In vitro screening and isolation of human aromatase inhibitors from Cicer arietinum by a novel continuous online method combining chromatographic techniques

Ultrafiltration liquid chromatography with mass spectrometry can efficiently and rapidly screen and identify ligands from the seeds of Cicer arietinum for human aromatase. Using this method, we identified 11 major compounds, including organic acids, organic acid glycosides, flavone glycosides, isoflavones, and isoflavone glycosides, as potent human aromatase inhibitors. A continuous online method, including pressurized liquid extraction, countercurrent chromatography, and preparative liquid chromatography, was developed for scaling up the production of these compounds with high purity and efficiency. The bioactivity of the separated compounds was assessed by an in vitro enzyme inhibition assay. This novel approach using a combination of ultrafiltration liquid chromatography with mass spectrometry and pressurized liquid extraction with countercurrent chromatography and preparative liquid chromatography as well as an in vitro enzyme inhibition assay could be applied to efficiently screen and isolate human aromatase inhibitors from complex samples and to the large‐scale production of functional food and nutraceutical ingredients.     

Separation of diarylethene-based photoswitchable isomeric compounds by high-performance liquid chromatography and supercritical fluid chromatography

Diarylethene-based photoswitches have become very popular over the last few decades for potential applications in chemistry, materials science, and biotechnology due to their unique physical and chemical properties. We report the isomeric separation of a diarylethene-based photoswitchable compound using high-performance liquid chromatography. The separated isomers were characterized by ultraviolet-visible spectroscopy and mass spectrometry confirmed the isomeric nature of the compounds. The isomers were purified by preparative high-performance liquid chromatography, providing fractionated samples to study the isomers individually. A total amount of 13 mg of an isomer of interest was fractionated from a solution of 0.4 mg/ml of the isomeric mixture. Because the preparative high-performance liquid chromatographic method required large quantities of solvent, we explored the use of supercritical fluid chromatography as an alternative separation mode which, to the best of our knowledge, is the first time this technique is used to separate diarylethene-based photoswitchable compounds. Supercritical fluid chromatography provided faster analysis times while maintaining sufficient baseline resolution for the separated compounds and consuming less organic solvent in the mobile phase compared to high-performance liquid chromatography. It is proposed that the supercritical fluid chromatographic method be upscaled and used in future fractionation of the diarylethene isomeric compounds, becoming a more environmentally benign approach for compound purification.     

Preparative mass‐spectrometry profiling of minor concentrated metabolites in Salicornia gaudichaudiana Moq by high‐speed countercurrent chromatography and off‐line electrospray mass‐spectrometry injection

Salicornia species have just been introduced to the European market as a vegetable named ‘samphire’, ‘green asparagus’, or ‘sea asparagus’. Due to its increasing attention, and associated value, minor compounds of Salicornia gaudichaudiana Moq were investigated. The use of countercurrent chromatography and mass spectrometry enabled the search for known, as well as potentially novel natural products. Their identification was achieved based on molecular weights and mass‐spectrometric fragmentation data. Low detection limits enabled the visualization of all compounds with their identification in almost real time close to the preparative countercurrent chromatography experiment. A list of known natural products from Salicornia genus guided the identification process of compounds occurring in Salicornia gaudichaudiana Moq by tandem mass spectrometry fragment comparison. The natural product classes were divided into four groups: chlorogenic acid derivatives; flavonoid derivatives; pentacyclic triterpenoid saponins; and other compounds.     

Studies on neurosteroids. IX. Characterization of estrogens in rat brains using gas chromatography-tandem mass spectrometry.

The characterization of the classical estrogens (estrone, estradiol, estriol) and guaiacol estrogens (2-hydroxyestrone 3-methyl ether, 4-hydroxyestrone 3-methyl ether) in rat brains was performed using gas chromatography-tandem mass spectrometry (GC-MS-MS). Estrogens were purified from Wistar strain rat brains by some chromatographic pre-treatments, such as solid-phase extraction, preparative thin-layer chromatography or preparative high-performance liquid chromatography. After the derivatization with O-methylhydroxylamine and/or N,O-bis(trimethylsilyl)trifluoroacetamide, estrogens were identified by comparison of their chromatographic behavior during GC-MS-MS operating in the product ion scan mode and comparison with the product ion MS spectra of an authentic sample. These evidences suggested that estrogens exist in rat brains as neurosteroids or neuroactive steroids.     

Characterization of N-terminal formaldehyde adducts to hemoglobin

A procedure to prepare and purify adducts of formaldehyde (FA) to the N-terminus of peptides was developed. FA-VHLTPEEK and FA-VLSPADK were produced with purities >95% upon incubation of the peptides with FA in phosphate-buffered saline (PBS) at a pH level of 7.4. The peptides were purified by preparative liquid chromatography and were characterized by their retention times in liquid chromatography, their fragmentation patterns obtained by tandem mass spectrometry, and their accurate mass and nuclear magnetic resonance measurements. This is the first time an imidazolidone-type structure has been reported for FA adducts. The same peptides were identified in tryptic digests of human hemoglobin incubated with FA at physiological conditions and in human hemoglobin specimens. These peptides are suitable for use as calibrators for the quantitative assessment of internal exposure to FA.     

Characterization of crude oil biomarkers using comprehensive two‐dimensional gas chromatography coupled to tandem mass spectrometry

Oil samples from Recôncavo basin (NE Brazil), previously analyzed by traditional techniques such as gas chromatography coupled to tandem mass spectrometry, were evaluated using comprehensive two‐dimensional gas chromatography coupled to quadrupole mass spectrometry and comprehensive two‐dimensional gas chromatography coupled to tandem mass spectrometry along with simplified methods of samples preparation to evaluate the differences and advantages of these analytical techniques to better understand the development of the organic matter in this basin without altering the normal distribution of the compounds in the samples. As a result, the geochemical parameters calculated by comprehensive two‐dimensional gas chromatography coupled to tandem mass spectrometry described better the origin, maturity, and biodegradation of both samples probably by increased selectivity, resolution, and sensitivity inherent of the multidimensional technique. Additionally, the detection of the compounds such as, the C(14α‐) homo‐26‐nor‐17α‐hopane series, diamoretanes, nor‐spergulanes, C₁₉–C₂₆ A‐nor‐steranes and 4α‐methylsteranes resolved and detected by comprehensive two‐dimensional gas chromatography coupled to tandem mass spectrometry were key to classify and differentiate these lacustrine samples according to their maturity and deposition conditions.     

Development of a method to screen and isolate bioactive constituents from Stellera chamaejasme by ultrafiltration and liquid chromatography combined with semi‐preparative high‐performance liquid chromatography and high‐speed counter current chromatography

A simple and efficient method based on ultrafiltration with liquid chromatography and mass spectrometry was used for the rapid screening and identification of ligands in the extracts of Stellera chamaejasme. The bound ligands, i.e. daphnoretin, isopimpinellin, chamaechromone, neochamaejasmin A, and chamaejasmine (purity of 96.8, 90.75, 91.41, 93.98, and 98.91%, respectively), were separated by semi‐preparative high‐performance liquid chromatography combined with high‐speed counter‐current chromatography. To the best of our knowledge, this is the first study to report the detection of potent lipoxidase and lactate dehydrogenase inhibitors in Stellera chamaejasme extracts. The results demonstrate that our method of ultrafiltration with liquid chromatography and mass spectrometry combined with mixed chromatography can be used to screen and confirm the bioactivity of all isolated compounds. This method also eliminates the need for separation of inactive compounds, thereby improving efficiency when studying bioactive substances. For some complex mixtures, neither semi‐preparative high‐performance liquid chromatography nor high‐speed counter‐current chromatography can purify all the target active compounds with high purity in a one‐step separation. The combination of the two methods allow for efficient purification of target bioactive compounds with different polarities and physicochemical properties based on their complementary properties.     

Lipid profile of fish species by liquid chromatography coupled to mass spectrometry and a novel linear retention index database

In a previous article, Rigano et al. established a new linear retention index system for the identification of triacylglycerols by liquid chromatography methods only on the basis of the retention behavior and independently from many experimental parameters. In that work, a database of 209 compounds was built, but only 54 of them, typical of vegetable oils, were confirmed by mass spectrometry. The aim of the present research is to extend the applicability of the novel approach to more complex samples, such as fish lipid extracts, and assess the complementarity between mass spectromtery and retention information to achieve univocal identification. With this purpose, a new software was implemented to make the identification process easy and automatic as in gas chromatography‐mass spectrometry where the retention index filter is added in the spectral search to discriminate between compounds with similar mass spectrometry spectra. A total of 69 species were identified and, thanks to their baseline separation obtained by an ultra high performance liquid chromatography method, a semiquantification was also performed. The species under investigation were Dicentrarchus labrax, coming from aquaculture and the wild. Some differences in their native lipid composition were observed, probably related to a different diet. A major number of samples would be necessary to confirm such a preliminary finding.     

Recent advances in liquid chromatography-mass spectrometry and capillary zone electrophoresis-mass spectrometry for protein analysis.

The utility of the combination of separations techniques, such as liquid chromatography and capillary zone electrophoresis, with mass spectrometry in applications involving protein analysis is discussed. The use of continuous-flow fast atom bombardment and electrospray ionization mass spectrometry is compared for the analysis of tryptic digests. For liquid chromatography, both microbore and slurry-packed capillary bore columns were used to separate peptides from proteolytic digests.     

An overview of the doping control analysis during the Olympic Games of 2004 in Athens, Greece

This study summarizes the results obtained from the doping control analysis during the period of the XXVIII summer Olympic Games (30 July-29 August 2004). The analysis of all doping control samples was performed at the Doping Control Laboratory (DCL)—the World Anti-Doping Agency (WADA) Accredited Laboratory of Athens. Three thousand six hundred and seventeen tests were conducted in total throughout the games. In 23 specimens the presence of a prohibited substance was confirmed. Sixteen of those were related to anabolic agents. The screened results were confirmed with various mass spectrometry analytical techniques, such as gas chromatography/high resolution mass spectrometry (GC/HRMS), gas chromatography/mass spectrometry (GC/MS), gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS) and liquid chromatography/mass spectrometry (ion trap) (LC/MS). The results of the first time applied screening and confirmatory procedures for the detection of recombinant human growth hormone in serum were also presented. Besides, 107 therapeutic use exemptions (TUE) were verified for glucocorticosteroid and beta2-agonist use.     

基于色谱方法的煤沥青指纹图谱构建

煤沥青成分极其复杂,其中含有分子量较大的多环芳烃及其氧化产物等组分。不同来源的原料煤及生产工艺得到的煤沥青在芳构化程度、组成、性质、分子结构等方面皆差别很大。该文分别采用气相色谱-质谱(GC-MS)、裂解/气相色谱-质谱(Py/GC-MS)和高效液相色谱(HPLC)手段,为煤沥青指纹图谱库的构建发展了普适、稳定、简单的分析方法。在GC-MS方法中采用常用的HP-5MS柱,在线性温度梯度条件下分析,得到33个组分的色谱图,方法对不同样品的稳定性良好。采用裂解/气相色谱-质谱方法研究探讨了较高温度下组分结构和组成的变化,对于沥青产品的加工以及老化研究具有借鉴意义。在HPLC方法建立过程中,采用最常用的C_(18)柱反相色谱分离模式,以甲醇/水为流动相,在线性梯度条件下建立方法,针对不同样品可以得到41个组分的稳定色谱峰。基于该文的方法,可为进一步建立煤沥青的指纹谱图库,以及进一步完善煤沥青的评价方法,开发煤沥青的新用途提供理论基础数据。     

山竹果皮中两种杂氧蒽酮化学成分的分析研究

建立了山竹果皮中杂氧蒽酮类化合物的快速提取、分离、鉴定方法。以无水乙醇为夹带剂,应用超临界萃取技术对山竹果皮中的杂氧蒽酮类活性化合物进行提取,快速制备色谱装置Isolera One对粗提物进行分离,得到2种杂氧蒽酮化合物,经质谱鉴定其为α-倒捻子素(α-Mangostin)和Gartanin,两者经高效液相色谱检测纯度均不低于90%,并辅以电喷雾多级串联质谱对两种化合物的碎裂规律进行了研究。     

Salbutamol extraction from urine and its stability in different solutions: identification of methylation products and validation of a quantitative analytical method

Salbutamol is commonly used in asthma treatment, being considered a short‐effect bronchodilator. This drug poses special interest in certain fields of chemical analysis, such as food, clinical and doping analyses, in which it needs to be analyzed with quantitative precision and accuracy. Salbutamol, however, is known to degrade under certain conditions and this is critical if quantitative results must be generated. The present work aimed to investigate salbutamol extraction from urine samples, to determine whether salbutamol is unstable in other solvents as well as in urine samples, to elucidate the structures of the possible degradation products and to validate an analytical method using the extraction procedure evaluated. Stability investigations were performed in urine at different pH values, in methanol and acetone at different temperatures. Semi‐preparative liquid chromatography was performed for the isolation of degradation products, and gas chromatography coupled to mass spectrometry as well as nuclear magnetic resonance were used for identification. Three unreported methylation products were detected in methanolic solutions and had their structures elucidated. Urine samples showed a reduction in salbutamol concentration of up to 25.8% after 5 weeks. These results show that special care must be taken regarding salbutamol quantitative analyses, since degradation either in standard solutions or in urine could lead to incorrect values. Copyright © 2013 John Wiley & Sons, Ltd.     

Multiple component isolation in preparative multidimensional gas chromatography with characterisation by mass spectrometry and nuclear magnetic resonance spectroscopy

The preparative scale isolation of multiple components from an essential oil matrix is described using multidimensional gas chromatography (prep-MDGC) which allows their further characterisation by mass spectrometry and nuclear magnetic resonance (NMR) spectroscopy. Menthol, linalyl acetate, carvone and geraniol were isolated individually, and were also collected in various combinations. It was demonstrated to be possible to collect multiple selected components from numerous repeat injections of the sample, to permit increased mass recovery from an external cryotrap collection device. Peak retention times remained reproducible (<0.3 s) over the repeated injections and switching events. This methodology may be utilised to confirm peak identity or to produce unique mixed-component reference standards, for instance to allow their identification in other samples using GC/MS, or identify them in comprehensive two-dimensional gas chromatography (GC × GC) analysis.     

Studies on neurosteroids. Part XIII. Characterization of catechol estrogens in rat brains using liquid chromatography-mass spectrometry-mass spectrometry

The existence of catechol estrogens in rat brains was clarified using liquid chromatography-atmospheric pressure chemical ionization-ion trap-mass spectrometry-mass spectrometry (LC-APCI-MS2). The catechol estrogens were extracted in the presence of ascorbic acid and then derivatized to acetates with acetic anhydride and pyridine. After a successive purification with silica gel mini-column chromatography, reversed-phase solid-phase extraction and preparative HPLC, the obtained fractions containing the catechol estrogen acetates were subjected to LC-APCI-MS2. 2-Hydroxyestrone, 2-hydroxyestradiol and their 4-hydroxy isomers were identified as acetates by comparison with authentic samples based on their chromatographic behavior and mass spectral data. The derivatization to acetate was useful for the treatment of labile catechol estrogens.     

Estimating molecular masses of petroleum-derived fractions: High mass (>2000 u) materials in maltenes and asphaltenes from Maya crude oil

Molecular mass ranges and average masses of fractions from a heavy Mexican crude oil (Maya) have been studied, using mainly size exclusion chromatography (SEC) and laser desorption-mass spectrometry (LD-MS). Method development focused on the use of planar chromatography and size exclusion chromatography (SEC), to isolate narrow bands of material from solubility-separated fractions of the crude oil. The procedure provides a planar chromatography based method for studying mass ranges in complex hydrocarbon mixtures. It allows the calculation of ‘best estimate’ values for number and mass-averages. These can then be used in average structural parameter (ASP) calculations, for studying structural features of the samples. The method is applicable to both coal and petroleum-derived samples. The molecular mass estimates arrived at in this work for petroleum-derived samples are considerably higher than those reported by other workers for similar samples. The results presented here provide strong evidence for the presence of ions approaching m/z 10,000 in the Maya asphaltene. The maltene fraction was found to contain a small amount of ions with mass (m/z) in excess of 2000.     

食品中邻苯二甲酸酯类化合物的分析方法研究进展

邻苯二甲酸酯是应用最广泛的增塑剂,具有生殖、发育毒性及致癌性,是近年来食品污染的一个重要来源。该类化合物种类多、同系物和同分异构体性质接近、在基体中含量范围宽,高效样品前处理、高选择性分离和高灵敏检测、降低本底干扰等技术是食品中邻苯二甲酸酯类化合物准确测定面临的挑战。本文综述了液液萃取、液液微萃取、固相萃取、固相微萃取、基质固相萃取等传统及新型的提取与净化技术在食品样品分析中的应用,比较分析了气相色谱、液相色谱、串联质谱、高分辨质谱以及酶联免疫、离子迁移谱等快速检测技术的特点,并展望了发展趋势。     

Isolation and characterization of methoxylated flavones in the flowers of Primula veris by liquid chromatography and mass spectrometry

Characterization of six flavones, which were named substances G1, G2, G3, G4, G5 and G6 according to their R_F values in normal-phase thin-layer chromatography, is reported. The pure flavones were purified after maceration with methanol by normal-phase solid-phase extraction, normal-phase medium-pressure liquid chromatography, normal-phase preparative thin-layer chromatography and preparative reversed-phase high-performance liquid chromatography (RP-HPLC). The collected fractions of several isolation steps were analyzed by normal-phase (NP) and RP-HPLC. Detection and identification of the substances G was accomplished by UV detection at 213–216 nm, diode array UV detection, or fluorescence detection (λ_ex=330 nm; λ_em=440 nm). The molecular mass, the elementary composition, and the structure of the six components was determined by electron-impact high-resolution mass spectrometry (EI-HRMS). Substance G4 was identified as 3′,4′,5′-trimethoxyflavone. The substances G1–G6 were shown to be mono-, di- tri- and pentamethoxyflavones. HPLC–electrospray ionization tandem mass spectrometry (ESI-MS–MS) of the flavones was carried out employing a 150×2 mm I.D. column packed with a 3 μm/100 Å octadecylsilica stationary phase and a mobile phase comprising 1.0% acetic acid in water–acetonitrile (50:50). Comparative RP-HPLC–ESI-MS of the raw methanol extract and the isolated substances G1–G6 proved that the isolated compounds were pure and were not artifacts. Finally, RP-HPLC–ESI-MS–MS was used to identify substances G1–G6 in phytopharmaceutical drugs.     

Identification of very long chain fatty acids by atmospheric pressure chemical ionization liquid chromatography‐mass spectroscopy from green alga Chlorella kessleri

A method is described for the enrichment of very long chain fatty acids (VLCFAs) from total fatty acids of heterotrophically cultivated green freshwater alga Chlorella kessleri and their identification as picolinyl esters by means of liquid chromatography‐mass spectrometry with atmospheric pressure chemical ionization (LC‐MS with APCI). The method is based on the use of preparative reversed phase HPLC of hundred‐milligram amounts and their subsequent identification by microbore APCI LC‐MS. A combination of these two techniques was used to identify unusual VLCFAs up to C₄₇, both saturated and monounsaturated, with two positional isomers (ω‐9 and ω‐26).     

Isolation,purification, and identification of the main phenolic compounds from leaves of celery (Apium graveolens L. var. dulce Mill./Pers.)

We developed a simple and meaningful preparative method for the separation and purification of the main phenolic compounds from the leaves of celery (Apium graveolens L. var. dulce Mill./Pers.) and we established an accurate and specific analytical method for the identification of the main phenolic compounds from celery leaves. The crude extract from celery leaves was prefractioned by polyamide resin to enrich the phenolic compounds. They were then purified further by preparative high‐performance liquid chromatography, and seven main phenolic compounds were obtained: including chlorogenic acid, luteolin 7‐O‐β‐d‐ apiofuranosyl(1→2)‐β‐d‐ glucopyranoside, luteolin 7‐O‐β‐d‐ glucopyranoside, apiin, chrysoeriol 7‐O‐β‐d‐ apiofuranosyl(1→2)‐β‐d‐ glucopyranoside, luteolin 7‐O‐[β‐d‐ apiofuranosyl(1→2)‐(6′′‐O‐malonyl)]‐β‐d‐ glucopyranoside, and apigenin 7‐O‐[β‐d‐ apiofuranosyl(1→2)‐(6′′‐O‐malonyl)]‐β‐d‐ glucopyranoside. Their purities were measured by using high‐performance liquid chromatography, and their chemical structures were confirmed using UV spectrophotometry, ultra high performance liquid chromatography with quadrupole time‐of‐flight tandem mass spectrometry, and NMR spectroscopy. Our studies indicate that preparative high‐performance liquid chromatography combined with polyamide resin is a simple and meaningful preparative method for the separation and purification of phenolic compounds from the leaves of celery or other plants, and the use of UV spectrophotometry, ultra high performance liquid chromatography with quadrupole time‐of‐flight tandem mass spectrometry, and NMR spectroscopy is an accurate and specific analytical method for the identification of phenolic compounds.