Correlation analysis between the HPLC fingerprints and in vitro antibacterial activity of ethyl acetate extracts from Radix isatidis

Aim To study the correlation between the HPLC fingerprints and in vitro antibacterial activities of EtOAc extracts of Radix isatidis from various sources. Methods Ten batches of Radix isatidis EtOAc extracts were analyzed with HPLC and the fingerprints were established. The influence of EtOAc extracts on the thermogenic curve of growth of Escherchia coli was obtained by microcalorimetry. The chemical differences of EtOAc extracts of Radix isatidis from various sources in the HPLC fingerprints were probed with hierarchical clustering analysis and similarity analysis. The correlation between the HPLC fingerprints and in vitro antibacterial activities was analyzed with multivafiant correlation analysis. Results Close correlation existed between the HPLC fingerprints and in vitro antibacterial activities of EtOAc extracts of Radix isatidis. Conlusion The combination of HPLC fingerprints and antibacterial activities can be used to discover principle components of Radix isatidis on bioactivity.

Applications of HPLC/MS in the analysis of traditional Chinese medicines

In China, traditional Chinese medicines （TCMs） have been used in clinical applications for thousands of years. The successful hyphenation of high-performance liquid chromatography （HPLC） and mass spectrometry （MS） has been applied widely in TCMs and biological samples analysis. Undoubtedly, HPLC/MS technique has facilitated the understanding of the treatment mechanism of TCMs. We reviewed more than 350 published papers within the last 5 years on HPLC/MS in the analysis of TCMs. The present review focused on the applications of HPLC/MS in the component analysis, metabolites analysis, and pharmacokinetics of TCMs etc. 50% of the literature is related to the component analysis of TCMs, which show that this field is the most popular type of research. In the metabolites analysis, HPLC coupled with electrospray ionization quadrupole time-of-flight tandem mass spectrometry has been demonstrated to be the powerful tool for the characterization of structural features and fragmentation behavior patterns. This paper presented a brief overview of the applications of HPLC/MS in the analysis of TCMs. HPLC/MS in the fingerprint analysis is reviewed elsewhere.

Establishment of HPLC Fingerprint, Cluster Analysis and Principle Component Analysis of Citri Reticulatae Pericarpium Viride

[Objectives] This study aimed to establish HPLC fingerprint and conduct cluster analysis and principle component analysis for Citri Reticulatae Pericarpium Viride. [Methods] Using the HPLC method, the determination was performed on XSelect~® HSS T3-C_(18) column with mobile phase of acetonitrile-0.5% acetic acid solution(gradient elution) at the flow rate of 1.0 mL/min. The detection wavelength was 360 nm. The column temperature was 25℃. The sample size was 10 μL. With peak of hesperidin as the reference, HPLC fingerprints of 10 batches of Citri Reticulatae Pericarpium Viride were determined. The similarity of the 10 batches of samples was evaluated by Similarity Evaluation System for Chromatographic Fingerprint of TCM(2012 edition) to determine the common peaks. Cluster analysis and principal component analysis were performed by using SPSS 17.0 statistical software. [Results] The HPLC fingerprints of the 10 batches of medicinal materials had total 11 common peaks, and the similarity was 0.919-1.000, indicating that the chemical composition of the 10 batches of medicinal materials was consistent. There were 11 common components in the 10 batches of medicinal materials, but their contents were different. When the Euclidean distance was 20, the 10 batches of samples were divided into two categories, S4 in the first category, and the others in the second one. When the Euclidean distance was 5, the second category could be further divided into two sub-categories, S1 and S10 in one sub-category, and S2, S3, S5, S6, S7, S8 and S9 in the other one. The principle component analysis showed that cumulative contribution rate of the two main component factors was 92.797%, and the comprehensive score of S7 was the highest with the best quality. [Conclusions] The results of HPLC fingerprinting, cluster analysis and principle component analysis can provide reference for the quality control of Citri Reticulatae Pericarpium Viride.

Reversed-Phase HPLC Analysis of Steviol Glycosides Isolated from Stevia rebaudiana Bertoni

High Performance Liquid Chromatography (HPLC) experiments have been performed on nine steviol glycosides namely rebaudioside A, steviolbioside, stevioside, rubusoside, rebaudioside B, rebaudioside C, rebaudioside D, rebaudioside F, and dulcoside A isolated from the leaves of Stevia rebaudiana using Reversed-Phase (RP) column. Using RP-HPLC method, the individual retention times for nine naturally occurring ent-kaurane diterpene glycosides of S. rebaudiana reported in JECFA have been determined at four different temperatures: 20℃, 40℃, 60℃, and 79℃. Also, calculated the relative retention times of the eight steviol glycosides steviolbioside, stevioside, rubusoside, rebaudioside B, rebaudioside C, rebaudioside D, rebaudioside F, and dulcoside A against the major steviol glycoside rebaudioside A. HPLC results suggested that temperatures 40℃ and 60℃ would be ideal conditions for better separation of steviol glycosides.

Phytochemical Analysis and Antimicrobial Activity of Lawsonia inermis Leaf Extracts from Burkina Faso

Lawsonia inermis is a hairless plant growing in various regions of North Africa, the Indian subcontinent, and the Middle East. It possesses many medicinal attributes, including curative properties against infectious dermatoses. This study was carried out to evaluate the phytochemical profile of the crude ethanolic extract of the plant leaves and its fractions as well as their antimicrobial activities. The phytochemical profile was performed using high-performance thin-layer chromatography (HPTLC), gas chromatography-mass spectrometry (GC-MS), and high-performance liquid chromatography (HPLC). Additionally, the phenolic and flavonoid contents were determined using the Folin-Ciocalteu spectrophotometric and the aluminum trichloride methods. Antimicrobial activity was tested using disc diffusion and microdilution methods. The presence of flavonoids, tannins, sterols, and triterpenes was revealed. GC-MS detected twelve compounds main compounds consisting of saturated and unsaturated fatty acids and phenolic and terpenoid compounds among twenty-seven components. HPLC also detected high contents of phenolic acids and flavonoids. The most abundant triterpene and sterols were ursolic acid (around 43.14 g/100g DW, 13.9 g/100g dry weight (DW), and 0.68 g/100g DW) in the crude ethanolic extract of leaves (FeLi), hexane fraction (FHLi) and dichloromethane fraction (FDLi), respectively and, β-sitosterol in FeLi (56.7 mg/100g DW), FHLi (10.55 g/100g DW), FDLi (106.1 mg/100g DW) and butanol fraction (FBLi) (357.4 mg/100g DW). Among the flavonoids, rutin = 3.24 g/100g and quercetin = 0.63 g/100g in the ethanolic extract, rutin = 15.73 g/100g in the dichloromethane fraction, and rutin = 0.23 g/100g) in the aqueous fraction;and among phenolic compounds, caffeic acid (37.65 g/100g DW) and vanillic acid (22.70 g/100g DW) were the most important in the ethyl acetate fraction (FAeLi). All organic fractions exhibited interesting antibacterial and antifungal activities against the tested strains, with the best activity recorded with the dichloromethane and ethyl acetate fractions. The leaf extracts’ phytochemical profile and antimicrobial activity support the use of Lawsonia inermis against infectious skin diseases.

HPLC/MS Fingerprint Analysis of Tangshenosides

Radix codonopsis(党参)，which is derived fromCodonopsis pilosula, C.pilosula subsp. Modesta andC. Tangshen(ChP,2000),has been used as a remedy fora decrease of appetite, psychoneurosis, fatigue, dyspepsiaetc.or as a substitute for Panax ginseng in traditionalChinese medicine. Relating to the constituents of theseherbs, triterpenes, phytosterols, furaldehyde, sesquitesand some phenolic glycosides have been reported, butnone of these compounds seems to be responsible for thebiological activities of this drug. As regards the quality evaluation of Radix Codonopsis, a HPLC analysis of atractylenolide Ⅲ has been described, but this sesquiterpene lactone exists in a very low content and is difficultfor conventional analysis. The determination of polysaccharide also met limitation because of its non-specialization. In this paper, we report the HPLC/MS fingerprintsanalysis of phelolic glycosides from Radix Codonopsis including three species.

Qualitative and Quantitative Analysis of Alkaloids in Cortex Phellodendri by HPLC-ESI-MS/MS and HPLC-DAD

A combined method of high performance liquid chromatograph-elecrtrospray-ionization mass spectrometer（HPLC-ESI-MS/MS） coupled with a photodiode array detector（HPLC-DAD） and principal component analysis（PCA） was applied to the qualitative and quantitative analyses of alkaloids in Cortex Phellodendri（CP） samples, and to the differentiation of two species of CP, Cortex Phellodendri Chinensis（CPC） and Cortex Phellodendri Amurensis（CPA）. Twenty-two peaks appeared in the HPLC-MS base peak chromatogram of CP detected by the HPLC-ESI-MS/MS analysis, and the alkaloids were identified according to the MSn data, the known MS fragmentation rules and the literature data. Five alkaloids including berberine, palmatine, jatrorrhizine, phellodendrine and magnoflorine were simultaneously determinated by the HPLC-DAD. Berberine was the primary component in all CP samples, and the contents of berberine and palmatine were exploited to be two critical parameters for effective discrimination between the two species of CP. The average content of berberine in CPC（58.75 mg/g） was higher than that in CPA（9.16 mg/g）, while the content of palmatine was less, only 0.25 mg/g in CPC and 4.19 mg/g in CPA. With the use of PCA, samples datasets were separated successfully into two different clusters corresponding to the two species, and berberine, pahnatine, phellodendrine and magnoflorine contribute most to the above mentioned calssifying . The proposed method oroved to be a useful tool in the aualitv control of Chinese herbal medicines.

Quantitative analysis of sialic acids in Chinese conventional foods by HPLC-FLD

Background: Sialic acids are a family of ninecarbon sugar compounds with carboxylic acyl derivatives. It exists in bacteria, fish, mammals and other living organisms, participates in and regulates many important life events, such as cell recognition, membrane flow, endocytosis and so on. Sialic acid is usually located in the outermost layer of the sugar part of the cell membrane and the key positions of secreted glycoconjugates (glycolipids, glycoprotein and lipopolysaccharide). Sialic acid (Sia) is an important material foundation for variety of the structure and founction of glycoconjugates. Sia has been known as nearly 50 members, including N-acetylneuraminic acid (Neu5Ac), N-glycoulylneuraminic acid (Neu5Gc) and deaminoneuraminic acid (KDN) as its core monomer. The rest of the sialic acids are derived from these three monomers. The contents of Sia in Chinese food products are unknown. Objective: To determine the contents of Sia in raw and cooked red meat, seafood, poultry and so on. Design: The following food products were purchased from a Chinese supermarket: pork, beef, lamb, salmon, cod, tuna, cow milk, cheese, butter, duck, chicken and chicken eggs. Human milk was collected from Xiamen Maternity and Child Health Care Hospital (Xiamen, China). All tissues were homogenized and hydrolyzed with 0.05 M, 0.1 M and 0.2 M TFA for 150 min at 80°C in dark, respectively. The concentrations of Neu5Ac, Neu5Gc and KDN were determined by using HPLC with fluorescence detector. Results: The contents of total Sia (μg/g tissue or μg/mL liquid sample) in Chinese raw meat were highest in lamb (269.60), followed by pork (254.88), duck (200.63), chicken (162.86) and beef (88.03). The percentages of Neu5Gc were 36.08%, 26.48%, 0%, 0% and 28.40%, respectively. Cod contained higher levels of Sia (171.63) than salmon (104.43) and tuna (77.98). Only Neu5Ac was 50 found in detected aquatic product. Egg yolk contained the highest level of Sia (682.04), and a higher level of Sia (390.67) was found in the egg white. Also our result showed that human milk contained extremely high level of Sia (602.55). Neu5Ac was the predominant form of Sia in all the deteced samples. KDN was found in cow milk only among the samples, the content was 1.14 μg/g. Conclusion: The highest content of Sia in examined Chinese foods was found in 56 eggs, followed by lamb, pork, duck, cod, chicken, salmon, beef and tuna. Knowledge of the Sia content in conventional foods may help us to better understand possible medical disorders involving the uptake of the “non-human” Neu5Gc from our diet.

HPLC Analysis of Monofluoro-S-Triazine Dye during the Dyeing Process

In the research, the HPLC technique was applied in order to monitor the hydrolysis and dye-fibre bond-forming during the dyeing process. The results show that using a proper execution of calibration curves of the active and hydrolyzed form of the dye and defined equations, HPLC technique enable determining the exact amount of both dye forms anytime during dyeing. Dyeing process was performed in dyeing machine with use of combination of both alkalis—Na2CO3 and NaOH—and with use of one alkali—Na2CO3. It was established that NaOH causes additional hydrolysis of the dye and that the use of Na2CO3 is more appropriate for Novacron Scarlet F-3G dye. The temperature of adsorption has no influence on dye fixation;an amount of fixed dye on fibre is up to 83.0%.

Qualitative and Quantitative Analysis for the Quality Control of Rhizoma Coptidis by HPLC-DAD and HPLC-ESI-MS

For quality control purpose, an approach of fingerprinting and simultaneous quantification of five major bioactive constituents of Rhizoma Coptidis was established via a high-performance liquid chromatograph coupled with a photodiode array UV detector（HPLC-DAD） and an electrospray ionization mass spectrometer（HPLC-ESI/MS） The compounds were identified on the basis of the comparison of their mass spectra with literature data and those of standard samples and quantified by the HPLC-DAD method. Baseline separation was achieved on an XTerra C18 column（5 μm, 250 mm×4.6 mm i. d.） with linear gradient elution of formate buffer（consisting of 0.5% formic acid, adjusted to pH=4.5 with ammonia） and acetonitrile（consisting of 0.2% formic acid and 0.2% triethylamine）. The me- thod was validated for linearity（r^2〉0.9995）, repeatability（RSD〈3.1%）, intra- and inter-day precision（RSD〈1.8%） with recovery（99.9%-105.1%）, limits of detection（0.15-0.35 μg/mL）, and limits of quantification（0.53-0.82 μg/mL）. The similarities of 32 batches of Rhizoma Coptidis and their classification according to their manufacturers were based on the retention time and peak areas of the characteristic compounds. The five compounds were selected for quality assessment ofRhizoma coptidis via partial least squares analysis（PLS）.

NON-AQUEOUS REVERSED PHASE HPLC WITH LOW-WAVELENGTH UV DETECTION FOR TRIGLYCERIDE ANALYSIS

Glycerides are first separated to classes of triglycerides(TGs), diglycerides(DGs) and monoglycerides(MGs) by normal phase HPLC on silica gel column. Individual triglyceride separation is then achieved by non-aqueous reversed phase(NARP) HPLC on C_(18) column with UV detection at 215nm.

Published the papers of HPLC-MS analysis—Chemical drug

Sun Guoxiang, Ding Nan, Song Yuqing, Wang Zhen, Song Liangwei. Determination of Guaiacol salicylate in Guacetisal by HPLCmethod and qualitative identification of relevant substances by HPLC-MS

Comparative Study on“Long-Dan”,“Qin-Jiao”and Their Adulterants by HPLC Analysis

“Long-Dan”and“Qin-Jiao”are two important TCM herbs since ancient times in China.In the Chinese Pharmacopoeia,the dried roots and rhizomes of four species from the genus Gentiana,e.g.Gentiana manshurica,G.scabra,G.triflora and G.rigescens,are recorded under the name of Gentianae Radix et Rhizoma(“Long-Dan”in Chinese),while the other four species from the same genus including G.macrophylla,G.crassicaulis,G.straminea and G.duhurica are recorded and used as the raw materials of Gentianae Macrophyllae Radix(“Qin-Jiao”in Chinese).On the basis of the establishment of a validated HPLC–UV method for quantifying simultaneously,five iridoid glycosides,e.g.loganic acid(1),swertiamarinin(2),gentiopicroside(3),sweroside(4)and 20-(o,m-dihydroxybenzyl)sweroside(5)have been used successfully as chemical markers for the comparison of the species used as“Long-Dan”,“Qin-Jiao”and their adulterants in the present study.The results suggested that four iridoid glycosides 1–4 commonly existed in both“Long-Dan”and“Qin-Jiao”,while 20-(o,m-dihydroxybenzyl)sweroside(5)also existed as one of the major components in“Dian-Long-Dan”species.Moreover,the contents of compounds 1–5 were various in different“LongDan”and“Qin-Jiao”species.Herein,we profiled and compared three“Long-Dan”species,four“Qin-Jiao”species and five adulterants by applying multivariate statistical techniques to their HPLC data sets to establish the differences and/or similarities.

Published the papers of HPLC-MS analysis—Traditional Chinese medicine

Medicinal materials Ma Chenchen, Li Bailin, Ou Jie, Wang Jing, Zhao Junhong. Detection of N-acyl-homoserine lactones class signal molecules of quorum sensing secreted by bacteria using high performance liquid chromatography-mass spectrometry/ mass spectrometry

ANALYSIS OF ETHACRYNIC ACID IN URINE BY HPLC-FLUORESEENCE DETECTOR

A sensitive and selective method has been developed for the detection of free ethacrynic acid in human urine. Using 4-(bromomethyl)-7-methoxycoumarin, ethacrynic acid was transformed into a fluorescent derivative, and was analysed by HPLC-fluorescence detector. The detection limit is 0.1ug/ml urine. The method is suitable for screening ethacrynic acid in doping control and studying its metabolism.

HPLC法同时测定脑脉泰胶囊中12种成分的含量

目的建立HPLC法同时测定脑脉泰胶囊中3′-羟基葛根素、2-羟基红花黄色素A、葛根素、葛根素芹菜糖苷、3′-甲氧基葛根素、大豆苷、二苯乙烯苷、木犀草苷、染料木苷、木犀草素、丹酚酸B、迷迭香酸的含量。方法分析采用KromasilC_(18)色谱柱(250mm×4.6mm,5μm);流动相乙腈-水(含1.0g/L磷酸),梯度洗脱;体积流量0.8mL/min;柱温30℃;检测波长280nm。再进行聚类分析、主成分分析。结果12种成分在各自范围内线性关系良好(r>0.9990),平均加样回收率97.07%~98.30%,RSD0.86%~1.82%。10批样品聚为4类,4种主成分累积方差贡献率达86.262%。结论该方法简便稳定,可靠准确,可为脑脉泰胶囊的质量控制提供科学依据。

基于HPLC-QAMS多组分定量联合多元统计分析及加权TOPSIS法的畲药树参综合品质评价

目的建立同步检测畲药树参中紫丁香苷、绿原酸、芥子醛葡萄糖苷、松柏醇、芦丁、山柰酚-3-O-芸香糖苷、3,4-O-二咖啡酰基奎宁酸、3,5-O-二咖啡酰基奎宁酸和4,5-O-二咖啡酰基奎宁酸含量的高效液相色谱一测多评(HPLC-QAMS)方法,并采用多元统计分析及加权优劣解距离(technique for order preference by similarity to ideal solution method,TOPSIS)法对其品质进行综合评价。方法以Waters Xbridge C 18色谱柱;乙腈-0.05%甲酸溶液为流动相,梯度洗脱;检测波长260 nm。以山柰酚-3-O-芸香糖苷为参照物,建立内参物与其他8个待测成分的相对校正因子(relative correction factor,RCF),进行RCF耐用性考察及色谱峰定位,同时与外标法实测结果进行对比,验证HPLC-QAMS法准确性和可靠性。运用主成分分析(principal component analysis,PCA)、正交偏最小二乘法-判别分析(orthogonal partial least squares-discriminant analysis,OPLS-DA)等多元统计分析以及W-TOPSIS法对9个成分HPLC-QAMS法含量结果的相关性进行分析,挖掘影响畲药树参产品质量的主要潜在标志物,建立畲药树参综合质量优劣评价方法。结果9种成分分别在3.27~81.75μg/mL、9.85~246.25μg/mL、0.43~0.75μg/mL、0.31~7.75μg/mL、1.58~39.50μg/mL、0.59~14.75μg/mL、1.26~31.50μg/mL、4.55~113.75μg/mL和1.98~49.50μg/mL范围内线性关系良好,平均加样回收率96.82%~100.07%(RSD<2.0%);HPLC-QAMS和外标法(ESM)含量测定结果差异无统计学意义(P>0.05),HPLC-QAMS法可用于畲药树参多组分定量控制;多元统计分析结果显示,前2个主成分累计方差贡献率89.589%,绿原酸、紫丁香苷、3,5-O-二咖啡酰基奎宁酸和4,5-O-二咖啡酰基奎宁酸是影响畲药树参产品质量的主要潜在标志物;加权TOPSIS法结果显示浙江地区所得畲药树参质量最优,其次为江西、安徽、湖南和湖北产树参,云南和贵州产树参位于排名后4位。结论所建立的HPLC-QAMS多组分定量控制方法,操作便捷、结果准确;多元统计分析联合加权TOPSIS法全面客观,可用于畲药树参品质的综合评价。

Analysis of three types of triterpenoids in tetraploid white birches(Betula platyphylla Suk.) and selection of plus trees

Betulin, oleanolic acid, and betulinic acid are naturally occurring pentacyclic triterpenoids that have significant medicinal value. Considerable amounts of these triterpenoids are available in the outer bark of white birch. In this study, we used ultrasound-assisted extraction （UAE） to extract triterpenoids from birch bark rapidly and with high efficiency. Using high performance liquid chro- matography （HPLC）, three types of triterpenoids were separated and detected. We examined the differences among triterpenoids extracted from diploid versus tetra- ploid white birch. Then, we used factor analysis to screen out tetraploid white birches with comprehensively excel- lent performance. The results indicate that the optimum conditions for extraction include the use of ethanol as an extraction solvent, a solid-to-liquid ratio of 0.1 g/10 ml, ultrasonic power set at 100 W, a temperature of 60 ℃ and an extraction time of 15 min. A reversed-phase C18 col- umn （4.6 mm × 250 mm × 5 μm） with a column tem- perature of 30 ℃ and the mobile phase composed of A （acetonitrile） and B （0.1% aqueous phosphoric acid, v/v） at a flow rate of 0.5 ml/min were used, and the detection wavelength was 195 nm. No significant difference wasobserved between diploid and tetraploid white birch in terms of the content of three types of triterpenoids （at a confidence level of 0.05）. As triterpenoid content, height, and DBH （diameter at breast height） are strongly interre- lated, we used factor analysis to evaluate all individuals, and we screened out six plus trees with excellent com- prehensive characters.

基于HPLC-UV-QDa检测不同剂型中噻唑膦和吡虫啉

建立了高效液相色谱-紫外检测器-质谱(HPLC-UV-QDa)测定5种不同剂型产品中噻唑膦和吡虫啉含量的分析方法。样品以乙腈+水(0.05%甲酸)为流动相,采用C18色谱柱进行梯度洗脱,结合紫外和质谱串联检测器进行定性和定量分析。研究结果显示,在质量浓度0.05~50 mg/L,噻唑膦、吡虫啉的线性关系良好,R2分别为0.9992~0.9999和0.9991~0.9996。在0.5、50、500 mg/kg等3个添加水平下,噻唑膦、吡虫啉的平均回收率分别为90.2%~103.9%和94.7%~104.1%,相对标准偏差(RSD)分别为1.04%~6.69%和1.27%~5.88%。噻唑膦和吡虫啉在5种基质中无明显基质效应,检出限分别为0.005和0.01 mg/L,定量限分别为0.02和0.04 mg/kg。方法灵敏度高、稳定性好、准确度高,适用于不同剂型产品中噻唑膦和吡虫啉的分析。

Purification and Structural Analysis of a Selective Toxin Fraction Produced by the Plant Pathogen Setosphaeria turcica

Thirteen fractions from the pathogenic plant fungus Setosphaeria turcica race 1 were separated and collected using high performance liquid chromatography （HPLC）. Their toxic activities were assayed through leaf puncturing on corn differentials （OH43, OH43Ht1, OH43Ht2, and OH43HtN）, and the results revealed that eight fractions were toxic and fraction 6 was specifically toxic to OH43Ht1, which could be taken as a gene-selective toxin fraction. Fraction 6 was finely purified via HPLC and condensed by freeze desiccation. Its chemical structure was analyzed with EI-MS, IR, HMBC, ^1H-NMR, and two-dimensional NMR. The results suggested that fraction 6 contained an unsaturated double bond, carbonyl and methylene groups with molecular weight of 142.