Analysis of isoflavones in soy drink by capillary zone electrophoresis coupled with electrospray ionization mass spectrometry

Capillary zone electrophoresis coupled with electrospray ionization mass spectrometry (CZE–ESI-MS) has been applied for the first time for the separation and quantification of isoflavones in soy products. The proposed method was successfully applied to the determination of seven isoflavones, including aglycones and glucosides, in soy drink. The target compounds were the glucosides daidzin and genistin, and the aglycones daidzein, genistein, formononetin, biochanin A and glycitein. During CE separation in positive mode, the analytes were present as anions, and MS detection was carried out in ESI positive-ion mode. To prevent the frequent drops in current and to improve the resolution in the separation of analytes in anionic form, a programmed nebulizing gas pressure (PNP) was applied along the analysis.     

Analysis of calcitonin and its analogues by capillary zone electrophoresis and matrix-assisted laser-desorption ionization time-of-flight mass spectrometry

Capillary zone electrophoretic (CZE) separations and mass spectrometric analysis of salmon calcitonin and related analogues were performed to generate electrophoresis and mass fingerprints for quality control of the recombinant polypeptide pharmaceutical salmon calcitonin. The calcitonins and their corresponding tryptic digests were successfully separated by CZE at low pH in fused silica capillaries dynamically modified with poly-cationic polymers. The poly-cationic modified inner surface of the fused silica capillaries generated a strong anionic electroosmotic flow (EOF). Analytes of negative, neutral, and positive charge were all swept through the capillary toward the positive electrode. Compared to Polybrene-coated capillaries, capillaries coated with PEI showed a markedly slower but much more stable electroosmotic flow. The migration order of the analytes was predicted by comparing approximate values of the charge to (molecular mass)2/3 ratios. The predicted migration order was confirmed by off-line analysis of CZE fractions with matrix-assisted laser-desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS).     

Characterization of industrial alkylpolyphosphonates by infusion electrospray ionization-ion trap mass spectrometry with identification of the impurities by tandem capillary zone electrophoresis

Technical grade diethylene-triaminepentakis(methylenephosphonic acid) (I), dihexamethylene-triaminepentakis(methylenephosphonic acid) (II), ethylene-diaminetetrakis(methylenephosphonic acid) (III), hexamethylene-diaminetetrakis(methylenephosphonic acid) (IV), amino-tris(methylenephosphonic acid) (V), hydroxyethyl-aminobis(methylenephosphonic acid) (VI), 1-hydroxyethylidene-1,1-diphosphonic acid (VII), and 2-phosphonobutane-1,2,4-tricarboxylic acid (VIII) were characterized by ion trap mass spectrometry with electrospray ionization (ESI-ITMS). Using the negative ion mode and acid and alkaline media, peak series corresponding to the nominal compounds and to impurities with a lower number of phosphonate groups were distinguished in I-V. Each series was constituted by [M - nH + (n - 1)Na](-) peaks and peaks produced from them by losses of water, H(3)PO(3)(or water plus HPO(2)), and combined losses. For each [M - nH + (n - 1)Na](-) peak, the number of losses coincided with the number of phosphonate groups not bound to sodium ions minus one (the group bearing the charge). Owing to the hydroxyethyl group, the spectrum of VI was dominated by the formation of intermolecular esters, with both losses and gains of water according to [nM - H +/- mH(2)O](-). A series of [M - nH + (n - 1)Na](-) peaks were observed for VII and VIII, showing in the latter case that the carboxylate groups may also form adducts with sodium ions. Losses of water and H(3)PO(3)were observed in VII, whereas losses of water, CO(2), and HPO(3) were seen in VIII. The reaction pathways leading to the production of the observed ions are described. The nominal compounds and the impurities were also separated and identified by capillary electrophoresis with ESI-ITMS detection.     

Determination of urinary androgen glucuronides by capillary electrophoresis with electrospray tandem mass spectrometry

Capillary electrophoresis–electrospray tandem mass spectrometry (CE‐ESI/MS/MS) is a simple and highly sensitive method for quantifying seven urinary androgen glucuronides. The urine samples were diluted and filtered through a membrane filter, and the filtrate was injected into a CE‐MS/MS system without further sample preparation steps such as extraction and derivatization. The calibration ranges were 0.01–5 µg/mL for glucuronides of androsterone and 11β‐OHAn‐3G, and 5–500 ng/mL for glucuronides of 11‐ketoAn, DHEA, testosterone, epitestosterone and DHT. The linearity of the method was 0.992–0.998, and the limits‐of‐detection at a signal‐to‐noise ratio of 3 were 5–10 ng/mL. The coefficients of variation were in the range of 4.0–9.0% for intra‐day assay and 4.1–9.8% for inter‐day assay. The proposed method may be applicable to metabolic profiling in both quantitative and qualitative analysis. Copyright © 2008 John Wiley & Sons, Ltd.     

Micelle to solvent stacking of organic cations in capillary zone electrophoresis with electrospray ionization mass spectrometry

The direction of the effective electrophoretic mobility of small organic cations in micellar electrokinetic chromatography using sodium dodecyl sulphate in a low-pH electrolyte can be reversed in the presence of organic solvent. This effective electrophoretic mobility change is presented here as a new dimension for on-line sample preconcentration of cations in capillary zone electrophoresis (CZE) using a background solution (BGS) modified by an organic solvent. The sample is prepared in a micellar solution without organic solvent. The focusing effect relies on the reversal in the effective electrophoretic mobility at the boundary zone between the micellar matrix and the BGS modified with organic solvent. This on-line sample preconcentration technique, called micelle to solvent stacking (MSS) afforded more than an order of magnitude improvement in concentration sensitivity compared to typical CZE-UV or CZE-electrospray ionization (ESI) MS analysis. The calculated limit of detection (S/N = 3) for pindolol and metoprolol analysed by MSS-CZE-ESI-MS was found to be 0.03 and 0.01 μg/mL, respectively.     

Ionene-dynamically coated capillary for analysis of urinary and recombinant human erythropoietin by capillary electrophoresis and online electrospray ionization mass spectrometry

In this article, a series of ionene polymers were synthesized and used to coat fused-silica capillaries for the separation of recombinant and urinary human erythropoietin (rhEPO and uEPO) standards by CE. The influence of the charge density of coatings on the separation of rhEPO and uEPO glycoforms was investigated. Then, we further studied the method for fast separation and detection of rhEPO and uEPO standards by CE-ESI-MS. The influence of several CE and MS operating parameters, such as the concentration of CE running buffer, applied external pressure, and the composition and flow rate of sheath liquid on CE-ESI-MS was studied. The results demonstrated that when the capillary was permanently coated with 6,6-ionene and the pH value of acetic acid-ammonium acetate running buffer was 4.80 and 5.50, respectively, a significantly reproducible separation was achieved for rhEPO and uEPO glycoforms. In the online CE-ESI-MS experiments, we not only achieved the online MS signal of uEPO, but also obtained baseline separation of three major rhEPO glycoforms successfully and reproducibly on the 6,6-ionene-coated capillaries. Furthermore, the standard mixture of rhEPO and uEPO was separated, and two incompletely resolved peaks that were identified to be rhEPO and uEPO by the unique MS "fingerprint" were obtained. Additionally, the molecular weight of rhEPO and uEPO were verified and compared to the results by MALDI-TOF-MS. It can be concluded that, in contrast to other indirect methods, the online CE-ESI-MS technique with the combination of the advantages of both CE and MS shows great potential for the separation and detection of rhEPO doping directly in competitive sports.     

Identification of anthraquinone coloring matters in natural red dyes by electrospray mass spectrometry coupled to capillary electrophoresis

Capillary electrophoresis with UV/visible diode-array detection (DAD) and electrospray mass spectrometric (ESI-MS) detection were used for the identification of anthraquinone color components of cochineal, lac-dye and madder, natural red dyestuffs often used by ancient painters. For the purpose of such analysis, ESI-MS was found to be a much more appropriate detection technique than DAD one owing to its higher sensitivity (detection limits in the range 0.1-0.5 micro g ml(-1)) and selectivity. The method developed made it possible to identify unequivocally carminic acid and laccaic acids A, B and E as coloring matters in the examined preparations of cochineal and lac-dye, respectively. In madder, European Rubia tinctorum, alizarin and purpurin were found. The method allows the rapid, direct and straightforward identification and quantification of components of natural products used in art and could be very helpful in restoration and conservation procedures.     

Structural characterization and identification of dibenzocyclooctadiene lignans in Fructus Schisandrae using electrospray ionization ion trap multiple-stage tandem mass spectrometry and electrospray ionization Fourier transform ion cyclotron resonance multiple-stage tandem mass spectrometry

The electrospray ionization ion trap multiple-stage tandem mass spectrometry (ESI-MSⁿ) and electrospray ionization Fourier transform ion cyclotron resonance multiple-stage tandem mass spectrometry (ESI-FT-ICR-MSⁿ) have been applied successfully to the direct investigation of a number of dibenzocyclooctadiene lignan constituents from the methanol extracts of the Fructus Schisandrae in the positive ion mode. The detailed structural characterization of the same skeleton and different peripheral substituents had been studied and the precise elemental compositions of ions at high mass resolution had been obtained. So the fragmentation mechanisms could be clarified. And the lignan components in Schisandra chinensis (Turcz.) Baill. fruits (SCF) and Schisandra sphenanthera Rehd. et Wils. fruits (SSF) were identified by comparing the structural information and fragmentation mechanisms. Then a pair of isobaric compounds was differentiated. Meanwhile these two similar fruits were distinguished. The research results demonstrated that ESI-MSⁿ technique is a sensitive, selective and effective tool for the direct analysis and rapid determination of constituents in complex mixtures from nature products. And these should be useful for the identification of similar compounds and differentiation of similar species from Chinese herbs.     

Analysis of native amino acids by liquid chromatography/electrospray ionization mass spectrometry: comparative study between two sources and interfaces

The analytical performances of two triple-quadrupole instruments, which differ in their atmospheric-pressure sources, were evaluated for native amino acid analysis. The Applied Biosystems/Sciex API 300 instrument was equipped with a turboIon Spray source and a curtain gas interface while the Waters/Micromass Quattro Ultima instrument was characterized by its Z-spray source. Liquid chromatography/mass spectrometry analysis of native amino acids requires volatile ion-pairing mobile phase additives (mainly perfluorinated carboxylic acids). The effects of the structure and concentration of the ion-pairing reagents as well as the organic modifier percentage on the electrospray response of amino acids were studied in detail. The most favourable chromatographic conditions depend strongly on the mass spectrometer used. Several instrumental parameters were also studied, including spray voltage, transmission lens voltages, temperature of desolvation and auxiliary gas flow rates. The results show substantial qualitative differences depending on the instrument geometry. The quantitative performances of the two triple-quadrupole mass spectrometers were evaluated in terms of limits of detection and quantification. The effects of the matrix on the analyte ionization were also examined, and the long-term stability of the electrospray performance was studied over 12 h using a mobile phase containing the perfluorinated ion-pairing reagents. The study provides information on the robustness of the MS instrument and its detection sensitivity towards native amino acid analysis. It appears that each instrument has its good and bad points since one provides higher sensitivity while another is more robust.     

Analysis of six active components in Radix tinosporae by nonaqueous capillary electrophoresis with mass spectrometry

Nonaqueous capillary electrophoresis with mass spectrometry has advantages for the analysis of active components in herbs. Here, a rapid nonaqueous capillary electrophoresis with mass spectrometry method was developed to separate, identify, and quantify palmatin, columbin, cepharanthine, menisperine, magnoflorine, and 20‐hydroxyecdysone in Radix tinosporae . Electrospray ionization MS^1‐3 spectra of the six components were collected and possible cleavage pathways of main fragment ions were elucidated. The conditions that could affect separation, such as the composition of running buffer and applied voltage, were studied, and the conditions that could affect the mass spectrometry detection, such as the composition and flow rate of sheath liquid, the pressure of nitrogen gas, and the temperature and flow rate of the dry gas, were also optimized. Under the optimized conditions, the correlation coefficient was >0.99. The relative standard deviations of migration time and peak areas were <10%. The recoveries were calculated to be 99.31–107.80% in real samples. It has been demonstrated that the proposed method has good potential to be applied to determine the six bioactive components in Radix tinosporae .     

Analysis of recombinant and modified proteins by capillary zone electrophoresis coupled with electrospray ionization tandem mass spectrometry.

A method for rapid characterization of recombinant and modified proteins with known sequences is described. The analytical system consists of a capillary zone electrophoresis (CZE) instrument coupled to an electrospray ionization ion trap tandem mass spectrometer via a sheath-flow interface. Following the procedure consists of proteolytic fragmentation, CZE peptide separation, tandem mass spectrometry (MS-MS) analysis of separated peptides, sequence database search and monitoring of the specific peptides, C 125 S mutated interleukin 2 (S-125-IL2) and bovine beta-casein were characterized as a model of recombinant protein and naturally modified protein, respectively. A tryptic peptide mixture derived from the synthetic salmon calcitonin (s-CT) was also analyzed to test the performance of the system. Although a conventional sheath-flow interface with much higher flow-rate compared to the microspray interface and nanospray interface was used, the proteins were identified at the low picomole level.     

毛细管电泳结合电喷雾质谱分析人类癌基因c-myb四链体核酸与活性天然产物的相互作用

药物与靶点间的作用关系直接影响到药理和药效。药物-靶点结合能力、结合计量关系等信息是药物研发过程中必需的表征数据。人类癌基因c-myb在结直肠癌等多种癌症组织中存在过度表达,目前已成为结直肠癌、白血病等癌症疾病潜在的治疗靶点。位于癌基因c-myb启动子区的一段富含鸟嘌呤(G)的DNA序列,通过阳离子的诱导可自发折叠形成分子内G-四链体,而小分子的特异性识别可以稳定该G-四链体,进而调节基因的转录和表达过程。该文采用压力辅助毛细管电泳前沿分析(PACE-FA)结合电喷雾质谱(ESI-MS)研究人类癌基因c-myb启动子G-四链体(G4)与天然产物分子间的相互作用。PACE-FA法在毛细管电泳前沿分析(CE-FA)过程中施加一个与分析物迁移同向的压力,在保证结果准确度的前提下,能够大大加快分析速度。同时结合ESI-MS,可快速解析结合分子与靶点的亲合力和化学计量关系。首先,利用ESI-MS快速筛选出3种有亲合力的天然产物,亲合力大小依次为:土荆皮乙酸>丁溴东莨菪碱>荷叶碱。考虑到溶液相中存在特异性与非特异性结合,接着用PACE-FA法准确分析溶液相中结合的特异性和结合常数。结果...     

Analysis of dinitrofluoranthenes by high resolution capillary gas chromatography/mass spectrometry

This paper reports the retention indices of eighteen of the possible twenty-five dinitrofluoranthene isomers.     

Recent developments in the characterization of nucleic acids by liquid chromatography,capillary electrophoresis,ion mobility,and mass spectrometry (2010–2020)

The development of new strategies for the analysis of nucleic acids has gained momentum due to the increased interest in using these biomolecules as drugs or drug targets. The application of new mass spectrometry ion activation techniques and the optimization of separation methods including liquid chromatography, capillary electrophoresis, and ion mobility have allowed more detailed characterization of nucleic acids and oligonucleotide therapeutics including confirmation of sequence, localization of modifications and interaction sites, and structural analysis as well as identification of failed sequences and degradation products. This review will cover tandem mass spectrometry methods as well as the recent developments in liquid chromatography, capillary electrophoresis, and ion mobility coupled to mass spectrometry for the analysis of nucleic acids and oligonucleotides.     

Separation and identification of oligomeric ethyl silicates by liquid chromatography with electrospray ionization mass spectrometry

Reversed‐phase liquid chromatography coupled with electrospray ionization mass spectrometry was used to study the molecular structures of components and molar mass distributions in ethyl silicate‐40, a versatile liquid precursor for silicon‐based materials. Identity testing by standard spectroscopic techniques showed that a commercial sample of ethyl silicate‐40 was composed of linear/branched ethoxysiloxane oligomers with the silicon atoms ranging from 2 to 12 together with minor monocyclic species. Analysis of the sample by liquid chromatography coupled with evaporative light scattering detection resulted in an elution profile consisting of a series of peak clusters. Peak identification showed that the linear/branched homologous series of oligomers were eluted in the order of increasing number of silicon atoms in the molecules and the time duration (width) of the resulting peak clusters increased in the same fashion corresponding to increasing number of geometric isomers. In addition, small amounts of monocyclic oligomers present in the sample were found to be less retained than each linear/branched counterpart. Finally, the molar mass distribution parameters for ethyl silicate‐40 determined by the developed method were in good agreement with the literature values. Overall, this work demonstrates that reversed‐phase liquid chromatography coupled with electrospray ionization mass spectrometry is an indispensable tool for the comprehensive characterization of complex mixtures of this type.     

Analysis of therapeutic peptides in human urine by combination of capillary zone electrophoresis-electrospray mass spectrometry with preparative capillary isotachophoresis sample pretreatment

The presented study deals with the off-line coupling of preparative isotachophoresis (pITP) with on-line combination of capillary zone electrophoresis with electrospray mass spectrometric detection (CZE-ESI-MS) used for the analysis of therapeutic peptides (anserine, carnosine, and buserelin) in complex matrix (urine). Preparative capillary isotachophoresis, operating in a discontinuous fractionation mode in column-coupling configuration, served as a sample pretreatment technique to separation, and fractionation of mixture of therapeutic peptides present in urine at low concentration level. The fractions isolated by pITP procedure were subsequently analyzed by capillary zone electrophoresis with electrospray mass spectrometric detection. Acetic acid at 200 mmol L(-1) concentration served as background electrolyte in CZE stage and it is compatible with MS detection in positive ionization mode. In pITP fractionation procedure, sodium cation (10 mmol L(-1) concentration) as leading ion and beta-alanine as terminating ion (20 mmol L(-1) concentration) were used. While using CZE-ESI-MS, the limits of detection were 0.18 μg mL(-1) for carnosine, 0.17 μg mL(-1) for anserine and 0.64 μg mL(-1) for buserelin in water and 0.19 μg mL(-1) for carnosine, 0.50 μg mL(-1) for anserine and 0.74 μg mL(-1) for buserelin in 10 times diluted urine, respectively. The cleaning power of pITP sample pretreatment was proved as the peptides provided the higher MS signals at lower concentration levels resulting from the minimized matrix effects. The quality of obtained MS/MS spectra was very good so that they can provide information about the structure of analytes, and they were used for verification of the analytes identities. The pITP pretreatment improved the detection limits of the analyzed therapeutic peptides at least 25 times compared to the CZE-ESI-MS itself.     

Measurement of equilin and estrone in human plasma by capillary gas chromatography-negative ion chemical ionization mass spectrometry

A procedure is described for the analysis of the estrogens equilin and estrone in human plasma following oral administration of conjugated estrogen preparations. After enzymatic hydrolysis of the sulfate conjugates, plasma proteins are precipitated with methanol and the estrogens extracted into ethyl acetate. Derivatization with the reagent flophemesylamine converts equilin and estrone into volatile pentafluorophenyldimethylsilyl ethers ideally suited to capillary gas chromatography-negative ion chemical ionization mass spectrometry. Using a 15 meter dimethyl silicone bonded phase fused silica capillary column separation of the estrone and equilin derivatives is achieved within 9 minutes. Selected ion monitoring of the intense negative molecular ions enables levels of 1 ng.ml^?1 to be measured with coefficients of variation of 9.3 % and 14.2 % for estrone and equilin respectively. Plasma levels of the compounds are reported in two male volunteers up to 24 hours after dosing with 5 milligrams of Premarin?. (? Ayerst Laboratories Inc., New York, USA.).