Subsequent HPLC Separation Research Articles

Preparative separation of ten flavonoids from Scutellaria baicalensis Georgi roots using two-dimensional countercurrent chromatography with an online-storage, dilution, and mixing interface

The process of counter-current chromatography (CCC) separation for natural products typically necessitates the use of multiple solvent systems to accommodate constituents with a wide range of polarities. However, the incompatibility between these different solvent systems often results in unsuccessful online 2D successive separations. In this study, a 2D CCC system was developed, featuring an interface for online-storage, dilution, and mixing. It facilitated the implementation of online 2D CCC using different solvent systems. The method was subsequently applied for the preparative isolation of flavonoids from Scutellaria baicalensis Georgi roots. For 1D CCC, n-heptane-ethyl acetate–methanol-water (HepEMWat, 5:5:4:6, v/v) was utilized, while for 2D CCC, ethyl acetate-n-butanol-water (EBuWat, 0:5:5, v/v) was employed. The eluent with low resolution in 1D CCC was stored online, diluted three times using the lower phase of EBuWat (0:5:5, v/v), and subsequently transferred into 2D CCC for further isolation utilizing the same EBuWat (0:5:5, v/v) system. As results, six lipophilic compounds were isolated in 1D CCC in a normal mode, while two major hydrophilic constituents were isolated in a pH-peak-focusing mode in 2D CCC. Additionally, two additional compounds were purified through subsequent semi-preparative HPLC separation in order to resolve co-elution in 2D CCC. The developed 2D CCC system with a multifunctional interface demonstrated to be an exceptionally efficient and promising approach for the high-throughput purification of complex natural products.

Synthesis of Optically Active N-(4-Hydroxynon-2-enyl)pyrrolidines: Key Building Blocks in the Total Synthesis of Streptomyces coelicolor Butanolide 5 (SCB-5) and Virginiae Butanolide A (VB-A)

AbstractStarting from 5-methylhexanal and (S)-configured N-propargylprolinol ethers, coupling delivered N-(4-hydroxynon-2-ynyl)prolinol derivatives as mixtures of C4 diastereomers. Resolution of the epimers succeeded after introduction of an (R)-mandelic ester derivative and subsequent HPLC separation. Alternatively, suitable oxidation gave the corresponding alkynyl ketone. Midland reagent controlled diastereoselective reduction afforded a defined configured propargyl alcohol with high selectivity. LiAlH4 reduction and Mosher analyses of the allyl alcohols enabled structure elucidation. The suitably protected products are used as key intermediates in enantioselective Streptomyces γ-butyrolactone signaling molecule total syntheses.

Determination of Twenty Proteinogenic Amino Acids and Additives in Cultural Liquid by High-Performance Liquid Chromatography

An HPLC procedure was developed to determine twenty proteinogenic amino acids, ammonium, cystine, and alanyl-glutamine dipeptide in liquid culture media. 6-Aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) was used as a fluorescent label for the derivatization of amino acids with the subsequent HPLC separation on a reversed-phase column. Specificity, linearity, correctness, precision, analytical range, and limit of quantification were evaluated for each particular component in the procedure validation tests. The analytical range for amino acids was 10–800 μM (20–800 μM for tryptophan and cystine), and the limit of quantification was no higher than 5 μM (15 μM for tryptophan and 8 μM for cystine).

Potential of Myrtus communis Linn. as a bifunctional food: Dual high-resolution PTP1B and α-glucosidase inhibition profiling combined with HPLC-HRMS and NMR for identification of antidiabetic triterpenoids and phloroglucinol derivatives

Abstract Myrtus communis Linn., an aromatic shrub widely distributed in Mediterranean littoral, is traditionally used as a culinary herb and for manufacturing of the local Sardinian liquor, Mirto. In this study, the potential for developing M. communis into a functional food to treat type-2-diabetes (T2D) was investigated. Antidiabetic constituents in crude defatted chloroform extract of M. communis were pinpointed by dual high-resolution PTP1B and α-glucosidase inhibition profiling. Subsequent analytical-scale HPLC separation led to isolation and identification of 14 triterpenoids and three phloroglucinol derivatives associated with PTP1B and/or α-glucosidase inhibitory activity with IC50 values in the range of 8.9–69.4 μ m and 34.3–88.5 μ m , respectively. We also report three previously undescribed phloroglucinol derivatives. This is the first report of PTP1B inhibitors in M. communis, and the identification of both PTP1B and α-glucosidase inhibitors, demonstrates the potential of M. communis as a bifunctional food for management of T2D.

Radical Scavenging Properties of Roasted Egoma (Perilla frutescens var. frutescens) Oils and Identification of Their Characteristic Scavengers.

The radical scavenging activity of commercially available roasted (deep colored) and unroasted (light colored) egoma (Perilla frutescens var. frutescens) oils was evaluated by the DPPH radical scavenging method. The antiradical activity of roasted oils was higher than that of unroasted oils, and the activity of methanol-water extracts from the roasted egoma oils was significantly higher than that of unroasted oils. The antiradical activity of the methanol-water fractions was strongly correlated to that of whole oils (r=0.72) and the color depth of oils (r=0.93), which was an index of roasting. Fractionation of the methanol-water extract of a roasted egoma oil according to molecular size using ultra membrane filters revealed that the fraction under 3 kDa had the strongest radical scavenging activity. Subsequent preparative HPLC separation using an ODS column also revealed that the second fraction was the most active. Our HPLC analytical method for DPPH radical scavengers in complex mixtures detected four strong radical scavenger peaks in the fraction. Among the detected peaks, two peaks were determined to be derived from rosmarinic acid and luteolin by comparison with the retention times and UV spectra of the authentic samples, and the other two compounds could not be identified because no characteristic UV spectra were observed. These identified polyphenols (rosmarinic acid and luteolin) have been reported to be present in the non-oily part of egoma seeds. They probably migrated to the oily part during the egoma oil roasting process.

Spatial profiling of stapled α–helical peptide ATSP-7041 in mouse whole-body thin tissue sections using droplet-based liquid microjunction surface sampling-HPLC-ESI–MS/MS

The application of a fully automated autosampler/HPLC-ESI–MS/MS system for spatially resolved quantitative droplet-based liquid extraction surface sampling/profiling of stapled α–helical peptide ATSP-7041 in mouse whole-body thin tissue sections is reported. 20-μm-thick serial tissue sections of an ATSP-7041 dosed mouse were prepared and the absolute concentration of the targeted peptide was first determined in different organs using 2.3-mm diameter tissue punches, standard bulk tissue extraction protocols, and subsequent HPLC separation and tandem mass spectrometric analysis. The same organs/locations were then analyzed in neighboring tissue sections using the droplet-based surface sampling approach. The observed ATSP-7041 concentration using this method was always significantly lower than that measured by the tissue punch workflow at the same tissue location of a serial section. Calculated extraction efficiencies were 10.7 ± 0.5% (brain), 11.0 ± 3.2% (liver spot 1), 10.7 ± 2.6% (liver spot 2), 15.0 ± 0.6% (lung) and 12.9 ± 0.7% (blood). While these extraction efficiency values were low, they were reproducible within a given organ. This suggests that once the extraction efficiency is established for a given tissue type and drug, the reproducibility of the droplet-based approach could provide a non-labor intensive and high-throughput means to acquire spatially resolved quantitative analysis of multiple samples of the same type.

Mass Spectrometric Quantification of N-Linked Glycans by Reference to Exogenous Standards.

Environmental and metabolic processes shape the profile of glycoprotein glycans expressed by cells, whether in culture, developing tissues, or mature organisms. Quantitative characterization of glycomic changes associated with these conditions has been achieved historically by reductive coupling of oligosaccharides to various fluorophores following release from glycoprotein and subsequent HPLC or capillary electrophoretic separation. Such labeling-based approaches provide a robust means of quantifying glycan amount based on fluorescence yield. Mass spectrometry, on the other hand, has generally been limited to relative quantification in which the contribution of the signal intensity for an individual glycan is expressed as a percent of the signal intensity summed over the total profile. Relative quantification has been valuable for highlighting changes in glycan expression between samples; sensitivity is high, and structural information can be derived by fragmentation. We have investigated whether MS-based glycomics is amenable to absolute quantification by referencing signal intensities to well-characterized oligosaccharide standards. We report the qualification of a set of N-linked oligosaccharide standards by NMR, HPLC, and MS. We also demonstrate the dynamic range, sensitivity, and recovery from complex biological matrices for these standards in their permethylated form. Our results indicate that absolute quantification for MS-based glycomic analysis is reproducible and robust utilizing currently available glycan standards.

A Validated HPLC Method for Simultaneous Determination of Caffeoyl Phenylethanoid Glucosides and Flavone 8-C-glycosides in Haberlea rhodopensis

A HPLC-UV method for analysis of the main compounds: caffeoyl phenylethanoid glucosides myconoside (1) and paucifloside (2) and flavone 8-C-glycosides: hispidulin 8-C-β-galactopyranoside (3), hispidulin 8-C-(2"-O-syringoyl-β-glucopyranoside) (4), hispidulin 8-C-(6-O-acetyl-β-glucopyranoside) (5) and hispidulin 8-C-(6-O-acetyl-2"-O-syringoyl--glucopyranoside) (6) in Haberlea rhodopensis leaves was developed and validated. Compound 3 was isolated for the first time from the title species. Ultrasound extraction with 80% methanol at room temperature allowed a good recovery of analytes (from 87.2 % for 1 to 109.8 % for 3) and the precision of the entire procedure was between 1.6% and 6.9%. The subsequent HPLC separation and quantification was achieved using a Hypersil ODS C18 column and UV detection at 280 nm. The mobile phase comprised methanol and 0.1 % o-phosphoric acid, and gradient elution mode was applied. The detection limits ranged from 0.042 μg/mL (6) to 0.18 μg/mL (5). The total amount in leaves of the assayed phenolic compounds was 374.2 mg/g. Myconoside was found to be the dominant compound in H. rhodopensis extract (332.2 ± 0.7 mg/g dw) and reached up to 88.8% of the analyzed mixture in leaves, while the total content of flavone C-glycosides was 17.1 mg/g dw.

Quantitation of repaglinide and metabolites in mouse whole-body thin tissue sections using droplet-based liquid microjunction surface sampling-high-performance liquid chromatography-electrospray ionization tandem mass spectrometry

Herein, quantitation aspects of a fully automated autosampler/HPLC-MS/MS system applied for unattended droplet-based surface sampling of repaglinide dosed thin tissue sections with subsequent HPLC separation and mass spectrometric analysis of parent drug and various drug metabolites were studied. Major organs (brain, lung, liver, kidney and muscle) from whole-body thin tissue sections and corresponding organ homogenates prepared from repaglinide dosed mice were sampled by surface sampling and by bulk extraction, respectively, and analyzed by HPLC-MS/MS. A semi-quantitative agreement between data obtained by surface sampling and that by employing organ homogenate extraction was observed. Drug concentrations obtained by the two methods followed the same patterns for post-dose time points (0.25, 0.5, 1 and 2h). Drug amounts determined in the specific tissues was typically higher when analyzing extracts from the organ homogenates. In addition, relative comparison of the levels of individual metabolites between the two analytical methods also revealed good semi-quantitative agreement.

HPLC separation and ultrasensitive optical quantification of ceramide species applying 7-(diethylamino)coumarin-3-carbonyl azide derivatisation.

Ceramides are derivatised using 7-(diethylamino)coumarin-3-carbonyl azide; subsequent gradient HPLC separation allows sensitive optical quantification of individual cellular ceramides. Compared to 9-anthracenecarbonyl cyanide (9-anthroyl nitrile) as derivatisation agent, the limit of detection could be improved 415-fold, respectively 10,000-fold (detection limit 0.6 pmol labelled ceramide/sample) when compared to benzoyl chloride-labelling. Acidic or alkaline catalysts are not required, enabling drying and storing of the labelled samples and a free choice of solvents for subsequent HPLC-separation. The quantitative method is characterised by high sensitivity, linearity and robustness in the pico- to nanomolar concentration range and does not require mass-spectrometry for quantification of cellular ceramides.

Analysis of glycosaminoglycan-derived, precolumn, 2-aminoacridone–labeled disaccharides with LC-fluorescence and LC-MS detection

Glycosaminoglycans (GAGs) possess considerable heterogeneity in average molecular mass, molecular mass range, disaccharide composition and content and position of sulfo groups. Despite recent technological advances in the analysis of GAGs, the determination of GAG disaccharide composition still remains challenging and provides key information required for understanding GAG function. Analysis of GAG-derived disaccharides relies on enzymatic treatment, providing one of the most practical and quantitative approaches for compositional mapping. Tagging the reducing end of disaccharides with an aromatic fluorescent label affords stable derivatives with properties that enable improved detection and resolution. HPLC with on-line electrospray ionization mass spectrometry (ESI-MS) offers a relatively soft ionization method for detection and characterization of sulfated oligosaccharides. GAGs obtained from tissues, biological fluids or cells are treated with various enzymes to obtain disaccharides that are fluorescently labeled with 2-aminoacridone (AMAC) and resolved by different LC systems for high-sensitivity detection by fluorescence, and then they are unambiguously characterized by MS. The preparation and labeling of GAG-derived disaccharides can be performed in ∼1-2 d, and subsequent HPLC separation and on-line fluorescence detection and ESI-MS analysis takes another 1-2 h.

HPLC Analysis of Flavonoids from the Aerial Parts of Bupleurum Species

Aim To establish a quantitative analysis method with HPLC-UV for simultaneous determination of flavonoids in the aerial parts of Bupleurum, to investigate the content and distribution of flavonoids in different species of Bupleurum with the established method for quality control. Method A rapid and accurate HPLC-UV method was developed and validated for the quantitative determination of twelve flavonoids, (+)-catechin (1), apigenin-6, 8-di- C-glucopyranoside (2), quercetin-3- O-rutinoside (3), kaempferol-3, 7-di- O-rhamnoside (4), quercetin-3- O-arabinoside (5), quercetin-3- O-rhamnoside (6), 7-hydroxyl-2, 5-dimethyl-4H-chromone (7), acacetin-7- O-rutinoside (8), kaempferol-7- O-rhamnoside (9), quercetin (10), kaempferol (11) and isoramnetin (12). The subsequent HPLC separation and quantification was achieved in 60 minutes using a Shimpack ODS C 18 column and at 254 nm. The mobile phase comprised solvent A (0.1% formic acid aqueous, V/ V, pH 2.6) and solvent B (acetonitrile) and gradient elution mode was applied. This established method was therefore applied to determine the amounts of flavonoids in twenty-eight samples collected from different regions in China. Result The linear regression analytical data for the calibration plots showed a good linear relationship with r 2 > 0.9995 within the test ranges. The average recovery was from 97.10 to 102.86% indicating good accuracy. It was successfully applied to quantify twelve flavonoids in different samples. Conclusion It is the first time to establish a quantitative method to investigate the content and distribution of flavonoids in different species of Bupleurum.

Simultaneous determination of benzophenones and gentisein in hypericum annulatum moris by high‐performance liquid chromatography

The content of the benzophenones, hypericophenonoside, neoannulatophenonoside, annulatophenonoside, annulatophenone, acetylannulatophenonoside and the xanthone derivative gentisein have been determined in aerial parts, leaves, flowers and stems of Hypericum annulatum Moris. Extraction of samples with methanol by magnetic stirring at room temperature allowed a good recovery of analytes (from 90.70% for gentisein to 103.81% for annulatophenonoside) and the precision of the entire procedure was < 6.05%. The subsequent HPLC separation and quantification was achieved using a Hypersil ODS C18 column and UV detection at 290 nm. The mobile phase comprised methanol and 20 mm potassium dihydrogen phosphate (adjusted to a pH of 3.19 with o-phosphoric acid), and gradient elution mode was applied. The detection limits were 0.03, 0.02 and 0.001 microg/mL for hypericophenonoside, acetylannulatophenonoside and gentisein, respectively. The total amounts of the phenolic compounds assayed ranged from 10.92 mg/g in stems to 82.86 mg/g in leaves. Hypericophenonoside was the dominant benzophenone present in the majority of the plant samples, being present in amounts between 7.54 +/- 0.25 mg/g in stems and 64.22 +/- 2.44 mg/g in leaves. Hypericophenonoside accounted for up to 77.50% of the components found in the leaves, whereas annulatophenonoside (6.29 +/- 0.15 mg/g) and acetylannulatophenonoside (8.95 +/- 0.09 mg/g) were detected in much lower quantities. In contrast to leaves, flowers showed a tendency towards higher contents of gentisein (9.35 +/- 0.07 mg/g) and neoannulatophenonoside (4.72 +/- 0.04 mg/g) than the other parts assayed.

Characterisation and partial purification of a white mustard kairomone that attracts the beet cyst nematode, Heterodera schachtii

AbstractA kairomone from white mustard, Sinapis alba , that attracts infective juveniles (J2) of the beet cyst nematode, Heterodera schachtii, was extracted from root exudates and enriched by rotary evaporation. The kairomone-containing root exudate fractions were then repeatedly enriched and separated on a combination of octyl and octadecyl columns, leaving a single UV-absorbing HPLC peak. Glucose and fructose were identified by a subsequent HPLC separation in the ion moderated partition chromatography mode. The kairomone appeared in the fructose-containing fraction. As fructose did not cause nematode attraction, the fructose fraction contained at least one further substance. HPLC separations showed that the kairomone shares several properties with fructose and that it is relatively more polar than those compounds giving an UV signal. The repeated reversed-phase and ion moderated partition chromatography separations of single kairomone-containing fractions indicate that the kairomone is composed of one single substance which could not be identified with the methods applied. Gas chromatographic and mass spectrometric analyses revealed the presence of glucose, fructose, myo-inositol, sucrose and xylofuranose/ribofuranose in S. alba root exudates.

Depletion of Bcl-2 by an antisense oligonucleotide induces apoptosis accompanied by oxidation and externalization of phosphatidylserine in NCI-H226 lung carcinoma cells.

Oxidant-induced apoptosis involves oxidation of many different and essential molecules including phospholipids. As a result of this non-specific oxidation, any signaling role of a particular phospholipid-class of molecules is difficult to elucidate. To determine whether preferential oxidation of phosphatidylserine (PS) is an early event in apoptotic signaling related to PS externalization and is independent of direct oxidant exposure, we chose a genetic-based induction of apoptosis. Apoptosis was induced in the lung cancer cell line NCI-H226 by decreasing the amount of Bcl-2 protein expression by preventing the translation of bcl-2 mRNA using an antisense bcl-2 oligonucleotide. Peroxidation of phospholipids was assayed using a fluorescent technique based on metabolic integration of an oxidation-sensitive and fluorescent fatty acid, cis-parinaric acid (PnA), into cellular phospholipids and subsequent HPLC separation of cis-PnA-labeled phospholipids. We found a decrease in Bcl-2 was associated with a selective oxidation of PS in a sub-population of the cells with externalized PS. No significant difference in oxidation of cis-PnA-labeled phospholipids was observed in cells treated with medium alone or a nonsense oligonucleotide. Treatment with either nonsensc or antisense bcl-2 oligonucleotides was not associated with changes in the pattern of individual phospholipid classes as determined by HPTLC. These metabolic and topographical changes in PS arrangement in plasma membrane appear to be early responses to antisense bcl-2 exposure that trigger a PS-dependent apoptotic signaling pathway. This observed externalization of PS may facilitate the 'labeling' of apoptotic cells for recognition by macrophage scavenger receptors and subsequent phagocytic clearance.

Determination of RS, E/Z-tocotrienols by HPLC

Synthetic α-tocotrienol was separated into four geometrical E/Z side chain isomers by preparative HPLC (permethylated β-cyclodextrin phase). The isolated isomers were resolved in ethylene glycol dimethyl ether, converted into the corresponding methyl ether using dimethyl sulfate, and the tocotrienol methyl ethers were extracted with n-hexane. A subsequent HPLC separation on a chiral phase (adsorbent cellulose derivated with 3,5-dimethyl phenyl carbamate) discriminates between the enantiomers of each E/Z side chain isomer, achieving the complete resolution of the eight occurring synthetic RS, E/Z-α-tocotrienols. The method can be shortened by omitting the preparative separation of the E/Z tocotrienol isomers prior to the chromatography on the chiral dimethyl phenyl carbamate phase. The simplified method achieved the following separation: RS, E/Z-α-tocotrienol separated into five peaks, RS, E/Z-β-tocotrienol into eight, RS, E/Z-γ-tocotrienol into six and RS, E/Z-δ-tocotrienol into eight peaks. The naturally occurring R, E-E-tocotrienol isomer could be identified within the synthetic RS, E/Z-isomers by co-chromatography with tocotrienol methyl ethers derived from natural sources, respectively.

Determination of Amphetamine by HPLC After Acetylation

An analytical procedure has been developed for the HPLC determination of amphetamine by off-line pre-column derivatization. The proposed procedure consists of sample preparation by acetylation of amphetamine with acetic anhydride and a subsequent reversed-phase HPLC separation on an octadecyl silica stationary phase with salt-free mobile phase (tetrahydrofuran, acetonitrile, 0.1% triethylamine in water, 15:15:70 v/v) applying UV-detection. The applicability of the elaborated procedure is demonstrated with results obtained by analysis of real samples seized in the Hungarian black market.

Trace enrichment, chromatographic separation and biodegradation of cytostatic compounds in surface water

A trace analytical procedure for the cytostatic drugs carmustine, chlorambucil, cisplatin, cyclophosphamide, cytarabine, etoposide, 5-fluorouracil, melphalan, methotrexate, and vinblastine was developed in order to evaluate the environmental hazards of these drugs in clinical waste water and sewage treatment plants. The analysis was performed using solid phase extraction with subsequent HPLC separation and quantitative determination by gradient elution techniques with DAD and fluorescence detection. Detection limits after the clean-up and enrichment procedure vary from 0.002 to 0.2 mg/L. A simulation of the degradation processes under conditions as close as possible to those in a real sewage plant showed that cisplatin and cyclophosphamide are not biodegradable, but cytarabine and 5-fluorouracil are biodegradable in different magnitudes. The biodegradation process of methotrexate is combined with the generation of the toxic and persistent degradation product 7-hydroxymethotrexate.

Soybean lipoxygenase: Substrate structure and product selectivity

To study the influence of structural properties of the substrate on the selectivity of the dioxygenation catalyzed by soybean lipoxygenase (LOX), a number of synthesized (Z,Z)-3,6-dienyl 1-adipates with various distal residues were used and the kinetic parameters, the regioselectivity, and the enantioselectivity of product formation determined. Product analysis comprised the reduction of hydroperoxides, derivatization to their methyl esters, purification by silica gel chromatography, and subsequent HPLC separation of (Z,E)-1,3-hydroxydiene methyl esters using both achiral and chiral phases. Enantioseparations were performed by HPLC on chiral phases employing both underivatized products and naphthoylate derivatives. The calculation of the hydrophobicity difference between the distal and the proximal residues of the substrate was confirmed to be a quantitative parameter to predict the positional specificity of the enzymic catalysis. Higher enantiomeric excess was observed only for 7-oxygenated products ; 3-oxygenated products were found to be racemic.

Determination of 2-alkylcyclobutanone using fluorescent labelling

2-Alkylcyclobutanones are formed under the influence of ionising radiation from triglycerides. The determination of cyclobutaneones could be a powerful means of identifying irradiated foods, not only from a legal viewpoint but also in food quality control. In this paper 2-dodecylcyclobutaneone was used as a model substance, synthesised by means of the key substance 1-bromo-1-ethoxycyclopropane. The determination was carried out by labelling the cyclic ketone with fluorescent dyes and subsequent HPLC separation. 1-Pyrenebutyrylhydrazide and 7-diethylamino-3-carbonylazide were used as labelling reagents. Dodecylcyclobutanone was sensitively determined in fat matrices. Labelling with 1-pyrenebutyrylhydrazide was unsuccessful because of its low reactivity. Medium- and short-chain aldehydes and ketones were easily labelled. After reduction to the corresponding alcohol, dodecylcyclobutanone, as well as aldehydes and ketones, were successfully labelled with 7-diethylamino-3-carbonylazide. Separation was carried out with good results on RP-18 reversed phases using methanol water mixtures as mobile phases. The detection limit of dodecylcyclobutanone is about 5 ng.