High performance liquid chromatography/electrospray ionization mass spectrometry for simultaneous analysis of alkamides and caffeic acid derivatives from Echinacea purpurea extracts

Extracts of the plant Echinacea purpurea are widely used for medicinal purposes. Effective quality control of these extracts requires rapid methods to determine their chemical composition. A new method for analysis of caffeic acid derivatives and alkamides from Echinacea extracts has been developed. With this method, isomeric isobutylamides and 2-methylbutylamides can be distinguished, a capability that previously published methods have lacked. Quantitative analyses carried out with this method on E. purpurea extracts that have been stored for 18 months indicate that they contain caftaric acid, cichoric acid, and undeca-2Z,4E-diene-8,10-diynoic acid isobutylamide at concentrations of 0.7, 0.71 and 2.0mg/mL, respectively.     

Simultaneous determination of water-soluble and fat-soluble synthetic colorants in foodstuff by high-performance liquid chromatography-diode array detection-electrospray mass spectrometry

An accurate method was developed for the simultaneous determination of water-Tartrazine, Amaranth, Ponceau 4R, Sunset Yellow FCF, and fat-Sudan (I-IV), synthetic soluble colorants in foodstuff. This method uses dimethylsulfoxide (DMSO) as the extraction solvent in the sample preparation process and high performance liquid chromatography (HPLC)-diode array detector (DAD)-electrospray mass spectrometry (ESI-MS), applying selected ion recording in positive/negative alternate mode to acquire mass spectral data, as the analytical technique. Linearity of around three orders in the magnitude of concentration was generally obtained. Detection and quantification limits of the investigated dyes, which were evaluated at signal to noise ratio of 3 for detection limit and 10 for quantification limit, were in the ranges of 0.01-4 and 0.03-11.2 ng, respectively. The recoveries of the eight synthetic colorants in four matrices ranged from 93.2 to 108.3%. Relative standard deviations of less than 8.2% were also achieved. This method has been applied successfully in the determination of water-soluble colorants in the soft drink and the delicious ginger, and fat-soluble dyes in chilli powders and chilli spices.     

Separation of alkamides from Echinacea purpurea extracts by cyclodextrin-modified micellar electrokinetic chromatography

Separation of nine important alkyl methylbutyl- and isobutylamides (known as alkamides) obtained from Echinacea purpurea extracts was investigated by using cyclodextrin-modified micellar electrokinetic chromatography (CD-MEKC). Hydrophobic alkamides interact strongly with the micelles from the most common surfactants used in MEKC and this lead to predominant partition of the analytes into the micellar phase, resulting in poor resolution. The addition of neutral CDs to the alkaline (10 mM phosphate buffer pH 8.0) micellar system of sodium dodecyl sulfate (SDS), sodium cholate (SC) and sodium deoxycholate (SDC) was found to improve the separation of the studied alkamides. Among the several combinations surfactant/CD, three different systems showed to be particularly effective: SDS/hydroxypropyl-beta-CD (110 mM/100 mM) and SC/heptakis (2, 3, 6-tri-O-methyl)-beta-CD (200 mM/40 mM) which provided a complete separation of the studied compounds, and SDC/heptakis (2, 6-di-O-methyl)-beta-CD. The importance of appropriate surfactant vs. CD concentration ratio as well as that of total concentration of both surfactant and CD was considered. The optimization of the separation was performed by focussing the need for a rapid separation of nine alkamides diagnostically useful to define the fingerprint of Echinacea species.     

Simultaneous analysis and identification of main bioactive constituents in extract of Zizyphus jujuba var. sapinosa (Zizyphi spinosi semen) by high-performance liquid chromatography-photodiode array detection-electrospray mass spectrometry

A high-performance liquid chromatography (HPLC) coupled with UV photodiode array detection and electrospray mass spectrometry (HPLC-DAD-ESI-MS) was developed for the simultaneous analysis and identification of main bioactive constituents in extract of zizyphi spinosi semen including three flavonoids, three alkaloids and four triterpene saponins. The separation was achieved on a C₁₈ column with a gradient solvent system of 0.08% aqueous formic acid solution-acetonitrile. The compounds were identified with MS-spectra and authentic standards. The contents of jujuboside A, jujuboside B, spinosin and 6?-feruloyl spinosin were quantified by using DAD in different extract of zizyphi spinosi semen.     

Quality control of medicinal herbs Fructus gardeniae, Common Andrographis Herb and their preparations for their active constituents by high-performance liquid chromatography-photodiode array detection-electrospray mass spectrometry

Dehydroandrographolide, andrographolide and geniposide are the main active constituents of many herbal medicines, e.g., Fructus gardeniae, Common Andrographis Herb. They are used as the markers to control the quality of such herbal medicines and their herbal preparations. In this paper, a simple and sensitive high-performance liquid chromatographic (HPLC) method coupled with photodiode array detection (DAD) and electrospray mass spectrometry (ESI/MS) were developed to determine the three compounds simultaneously in extracts of medicinal herbs and herbal preparations produced by different companies. The extracts were separated on a C₁₈ reversed phase HPLC column, with a gradient solvent system, the time for the separation of the three target analytes was 10 min. The abundance ions were recorded using selected ion monitoring (SIM) mode with m/z 297.3, 297.3 and 411.1 for dehydroandrographolide, andrographolide and geniposide, respectively. The limit of detection for dehydroandrographolide, andrographolide and geniposide were 20, 30 and 150 ng mL⁻¹, respectively. The proposed method was successfully applied to the determination of the contents of the compounds in related to medicinal herbs and preparations.     

Simultaneous analysis of anthocyanins and flavonols in petals of lotus (Nelumbo) cultivars by high-performance liquid chromatography-photodiode array detection/electrospray ionization mass spectrometry

A fast and reliable HPLC method for the simultaneous separation of anthocyanins and flavonols in lotus petals was developed based on the study of four candidate solvent systems. Fifteen flavonoids were identified by high-performance liquid chromatography with photodiode array detection/mass spectrometry. Among them, two anthocyanins and nine flavonols were discovered in lotus petals for the first time. This work is valuable for both the hybrid breeding on lotus oriented to flower color and the utilization of lotus petals as functional food materials.     

Determination of galacturonic acid content in pectin from fruit juices by liquid chromatographydiode array detection-electrospray ionization tandem mass spectrometry

A reversed-phase high-performance liquid chromatographic (HPLC) method is applied for the determination of galacturonic acid (GA) of pectins in different commercial fruit juices. The separation was carried out on a C₁₈ column using precolumn derivatization with p-aminobenzoic acid (p-ABA) and UV detection at 304 nm. The identification of GA was confirmed by high-performance liquid chromatography/electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS) in positive ion mode. The concentration of GA in the samples analyzed ranged from 12.9 ± 0.5 to 49.4 ± 0.5 mg_GA L⁻¹. Amongst the samples analyzed, mango juice was found to be richest in GA content, and therefore a good source of pectins. Detection and quantification limits of the described methodology were 1.2 and 3.9 mg L⁻¹, respectively. Quantitative GA recoveries in the beverages had a range between 90 and 98%. The results showed that the HPLC method proposed was precise and suitable for the identification and quantification of GA in commercial fruit juices.     

Fingerprint quality control of Tianjihuang by high-performance liquid chromatography-photodiode array detection

A novel, simple and accurate fingerprint method was developed using high-performance liquid chromatography-photodiode array detection (HPLC-DAD) for the quality control of Hypericum japonicum thunb (Tianjihuang), a Chinese herbal medicine used for the treatment of several bacterial diseases, infectious hepatitis, gastrointestinal disorder, internal hemorrhage and tumors. For the first time, the feasibility and advantages of employing chromatographic fingerprint were investigated and demonstrated for the evaluation of Tianjihuang by systematically comparing chromatograms with a professional analytical software recommended by State Food and Drug Administration (SFDA). Our results revealed that the chromatographic fingerprint combining similarity evaluation could efficiently identify and distinguish raw herbs of Tianjihuang from different sources. The effects resulted from collecting locations, harvesting time and storage time on herbal chromatographic fingerprints were also examined.     

Chemometric analysis of high performance liquid chromatography-diode array detection-electrospray mass spectrometry of 2- and 3-hydroxypyridine

Triply coupled high performance liquid chromatography using diode array detection and positive ion electrospray mass spectrometry of 2- and 3-hydroxypyridine is presented. Considerations of the physical method for coupling the two detectors, the influence of pH on retention times, the cone voltage of the mass spectrometer and the linear concentration ranges are described. Data from both detectors are aligned and interpolated. The analyte mass spectra are reduced to 20 significant masses. Principal components plots on the raw, normalised and standardised data, derivatives to determine composition 1 regions, deconvolution and procrustes analysis to compare data from both detectors are discussed. Common trends in both mass spectral and diode array chromatograms are interpreted. This paper represents a new approach to common processing of chromatographic data from two detectors.     

液相色谱-二极管阵列检测/质谱联用在活性染料结构鉴定中的应用

基于多年运用液相色谱-二极管阵列检测/质谱(HPLC-DAD/MS)联用技术分析活性染料的实验积累,总结了HPLC-DAD/MS联用方法在商品活性染料结构鉴定中的重要应用。商品染料不需要纯化即可通过单次进样达到复杂组分的分离并得到光谱和质谱信息;结合衍生物质量差值法可推定染料的活性基种类和数目;采用二极管阵列检测器的全光谱扫描功能可以得到染料的发色体类别等有用的信息。HPLC-DAD/MS联用方法简便、快速、准确,尤其适用于多组分拼混的商品染料的结构鉴定,有较大的实用价值。通过对多组分商品染料活性灰、活性深黑、海洋蓝中关键组分的结构分析实例,介绍了HPLC-DAD/MS联用分析技术在未知商品活性染料结构分析中的应用。     

超高效液相色谱-二极管阵列检测-串联质谱法测定茶叶中15种黄酮醇糖苷类化合物

采用ACQUITY UPLC HSS T3色谱柱,以含0.1%(v/v)甲酸的乙腈-水为流动相,梯度洗脱,建立了超高效液相色谱-二极管阵列检测-串联质谱(UPLC-PDA-MS/MS)联用技术测定茶叶中黄酮醇糖苷类化合物的方法。结合色谱保留时间、紫外光谱、一级和二级质谱参数等信息,在绿茶和红茶中共识别了15种黄酮醇糖苷类化合物,包括3种杨梅素糖苷、6种槲皮素糖苷和6种山柰素糖苷类化合物。定量分析中采用串联四极杆质谱检测,以槲皮素-3-葡萄糖-鼠李糖二糖糖苷(Q-GRh)为标准品,其他黄酮醇糖苷进行相对定量。结果表明,绿茶和红茶中黄酮醇糖苷类化合物的含量和分布差异显著,绿茶中的黄酮醇糖苷总量是红茶的1.7倍,绿茶中的黄酮醇糖苷主要以杨梅素-3-半乳糖糖苷(M-Ga)、杨梅素-3-葡萄糖糖苷(M-G)、槲皮素-3-葡萄糖-鼠李糖-葡萄糖三糖糖苷(Q-GaRhG)、槲皮素-3-半乳糖-鼠李糖-葡萄糖三糖糖苷(Q-GRhG)、山柰素-3-半乳糖-鼠李糖-葡萄糖三糖糖苷(K-GaRhG)和山柰素-3-葡萄糖-鼠李糖-葡萄糖三糖糖苷(K-GRhG)为主,而红茶中主要以Q-GRh、槲皮素-3-葡萄糖糖苷(Q-G)、山柰素-3-葡萄糖-鼠李糖二糖糖苷(K-GRh)和山柰素-3-半乳糖糖苷(K-Ga)为主。本方法简单快速,准确性好,可用于茶叶中黄酮醇糖苷类化合物的分析。     

Analysis of xanthohumol and isoxanthohumol in different hop products by liquid chromatography-diode array detection-electrospray ionization tandem mass spectrometry

An analytical methodology based on the sample extraction with methanol/formic acid by ultra-sonication and subsequent analysis by high-performance liquid chromatography with diode array detection is proposed for the determination of xanthohumol (XN) and isoxanthohumol (IXN) in different hop products. The identity of the compounds was confirmed by high-performance liquid chromatography/electrospray ionization tandem mass spectrometry in positive ion mode. The performance of the method was assessed by the evaluation of parameters such as absolute recovery, repeatability, linearity and limits of detection and quantitation. This methodology was applied to investigate the impact of the extraction process of the hop products on the amount of xanthohumol and isoxanthohumol. The ethanolic extract revealed to be the hop product richest in xanthohumol (3.75+/-0.05 g/100 g) relatively to the pellets (0.62+/-0.01 g/100 g) and supercritical CO2 extract (0.089+/-0.001 g/100 g).     

Analysis of alkannin derivatives from Alkanna species by high-performance liquid chromatography/photodiode array/mass spectrometry

Alkannin, shikonin (A/S) and their derivatives are enantiomeric hydroxynaphthoquinone red pigments found in the roots of almost 150 species of the Boraginaceae family. A/S have been shown to exhibit strong wound healing, antimicrobial, anti-inflammatory and antioxidant activities and recent extensive research has well established their antitumor properties. A/S and their derivatives comprise the active ingredients of several pharmaceutical and cosmetic preparations. Although A/S have been efficiently synthesized and have been produced by cell tissue cultures in high yield, most of the pharmaceutical preparations worldwide contain A/S extracted from the roots of Boraginaceous species, found in nature. In the present study, a high-performance liquid chromatography/photodiode array/mass spectrometry (HPLC/PDA/MS) method was established to identify monomeric hydroxynaphthoquinones of the alkannin series and other metabolites from Boraginaceous root extracts. This method can be applied for the identification of alkannin derivatives and other metabolites from Boraginaceous cell cultures, and also to determine active ingredients in pharmaceutical preparations containing A/S derivatives. A phytochemical investigation of the Alkanna genus grown in Greece was also performed. Fifty-three root samples belonging to 10 species of the genus Alkanna (A. calliensis, A. corcyrensis, A. graeca, A. methanaea, A. orientalis, A. pindicola, A. primuliflora, A. sieberi, A. stribrnyi and A. tinctoria) were collected from several regions of the Greek flora and analyzed for their constituent hydroxynaphthoquinones and other metabolites. In most of the above Alkanna samples tested, the main hydroxynaphthoquinones were determined to be beta,beta-dimethylacrylalkannin, isovalerylalkannin + alpha-methyl-n-butylalkannin and acetylalkannin. The hydroxynaphthoquinone constituents and their proportions were found to vary among Alkanna species. Unknown metabolites (not monomeric hydroxynaphthoquinones) were detected by HPLC-PDA-MS, while in several Alkanna species hydroxynaphthoquinones were detected for the first time.     

Quantitative analysis of caffeic acid phenethyl ester in crude propolis by liquid chromatography-electrospray ionization mass spectrometry

Caffeic acid phenethyl ester (CAPE), an active component of propolis from honeybee hives, is known to have antimitogenic, anticarcinogenic, antinflammatory, and immunomodulatory properties. The paper describes a rapid and simple liquid chromatography-electrospray ionisation mass spectrometry method for qualitative and quantitative determination of CAPE. The chromatographic separation was performed with a Luna RP-C18 column using a water-acetonitrile linear gradient. The method was linear over a 0.125-80 ng/mL range (LOD = 62.5 pg/mL). The method was applied for the quantitation of caffeic acid phenethyl ester in crude propolis samples, which were analysed directly after extraction with ethyl acetate solution.     

Characterization and determination of six aristolochic acids and three aristololactams in medicinal plants and their preparations by high-performance liquid chromatography-photodiode array detection/electrospray ionization mass spectrometry

Aristolochic acid derivatives (AAs) and aristolactam derivatives (ALs) have been characterized by electrospray ionization mass spectrometry, and their fragmentation pathways are proposed. ALs exhibit a single ionization product [M+H]+, whereas AAs show multiple ionization products. By optimizing the chromatographic separation and mass spectrometric parameters, the precursor ions of the derivatives with the best responses were found, and the sensitivities in the determination of the nine derivatives were improved. Based on the investigation of ionization behaviour, a HPLC-DAD/ESI-MS (high-performance liquid chromatography-photodiode array detection/electrospray ionization mass spectrometry) method has been developed for simultaneous analysis of nine derivatives, i.e., AA I, AA II, AA C, AA D, 7-OH AA I, aristolic acid I, AL AII, AL IIIa and AL IVa, in nine medicinal herbs and two preparations. The method appears to be suitable for safety assurance and quality control of commercially available samples with good selectivity and suitable sensitivity.     

Speciation of mercury by ionic liquid-based single-drop microextraction combined with high-performance liquid chromatography-photodiode array detection

Room temperature ionic liquids can be considered as environmentally benign solvents with unique physicochemical properties. Ionic liquids can be used as extractant phases in SDME, being compatible with chromatographic systems. A single-drop microextraction method was developed for separation and preconcentration of mercury species (MeHg⁺, EtHg⁺, PhHg⁺ and Hg²⁺), which relies on the formation of the corresponding dithizonates and microextraction of these neutral chelates onto a microdrop of an ionic liquid. Afterwards, the separation and determination were carried out by high-performance liquid chromatography with a photodiode array detector. Variables affecting the formation and extraction of mercury dithizonates were optimized. The optimum conditions found were: microextraction time, 20 min; stirring rate, 900 rpm; pH, 11; ionic liquid type, 1-hexyl-3-methylimidazolium hexafluorophosphate ([C₆MIM][PF₆]); drop volume, 4 μL; and no sodium chloride addition. Limits of detection were between 1.0 and 22.8 μg L⁻¹ for the four species of mercury, while the repeatability of the method, expressed as relative standard deviation, was between 3.7 and 11.6% (n = 8). The method was finally applied to the determination of mercury species in different water samples.     

Simultaneous qualitation and quantification of thirteen bioactive compounds in Flos lonicerae by high-performance liquid chromatography with diode array detector and mass spectrometry

A high-performance liquid chromatography (HPLC) with diode array detector (DAD) and electrospray ionization mass spectrometry (ESI-MS) was established for the simultaneous determination of thirteen bioactive compounds in Flos Lonicerae. The optimal chromatographic conditions were obtained on a C(18) column (250x4.6 mm, 5.0 microm) with the column temperature at 30 degrees C. The mobile phase was composed of (A) acetic acid aqueous (0.4%, v/v) and (B) acetonitrile using a gradient elution, the flow rate was 1 ml/min. Detection wavelengths were set at 240 nm for iridoids (loganin, sweroside, secoxyloganin and centauroside), 330 nm for phenolic acids (chlorogenic acid, caffeic acid, 4,5-di-O-caffeoyl quinic acid and 3,4-di-O-caffeoyl quinic acid) and 360 nm for flavonoids (rutin, hyperoside, quercetin-3-O-glucoside, luteolin-7-O-glucoside and lonicerin). The identities of the peaks were accomplished by comparing retention times, UV and mass data with reference compounds under the same conditions. All calibration curves showed good linear regression (r(2)>0.9983) within test ranges. The developed method provided satisfactory precision and accuracy with overall intra-day and inter-day variations of 0.78--1.85% and 1.13--2.36%, respectively, and the overall recoveries of 91.3--104.2% for the thirteen compounds analyzed. The verified method was successfully applied to quantitative determination of the three types of bioactive compounds in ten commercial Flos Lonicerae samples from different markets in China. The analytical results demonstrated that the contents of the thirteen analytes were relatively variant.     

Analysis of antioxidant compounds in sweet orange peel by HPLC-diode array detection-electrospray ionization mass spectrometry

HPLC-diode array detection-electrospray ionization mass spectrometry was used to determine qualitatively and quantitatively the flavonoid content of several fractions and residues of extracts of Greek navel sweet orange peel (Citrus sinensis) from the region of southern Greece (Leonidi-Tripoli). The main groups of flavonoids found according to HPLC retention times, spectral data and literature references were polymethoxylated flavones, C-glycosylated flavones, O-glycosylated flavones, O-glycosylated flavanones, flavonols and phenolic acids and their derivatives. The ethyl acetate fraction which has been shown in previous work to possess the best radical scavenging activity among the others was found to contain C-glycosylated flavones, polymethoxylated flavones, O-glycosylated flavones, O-glycosylated flavanones, two phenolic acid derivatives and two unknown compounds, all in low concentrations. The group of C-glycosylated flavones was reported for the first time in the peel of Navel sweet orange. The C-glycosylated flavones found according to their spectral characteristics and literature were 6-C-beta-glucosyldiosmin, 6,8-di-C-glucopyranosylapigenin, 6,8-di-C-beta-glucosyldiosmin and two unknown. The results suggest that the ethyl acetate fraction of navel Citrus sinensis peel consists of significant antioxidant compounds and can be used as a food additive of natural origin or a pharmaceutical supplement using as a source of peel the byproducts of the orange juice industry.     

Simultaneous determination of monomeric and oligomeric alkannins and shikonins by high-performance liquid chromatography-diode array detection-mass spectrometry

Alkannin and shikonin (A/S) derivatives have been found in the roots of several Boraginaceous species and are produced through plant tissue cultures. The chiral compounds alkannins and shikonins are potent pharmaceutical substances with a wide spectrum of pharmacological activities such as wound healing, antimicrobial, anti-inflammatory, anticancer and antioxidant. Although oligomeric A/S derivatives have been detected in root extracts and commercial samples their detection and determination through high-performance liquid chromatography has not been reported. Therefore, in the present study a rapid, simple high-performance liquid chromatography-diode array detection-mass spectrometry (HPLC-DAD-MS) method was developed to detect, separate and determine monomeric and oligomeric/polymeric derivatives of alkannin/shikonin simultaneously for the first time. An optimization of HPLC-DAD parameters was performed. Both atmospheric pressure chemical ionization (APCI) and electrospray ionization (ESI) modes were applied, in order to compare detection of monomeric and oligomeric A/S. Additionally, oligomeric A/S constituents in several samples were identified and the mode of A/S polymerization was proposed.