二醇基柱高效液相色谱-蒸发光散射检测法测定饮料中的糖

采用二醇基柱分离、蒸发光散射检测法测定了饮料中的糖。比较了乙腈-水、二氯甲烷-甲醇两种流动相分析糖的特点。采用二氯甲烷-甲醇为流动相,果糖、葡萄糖、蔗糖、麦芽糖和棉子糖的峰面积与绝对进样量的标准曲线在绝对进样量为1.6μg-40μg时均具有良好的线性关系。该方法对上述5种糖的检测限分别为0.20μg,0.16μg,0.16μg,0.20μg和0.20μg。     

CEC separation of peptides using a poly(hexyl acrylate-co-1,4-butanediol diacrylate-co-[2-(acryloyloxy)ethyl]trimethyl ammonium chloride) monolithic column

A polyacrylate-based monolithic column bearing cationic functionalities and designed for capillary electrochromatography (CEC) has been prepared via photopolymerization of a mixture of hexyl acrylate, butanediol diacrylate, 2-(acryloyloxy) ethyltrimethyl ammonium chloride (monomers), azobisisobutyronitrile (photoinitiator), acetonitrile, phosphate buffer, and ethanol (porogens). The polymerization process was initiated with UV light at 360 nm. The column performance was evaluated via the separations of alkylbenzenes, substituted anilines, basic drugs, peptides, and a protein digest. The separation of complex peptide mixtures was then studied since such separations constitute a promising application of capillary electrochromatography. In particular, the effects of mobile phase composition, including ionic strength of the buffer solution and the percentage of acetonitrile on the retention factor, the column efficiency, and the resolution were determined. The separations were affected by both interaction of the peptides with the stationary phase and their own electrophoretic mobility. Excellent separations with column efficiencies of up to 160 000 plates/m were achieved for both a mixture of ten well-defined peptides and a tryptic digest of cytochrome c. The fractions of eluent containing peptides of the digest separated in the monolithic column were collected and characterized using matrix-assisted laser desorption ionization mass spectrometry.     

Monolithic columns with optimized pore structure for molecular size-based separations of synthetic polymers

Monolithic capillary columns based on divinylbenzene were synthesized using different alcanols as porogens. Prepared columns were tested in separation of polystyrene standards according to their molar mass (MM) and were characterized by corresponding calibration graphs. It was demonstrated that a decrease of alcanol chain length from dodecanol to octanol resulted in a decrease of column permeability and in an improved column ability to separate polystyrene standards. In contrast, removing a good solvent from porogen mixture results in an increase of column permeability and in a decrease of column performance toward polystyrene standards. Optimized synthetic conditions included porogen composed of nonanol and toluene or mesytilene, and the column prepared with this porogen was capable of separating a mixture of 14 polystyrene standards with MM ranged from several millions to oligomers.     

A shotgun tandem mass spectrometric analysis of phospholipids with normal-phase and/or reverse-phase liquid chromatography/electrospray ionization mass spectrometry

Electrospray ionization mass spectrometry is used in lipidomics studies. The present research established a top-down liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) shotgun analysis method for phospholipids (PLs) using a normal-phase column or a C30 reverse-phase column with the data-dependent MS/MS scanning mode. A normal-phase column can separate most of the major different classes of PLs. By using LC/ESI-MS/MS with a normal-phase column, approximately 50 molecular species were identified in a PL mixture from rat liver. When the reverse-phase column was used, the PLs could be separated depending on their hydrophobicity, essentially the length of their fatty acyl chains and the number of unsaturated bonds in them. The LC/ESI-MS/MS method using a C30 reverse-phase column was applied to phosphatidylcholine (PC) and phosphatidylethanolamine (PE) mixtures as test samples. Molecular species with the same molecular mass but with different pairs of fatty acyl chains were separately identified. As a result, about 60 PC and 50 PE species were identified. PLs from rat liver were subjected to LC/ESI-MS/MS using the C30 reverse-phase column and about 110 molecular species were identified. Off-line two-dimensional LC/ESI-MS/MS with the normal-phase and C30 reverse-phase columns allowed more accurate identification of molecular species by using one-dimensional C30 reverse-phase LC/ESI-MS/MS analysis of the collected fractions.     

Potential of poly(styrene-<Emphasis Type="Italic">co</Emphasis>-divinylbenzene) monolithic columns for the LC-MS analysis of protein digests

Two polystyrene-based capillary monolithic columns of different length (50 and 250 mm) were used to evaluate the effects of column length on gradient separation of protein digests. A tryptic digest of a 9-protein mixture was used as a test sample. Peak capacities were determined from selected extracted ion chromatograms, and tandem mass spectrometry data were used for database matching using the MASCOT search engine. Peak capacities and protein identification scores were higher for the long column with all gradients. Peak capacities appear to approach a plateau for longer gradient times; maximum peak capacity was estimated to be 294 for the short column and 370 for the long column. Analyses with similar gradient slope produced a ratio of the peak capacities of 3.36 for the long and the short column, which is slightly higher than the expected value of the square root of the column length ratio. The use of a longer monolith improves peptide separation, as reflected by higher peak capacity, and also increases protein identification, as observed from higher identification scores and a larger number of identified peptides. Attention has also been paid to the peak production rate (PPR, peak capacity per unit time). For short analysis times, the short column produces a higher PPR, while for analysis times longer than 40 min, the PPR of the 250-mm column is higher.     

Liquid chromatographic separation of proteins derivatized with a fluorogenic reagent at cysteinyl residues on a non-porous column for differential proteomics analysis

A wide-pore (30 nm) reversed-phase column (Intrada WP-RP, particle size 3 μm) was recently utilized for protein separation in differential proteomics analysis with fluorogenic derivatization-liquid chromatography-tandem mass spectrometry (FD-LC-MS/MS), and exerted a tremendous effect on finding biomarkers (e.g., for breast cancer). Further high-performance separation is required for highly complex protein mixtures. A recently prepared non-porous small-particle reversed-phase column (Presto FF-C18, particle size: 2 μm) was expected to more effectively separate derivatized protein mixtures than the wide-pore column. A preliminary experiment demonstrated that the peak capacity of the former was threefold greater than that of the latter in gradient elution of a fluorogenic derivatized model peptide, calcitonin. The FD-LC-MS/MS method with a non-porous column was then optimized and applied to separate liver mitochondrial proteins that were not efficiently separated with the wide-pore column. As a result, high-performance separation of mitochondrial proteins was accomplished, and differential proteomics analysis of liver mitochondrial proteins in a hepatitis-infected mouse model was achieved using the FD-LC-MS/MS method with the non-porous column. This result suggests the non-porous small-particle column as a replacement for the wide-pore column for differential proteomics analysis in the FD-LC-MS/MS method.     

Analysis of carbohydrates in drinks by high-performance liquid chromatography with a dynamically modified amino column and evaporative light scattering detection

A high-performance liquid chromatographic method with a dynamically modified amino column and evaporative light-scattering detector (ELSD) was established for the direct analysis of the carbohydrates in some drinks. A separation column (Zorbax Rx-SIL, 250 mm x 4.6 mm I.D., 5 microm, Hewlett-Packard, USA) which was modified by ethylenediamine and a guard column (Zorbax Rx-SIL, 12.5 mm x 4.6 mm I.D., 5 microm) were used. The mobile phase was a mixture of water-acetonitrile (1:2.6, v/v) containing 0.03% (v/v) ethylenediamine. Regression equations revealed linear relationship (correlation coefficients=0.996-0.999) between the mass of carbohydrates injected and the carbohydrates peak areas detected by ELSD. The detection limits of ELSD (S/N=3) were between 0.2 and 1.2 microg for different carbohydrates. This method is simple and sensitive.     

基质固相分散萃取-高效液相色谱-串联质谱法分析拟南芥中的赤霉素

建立了基质固相分散萃取-高效液相色谱-串联质谱法(MSPD-HPLC-MS/MS)同时测定拟南芥中3种赤霉素GA1, GA3和GA4的分析方法。将拟南芥样品与C18填料混合研磨制成MSPD柱,并采用80%冷甲醇洗脱。采用反相C18色谱柱进行分离,以0.05%甲酸水溶液和乙腈为流动相进行梯度洗脱,采用电喷雾离子源负离子模式(ESI~)电离,多反应监测模式检测。对样品前处理条件、色谱分离条件和质谱检测条件进行了优化,结果表明,在最优条件下,3种赤霉素在10～300 ng/g范围内均呈良好线性关系,相关系数(r2)均大于0.98,检出限在1.1～4.1 ng/g之间。在10～50 ng/g添加水平下,平均回收率范围为54.7%～102.6%,相对标准偏差（RSD,n=3)为3.2%～12.8%。该方法操作简单、灵敏度高、选择性好、回收率高,适合拟南芥中GA1、GA3、GA4含量的测定。     

Separation and characterization of three positional isomers of dimaltosyl-cyclomaltoheptaose (dimaltosyl-beta-cyclodextrin).

A mixture of maltosylcyclomaltoheptaoses (maltosyl-beta-cyclodextrins, G2-beta CDs) was prepared from maltose and beta-cyclodextrin (beta CD) through the reverse action of Klebsiella pneumoniae pullulanase. Three positional isomers of dimaltosyl-beta CD in the mixture were separated by high-performance liquid chromatography on a reversed phase column and a graphitized carbon column. Their molecular weights were measured by fast-atom bombardment mass spectrometry, and the structures were established by methylation analysis, hydrolysis with glucoamylase to the known compounds, three positional isomers of diglucosyl-beta CD, and 13C-nuclear magnetic resonance spectroscopy.     

Reversed-phase liquid chromatography of immunoglobulin G molecules and their fragments with the diphenyl column

A diphenyl column was able to resolve two closely related monoclonal IgG2 molecules, while a C8 column failed to separate these IgGs under identical chromatographic conditions. The diphenyl column also showed a better separation of a mixture of two light and two heavy chains than the C8 column. The influence of amino acid side chains from protein sequences in binding to the diphenyl and C8 stationary phases was studied by using a set of synthetic peptides with the sequence GXXLLLKK, where X represents substitution with all of the 20 amino acids. Peptides containing aromatic amino acids showed a greater binding on the diphenyl column than on the C8 column. This increase in retention was attributed to pi-pi interactions between the aromatic amino acid side chains and the diphenyl ligand. Based on the retention of peptides on the diphenyl column, new retention coefficients were assigned for the separation of proteins. A good correlation was observed between the sum of retention coefficients (SigmaRc) for IgGs and their retention time on the diphenyl column. On-column hydrogen-deuterium exchange showed that the diphenyl column had a larger surface of interaction with protein than the C8 column. pi-pi interactions and the large contact surface resulted in improved resolution of IgGs and their fragments on the diphenyl column.     

超高压液相色谱-飞行时间质谱法测定食品中19种非法染料

建立了同时测定食品中19种非法染料的方法。样品经丙酮/正己烷提取后,采用氧化铝柱层析净化,超高压液相色谱C18短柱分离,流动相用乙腈:0.1%甲酸溶液梯度淋洗,采用电喷雾离子源,在正离子模式下以飞行时间质谱检测,质量偏差小于1毫道尔顿。19种非法染料加标回收率在76%～108%,之间,方法的检测限为0.34～14μg/kg,精密度RSD在0.3%～12%之间。     

固相萃取-高效液相色谱法测定人血浆中的川芎嗪

建立了高效液相色谱测定人血浆中川芎嗪浓度的方法。色谱条件:分析柱为Luna C18(150 mm×4.6 mm i.d.,5 μ m),流动相为甲醇-乙腈-醋酸盐缓冲液(pH 5.0)(体积比为50∶8∶42),流速1.0 mL/min,柱温40 ℃,检测波长280 nm。 血浆样品预处理采用C8固相小柱萃取法。方法的线性范围为25～5000 μg/L,线性相关系数为0.9999。高、中、低浓度 的川芎嗪在标准血浆样品中的平均提取回收率为96.72％～100.90％,日内和日间相对标准偏差(RSD)小于8.64％,准确度 为99.59%~103.26%,检测限为10 μg/L。该方法的各项效能指标符合生物样品的分析要求,可用于川芎嗪制剂的人体药 代动力学研究。     

L-羟脯氨酸寡肽混合物的高效液相色谱分离与质谱分析

研究了三氯氧磷辅助下L-羟脯氨酸的成肽反应,建立了采用反相高效液相色谱-质谱/质谱联用技术分离鉴定羟脯氨酸寡肽混合物的方法,优化了L-羟脯氨酸寡肽混合物的色谱分离条件。实验以YWG C8柱(10 μm,250 mm×10 mm)为分离柱,以乙腈-0.06%三氟乙酸水溶液(体积比为2∶98)为流动相进行等度洗脱,在正离子模式下对洗脱物进行了电喷雾电离串联质谱鉴定。结果显示,分离出的各组分分别为L-羟脯氨酸二肽、L-羟脯氨酸环二肽和L-羟脯氨酸三肽。     

Ultra-fast high-performance capillary sodium dodecyl sulfate gel electrophoresis of proteins

An ultra-fast analysis of proteins, based on sodium dodecyl sulfate (SDS)-mediated gel electrophoresis was developed, in which protein molecular mass standards ranging from M_r 14 200 to 94 700 were separated within 3 min. A 50 μm diameter uncoated fused-silica capillary column and a high field strength are used. The effects of the SDS concentration in the separation gel buffer and in the sample buffer on the resolution of protein test mixture were studied. The influence of the heat treatment of the sample prior analysis is also discussed.     

Analysis of hydroxamate siderophores in soil solution using liquid chromatography with mass spectrometry and tandem mass spectrometry with on‐line sample preconcentration

A liquid chromatography with electrospray ionization mass spectrometry method was developed to quantitatively and qualitatively analyze 13 hydroxamate siderophores (ferrichrome, ferrirubin, ferrirhodin, ferrichrysin, ferricrocin, ferrioxamine B, D₁, E and G, neocoprogen I and II, coprogen and triacetylfusarinine C). Samples were preconcentrated on‐line by a switch‐valve setup prior to analyte separation on a Kinetex C₁₈ column. Gradient elution was performed using a mixture of an ammonium formate buffer and acetonitrile. Total analysis time including column conditioning was 20.5 min. Analytes were fragmented by applying collision‐induced dissociation, enabling structural identification by tandem mass spectrometry. Limit of detection values for the selected ion monitoring method ranged from 71 pM to 1.5 nM with corresponding values of two to nine times higher for the multiple reaction monitoring method. The liquid chromatography with mass spectrometry method resulted in a robust and sensitive quantification of hydroxamate siderophores as indicated by retention time stability, linearity, sensitivity, precision and recovery. The analytical error of the methods, assessed through random‐order, duplicate analysis of soil samples extracted with a mixture of 10 mM phosphate buffer and methanol, appears negligible in relation to between‐sample variations.     

Preparation and characterization of alkylated polymethacrylate monolithic columns for micro-HPLC of proteins

Protein separations were carried out by micro-high performance liquid chromatography (micro-HPLC) with surface alkylated monolithic columns, which were prepared by in situ copolymerization of glycidyl methacrylate (GMA) and ethylene glycol dimethacrylate (EGDMA) in the presence of dodecanol and toluene as porogens. First, glycidyl groups at the surface of the porous monolith were hydrolyzed with sulfuric acid. The hydroxyl groups thus formed were then reacted with n-alkyl chloride to form alkylated stationary phase. Separation performance for proteins on columns with C18 and C8 stationary phases was compared. The results showed that a poly(GMA-EGDMA) support derivatized with octadecyl moieties could achieve much better resolution than one with octyl groups. A protein mixture was separated with the octadecylated poly-(GMA-EGDMA) monolithic column, and the effluent peaks were collected and analyzed by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). The physical properties of the monolithic columns such as column morphology, surface area, mesopore size distribution, and column permeability were further characterized by scanning electron microscopy (SEM), multipoint BET nitrogen adsorption/desorption, and Darcy's law, respectively.     

液相色谱串联质谱法测定蔬菜中四聚乙醛残留量

建立了测定蔬菜中四聚乙醛残留量的液相色谱串联质谱方法.蔬菜样品经乙腈提取,盐析后吹干乙腈提取液,再用氟罗里硅土固相萃取小柱净化,用正已烷/丙酮(80:20,V/V)混合溶剂洗脱,氮吹后用乙腈-20mmol/L,乙酸铵溶液(70:30,V/V)溶解后进行仪器分析.分析采用XBridgeTM C18色谱柱分离,乙腈-20m...     

Capillary column high-performance liquid chromatographic-electrospray ionization triple-stage quadrupole mass spectrometric analysis of proteins separated by two-dimensional polyacrylamide gel electrophoresis application to cerebellar protein mapping

A method is presented for the structural characterization of proteins separated by two-dimensional poly-acrylamide gel electrophoresis (2D-PAGE). The method includes separation of a protein mixture by 2D-PAGE, recovery of proteins from the gel spots revealed by copper staining and analysis of the proteins by triple-stage quadrupole mass spectrometry using an electrospray ionization interface (ESI-TSQMS). Prior to the mass spectrometric analysis, the extracted proteins were passed through a small reversed-phase column (10 × 4.0 mm I.D.) to remove salts and gel-derived contaminants and then introduced into the mass spectrometer through a reversed-phase capillary column with 0.25 mm I.D. Application of the method to the analysis of rat cerebellar proteins suggests that the molecular mass could be accurately determined with sub-picomole amounts of protein samples derived from one or two 2D gels. The method was also useful for peptide mapping and determination of amino acid sequences of proteins micro-prepared from the 2D gel. Because 2D-PAGE has an excellent resolving power in protein separation and because capillary LC-ESI-TSQMS provides structural information with very small amounts of samples, the combined system of 2D-PAGE and capillary LC-ESI-TSQMS described here should allow wide applications to molecular studies of genes and proteins, such as identifications of protein spots on 2D gels, confirmation of gene/protein sequences and analysis of post-translational modification of proteins present naturally in tissue/cell extracts or expressed by recombinant DNA techniques.     

Protein identification with Teflon as matrix-assisted laser desorption/ionization sample support

Protein identification is a critical step in proteomics, and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) plays an important role in that identification. Polytetrafluoroethylene (Teflon) was tested as a new MALDI sample support to improve protein identification. The tryptic peptides obtained from a model protein were bound to the surface of a modified MALDI sample holder via the hydrophobic interactions that occur between the Teflon surface and the peptide ion-pairs, and the affinity of alpha-cyano-4-hydroxycinnamic acid for the peptides. During that surface-binding step, the peptide mixture was also desalted and concentrated. A greater number of matched peptides and a larger sequence coverage were obtained for the proteins when Teflon was used as the sample support compared with conventional sample preparation methods and a stainless-steel surface. In addition, the characterization of a small amount of protein was improved with Teflon. Nine silver-stained protein spots obtained from 2-D gel of a human cerebrospinal fluid (CSF) proteome were identified by this method. Among the nine protein spots, peptide 6:c3c fragment and procollagen c-proteinase enhancer were not annotated in any published 2-D map of human CSF. A Teflon MALDI sample support is a low-cost, simple, and effective method that can be used to improve the quality of the MALDI mass spectrum of a complex tryptic peptide mixture, and to achieve a higher level of reliability and success in protein identification.     

液相色谱串联质谱法同时测定饲料中8种苯并咪唑类药物

建立了同时测定饲料中8种苯并咪唑类药物(噻苯咪唑、丙硫咪唑、硫苯咪唑、苯硫氧咪唑、氟苯咪唑、甲苯咪唑、丙氧苯唑和三氯苯唑)的液相色谱串联质谱分析方法。饲料样品用酸化乙腈直接提取,提取液用甲酸溶液稀释后进行分析。分析时用XBridgeTMC18色谱柱,以甲酸溶液-乙腈体系进行梯度洗脱,MRM方式测定,基质外标法定量。8种苯并咪唑类药物均在0.02~10.0 mg.L-1范围内呈良好的线性关系,相关系数(r2)均不低于0.990,在饲料样品中的检出限为2.1~63.0μg.kg-1。饲料中苯并咪唑类药物在0.50、30、200 mg.kg-13种加标水平下的回收率为84%~104%,相对标准偏差均小于10.0%。方法分析单个样品约需30 min,该方法适合饲料中8种苯并咪唑类药物的同时分析。