Simultaneous determination of five triterpene acids in rat plasma by liquid chromatography–mass spectrometry and its application in pharmacokinetic study after oral administration of Folium Eriobotryae effective fraction

Folium Eriobotryae effective fraction (FEA), the extract of Folium Eriobotryae, had been used as anti‐hyperglycemia and anti‐hyperlipemia medicine in China. A previous study indicated that euscaphic acid, maslinic acid, corosolic acid, oleanolic acid and ursolic acid, the five structurally similar triterpene acids (containing two groups of structural isomers), are the major components of FEA. In the present study, we developed a specific and reliable LC‐MS method for simultaneous determination of the five triterpene acids in rat plasma, and further investigated their pharmacokinetic properties after oral administration of FEA. Following a simple sample preparation, chromatographic separation was achieved on a C₁₈ column with a mobile phase composed of methanol–0.1% ammonium acetate (80:20, v/v). Quantification was achieved by monitoring the selected ions at m/z 487.6 for euscaphic acid, m/z 471.5 for maslinic acid and corosolic acid, m/z 455.5 for oleanolic acid and ursolic acid and m/z 469.5 for internal standard. The method was validated to be specific, accurate and precise over the concentration ranges of 10–3000 ng/mL with limits of detections of 5 ng/mL for the five triterpene acids. Finally, the method was successfully applied to the pharmacokinetic study of the five structurally similar triterpene acids in rats after oral administration of FEA. Copyright © 2015 John Wiley & Sons, Ltd.     

Separation of six compounds from pigeon pea leaves by elution–extrusion counter‐current chromatography

A valid and reliable method was established to separate six compounds from pigeon pea leaves via elution‐extrusion counter‐current chromatography. A solvent system composed of n‐hexane/methanol/formic acid aqueous solution with pH = 3 (10:6:4, v/v) was screened to achieve satisfactory isolation from the ethanol extract of pigeon pea leaves. Four compounds, 9.2 mg of compound 1 (96.8%), 3.2 mg of 2 (88.0%), 6.2 mg of 4 (94.2%) and 25.2 mg of 5 (94.2%), were obtained by conventional elution from 100 mg of the precipitation fraction, respectively. Two compounds, 14.4 mg of 3 (96.3%) and 28.1 mg of 6 (96.6%), with high K values were obtained by the subsequent extrusion procedure. The compounds 1 – 6 were identified as 3‐methoxy‐5‐(2‐phenylethenyl)‐phenol, pinostrobin chalcone, pinostrobin, 2‐hydroxy‐4‐methoxy‐6‐(2‐phenylvinyl)‐benzoic acid, longistylin C and cajaninstilbene acid by quadrupole time‐of‐flight mass spectrometry, and ¹H and ¹³C NMR spectroscopy. The in vitro antiproliferation activities of compounds 1 , 5 and 6 against human hepatoma cell were evaluated and the half‐maximum inhibitory concentrations were acquired.     

Surface molecularly imprinted polymers prepared by two‐step precipitation polymerization for the selective extraction of oleanolic acid from grape pomace extract

Surface molecularly imprinted polymers were successfully prepared by a novel two‐step precipitation polymerization method. The first‐step allowed the formation of 4‐vinylpyridine divinylbenzene and trimethylolpropane trimethacrylate copolymeric microspheres. In the second‐step precipitation polymerization, microspheres were modified with a molecularly imprinting layer of oleanolic acid as template, methacrylic acid as functional monomer, and divinylbenzene/ethylene glycol dimethacrylate as cross‐linker. The obtained polymers had an average diameter of 4.43 μm and a polydispersity index of 1.011; adsorption equilibrium was achieved within 40 min, with adsorption capacity reaching 27.4 mg/g. Subsequently, the polymers were successfully applied as the adsorbents of molecularly imprinted solid‐phase extraction to separate and purify the oleanolic acid from grape pomace. The content of oleanolic acid in the grape pomace extract was enhanced from 13.4 to 93.2% after using the molecularly imprinted solid‐phase extraction process. This work provides an efficient way for effective oleanolic acid separation and enrichment from complex matrices, which is especially valuable in industrial production.     

Microwave‐assisted extraction versus Soxhlet extraction to determine triterpene acids in olive skins

Microwave‐assisted extraction is compared with a more classical technique, Soxhlet extraction, to determine the content of triterpene acids in olive skins. The samples used in their original unmilled state and milled were extracted with ethyl acetate or methanol as solvents. The optimized operating conditions (e.g., amount and type of solvent, and time and temperature of extractions) to attain the better extraction yields have been established. For the identification and quantitation of the target compounds, an ultra high performance liquid chromatography with tandem mass spectrometry method was employed. The best results were achieved using the microwave‐assisted extraction technique, which was much faster than the Soxhlet extraction method, and showed higher efficiency in the extraction of the triterpenic acids (oleanolic and maslinic).     

Separation of phenolic acids from sugarcane rind by online solid‐phase extraction with high‐speed counter‐current chromatography

Sugarcane rind contains some functional phenolic acids. The separation of these compounds from sugarcane rind is able to realize the integrated utilization of the crop and reduce environment pollution. In this paper, a novel protocol based on interfacing online solid‐phase extraction with high‐speed counter‐current chromatography (HSCCC) was established, aiming at improving and simplifying the process of phenolic acids separation from sugarcane rind. The conditions of online solid‐phase extraction with HSCCC involving solvent system, flow rate of mobile phase as well as saturated extent of absorption of solid‐phase extraction were optimized to improve extraction efficiency and reduce separation time. The separation of phenolic acids was performed with a two‐phase solvent system composed of butanol/acetic acid/water at a volume ratio of 4:1:5, and the developed online solid‐phase extraction with HSCCC method was validated and successfully applied for sugarcane rind, and three phenolic acids including 6.73 mg of gallic acid, 10.85 mg of p‐coumaric acid, and 2.78 mg of ferulic acid with purities of 60.2, 95.4, and 84%, respectively, were obtained from 150 mg sugarcane rind crude extracts. In addition, the three different elution methods of phenolic acids purification including HSCCC, elution–extrusion counter‐current chromatography and back‐extrusion counter‐current chromatography were compared.     

Elution–extrusion counter‐current chromatography for the separation of two pairs of isomeric monoterpenes from Paeoniae Alba Radix

In this work, a simple and efficient protocol for the rapid separation of two pairs of isomeric monoterpenes from Paeoniae Alba Radix was developed by combining macroporous resin and elution–extrusion counter‐current chromatography. The crude extract was firstly subjected to a D101 macroporous resin column eluted with water and a series of different concentrations of ethanol. Then, effluents of 30 and 95% ethanol were collected as sample 1 and sample 2 for further counter‐current chromatography purification. Finally, a pair of isomers, 96 mg of compound 1 and 48 mg of compound 2 with purities of 91.1 and 96.2%, respectively, was isolated from 200 mg of sample 1. The other pair of isomers, 14 mg of compound 3 and 8 mg of compound 4 with purities of 93.6 and 88.9%, respectively, was isolated from 48 mg of sample 2. Their purities were analyzed by high‐performance liquid chromatography, and their chemical structures were identified by mass spectrometry and ¹H NMR spectroscopy. Compared to a normal counter‐current chromatography separation, the separation time and solvent consumption of elution–extrusion counter‐current chromatography were reduced while the resolutions were still good. The established protocol is promising for the separation of natural products with great disparity of content in herbal medicines.     

An efficient strategy based on liquid‐liquid extraction and pH‐zone‐refining counter‐current chromatography for selective enrichment,separation, and purification of alkaloids and organic Acids from natural products

This study presents an efficient strategy based on liquid‐liquid extraction and pH‐zone‐refining counter‐current chromatography for selective enrichment, separation, and purification of alkaloids and organic acids from natural products. First, an acid or base modified two‐phase solvent system with maximum or minimum partition coefficient was developed for the liquid‐liquid extraction of the crude extract. As a result, alkaloids or organic acids could be selectively enriched in the upper or lower phase. Then pH‐zone‐refining counter‐current chromatography was employed to separate and purify the selectively enriched alkaloids or organic acids efficiently. The selective enrichment and separation of five bufadienolide from toad venom of Bufo marinus was used as an example to show the advantage of this strategy. As a result, 759 mg of selectively enriched bufadienolide was obtained from 2 g of crude extract and the total content of five targets was increased from 14.64 to 83%. A total of 31 mg of marinobufagin‐3‐adipoyl‐l ‐arginine, 42 mg of telocinobufagin‐3‐pimeloyl‐l ‐arginine, 51 mg of telocinobufagin‐3‐suberoyl‐l ‐arginine, 132 mg of marinobufagin‐3‐suberoyl‐l ‐arginine, and 57 mg of bufalin‐3‐suberoyl‐l ‐arginine were all simultaneously separated from 500 mg of selectively enriched sample, with the purity of 92.4, 97.5, 90.3, 92.1, and 92.8%, respectively.     

pH‐zone‐refining elution–extrusion countercurrent chromatography: Separation of hydroxyanthraquinones from Cassiae semen

Seven hydroxyanthraquinones were successfully separated from the traditional Chinese medicinal herb Cassiae semen by conventional and pH‐zone‐refining countercurrent chromatography with an environmentally friendly biphasic solvent system, in which elution–extrusion mode was investigated for pH‐zone‐refining countercurrent chromatography for the first time. A two‐phase solvent system composed of n‐hexane/ethyl acetate/ethanol/water (5:3:4:4, v/v/v/v) was used for the conventional countercurrent chromatography while the same system with a different volume ratio n‐hexane/ethyl acetate/ethanol/water (3:5:2:6, v/v/v/v) was used for pH‐zone‐refining countercurrent chromatography, in which 20 mmol/L of trifluoroacetic acid was added in the organic phase as a retainer and 15 mmol/L of ammonia was added to the aqueous phase as an eluter. A 400 mg crude sample could be well separated by pH‐zone‐refining countercurrent chromatography, yielding 53 mg of aurantio‐obtusin, 40 mg of chryso‐obtusin, 18 mg of obtusin, 24 mg of obtusifolin, 10 mg of emodin, and 105 mg of the mixture of chrysophanol and physcion with a purity of over 95.8, 95.7, 96.9, 93.5, 97.4, 77.1, and 19.8%, as determined by high‐performance liquid chromatography. Furthermore, the difference in elution sequence between conventional and pH‐zone‐refining mode was observed and discussed.     

Separation of high‐purity eicosapentaenoic acid and docosahexaenoic acid from fish oil by pH‐zone‐refining countercurrent chromatography

The evidence for unique effects of eicosapentaenoic acid and docosahexaenoic acid is growing. Further understanding and exploration of their independent effects in the nutraceutical and pharmaceutical industry is calling for the more efficient separation techniques to overcome the equivalent chain length rule of fatty acids. In this study, free eicosapentaenoic and docosahexaenoic acid were successfully separated by pH‐zone‐refining countercurrent chromatography for the first time. The different solvent systems and the influence of retainer and eluter concentration on the separation efficiency were investigated. A two‐phase solvent system composed of n‐heptane/methanol/water (100:55:45, v/v) was selected with 50 mM of trifluoroacetic acid as retainer in the organic phase and 40 mM of ammonium hydroxide as an eluter in the aqueous phase for the separation of 500 mg of free fatty acids from a refined fish oil sample. 79.6 mg of eicosapentaenoic acid and 328.3 mg docosahexaenoic acid were obtained with the purities of 95.5 and 96.9% respectively determined by gas chromatography with mass spectrometry after methyl esterification. The scale‐up separation of 1 g of samples from both refined and crude fish oil after urea complexation were also achieved successfully with a markedly increased concentration 150 mM of retainer, producing satisfactory yields and purities of targets.     

Isolation of aristolochic acid I from herbal plant using molecular imprinted polymer composited ionic liquid‐based zeolitic imidazolate framework‐67

Aristolochic acid I is a toxic compound found in the genus of Aristolochia plants, which are commonly used as herbal cough treatment medicines. To remove the aristolochic acid I in extract efficiently and selectively, a molecularly imprinted polymer composed of ethylimidazole ionic liquid‐based zeolitic imidazolate framework‐67 was synthesized and used as the adsorbent. Under the conditions optimized by the software design expert, the sorbent showed highest adsorption amount of 34.25 mg/g in methanol/water (95:5, v/v) at 39°C for 138 min. The sorbent was then applied to solid phase extraction to isolate aristolochic acid I from the extract of the herbal plant Fibraurea Recisa Pierre. 0.043 mg/g of aristolochic acid I was obtained after the loading, washing, and elution processes. The limit of detection of 2.41 × 10^?5 mg/mL and good recoveries provided evidence for the accuracy of this method.     

Preparative and scaled‐up separation of high‐purity α‐linolenic acid from perilla seed oil by conventional and pH‐zone refining counter current chromatography

α‐Linolenic acid is an essential omega‐3 fatty acid needed for human health. However, the isolation of high‐purity α‐linolenic acid from plant resources is challenging. The preparative separation methods of α‐linolenic acid by both conventional and pH‐zone refining counter current chromatography were firstly established in this work. The successful separation of α‐linolenic acid by conventional counter current chromatography was achieved by the optimized solvent system n‐heptane/methanol/ water/acetic acid (10:9:1:0.04, v/v), producing 466 mg of 98.98% α‐linolenic acid from 900 mg free fatty acid sample prepared from perilla seed oil with linoleic acid and oleic acid as by‐products. The scaled‐up separation in 45× is efficient without loss of resolution and extension of separation time. The separation of α‐linolenic acid by pH‐zone refining counter current chromatography was also satisfactory by the solvent system n‐hexane/methanol/water (10:5:5, v/v) and the optimized concentration of trifluoroacetic acid 30 mM and NH₄OH 10 mM. The separation can be scaled up in 180× producing 9676.7 mg of 92.79% α‐linolenic acid from 18 000 mg free fatty acid sample. pH‐zone refining counter current chromatography exhibits a great advantage over conventional counter current chromatography with 20× sample loading capacity on the same column.     

13C nuclear magnetic resonance spectroscopy for determining the different components of epicuticular waxes of olive fruit (Olea europaea) Dritta cultivar

¹³C nuclear magnetic resonance spectroscopy (NMR) was applied to determine the different components of apolar and polar fractions which were isolated by column chromatography from the crude chloroform-soluble waxes of olive fruits (Olea europaea) Dritta cultivar.¹³C NMR enabled the determination in the wax apolar fraction, of aliphatic aldehydes, and of benzyl, alkyl and glyceryl esters. In particular, the fatty acid composition of alkyl esters, comprising saturated and unsaturated oleic and linoleic acids, was determined. Acyl chain composition and the chain composition of 1,3- and 2-glycerol positions were also determined for triacylglycerols of olive fruit waxes.Oleanolic and maslinic acids were confirmed to be the major components of wax polar fraction. Complete assignments of ¹³C NMR chemical shifts of oleanolic and maslinic acids as a mixture were achieved by using homonuclear correlation spectroscopy with gradient (g-COSY), attached proton test (APT), inverse-detected heteronuclear single-quantum coherence with gradient (g-HSQC), high-resolution heteronuclear correlation spectroscopy (HETCOR) for C-H directly attached pairs and C-H long-range-coupled experiments.     

Separation and purification of lanosterol,dihydrolanosterol, and cholesterol from lanolin by high‐performance counter‐current chromatography dual‐mode elution method

Lanosterol is a potential drug for cataracts treatment, which can reverse the aggregation of intracrystalline proteins. The low concentration in lanolin calls for high‐performance separation methods. In this study, a counter‐current chromatography dual‐mode elution method was developed for the first time to separate and purify lanosterol from hexane extract of lanolin after saponification, in which the column was first eluted with the lower phase as mobile phase in head‐to‐tail mode, followed by the upper phase in the tail‐to‐head mode. High purity of lanosterol, dihydrolanosterol, and cholesterol can be obtained simultaneously. A solvent system composed of n‐heptane/acetonitrile/ethyl acetate (5:5:1, v/v/v) was selected and optimized via partition coefficient determination. Compounds such as 111 mg lanosterol, 84 mg dihydrolanosterol, and 183 mg cholesterol with high purity of 99.77, 95.71, and 91.43%, respectively, analyzed by high‐performance liquid chromatography were obtained within 80 min from 700 mg crude extract from 1.78 g lanolin. The method was also used to improve the purity of commercial lanosterol product from 66.97 to above 99%. Counter‐current chromatography could serve as a potential and powerful technique for commercial production of highly pure lanosterol.     

Effective on‐line high‐speed shear dispersing emulsifier technique coupled with high‐performance countercurrent chromatography method for simultaneous extraction and isolation of carotenoids from Lycium barbarum L. fruits

An efficient combination strategy based on high‐speed shear dispersing emulsifier technique and high‐performance countercurrent chromatography was developed for on‐line extraction and isolation of carotenoids from the fruits of Lycium barbarum. In this work, the high‐speed shear dispersing emulsifier technique has been employed to extract crude extracts using the upper phase of high‐performance countercurrent chromatography solvent system composed of n‐hexane?dichloromethane?acetonitrile (10:4:6.5, v/v) as the extraction solvent. At the separation stage, the high‐performance counter‐current chromatography process adopts elution–extrusion mode and the upper phase of the solvent system as stationary phase (reverse‐phase mode). As a result, three compounds including zeaxanthin, zeaxanthin monopalmitate, and zeaxanthin dipalmitate with purities of 89, 90, and 93% were successfully obtained in one extraction‐separation operation within 120 min. The targeted compounds were analyzed and identified by high‐performance liquid chromatography, mass spectrometry, and NMR spectroscopy. The results indicated that the present on‐line combination method could serve as a simple, rapid, and effective way to achieve weak polar and unstable compounds from natural products.     

Screening of antioxidant phenolic compounds in Chinese Rhubarb combining fast counter‐current chromatography fractionation and liquid chromatography/mass spectrometry analysis

In this paper, an effective method combing fast elution‐extrusion counter‐current chromatography (CCC) and LC/MS for rapid screening of antioxidative phenolic compounds in Chinese Rhubarb is presented. An integrated three‐coil CCC column (40 mL each coil) was used to accomplish the optimization of biphasic liquid system. In a single run (approximately 40 min), the solvent system composed of n‐hexane/ethyl acetate/methanol/water (1:1:1:1, v/v) was selected as optimum CCC liquid system for fast fractionation of the crude ethanol extract. With a 140 mL‐capacity CCC instrument, 100 mg Chinese Rhubarb extract was separated under the optimized conditions, producing six fractions in only 100 min. The quantities of each fraction were ～15 mg. In addition, each fraction was subjected to antioxidant activity assay and characterized by LC/MS analysis. Fifty compounds, including phenolic acids, phenolic glucosides and hydroxyanthraquinones, were detected by LC/MS/MS analysis. As a result, gallic acid together with Fr I showed excellent antioxidant activity, which was well consistent with previous studies and exhibited great potential for natural drug discovery program of the present method.     

5‐O‐Caffeoylshikimic acid from Solanum somalense leaves: Advantage of centrifugal partition chromatography over conventional column chromatography

Solanum somalense leaves, used in Djibouti for their medicinal properties, were extracted by MeOH. Because of the high polyphenol and flavonoid contents of the extract, respectively, determined at 80.80 ± 2.13 mg gallic acid equivalent/g dry weight and 24.4 ± 1.01 mg quercetin equivalent/g dry weight, the isolation and purification of the main polyphenols were carried out by silica gel column chromatography and centrifugal partition chromatography. Column chromatography led to 11 enriched fractions requiring further purification, while centrifugal partition chromatography allowed the easy recovery of the main compound of the extract. In a solvent system composed of CHCl₃/MeOH/H₂O (9.5:10:5), 21.8 mg of this compound at 97% purity was obtained leading to a yield of 2.63%. Its structure was established as 5‐O‐caffeoylshikimic acid by mass spectrometry and NMR spectroscopy. This work shows that S. somalense leaves contain very high level of 5‐O‐caffeoylshikimic acid (0.74% dry weight), making it a potential source of production of this secondary metabolite that is not commonly found in nature but could be partly responsible of the medicinal properties of S. somalense leaves.     

Analysis of 16 phthalic acid esters in food simulants from plastic food contact materials by LC‐ESI‐MS/MS

An RP LC‐ESI‐MS/MS method for the determination of the migration of 16 primary phthalic acid esters from plastic samples has been developed using distilled water, 3% acetic acid, 10% alcohol, and olive oil as food simulants. Detection limits were 1.6–18.5 μg/kg in distilled water, 1.4–17.3 μg/kg in 3% acetic acid, 1.4–19.2 μg/kg in 10% alcohol, and 31.9–390.8 μg/kg in olive oil. The RSDs were in the range of 0.07–11.28%. The real plastic products inspection showed that only few analyzed samples were phthalates contaminated. Bis‐2‐ethylhexyl ester and dibutyl phthalate were the common items migrated from the plastic products into food and feeds, but the migration concentrations were far below the limits set by European Union (1.5 mg/kg for bis‐2‐ethylhexyl ester and 0.3 mg/kg for dibutyl phthalate).     

Rapid isolation of omega‐3 long‐chain polyunsaturated fatty acids using monolithic high performance liquid chromatography columns

Eicosapentaenoic and docosahexaenoic acids are important bio‐active fatty acids in fish oils. Monolithic HPLC columns both in the polymeric cation exchange (silver‐ion) and RP formats were compared with corresponding packed columns for the isolation of these acids from tuna oil ethyl esters. Monolithic columns in both formats enabled rapid (typically 5–10 min) separations compared with packed columns (30 min). Polymeric monolithic silver‐ion disc column rapidly furnished mixtures of eicosapentaenoic and docosahexaenoic esters (90% purity) within 5–10 min, but was unable to resolve individual esters. A preparative version of the same column (80 mL bed volume) enabled isolation (>88% purity) of 100 mg quantities of eicosapentaenoic and docosahexaenoic esters from esterified tuna oil within 6 min. Baseline separation of eicosapentaenoic and docosahexaenoic esters was achieved on all RP columns. The results show that there is potential to use polymeric monolithic cation exchange columns for scaled‐up preparation of eicosapentaenoic and docosahexaenoic ester concentrates from fish oils.     

Determination of major phenolic compounds in apples: Part I—Optimization of high‐performance liquid chromatography separation with diode array detection

The separation of seven phenolic compounds including gallic acid, chlorogenic acid, epicatechin, quercitrin, rutin, phloridzin, and phloretin present in apple peel and pulp and differing in elution properties has been optimized using high‐performance liquid chromatography with diode array detection. Several stationary phases were tested to achieve the efficient separation of phenolic compounds in fruit extracts and C18 was found to be the most efficient. Core–shell and fully porous C18 packings were assessed with respect to the complex composition of the fruit extracts. The developed high‐performance liquid chromatography method comprised gradient elution in which mobile phase A was water at pH 2.8 adjusted with acetic acid and B was acetonitrile. The gradient shape was the following: 0 min 95% A/5% B, 2.5 min 85% A/15% B, 12 min 50% A/50% B, 15 min 95% A/5% B. The flow rate was 1 mL/min, injection volume 10 μL, and UV detection at 255, 280, 320, and 365 nm was applied. Our method was validated for both C18 core–shell and fully porous packings. The resolution 6.2–14.8, symmetry 0.99–1.34, peak capacity 18–60, peak area repeatability 0.45–1.00% relative standard deviation, calibration range 0.125–5 mg/mL (0.25–10 mg/mL for chlorogenic acid and rutin), correlation coefficients of calibration curve 0.9976–0.9997, and accuracy evaluated as recovery 95.56–107.54% were determined for the core–shell column.     

Clay‐Na as a sorbent for the extraction of anti‐inflammatory compounds in water samples

In this work, clay‐Na particles are used as the adsorbent for the solid‐phase extraction of acidic compounds. The novel sorbent under study is based on high‐specific surface area, cation‐exchange capacity designed specifically to offer ion‐exchange properties with the goal being to selectively extract a group of acidic compounds. The effects of the extraction parameters including extraction elution solvent, sample volume and pH. In optimum conditions, the repeatability for one fiber (n = 3), expressed as % relative standard deviation, was between 0.3 and 4.3% for the acid compounds. The detection limits for the studied acidic compounds were between 0.1–0.6 μg/L. The developed method offers the advantages of being simple to use and having a low cost of equipment.