Validation of a quantitative assay of arbutin using gas chromatography in Origanum majorana and Arctostaphylos uva‐ursi extracts

Introduction – Arbutin is a skin‐whitening agent that occurs naturally in the bark and leaves of various plants. It is commonly quantified in plant extracts and skin‐whitening products by HPLC. Objective – To develop an alternative gas chromatographic method for the separation and quantification of arbutin in Origanum majorana and Arctostaphylos uva‐ursi extracts. Methodology – N,O‐Bis(trimethylsilyl)acetamide and trimethylchlorosilane were used as silylation reagents, and the gas chromatographic separation of silylated extracts and standards was performed using a DB‐5 narrow bore column. GC‐MS was used for the compound identification, and the quantification was carried out by GC‐FID. The quantitative results were compared with those of HPLC analysis. Results – The developed method gave a good sensitivity with linearity in the range 0.33–500 mg/mL and recovery >98%, allowing the quantification of arbutin in O. majorana and A. uva‐ursi extracts. The relative standard deviations (RSD) relating to intra‐day and inter‐day precision were <0.002% and <4.8%, respectively. The GC results correlated well with those obtained by HPLC analysis. Conclusion – The analysis of marjoram and bearberry samples showed that the established GC method was rapid, selective, and demonstrated that arbutin could be screened alternatively by gas chromatography. Copyright © 2009 John Wiley & Sons, Ltd.     

An efficient strategy based on MAE, HPLC-DAD-ESI-MS/MS and 2D-prep-HPLC-DAD for the rapid extraction, separation, identification and purification of five active coumarin components from Radix Angelicae Dahuricae

Introduction – Further studies of active coumarin components in Radix Angelicae Dahuricae (AE) are absolutely essential to provide data on pharmacology, toxicology and quality for innovative drug candidates. Thus, the preparation of active component standards and the administration of coumarin monomers should be carried out. The isolation of the low‐level active components from complex Traditional Chinese Medicine (TCM) samples necessitates the development of rapid, simple and economical modern extraction, separation, identification and purification methods. Objective – To develop an efficient strategy for the rapid extraction, separation, identification and purification of coumarins from AE. Methodology – First, active coumarins in AE were extracted with microwave‐assisted extraction (MAE) after the extraction conditions were optimised. Second, gradient extraction methods with MAE were used to partially purify AE. Third, a high‐performance liquid chromatography–diode array detection‐electrospray ionisation tandem mass spectrometry (HPLC‐DAD‐ESI‐MS/MS) method was applied for the preliminary on‐line identification and screening of the main coumarins in AE extract. Finally, a two‐dimensional preparative high‐performance liquid chromatography–diode array detection (2D‐prep‐HPLC‐DAD) system was developed for further preparative separation of those target components. Results – Altogether 10 coumarins have been identified and five of them including xanthotoxol, osthenol, oxypeucedanin hydrate, byakangelicin and imperatorin were deemed as target components for the preparative isolation. All of the five isolated coumarins were at high purities of over 99% and the production rate was much higher than the traditional methods. Conclusion – The present paper demonstrates that these consecutive approaches are very useful for to isolate chemical constituents from TCM.     

土茯苓中总黄酮的含量测定方法研究

目的：建立土茯苓总黄酮的含量测定方法。方法：采用70%乙醇超声提取工艺和可见分光光度法测定土茯苓中总黄酮的含量,以芦丁为对照品,在500 nm波长处测量。结果：芦丁在0.008～0.048 mg.mL-1范围内线性关系良好（r=1.000）,平均加样回收率（n=6）以芦丁计为98.34%,RSD为3.4%。结论：此方法操作简单,精密度、稳定性和重复性良好,可用于土茯苓总黄酮含量测定。     

Comparative Investigation for Raw and Processed Products of Euodiae Fructus Based on High-Performance Liquid Chromatography Fingerprints and Chemical Pattern Recognition

As a traditional Chinese medicine, Euodiae Fructus is widely used due to its analgesic, anti-inflammatory, and antihypertensive effects. However, Euodiae Fructus has also been documented to be toxic, and the toxic effects can be reduced by processing. To distinguish Euodiae Fructus from its processes products and study the changes of raw and processed products before and after processing, we evaluated four auxiliary material processing methods including vinegar, Zingiberis Rhizoma, Coptidis Rhizoma, and Glycyrrhizae Radix et Rhizoma. The raw Euodiae Fructus and four processed Euodiae Fructus samples were analyzed and compared based on the high-performance liquid chromatography (HPLC) fingerprints combined with chemometrics, including principal component analysis (PCA), partial least squares-discriminant analysis (PLS-DA), and principal component analysis-class (PCA-Class). A total of 27 common peaks were obtained by fingerprint analysis. The fingerprint similarity of raw and processed samples was between 0.86–0.999. We also determined the contents of the main active ingredients - Evodiamine and Rutaecarpine. PCA and PLS-DA analyses were used to distinguish between the raw and processed samples of Euodiae Fructus, and 14 chemical markers were screened out. Four kinds of processed products were further analyzed and the results showed that they could be successfully distinguished under the established models, and 12 chemical markers were labeled. PCA-Class results revealed that the classification models constructed in this study had adequate discrimination ability. The method combined with HPLC fingerprinting and multi-component chemical pattern recognition technology could be used to differentiate raw and processed Euodiae Fructus with adequate predictive power. Our findings confirmed the rationality of the pharmacopoeial method and provided a reference for the quality control of the Glycyrrhizae Radix et Rhizoma processed Euodiae Fructus.     

Quantitative determination of lanostane triterpenes in Fomes officinalis and their fragmentation study by HPLC-ESI

Introduction – The fruit bodies of Fomes officinalis are used for the treatment of coughs, gastric cancer, rheumatism and hydropsia; however, no method is currently available to assess the quality of this medicinal fungus based on quantitative profile of its main triterpenes. Objective – To develop a simple and accurate HPLC‐UV method for the simultaneous quantification of five lanostane‐type triterpenes in the fruit bodies of F. officinalis. Method – Separations were performed on an Agilent Zorbax Eclipse XDB‐C₁₈ column by gradient elution using acetonitrile : formic acid. Analytes were identified by HPLC coupled with electrospray ionisation mass spectrometry experiments. The quantitative HPLC‐UV method was validated for linearity, precision, accuracy and limits of detection and quantification. Results – Calibration curves presented good linear regression (r > 0.9996) within test ranges. The relative standard deviation of this method was less than 1.7% for intra‐ and inter‐day assays and overall recoveries were 96.4–104.1% for the five compounds analysed. The method was successfully applied to the quantification of five triterpenes in 16 samples of F. officinalis collected from different regions. Conclusion – The developed assay could be considered as a suitable quality control method for F. officinalis. Copyright © 2010 John Wiley & Sons, Ltd.     

Fingerprint analysis of Flos Carthami by capillary electrophoresis

Capillary electrophoresis (CE) was employed in fingerprint analysis of Flos Carthami. A standardized procedure was used to develop the CE fingerprint. An electrophoretic profile of genuine Flos Carthami from Fengqiu, He'nan, China, was first established as the characteristic fingerprint. This profile was then used to identify and assess the consistency of the herb. A study with a limited number of samples from nine sources showed a fair consistency in their CE fingerprints with that of the genuine sample. Flos Carthami was well distinguished from Stigma Croci, a possible substitute in traditional Chinese medicine, and Flos Hemerocallis, a commercial adulterant, by comparing the fingerprints of each herb.     

Analysis of marker compounds with anti‐platelet aggregation effects in mailuoning injection using platelet binding assay combined with HPLC‐DAD‐ESI‐MS and solid‐phase extraction technique

Introduction – Mailuoning is prepared from a traditional formula of Chinese medicines and widely used as an antithrombotic agent. In this study, the platelet binding assay was used as a novel biospecific separation and analysis method to explore its active constituents, which could be considered as marker compounds for quality control. Objective – To establish a rapid and simple method to predict marker compounds in herbal medicine injection and evaluate the effects of those compounds. Material and methods – Platelets were used to bind and separate constituents. Binding constituents were analysed and taken as potential active compounds for further evaluation. Solid‐phase‐extraction was adopted to improve sensitivity. HPLC‐DAD and ESI‐MS were used to determine the binding constituents. Results – Five compounds were extracted through the platelet binding process and identified as neochlorogenic acid, caffeic acid, isochlorogenic acid and their isomers. Caffeic acid was selected for the flow cytometric assay to test its effect on platelets activation, which was determined by CD62P (P‐selectin) expression. The results indicated that caffeic acid could significantly inhibit platelet activation while chlorogenic acid did not. Conclusion – Caffeic acid could be considered as a marker compound of Mailuoning injection due to its anti‐platelet effect. The study also suggested that platelet binding assay combined with some preconcentration technique could be efficiently used to predict anti‐platelet compounds in complicated herbal medicines. Copyright © 2010 John Wiley & Sons, Ltd.     

萆薢类药材的HPLC指纹图谱研究

应用HPLC方法测定了薯蓣属根状茎组10种1亚种1变种植物23个样本,建立了萆薢类药材总皂苷元粗提物的HPLC指纹图谱.色谱柱为Zorbax Eclipse XDB-C_(18)柱(4.6×250 mm,5 μm),流动相为乙腈-水,柱温30℃,检测波长为203 nm.结果表明,用上述条件所建立的指纹图谱共标示出7个共有峰,且可较全面地反映萆薢类药材的皂苷元类成分,为萆薢类药材薯蓣属根状茎组植物鉴别及质量控制提供一种方法.     

Qualitative analysis and simultaneous quantification of phenolic compounds in the aerial parts of Salvia miltiorrhiza by HPLC-DAD and ESI/MS(n)

Introduction – The increasing demands of roots and rhizomes of Salvia miltiorrhiza almost exhausted the wild Salvia sources in China. However, the content and composition of phenolic acids in the aerial parts of the plant and their potential to be used as a substitute has not been explored. Objective – To evaluate the potential of the aerial parts of Salvia miltiorrhiza as new natural sources of phenolic acids. Methodology – HPLC coupled with diode array detection (DAD) and electrospray ionization multistage mass spectrometry (ESI/MSⁿ) has been used for qualitative and quantitative analysis of phenolic compounds. Results – A total of 38 phenolic compounds were identified or tentatively characterized. A quantitative HPLC‐DAD method allowing the simultaneously quantification of six phenolic acids was optimized and validated for linearity, precision, accuracy, and limits of detection and quantification. Calibration curves showed good linear regression (r² > 0.9991) within test ranges; the recoveries ranged between 95.64 and 101.67% and the RSDs were less than 3.01%. Conclusion – The developed methods have been proved to be effective for the identification and quantification of phenolic acids in S. miltiorrhiza. The results obtained suggest that the aerial parts of the plant could be used as an alternative source of sage phenolics. Copyright © 2010 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.     

猫须草HPLC指纹图谱研究

为建立猫须草药材HPLC指纹图谱分析方法,采用高效液相色谱法,以Phenomenex Synergi 4u hydro-RP 250×4.60 mm为色谱柱,以甲醇-0.1%甲酸溶液为流动相梯度洗脱,检测波长254 nm,流速1.0 mL·min^-1,柱温40 ℃。结果表明,建立的猫须草药材HPLC指纹图谱,确定了15个共有峰,各猫须草样品指纹图谱与对照指纹图谱的相似度均在0.9以上。该方法简单、准确、重复性好,为更好地控制猫须草药材质量提供有效可靠的方法。     

Systematic characterisation of secondary metabolites from Ixeris sonchifolia by the combined use of HPLC‐TOFMS and HPLC‐ITMS

Introduction – Ixeris sonchifolia (Bunge) Hance, a folk medicine, has been widely used in China for its anti‐inflammatory and haemostatic effects. However, the miscellaneous component composition of this herbal medicine is not well known. Objective – To develop a fast and comprehensive analytical method for the characterisation of various components from I. Sonchifolia, as a tool for the quality control of the herb and its related preparations. Methodology – Ixeris sonchifolia samples were extracted with 60% aqueous methanol, purified by solid‐phase extraction and then analysed by the combinatorial use of HPLC‐TOFMS and HPLC‐ITMS. Results – A total of six sesquiterpene lactones, six phenolic acids and seven flavonoids were identified or tentatively characterised. Five of them were reported for the first time in I. sonchifolia and, in particular, two amino acid‐sesquiterpene lactone conjugates, 11,13‐dihydro‐13‐prolyl‐ixerin Z and 11,13‐dihydro‐13‐prolyl‐ixerin Z₁, that were first found in this plant source. Conclusion – A global profile of I. sonchifolia constituents was described, which could be useful for the quality control of this herb and its related preparations. The employed combination of HPLC‐TOFMS and HPLC‐ITMS could also be a promising tool for the analysis of other herbal medicines containing sesquiterpene lactones, phenolic acids or flavonoids. Copyright © 2010 John Wiley & Sons, Ltd.     

Simultaneous determination of five characteristic stilbene glycosides in root bark of Morus albus L. (Cortex Mori) using high-performance liquid chromatography

Introduction – Cortex Mori, one of the well‐known traditional Chinese herbal medicines, is derived from the root bark of Morus alba L. according to the China Pharmacopeia. Stilbene glycosides are the main components isolated from aqueous extracts of Morus alba and their content varies depending on where Cortex Mori was collected. We have established a qualitative and quantitative method based on the bioactive stilbene glycosides for control of the quality of Cortex Mori from different sources. Objective – To develop a high‐performance liquid chromatography coupled with ultraviolet absorption detection for simultaneous quantitative determination of five major characteristic stilbene glycosides in 34 samples of the root bark of Morus alba L. (Cortex Mori) from different sources. Methodology – The analysis was performed on an ODS column using methanol‐water‐acetic acid (18: 82: 0.1, v/v/v) as the mobile phase and the peaks were monitored at 320 nm. Results – All calibration curves showed good linearity (r ≥ 0.9991) within test ranges. This method showed good repeatability for the quantification of these five components in Cortex Mori with intra‐ and inter‐day standard deviations less than 2.19% and 1.45%, respectively. Conclusion – The validated method was successfully applied to quantify the five investigated components, including a pair of cis‐trans‐isomers 1 and 2 and a pair of isomers 4 and 5 in 34 samples of Cortex Mori from different sources. Copyright © 2010 John Wiley & Sons, Ltd.     

Glycosidase activity profiling for bacterial identification by a chemical proteomics approach

Genome sequencing projects are suggesting there are dozens of glycosidase sequences that could be used to fingerprint cell types and serve as starting points for biocatalyst discovery. Herein, we present a simple chemical proteomics approach to profile intracellular glycosidase activities of three different bacterial cell extracts using a synthetic α- and β-linked library of 18 representative substrates with electrospray ionization-mass spectrometry (ESI-MS) reaction monitoring. Three target bacteria – Escherichia coli K12, Bacillus cereus and Pseudomonas aeruginosa – can be easily differentiated by this method. Compared with traditional chromogenic and fluorogenic methods to profile bacterial enzyme activities individually, this MS-based method can detect multiple enzyme activities in one reaction and easily highlight activity differences between whole cell extracts.     

Fingerprint analysis of Psoralea corylifolia L. by HPLC and LC-MS

High-performance liquid chromatography (HPLC) was developed for fingerprint analysis of Psoralea corylifolia. Liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MSn) technique was first employed to identify the components of the fingerprint. The samples were separated with an Alltima C18 column (250 mm x 4.6 mm, 5 microm) by linear gradient elution using water-acetic acid (A; 100:0.1, v/v) and acetonitrile (B; 0 min, 40%; 15 min, 50%; 35 min, 60%; 45 min, 70%; 55 min, 80%; and maintained for 5 min) as mobile phase at a flow rate of 1.0 ml/min and detector wavelength at 245 nm. A standard procedure was developed for HPLC fingerprint analysis. Average chromatogram of 10 batches of P. corylifolia L. from Sichuan and Henan Provinces, PR China, which has been considered as the original and genuine herbal medicine for a long time, was first established as the characteristic fingerprint. There are 12 common peaks in this fingerprint. Ten of these common peaks were identified by MS data. This profile was then used to identify and assess the differences among the herb grown in various areas of China. The HPLC fingerprint analysis is specific and may serve for quality identification and comprehensive evaluation of P. corylifolia.     

Comparative quantitative analysis of artemisinin by chromatography and qNMR

Introduction – Since the discovery of artemisinin in the 1970s, many techniques based on diverse chromatography techniques have been developed to detect and quantify this important antiplasmodial compound. The accurate quantification of this compound in the Artemisia annua plant material is mainly needed for breeding purposes in order to cultivate higher yielding varieties. It is also important for the quality control of herbal preparations containing A. annua plant material. Objective – To evaluate the most common validated quantification techniques (LC‐MS, HPLC‐ELSD and TLC) and compare the results to quantitative nuclear magnetic resonance spectroscopy (qNMR) in eight different A. annua samples collected from around the world. Methodology – The leaf material were extracted according to standard procedures and analysed with the validated quantification techniques. For the qNMR analysis we did not employ a standard curve but instead used an internal standard (maleid acid) which is not chemically related to artemisinin. Results – We found a significant difference between the results in this study. Compared with the qNMR results the HPLC‐ELSD corresponded closely, followed by LC‐MS. Quantitation with TLC led to an estimation range of ?0.5 to +3.2 mg artemisinin/g of A. annua. Conclusion – These results imply that qNMR, with the addition of an internal standard, can be used to quantify artemisinin in A. annua samples in a rapid and reproducible manner. Copyright © 2010 John Wiley & Sons, Ltd.     

Effect of N source on concentration of Rubisco in Eucalyptus diversicolor, as measured by capillary electrophoresis

Proteins in plant tissues have been extensively characterised by conventional methods such as liquid chromatography and polyacrylamide gel electrophoresis – methods that are tedious and time‐consuming. Capillary electrophoresis is potentially a more simple and cost‐effective method (with respect to time and consumables) but needs substantial development, especially for native plants which are frequently poor in protein and rich in interfering substances (oils, tannins, phenols). We report here the development of capillary electrophoresis (CE) for the separation of SDS‐protein complexes (by molecular mass) and their quantification in plant tissues. In leaf extracts, two peaks dominated the electropherograms, these peaks had migration times corresponding to the small and large subunits of Rubisco (ribulose‐1,5‐bisphosphate carboxylase/oxygenase; EC 4.1.1.39) and co‐migrated with added purified Rubisco. Linearity of peak area, reproducibility of migration time and peak areas for the small and large subunit were excellent, suggesting Rubisco could be quantified with a high degree of accuracy. We determined how the concentration (0.5 or 4 mM) and form of N applied (nitrate versus ammonium) affects partitioning of N to Rubisco in seedlings of Eucalyptus diversicolor. Analysis of extracts from leaves of Eucalyptus diversicolor was only possible after precipitation of proteins with trichloroacetic acid (TCA). Precipitation with TCA was highly reproducible and recovery of added Rubisco through procedures of extraction, precipitation and analysis were close to 100% for both subunits. An 8‐fold difference in the concentration of N applied did not affect total N, the concentration of Rubisco or the fraction of N present as Rubisco. The similarity of total N may well reflect faster rates of growth in those plants receiving 4 mM N, and a subsequent ‘dilution’ of tissue N. The N source did not affect total N, the concentration of Rubisco or the fraction of N present as Rubisco. Despite similar Rubisco concentrations, the total concentration of soluble proteins was greater in ammonium‐grown plants.     

Quantitative and chemical fingerprint analysis for quality control of Rhizoma Coptidischinensis based on UPLC-PAD combined with chemometrics methods

To control the quality of Rhizoma Coptidis, a method based on ultra performance liquid chromatography with photodiode array detector (UPLC-PAD) was developed for quantitative analysis of five active alkaloids and chemical fingerprint analysis. In quantitative analysis, the five alkaloids showed good regression (R > 0.999 2) within test ranges and the recovery of the method was in the range of 98.4- 100.8%. The limit of detections and quantifications for five alkaloids in PAD were less than 0.07 and 0.22 μg/ml, respectively. In order to compare the UPLC fingerprints between Rhizoma Coptidis from different origins, the chemometrics procedures, including similarity analysis (SA), hierarchical clustering analysis (HCA), principal component analysis (PCA) were applied to classify the Rhizoma Coptidis samples according to their cultivated origins. Consistent results were obtained to show that Rhizoma Coptidis samples could be successfully grouped in accordance with the province of origin. Furthermore, five marker constituents were screened out to be the main chemical marker, which could be applied to accurate discrimination and quality control for Rhizoma Coptidis by quantitative analysis. This study revealed that UPLC-PAD method was simple, sensitive and reliable for quantitative and chemical fingerprint analysis, moreover, for the quality evaluation and control of Rhizoma Coptidis.     

Comparative Study of Fatty Acids Profile in Eleven Wild Mushrooms of Boletacea and Russulaceae Families

Eleven species of wild mushrooms which belong to Boletaceae and Russulaceae families were examined by gas chromatography (GC) and gas chromatography–mass spectrometry (GC/MS) analysis for the presence of fatty acids. As far as we know, the fatty acid profiles of B. purpureus and B. rhodoxanthus were described for the first time. Twenty‐six fatty acids were determined. Linoleic (19.5 – 72%), oleic (0.11 – 64%), palmitic (5.9 – 22%) and stearic acids (0.81 – 57%) were present in the highest contents. In all samples, unsaturated fatty acids dominate. Agglomerative hierarchical clustering was used to display the correlation between the fatty acids and their relationships with the mushroom species. Based on the fatty acids profile in the samples, the mushrooms can be divided into two families: Boletaceae and Russulaceae families, using cluster analysis.