Simultaneous quantification of flavonoids and triterpenoids in licorice using HPLC

Numerous bioactive compounds are present in licorice (Glycyrrhizae Radix), including flavonoids and triterpenoids. In this study, a reversed-phase high-performance liquid chromatography (HPLC) method for simultaneous quantification of three flavonoids (liquiritin, liquiritigenin and isoliquiritigenin) and four triterpenoids (glycyrrhizin, 18alpha-glycyrrhetinic acid, 18beta-glycyrrhetinic acid and 18beta-glycyrrhetinic acid methyl ester) from licorice was developed, and further, to quantify these 7 compounds from 20 different licorice samples. Specifically, the reverse-phase HPLC was performed with a gradient mobile phase composed of 25 mM phosphate buffer (pH 2.5)-acetonitrile featuring gradient elution steps as follows: 0 min, 100:0; 10 min, 80:20; 50 min, 70:30; 73 min, 50:50; 110 min, 50:50; 125 min, 20:80; 140 min, 20:80, and peaks were detected at 254 nm. By using our technique, a rather good specificity was obtained regarding to the separation of these seven compounds. The regression coefficient for the linear equations for the seven compounds lay between 0.9978 and 0.9992. The limits of detection and quantification lay in the range of 0.044-0.084 and 0.13-0.25 microg/ml, respectively. The relative recovery rates for the seven compounds lay between 96.63+/-2.43 and 103.55+/-2.77%. Coefficient variation for intra-day and inter-day precisions lay in the range of 0.20-1.84 and 0.28-1.86%, respectively. Based upon our validation results, this analytical technique is a convenient method to simultaneous quantify numerous bioactive compounds derived from licorice, featuring good quantification parameters, accuracy and precision.     

Matrix solid‐phase dispersion with sand in chromatographic analysis of essential oils in herbs

Introduction – Matrix solid‐phase dispersion (MSPD) is a very simple, cheap and relatively quick sample preparation procedure which involves simultaneous disruption and extraction of various solid and semi‐solid samples due to the direct mechanical blending of the sample with a SPE sorbent, mainly C₁₈. Little is known about MSPD application as a sample preparation method for the analysis of essential oil components in herbs. Objective – To evaluate if C₁₈ sorbent, commonly used in MSPD process, can be substituted with sand in the procedure of essential oil analysis. Methodology – Essential oil extracts were obtained from mint, sage, chamomile, marjoram, savory and oregano using MSPD with C₁₈ sorbent or sand, pressurised liquid extraction and steam distillation. Their qualitative and quantitative compositions ware established by GC‐MS and GC‐FID. Results – The results prove that C₁₈ sorbent can be substituted with sand in the procedure of essential oil analysis in herbs. The recoveries of essential oil components estimated using MSPD/sand are almost equal to those using pressurised liquid extraction. Conclusion – The results presented in the paper reveal that MSPD with sand is suitable for the isolation of essential oil components from herbs. Its extraction efficiency is equivalent to pressurised liquid extraction, recognised as one of the most efficient extraction methods. The cost of MSPD procedure for essential oil analysis can be significantly diminished by substituting C₁₈ with sand. Copyright © 2010 John Wiley & Sons, Ltd.     

Simultaneous extraction and separation of liquiritin,glycyrrhizic acid,and glabridin from licorice root with analytical and preparative chromatography

Simultaneous extraction and separation of liquiritin, glycyrrhizic acid, and glabridin from licorice were developed by liquidliquid extraction with liquid chromatography separation. By utilizing different extraction solvents, procedures, and times, the optimum extraction conditions were established. The extracts of licorice were separated and determined using a C₁₈ column with a mobile phase consisting of acetonitrile-water (containing 1.0% acetic acid) with a gradient elution of 0∼10 min from 20:80 to 60:40 (v/v). Preparative columns with different packing sizes were investigated to isolate the three compounds from the extracts of licorice. The 12 μm chromatographic column showed better separation for the three compounds from licorice. 0.29 mg/g for liquiritin, 1.43 mg/g for glycyrrhizic acid, and 0.07 mg/g for glabridin were obtained and the recoveries were 80.8, 89.7, and 72.5%, respectively.     

Solid-phase extraction on Styrosorb cartridges as a sample pretreatment method in the stereoselective analysis of propranolol in human serum

Sample pretreatment using solid-phase extraction (SPE) on cartridges filled with small-particle Styrosorb porous polystyrene-based sorbent has been used in the analysis of propranolol enantiomers in human serum by high-performance liquid chromatography (HPLC) with fluorescent detection. SPE on Sep-Pak C₁₈ cartridges was used as a reference pretreatment method. The propranolol content of the samples was determined by achiral normal-phase HPLC and the enantiomeric ratio of propranolol (S/R) was then determined by chiral HPLC on a column with silica-bonded cellulose-tris(3,5-dimethylphenyl carbamate). Recoveries of propranolol from serum using SPE on Styrosorb and C₁₈ phases were 97±5% and 96±5%, respectively. Detection and quantification limits for propranolol enantiomers were 4 and 7 ng/ml, respectively.     

Optimised separation procedures for the simultaneous assay of three plant hormones in liquid biofertilisers

Introduction – The overuse of petrochemical‐based synthetic fertilisers has caused detrimental effects to soil, water supplies, foods and animal health. This, in addition to increased awareness of organic farming, has generated considerable interest in the evaluation of renewable biofertilisers. Objective – The three objectives of the current research were: (1) to evaluate and optimise a solid phase extraction procedure for extraction of three plant hormones, IAA, GA₃ and ABA from two model biofertilisers produced from coconut shells and pineapple peels; (2) to develop an HPLC analysis procedure for the simultaneous separation and quantification of three plant hormones (IAA, GA₃ and ABA); and (3) to evaluate the changes in three plant hormones levels at four different fermentation time periods and varying number of general bacteria, lactic acid bacteria and yeast. Result – An optimised procedure for sample preparation, separation and simultaneous analysis of three plant hormones [indole‐3‐acetic acid (IAA), gibberellic acid (GA₃) and abscisic acid (ABA)] produced in liquid biofertilisers was developed. This method involves sample cleanup using a Sep‐pack Oasis^®MAX cartridge containing mixed‐mode anion‐exchange and reverse‐phase sorbents that provided optimum recovery of 85.6, 91.9 and 94.3%, respectively, for the three hormones, IAA, GA₃, and ABA. Baseline separation of three hormones was achieved using mobile phase consisting of 1% acetic acid and acetonitrile (75:25, v/v) at pH 4.0. The amounts of hormones produced in liquid biofertilisers were influenced by fruit types, fermentation time and total number of general bacteria, lactic acid bacteria and yeasts. The quantities of three plant hormones produced during fermentation correlated well with the total number of microorganisms present in the liquid biofertilisers. Conclusion – A simple and rapid sample preparation procedure followed by RP‐HPLC with UV detection was optimised and developed for simultaneous quantification and identification of three plant hormones namely, IAA, GA₃ and ABA in the liquid biofertilisers. This procedure allows quantification of the three plant hormones in their natural states without any prior derivatisation step. The results presented illustrate that the contents of the three plant hormones depended on the type of fruit wastes, fermentation time and the number of microorganisms found in liquid biofertilisers. This method can be extended to determine the quantity of three hormones in other matrices. This assay procedure will aid in the development of liquid biofertilisers, a valuable alternative fertilisers to promote plant growth. This process will help farmers to reduce production cost and pollution problems. Copyright © 2009 John Wiley & Sons, Ltd.     

Effects of modifiers on the supercritical CO<Subscript>2</Subscript> extraction of glycyrrhizin from licorice and the morphology of licorice tissue after extraction

Optimal conditions for the supercritical carbon dioxide (scCO₂) extraction of glycyrrhizin from licorice (Glycyrrhiza glabra) were investigated, with an emphasis on the types and levels of modifiers. The morphology of the licorice tissue remaining after the scCO₂ extraction of glycyrrhizin was examined by scanning electron microscopy, coupled with measurements of absolute density. Conventional organic solvent extraction was also carried out for purpose of quantitative comparison. At 50 MPa and 60°C glycyrrhizin could not be extracted with pure scCO₂, while a considerable amount of glycyrrhizin was extracted when water was added to scCO₂ as a modifier. The highest recovery was found to be about 97% when 70% aqueous methanol was added to scCO₂ at a concentration of 15%. The optimal pressure and temperature for the supercritical fluid extraction of glycyrrhizin were observed to be 30 MPa and 60°C, respectively. Under these conditions, the percentage recovery of glycyrrhizin attained a maximum value of 102.67±1.13% within 60 min. Furthermore, in the case of scCO₂ modified with 70% aqueous methanol, the licorice tissue obtained after extraction was found to be severely degraded by excessive swelling, and the absolute density of the licorice residues was observed to be the highest.     

Development of an LC‐MS method for simultaneous quantitation of amentoflavone and biapigenin,the minor and major biflavones from Hypericum perforatum L., in human plasma and its application to real blood

Introduction – Biflavones of Hypericum perforatum L. are bioactive compounds used in the treatment of inflammation and depression. Determination of amentoflavone and biapigenin from blood is challenging owing to their similar structures and low concentrations. Objective – To develop a rapid, sensitive and accurate method based on liquid‐phase extraction followed by high‐performance liquid chromatography and electrospray ionisation mass spectrometry (HPLC‐ESI‐MS) for quantification of biflavones in human plasma. Methodology – After extraction from blood, the analytes were subjected to HPLC with an XTerra® MS C₁₈ column and a binary mobile phase consisting of 2% formic acid in water and acetonitrile under isocratic elution conditions, with ESI‐MS detection in the negative ion mode and multiple reaction monitoring (MRM). Results – Both calibration curves showed good linearity within the concentration range 1–500 ng/mL. Limits of detection (S/N = 3) were 0.1 ng for pure substances and the limits of quantitation (S/N = 5) were 1.0 ng/mL from analyte‐spiked serum. The grand mean recovery was 90% from several subsamples of each biflavone. The imprecision (RSD) of peak areas was between 5% (intraday) and 10% (interday) for high concentrations (250 ng/mL) and between 10% (intraday) and 15% (interday) for low concentrations (1 ng/mL). Inaccuracy of the mean was less than 20% at the lower limit of quantitation. Conclusion – The developed and validated method for determination of biflavones from human plasma was effectively applied to pharmacokinetic studies of 13 probands and preliminary results indicate biphasic concentration–time curves. Copyright © 2010 John Wiley & Sons, Ltd.     

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.     

Selective extraction of polyunsaturated triacylglycerols using a novel ionic liquid precursor immobilized on a mesoporous complexing adsorbent

Mesoporous silica (SBA‐15) synthesized by using Pluronic123 as the structure‐directing template was functionalized by imidazolium‐based ionic liquid precursors. Silver salts were then immobilized onto the supported ionic liquids using the incipient wetness impregnation technique. The separation of unsaturated species was achieved through the reversible and specific interaction between silver ions and carbon–carbon double bonds. This adsorbent was examined for the selective separation of polyunsaturated triacylglycerols (PUTAG) using High Pressure Liquid Chromatography (HPLC) with Evaporative Light Scattering Detection (ELSD) as the quantification methodology. AgBF₄/SBA15 · HPSiOEtIM · PF₆ showed an adsorption capacity for linolenin of about 217 mg adsorbed/gram of sorbent. This adsorbent had good selectivity and a high capacity for the most highly unsaturated triacylglycerol when applied to a mixture of triacylglycerols with varying degrees of unsaturation. Consequently, a stepwise methodology was also developed to increase the recovery of the adsorbed components. This adsorbent retained its selectivity and capacity when recycled up to five times. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Simultaneous analysis of diosgenin and sarsasapogenin in Asparagus officinalis byproduct by thin‐layer chromatography

Introduction – Asparagus officinalis L. has several biological activities including antifungal, antiviral and antitumoral activities due to the steroidal saponins. Normally diosgenin and sarsasapogenin are analysed separately by thin‐layer chromatography or high‐performance liquid chromatography (HPLC‐UV or HPLC‐ELSD), which is time‐consuming and expensive, so we need to find a rapid solution to this problem. Objective – To develop a sensitive, rapid and validated TLC method for simultaneous detection and quantification of diosgenin and sarsasapogenin. Methodology – Samples were prepared by extraction of A. officinalis with 70% aqueous ethanol to get steroidal saponins, and then hydrolysed using 36 mL 2 m hydrochloric acid for 3 h. The hydrolysis product was extracted with chloroform, and then analysed by TLC, the results of which were verified by HPLC and HPLC‐MS. Results – The retention factor (R_f) of diosgenin and sarsasapogenin on TLC plate were 0.49 and 0.6, respectively. After calculation from the regression equation of the standard curve, the contents of diosgenin and sarsasapogenin in the A. officinalis extract were 0.27–0.46 and 0.11–0.32%, respectively. Conclusion – The study showed that thin‐layer chromatography can be applied for the determination of diosgenin and sarsasapogenin in the oldest tissue of A. officinalis, and also can be conducted for screening of sapogenin in other plant or extracts. Copyright © 2010 John Wiley & Sons, Ltd.     

Rapid screening of lignans from Phyllanthus myrtifolius and stilbenoids from Syagrus romanzoffiana by HPLC-SPE-NMR

Introduction – Application of on‐line solid‐phase extraction (SPE) as an interface between HPLC and NMR has gained great improvement in solving sensitivity problems and signal interferences by the eluents. Objective – Rapid analysis and characterisation by HPLC‐SPE‐NMR and LC/MS of the arylnaphthalene‐type lignans present in Phyllanthus myrtifolius and the minor stilbenoids present in the polyphenol‐rich fraction from the ethanol extract of the seeds of Syagrus romanzoffiana. Methodology – Pretreatment of fractions by liquid–liquid partitioning, followed by Sephadex LH‐20 fractionation, was found very useful to facilitate the focusing and analysis of the polyphenolic fraction. HPLC‐DAD‐SPE‐NMR (400 MHz and 600 MHz) analysis was carried out using an Agilent 1100 liquid chromatography, followed by a Prospekt 2 automated solid‐phase extraction unit, containing 96 HySphere‐Resin GP cartridges (10 × 2 mm, 10–12 µm), which was connected to a 120 or 60 µL LC probe. Results – Seven arylnaphthalene‐type lignans from the chloroform‐soluble fraction of P. myrtifolius and nine stilbenoids from a polyphenol‐rich butanol‐soluble fraction of the seeds of S. romanzoffiana were characterised. Conclusion – HPLC‐SPE‐NMR associated with HR‐ESI/MS, which consumed only analytical amounts of partially purified mixtures, was demonstrated to be a good tool for rapid screening of both known and new natural products. Copyright © 2011 John Wiley & Sons, Ltd.     

Determination of urinary 18β-glycyrrhetinic acid by gas chromatography and its clinical application in man

A sensitive and quantitative gas chromatographic assay for the determination of 18β-glycyrrhetinic acid (18β-GA), the main metabolite of glycyrrhizin after oral licorice consumption in human urine, has been developed and validated. For the extraction of 18β-GA from urine two Sep-Pak C₁₈ extractions, hydrolysis with Helix pomatia and three liquid–liquid extractions were performed, using 18α-glycyrrhetinic acid (18α-GA) as internal standard. Both 18β-GA and internal standard were converted into their pentafluorobenzyl-ester/trimethylsilyl-ether derivatives and detected by flame ionization detection using a WCOT-fused-silica capillary column. Good quality control data were obtained in precision and accuracy tests. The detection limit of the gas chromatographic method was 10 μg/l with a urine volume of 10 ml. A detection limit of 3 μg/l was obtained by performing GC–MS. The GC method was used to monitor the urinary excretion of 18β-GA after licorice consumption by two healthy volunteers and a patient suspected of licorice abuse. Furthermore, it was shown that this GC assay enables to detect other metabolites related to licorice consumption.     

Effects of space flight on DNA mutation and secondary metabolites of licorice (Glycyrrhiza uralensis Fisch.)

Licorice (Glycyrrhiza uralensis Fisch.) seeds were flown on a recoverable satellite for 18 days(the average radiation dose in the flight recovery module was 0.102 mGy/d, the distance from flight apogee to earth was 350 km, gravity 10^-6). After returning to earth, the seeds were germinated and grown to maturity. The parallel ground-based seeds were also planted under the same conditions. The leaves of licorice were used for inter-simple sequence repeat (ISSR) analysis and the two main secondary metabolites in one-year-old roots were analyzed by high performance liquid chromatography (HPLC). Among 22 random primers used in this experiment, 6 primers generated different DNA band types. Analysis of HPLC showed that the content of glycyrrhizic acid (GA) and liquiritin (LQ) in the roots from seeds flown in space was respectively 2.19, 1.18 times higher than that of the control group. The results demonstrated that the extraterrestrial environment induced mutagenic effects on licorice and affected its secondary metabolites. These changes indicated that extraterrestrial orbit is possible means of breeding of licorice so as to preserve this endangered medicinal plant.     

Studies on the stability of salvianolic acid B as potential drug material

Introduction – Salvianolic acid B (Sal B) is one of the major water‐soluble compounds isolated from the roots of Salvia miltiorrhiza, which is widely used as a traditional Chinese medicine. Although much research on the general stability of Sal B has been undertaken and reported, there is still a need for further study of the stability required as a potential drug material. Objective – To study the stability of Sal B in the solid state and in normal saline (NS) solution during storage, as required in the ICH guidelines (2003) and Chinese Pharmacopoeia (2005). Methodology – Sal B stability was analysed using the high‐performance liquid chromatography (HPLC) method described in the Chinese Pharmacopoeia. HPLC coupled with time‐of‐flight mass spectrometry (HPLC‐TOFMS) was applied for the separation and identification of the degradation products of Sal B. Results – In the solid state, Sal B packaged in aluminium foil bags was stable for 6 months under ‘accelerated conditions’ (40°C, 75% relative humidity, RH). However, solid Sal B degradation was observed under open exposure to stress conditions of high temperature (60°C) or high humidity (92.5 or 75% RH). In NS solution, Sal B underwent severe degradation under accelerated conditions. Through HPLC‐TOFMS, nine degradation products were identified and the possible degradation pathway was deduced. Conclusion – The results demonstrate that the potential drug material Sal B could be used in a solid formulation, but is not suitable for use as a liquid formulation. Copyright © 2011 John Wiley & Sons, Ltd.     

Application of microwave‐assisted extraction coupled with high‐speed counter‐current chromatography for separation and purification of Dehydrocavidine from Corydalis saxicola Bunting

Introduction – Dehydrocavidine is a major component of Corydalis saxicola Bunting with sedative, analgesic, anticonvulsive and antibacterial activities. Conventional methods have disadvantages in extracting, separating and purifying dehydrocavidine from C. saxicola. Hence, an efficient method should be established. Objective – To develop a suitable preparative method in order to isolate dehydrocavidine from a complex C. saxicola extract by preparative HSCCC. Methodology – The methanol extract of C. saxicola was prepared by optimised microwave‐assisted extraction (MAE). The analytical HSCCC was used for the exploration of suitable solvent systems and the preparative HSCCC was used for larger scale separation and purification. Dehydrocavidine was analysed by high‐performance liquid chromatography (HPLC) and further identified by ESI‐MS and 1H NMR. Results – The optimised MAE experimental conditions were as follows: extraction temperature, 60°C; ratio of liquid to solid, 20; extraction time, 15 min; and microwave power, 700 W. In less than 4 h, 42.1 mg of dehydrocavidine (98.9% purity) was obtained from 900 mg crude extract in a one‐step separation, using a two‐phase solvent system composed of chloroform–methanol–0.3 m hydrochloric acid (4 : 0.5 : 2, v/v/v). Conclusion – Microwave‐assisted extraction coupled with high‐speed counter‐current chromatography is a powerful tool for extraction, separation and purification of dehydrocavidine from C. saxicola. Copyright © 2009 John Wiley & Sons, Ltd.     

Polymer‐in‐“Quasi‐Ionic Liquid” Electrolytes for High‐Voltage Lithium Metal Batteries

Due to the limited oxidation stability (<4 V) of ether oxygen in its polymer structure, polyethylene oxide (PEO)‐based polymer electrolytes are not compatible with high‐voltage (>4 V) cathodes, thus hinder further increases in the energy density of lithium (Li) metal batteries (LMBs). Here, a new type of polymer‐in‐“quasi‐ionic liquid” electrolyte is designed, which reduces the electron density on ethereal oxygens in PEO and ether solvent molecules, induces the formation of stable interfacial layers on both surfaces of the LiNi_1/3Mn_1/3Co_1/3O₂ (NMC) cathode and the Li metal anode in Li||NMC batteries, and results in a capacity retention of 88.4%, 86.7%, and 79.2% after 300 cycles with a charge cutoff voltage of 4.2, 4.3, and 4.4 V for the LMBs, respectively. Therefore, the use of “quasi‐ionic liquids” is a promising approach to design new polymer electrolytes for high‐voltage and high‐specific‐energy LMBs.     

Analysis of sesquiterpene lactones in Eupatorium lindleyanum by HPLC‐PDA‐ESI‐MS/MS

Introduction – The aerial part Eupatorium lindleyanum is commonly used as an antipyretic and detoxicant clinically in traditional Chinese medicine. Our previous research showed that germacrane sesquiterpene lactones were its main active constituents, so the development of rapid and accurate methods for the identification of the sesquiterpene lactones is of great significance. Objective – To develop an HPLC‐PDA‐ESI‐MS/MS method capable for simple and rapid analysis of germacrane sesquiterpene lactones in the aerial part E. lindleyanum. Methodology – High‐performance liquid chromatography‐photodiode array detection‐electrospray ionization‐tandem mass spectrometry was used to analyze germacrane sesquiterpene lactones of Eupatorium lindleyanum. The fragmentation behavior of germacrane sesquiterpene lactones in a Micromass Q/TOF Mass Spectrometer was discussed, and 9 germacrane sesquiterpene lactones were identified by comparison of their characteristic data of HPLC and MS analyses with those obtained from reference compounds. Results – The investigated germacrane sesquiterpene lactones were identified as eupalinolides C (1), 3β‐acetoxy‐8β‐(4′‐hydroxy‐tigloyloxy)‐14‐hydroxy‐costunolide (2), eupalinolides A (3), eupalinolides B (4), eupalinolides E (5), 3β‐acetoxy‐8β‐(4′‐oxo‐tigloyloxy)‐14‐hydroxy‐heliangolide (6), 3β‐acetoxy‐8β‐(4′‐oxo‐ tigloyloxy)‐14‐hydroxy‐costunolide (7), hiyodorilactone B (8), and 3β‐acetoxy‐8β‐(4′‐hydroxy‐tigloyloxy)‐ costunolide (9). Compounds 6, 7 and 9 were reported for the first time. Conclusion – HPLC‐PDA‐ESI‐MS/MS provides a new powerful approach to identify germacrane sesquiterpene lactones in E. lindleyanum rapidly and accurately. Copyright © 2009 John Wiley & Sons, Ltd.     

Preparative isolation and purification of four flavonoids from the petals of nelumbo nucifera by high‐speed counter‐current chromatography

Introduction – Flavonoids, the primary constituents of the petals of Nelumbo nucifera, are known to have antioxidant properties and antibacterial bioactivities. However, efficient methods for the preparative isolation and purification of flavonoids from this plant are not currently available. Objective – To develop an efficient method for the preparative isolation and purification of flavonoids from the petals of N. nucifera by high‐speed counter‐current chromatography (HSCCC). Methodology – Following an initial clean‐up step on a polyamide column, HSCCC was utilised to separate and purify flavonoids. Purities and identities of the isolated compounds were established by HPLC‐PAD, ESI‐MS, ¹H‐NMR and ¹³C‐NMR. Results – The separation was performed using a two‐phase solvent system composed of ethyl acetate–methanol–water–acetic acid (4 : 1 : 5 : 0.1, by volume), in which the upper phase was used as the stationary phase and the lower phase was used as the mobile phase at a flow‐rate of 1.0 mL/min in the head‐to‐tail elution mode. Ultimately, 5.0 mg syringetin‐3‐O‐β‐d‐glucoside, 6.5 mg quercetin‐3‐O‐β‐d‐glucoside, 12.8 mg isorhamnetin‐3‐O‐β‐d‐glucoside and 32.5 mg kaempferol‐3‐O‐β‐d‐glucoside were obtained from 125 mg crude sample. Conclusion – The combination of HSCCC with a polyamide column is an efficient method for the preparative separation and purification of flavonoids from the petals of N. nucifera. Copyright © 2009 John Wiley & Sons, Ltd.     

Validated Method for the Quantification of Atractylenolide III in Different Processed Products of Rhizoma Atractylodes Macrocephalae

Introduction – Rhizoma Atractylodes Macrocephalae (RAM) contains several sesquiterpene compounds including atractylenolide III (AO‐III). This bioactive compound may be used as a chemical marker for the quality control of different processed RAM products. Objective – To develop and validate an RP‐HPLC method for the quantitative determination of AO‐III in RAM and in a variety of processed RAM products. Methodology – HPLC was carried out using a Kromssil C₁₈ RP‐column eluted with methanol–water (70:30) at a flow rate of 1.0 mL/min and with UV detection at 220 nm. Full validation was performed using standard methods. Results – The linear range of AO‐III was 5–50 µg/mL; the regression equation was y = 10210x + 11194 (r = 0.9994) and the average recovery was 101.08% (RSD = 0.98%). The detection and quantification limits for AO‐III were determined to be 0.005 and 0.018 µg/mL at signal‐to‐noise ratios of approximately 3:1 and 10:1, respectively. Conclusion – The described HPLC method is appropriate for quality assurance and differentiation of AO‐III in RAM and different processed products.