Isolation and purification of series bioactive components from Hypericum perforatum L. by counter-current chromatography

Counter-current chromatography (CCC) combined with pre-separation by ultrasonic solvent extraction was successively used for the separation of series bioactive compounds from the crude extract of Hypericum perforatum L. The petroleum ether extract was separated by the solvent system of n-heptane-methanol-acetonitrile (1.5:0.5:0.5, v/v) and n-heptane-methanol (1.5:1, v/v) in gradient elution, yielding a phloroglucinol compound, hyperforin with HPLC purity over 98%. The ethyl acetate extract was separated by using the solvent system composed of hexane-ethyl acetate-methanol-water (1:1:1:1 and 1:3:1:3, v/v) in gradient through both reverse phase and normal phase elution mode, yielding a naphthodianthrone compound, hypericin with HPLC purity about 95%. The n-butanol extract was separated with the solvent system composed of n-butanol-ethyl acetate-water (1:4:5 and 1.5:3.5:5, v/v) in elution and back-extrusion mode, yielding two of flavones, rutin and hyperoside, with HPLC purity over 95%. HPLC-MS, reference sample and UV spectrum were selectively used in separation to search for target compounds from HPLC-DAD profiles of different sub-extracts. The structures of isolated compounds were further identified by ESI-MS, 1HNMR and 13CNMR.     

Combined microwave-assisted extraction and high-speed counter-current chromatography for separation and purification of xanthones from Garcinia mangostana

A microwave-assisted extraction (MAE) method is presented for the extraction of xanthones, α-mangostin and γ-mangostin from Garcinia mangostana. The MAE conditions including extraction temperature, liquid/solid ratio, extraction time and concentration of ethanol were optimized with an orthogonal test, and 5 g sample was extracted with the optimized conditions. The crude extraction of MAE was successfully isolated and purified by high-speed counter-current chromatography (HSCCC) with a two-phase solvent system composed of petroleum ether-ethyl acetate-methanol-water (0.8:0.8:1:0.6, v/v) in one-step separation. The separation yielded 75 mg of α-mangostin at 98.5% purity, and 16 mg of γ-mangostin at 98.1% purity from 360 mg crude extract of G. mangostana in less than 7h. The purity of the two xanthones was determined by HPLC. Their structures were further identified by ESI-MS, (1)H NMR and (13)C NMR.     

Effective recovery of poly‐β‐hydroxybutyrate (PHB) biopolymer from Cupriavidus necator using a novel and environmentally friendly solvent system

This work demonstrates a significant advance in bioprocessing for a high‐melting lipid polymer. A novel and environmental friendly solvent mixture, acetone/ethanol/propylene carbonate (A/E/P, 1:1:1 v/v/v) was identified for extracting poly‐hydroxybutyrate (PHB), a high‐value biopolymer, from Cupriavidus necator. A set of solubility curves of PHB in various solvents was established. PHB recovery of 85% and purity of 92% were obtained from defatted dry biomass (DDB) using A/E/P. This solvent mixture is compatible with water, and from non‐defatted wet biomass, PHB recovery of 83% and purity of 90% were achieved. Water and hexane were evaluated as anti‐solvents to assist PHB precipitation, and hexane improved recovery of PHB from biomass to 92% and the purity to 93%. A scale‐up extraction and separation reactor was designed, built and successfully tested. Properties of PHB recovered were not significantly affected by the extraction solvent and conditions, as shown by average molecular weight (1.4 × 10⁶) and melting point (175.2°C) not being different from PHB extracted using chloroform. Therefore, this biorenewable solvent system was effective and versatile for extracting PHB biopolymers. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:678–685, 2016     

Application of analytical and preparative high-speed counter-current chromatography for the separation of Z-ligustilide from a crude extract of Angelica sinensis

Z-Ligustilide was separated and purified from the traditional Chinese medicinal plant Angelica sinensis by high-speed counter-current chromatography (HSCCC). Analytical HSCCC was first used for the systematic selection of the two-phase solvent system. Preparative HSCCC separation was performed with a two-phase solvent system composed of petroleum ether (60-90 degrees C)-ethanol-water at an optimum volume ratio of 10:17:10 (v/v). A total of 38 mg Z-ligustilide at 98.8% purity was obtained in one step from 200 mg crude extract as determined by HPLC analysis. The structure of the target compound was identified by electron impact ionisation mass spectrometry.     

高速逆流色谱分离制备蛹虫草中虫草素和N6-(2-羟乙基)-腺苷

采用高速逆流色谱（HSCCC）技术从蛹虫草子实体粗提物中分离制备高纯度虫草素和N⁶-(2-羟乙基)-腺苷。利用高效液相色谱（HPLC）测定目标产物在溶剂体系中的分配系数,优化HSCCC分离虫草素和N⁶-(2-羟乙基)-腺苷的溶剂体系,确定了以乙酸乙酯-正丁醇-1.5%氨水（1:4:5,V/V/V）为HSCCC的两相溶剂体系,并运用此溶剂体系,上相为固定相,下相为流动相,主机转速850r/min,流动相流速为1.5mL/min,检测波长为254nm条件下进行分离制备,在250min内从200mg蛹虫草子实体粗提物中一步分离得到10.8mg纯度99%的虫草素和6.1mg 纯度98%的N⁶-(2-羟乙基)-腺苷。该方法简便、快速,为虫草素和N⁶-(2-羟乙基)-腺苷的大量制备建立了基础。     

Preparative isolation and purification of harpagoside from Scrophularia ningpoensis hemsley by high-speed counter-current chromatography

The bioactive component harpagoside was successfully separated from the crude extract of Scrophularia ningpoensis Hemsley by one-step purification using high-speed counter-current chromatography (HSCCC). A two-phase solvent system containing n-butanol:ethyl acetate:water (1:9:10) was selected following consideration of the partition coefficient of the target compound. A 276 mg quantity of the crude extract was loaded onto a 250 mL HSCCC column and yielded 11 mg harpagoside at over 97% purity. The chemical structure of harpagoside was determined by HPLC-ESI/MS and 1H-NMR.     

Modeling the performance of pilot-scale countercurrent chromatography: scale-up predictions and experimental verification of erythromycin separation

Biosynthesis of polyketide antibiotics, such as erythromycin A (EA), can result in the formation of analogues of the main compound that are chemically and structurally extremely similar. The large-scale purification of these antibiotics by conventional high-performance liquid chromatography (HPLC) can be prohibitively expensive due to the large volume of both solvent and adsorbent required. This study examines the feasibility of using a novel pilot-scale countercurrent chromatography (CCC) machine as an alternative to HPLC. CCC is a low-pressure (typically <4000 kN m(-2)) liquid-liquid chromatographic technique that allows the separation of solutes on the basis of their partitioning between two immiscible liquid phases. The effects of mobile phase flow rate, column rotational speed, and sample injection volume on the attainable yield and purity of EA were investigated. Our results show that, at a mobile phase flow rate of 40 mL min(-1), a rotational speed of 1200 rpm, and an injection volume of 100 mL (10 g total erythromycin), EA could be satisfactorily fractionated with a purity of approximately 92% (w/w) and a recovery yield of approximately 100% (w/w). The total solute throughput was estimated to be 0.41 kg day(-1). More importantly, we demonstrated simple and predictive linear scale-up of the CCC separation based on data obtained from a single laboratory-scale CCC chromatogram, and verified this experimentally. The retention time and peak width of the target compound at the pilot scale could be predicted to within 4% for operation at a range of mobile-phase flow rates and injection volumes. This predictable nature of CCC separations, unlike HPLC methods, can greatly reduce process development times and enable a complete process-scale operating scenario to be planned.     

One‐step separation and purification of rupestonic acid and chrysosptertin B from Artemisia rupestris L. by high‐speed counter‐current chromatography

Introduction – Artemisia rupestris L. is a well‐known traditional Chinese medicinal plant in Xinjiang. Rupestonic acid is the main active ingredient of A. rupestris L., and has been chosen as a ‘marker compound’ for the chemical evaluation or quality control of A. rupestris L. and its products. Although HSCCC separation method was developed before, the separation was performed with two steps using the same solvent system, which were time‐consuming and waste of the solvents. Objective – To develop a simple HSCCC method for the separation and purification of rupestonic acid in a single run. Methodology – The measurement of partition coefficient (K) was introduced to select the two‐phase solvent system. The simple HSCCC method was established according to the selected solvent system for separation and purification of rupestonic acid. The purity of target compound was test by HPLC and the structure was identified by MS, ¹H NMR and ¹³C NMR. Results – A total of 72.3 mg of rupestonic acid and 53.5 mg of chrysosptertin B with over 95% purity were yielded from 500 mg extracts of Artemisia rupestris L. in one‐step separation. Conclusion – The rupestonic acid was separated in a single run by HSCCC. Copyright © 2009 John Wiley & Sons, Ltd.     

微生物发酵产辅酶Q10的高速逆流色谱法分离纯化

本文首次将高速逆流色谱法应用于微生物发酵液提取物中辅酶Q10的分离纯化,建立了一套可用于其制备分离的逆流色谱溶剂体系正庚烷－乙睛－二氯甲烷(12：7：3.5, v/v/v)。500mg发酵液粗提物经一步制备分离,可得到绝对纯度在98％以上辅酶Q10130mg。比较表明,该方法较传统的硅胶柱层析和结晶相结合的纯化方法在产物纯度、回收率及产率等方面都有一定的优势。     

Predictable and linear scale-up of four phenolic alkaloids separation from the roots of Menispermum dauricum using high-performance counter-current chromatography

This paper describes how distribution ratios were used for prediction of peak elution in analytical high-performance counter-current chromatography (HPCCC) to explore the method for separation and purification of bioactive compounds from the roots of Menispermum dauricum. Then important parameters related to HPCCC separations including solvent systems, sample concentration, sample loading volume and flow rate were optimized on an analytical Mini-DE HPCCC and finally linearly scaled up to a preparative Midi-DE HPCCC with nearly the same resolutions and separation time. Four phenolic alkaloids were for the first time obtained by HPCCC separation with a two-phase solvent system composed of petroleum ether–ethyl acetate–ethanol–water (1:2:1:2, v/v). This process produced 131.3 mg daurisolin, 197.1 mg dauricine, 32.4 mg daurinoline and 14.7 mg dauricicoline with the purity of 97.6%, 96.4%, 97.2% and 98.3%, respectively from 500 mg crude extract of the roots of M. dauricum in a one-step separation. The purities of compounds were determined by high-performance liquid chromatography (HPLC). Their structures were identified by electrospray ionization mass spectrometer (ESI-MS) and nuclear magnetic resonance (NMR).     

A novel design of simulated moving bed (SMB) chromatography for separation of ketoprofen enantiomer

A simulated moving bed (SMB) chromatography system is a powerful tool for preparative scale separation, which can be applied to the separation of chiral compound. We have designed our own lab-scale SMB chromatography using 5 HPLC pumps, 6 stainless steel columns and 4 multi-position valves, to separate a racemic mixture of ketoprofen in to its enantiomers. Our design has the characteristics of the low cost for assembly for the SMB chromatography and easy repair of the unit, which differs from the designs suggested by other investigators. It is possible for the flow path through each column to be independently changed by computer control, using 4 multi-position rotary valves and 5 HPLC solvent delivery pumps. In order to prove the operability of our SMB system, attempts were made to separate the (S)-ketoprofen enantiomer from a ketoprofen racemic mixture. The operating parameters of the SMB chromatography were calculated for ketoprofen separation from a batch chromatography experiment as well as by the triangle theory. With a feed concentration of 1 mg/mL, (S)-ketoprofen was obtained with a purity of 96% under the calculated operating conditions.     

Validation of a UHPLC-FLD analytical method for the simultaneous quantification of aflatoxin B1 and ochratoxin a in rat plasma, liver and kidney

A rapid and simple method for the simultaneous quantification of AFB1 and OTA in rat plasma, liver and kidney by UHPLC-FLD has been successfully validated according to the following criteria: selectivity, stability, linearity, precision, accuracy, recovery, robustness and limits of quantification and detection. The extraction method, calibration curves and chromatographic conditions are common for the three matrices. Plasma and homogenized tissue samples (100 μL) were extracted with acetonitrile:formic acid mixture (99:1) (300 μL). Chromatographic separation was performed with a mixture of water and acetonitrile:methanol (50:50), both acidified with 0.5% of formic acid using a gradient profile. The method avoids the use of immunoaffinity columns and allows reduction of sample and solvent volumes as well as toxic wastes. The detection is based on a photochemical reaction which enhances the AFB1 response without affecting the OTA signal before reaching the fluorescent detector. The mycotoxin recovery for each matrix was very efficient, between 93% and 96% for AFB1 and between 94% and 96% for OTA. For both mycotoxins the LOQs were 2μg/L in plasma and 8μg/kg in liver and kidney. The method has successfully been applied to rat samples after a single oral administration of a mixture of AFB1 and OTA and it could be a useful tool in toxicokinetic and toxicological studies.     

Purification of betulinic acid from Eugenia florida (Myrtaceae) by high-speed counter-current chromatography

A high yield of betulinic acid (up to 17% from the ethanolic extract) was found in the leaves of Eugenia florida collected in south-eastern Brazil, making this species a potential commercial source of the title compound. Extracts of E. florida were subjected to solvent partition, and rapid high-speed counter-current chromatography (HSCCC) was applied to the semi-crude extracts to afford betulinic acid in high purity. The mobile and stationary phases were derived from the two-phase solvent system composed of n-hexane-ethyl acetate-methanol-water (10:5:2.5:1). The developing solvent system (stationary and mobile phases) for optimum HSCCC separation was chosen by dissolving the fraction to be chromatographed in the proposed solvent mixture and determining the amount of betulinic acid in each phase by densitometric TLC. Purified betulinic acid was characterized by 13C-NMR, GC-MS and co-injection of its methyl ester with standards in GC-FID. The HSCCC technique is commonly employed to isolate triterpene glycosides, but is applied in this study to an aglycone.     

Isolation of four high-purity dammarane saponins from extract of Panax notoginseng by centrifugal partition chromatography coupled with evaporative light scattering detection in one operation

Introduction – Centrifugal partition chromatography (CPC), as a continuous liquid–liquid partition chromatography with no solid support matrix, combined with evaporative light scattering detection (ELSD) was employed for systematic separation and purification of weak‐chromophoric saponins from a highly valued and important traditional Chinese herbal medicine, Panax notoginseng. Objective – To separate and isolate high‐purity saponins from extract of Panax notoginseng using CPC‐ELSD with a simple and low toxicity solvent system. Methodology – Samples were preparaed by extracting the root material with acetone, treated with n‐butanol and then freeze‐dried. CPC‐ELSD was applied in the separation and detection of notoginsenoside and ginsenosides from extract of Panax notoginseng using a solvent system composed of ethyl acetate–n‐butanol–water (1:1:2, v/v/v). The saponins were analysed and identified by their retention time with high‐performance liquid chromatography (HPLC) coupled with ELSD, as well as electrospray ionisation tandem mass spectrometry (ESI‐MSⁿ ) in the negative and positive ion modes with the authentic standards. Results – A total of 9.6 mg of notoginsenoside R₁, 67.8 mg of ginsenoside Rg₁, 2.3 mg of Re and 286.5 mg of Rb₁ were purified from 487.2 mg of n‐butanol extract of P. notoginseng. The purities of obtained saponins in a single run were assessed to be over 98% by HPLC‐ELSD. Conclusion – CPC‐ELSD was proved to be a very fast and efficient tool for separation of high‐purity dammarane saponins. Copyright © 2011 John Wiley & Sons, Ltd.     

Preparative isolation and purification of geniposide from gardenia fruits by centrifugal partition chromatography

The iridoid glycoside, geniposide was purified by centrifugal partition chromatography (CPC) with a two-phase solvent system composed of ethyl acetate:isopropanol:water (3:2:5, v/v) from an 80% methanolic extract of fruits of Gardenia jasminoides. Preparative CPC yielded 56.2 mg of geniposide in a one-step separation of 500 mg of extract, with a purity of 95% as determined by HPLC. Isolated geniposide was identified from its 1H-NMR, 13C-NMR and MS spectra.     

高速逆流色谱纯化二氢杨梅素

本实验采用高速逆流色谱纯化二氢杨梅素。结果表明：选用溶剂系统为石油醚：乙酸乙酯：甲醇：水(1：3：2：2),在转速为800r／min,进样量为200mg、进样速度为1．5ml／min的条件下,能很好地将80％左右的粗二氢杨梅素提纯到90％以上,进样速度、进样量对二氢杨梅素的分离有一定影响。     

Screening method for benzodiazepines and hypnotics in hair at pg/mg level by liquid chromatography-mass spectrometry/mass spectrometry

A procedure is presented for the screening of 16 benzodiazepines and hypnotics in human hair by LC-MS/MS (alprazolam, 7-aminoclonazepam, 7-aminoflunitrazepam, bromazepam, clobazam, diazepam, lorazepam, lormetazepam, midazolam, nordiazepam, oxazepam, temazepam, tetrazepam, triazolam, zaleplon and zolpidem). The method involves decontamination of hair with methylene chloride, hair cut into small pieces, incubation of 20 mg in phosphate buffer (pH 8.4) in the presence of 1 ng diazepam-d5 used as internal standard, liquid-liquid extraction with diethyl ether/methylene chloride (10/90) and separation using liquid chromatography-tandem mass spectrometry. The limits of quantification for all benzodiazepines and hypnotics range from 0.5 to 5 pg/mg using a 20-mg hair sample. Linearity is observed from the limit of quantification of each compound to 200 pg/mg (r2 > 0.99). Coefficients of variation measured on six points and at two concentrations (10 and 50 pg/mg) range from 5 to 20% for all drugs but one. Extraction recovery, measured at the two same concentrations range from 32 to 76%. These results were found suitable to screen for 16 benzodiazepines in hair and detect them at very low concentrations, making this method suitable to monitor single dose.     

Development and validation of a method for determination of corticosteroids in pig fat using liquid chromatography-tandem mass spectrometry

A new method was developed to determine five corticosteroids (prednisolone, methylprednisone, flumethasone, dexamethasone, and methylprednisolone) in pig fat samples by liquid chromatography-tandem mass spectrometry (LC-MS/MS) utilizing an optimized liquid-liquid extraction (LLE) and subsequent solid-phase extraction (SPE) for sample clean-up. In the sample preparation, a pig fat sample was dissolved in n-hexane and then extracted into the methanol-water (50/50, v/v) mixture that enabled extraction of only medium polar corticosteroids and not the non-polar components of matrices. This extract was cleaned-up and concentrated on polymeric Oasis HLB SPE cartridge. Separation involved isocratic solvent (methanol-acetate buffer, pH 5.4) and Ascentis Express Fused-Core type HLPC column; reduced the analysis time to 7.5 min, which is at least two times lower than time required for separation using conventional techniques. Other advantage of the developed method is the minimized ion suppression of LC-MS/MS analysis, which allowed detection of corticosteroids in sub μg/kg. Method was validated according to European Union (EU) Commission Decision 2002/657/EC. Measured parameters such as selectivity, linearity, recovery, within-laboratory reproducibility, decision limit, and detection capability satisfied the EU Directive. Ranges of mean recoveries and within-laboratory reproducibility were 81-100% and 8.0-20.5%, respectively. Decision limits were calculated in the range from 4.5 to 11.9 μg/kg for MRL compounds and varied from 0.1 to 0.2 μg/kg for banned substances. Limit of detections (LODs), calculated as three time signal-to-noise ratio, were in the range of 0.1-0.3 μg/kg.     

Combinative application of pH-zone-refining and conventional high-speed counter-current chromatography for preparative separation of alkaloids from Stephania kwangsiensis

A method which involves the combination of pH-zone-refining counter-current chromatography (pH-zone-refining CCC) and conventional high-speed counter-current chromatography (HSCCC) was established for the preparative separation of alkaloids from the crude extracts of Stephania kwangsiensis. pH-zone-refining CCC was first performed with the solvent system composed of n-hexane-ethyl acetate-methanol-water (3:7:1:9, v/v), where triethylamine (10 mM) was added to the upper organic stationary phase as a retainer and hydrochloric acid (5 mM) to the aqueous mobile phase as an eluter. From 2.0 g of crude extract, 370 mg of sinoacutine and 600 mg of a mixture of three other alkaloids were obtained. Then, the mixture was further separated by conventional HSCCC with the solvent system composed of n-hexane-ethyl acetate-methanol-water (7:3:6:4, v/v), yielding 42 mg of (-)-crebanine, 50 mg of (-)-stephanine and 30 mg of l-romerine from 150 mg mixture of three other alkaloids, respectively. The purities of the four compounds were all over 98% as determined by HPLC, and the chemical structures of the four compounds were confirmed by positive ESI-MS and (1)H NMR data. Results of the present study successfully indicated that this method was efficient for the preparative separation of alkaloids from natural plants.     

Preparation of highly pure daidzin on oligo-β-cyclodextrin-Sepharose HP and investigation of chromatographic behavior of isoflavones by molecular docking

A novel method using column chromatography on oligo-β-cyclodextrin-Sepharose HP for the preparation of high purity daidzin from crude soybean samples was proposed in this work. The isoflavone of daidzin in sample A and B was purified under the optimum mobile phase composed of methanol/acetic acid/water=20.0/8.0/72.0 (v/v/v) at a flow-rate of 1.0 mL/min in one-step operation with a purity of 97.2% and 98.1%, a recovery of 95.3% and 96.3% respectively. The target products in isolated fraction were detected and characterized by HPLC analysis and ESI-MS spectrum. Preparative separation with sample-load of up to 2.42 mg/mL medium gave satisfactory results for daidzin with the purities over 97% and recoveries approximately 90%. Molecular docking simulations were utilized to help demonstrate the inclusion complexation between β-cyclodextrin and the isoflavones in samples through inclusion geometries and calculations of the binding energies. The prediction of the elution orders with AUTODOCK and SURFLEX-DOCK were validated by the chromatographic results.