SEC (Superdex 75 HR 10/30) column. Isocratic elution using 0.05 M TRIS-HCl+0.03 M NaHCO(3) was applied and separation of proteins was followed by UV detection at 278 nm. It was experimentally proven that proteins were eluted in 5.5 mL peak that was collected into a polyethylene cup. A 0.1 mL of the sample aliquot was then injected onto the CIM DEAE disk. The separation of serum proteins was obtained in 10 min by applying linear gradient elution from 100% buffer A (0.05 M TRIS-HCl+0.03 M NaHCO(3)) to 100% buffer B (A+1M NH(4)Cl) and followed by UV detection at 278 nm. Separated Al species were detected on-line by inductively coupled plasma mass spectrometry (ICP-MS). Well-resolved protein peaks were obtained. It was experimentally proven that 90+/-3% of Al in spiked serum of renal patient was eluted under the transferrin peak. The proposed speciation procedure removes LMM-Al species and enables reliable determination of the concentration and composition of Al bound to proteins by CIM DEAE-ICP-MS when the concentration of Al in serum is higher than 5 ng mL(-1). In comparison to chromatographic columns CIM disks enable faster separation and simpler manipulation during cleaning procedure and coupling to ICP-MS.     

应用高效体积排阻色谱偶联多角度激光散射鉴定猪圆环病毒2型灭活疫苗及病毒样颗粒疫苗抗原

本文旨在建立基于高效体积排阻色谱(high-performance size-exclusion chromatography,HPSEC)偶联多角度激光散射仪(multi-angle laser light scattering,MALLS)的猪圆环病毒2型(porcine circovirus type 2,PCV2)疫苗抗原检测方法。以纯化的PCV2灭活病毒及病毒样颗粒(virus-like particles,VLP)为参照,对4家生产企业的2种PCV2灭活病毒疫苗(a、b)及VLP疫苗(c、d)破乳后进行HPSEC-MALLS检测及分子量分析;结合PCV2抗原检测卡、Western blotting和透射电子显微镜(transmission electron microscope,TEM),鉴定了特征色谱峰;考察了方法的重复性和检测线性。结果表明,两家企业生产的PCV2灭活病毒疫苗破乳液水相经HPSEC分离,在保留时间约13.3 min处出现抗原特征峰;MALLS计算该色谱峰分子量分别为2.61×10⁶(±4.34%) Da和2.40×10⁶(±2.51%) Da。两种VLP疫苗也在13.3 min处出现抗原特征峰,分子量分别为2.09×10⁶(±2.94%) Da和2.88×10⁶(±11.85%) Da,接近PCV2的理论分子量;同时在保留时间约11.4 min处也出现色谱峰,经检测分子量为4.37×10⁶(±0.42%) Da,TEM表征显示为VLP二聚体。取疫苗d和PCV2 VLP纯品进行重复检测,抗原色谱峰面积的RSD(n=3)均小于1.5%,重复性好;将PCV2 VLP纯品梯度稀释检测,VLP及其多聚体的色谱峰面积与浓度均呈良好的线性关系,R²分别为0.999及0.997,能够满足定量及多聚体含量分析。该方法有望成为一种准确、高效的PCV2疫苗的体外评价方法,用于质量评价与提升。     

Isolation of kappa-carrageenan oligosaccharides using ion-pair liquid chromatography--characterisation by electrospray ionisation mass spectrometry in positive-ion mode

Oligo-kappa-carrageenans participate as elicitors in the cell-cell recognition process in marine plants. Analytical methods can be usefully applied to gain insight into the biochemistry of these biological processes. Therefore, enzymatically digested oligomers of kappa-carrageenans have been separated and isolated on a Spherisorb ODS1 (250 x 4 mm i.d., particle size 5 microm) column using ion-pair liquid chromatography coupled with an evaporative light scattering detector. Heptylamine (5 mM, pH4) has been selected as the ion-pairing agent and MeOH as the organic modifier in a gradient mode. Overloading the column with 1mg of the mixture, the chromatographic mechanism presented adequate stability. The mobile phase of each isolated oligomer was evaporated and the residue was infused into an electrospray ionisation mass spectrometry (ESIMS) in positive-ion mode with 4:1 MeCN-water as mobile phase. Each ESIMS spectrum presented ions consisting of the oligomer attached with a number of heptylammonium ions depending on the molecule size. In addition, the different m/z values permitted direct detection of the oligomers in ESIMS positive-ion mode. The analytical method developed separated the oligomers up to dotriacontasaccharide.