高效液相色谱-蒸发光散射检测法测定藜芦中的介藜芦碱和藜芦胺

建立了高效液相色谱-蒸发光散射检测(HPLC-ELSD)测定藜芦中介藜芦碱、藜芦胺含量的方法,并对4种藜芦属药材样品进行了测定。采用的色谱柱为Kromasil C18柱(250 mm×4.6 mm, 5 μm),以乙腈和0.1%三氟乙酸水溶液为流动相进行梯度洗脱,洗脱程序为:0～5 min, 20%乙腈; 5～30 min, 20%乙腈～40%乙腈, 30～40 min, 40%乙腈～20%乙腈; 40～45 min, 20%乙腈;流速为0.8 mL/min;柱温为35 ℃;采用ELSD检测,漂移管温度为98 ℃,载气流速2.2 L/min 。介藜芦碱和藜芦胺的线性范围分别为42.05～980 mg/L和43.77～1020 mg/L;平均回收率分别为99.2%和101.4%,相对标准偏差分别为1.7%和2.1% (n=6);信噪比为3时,测得介藜芦碱和藜芦胺最低检测限分别为18.37 mg/kg和21.50 mg/kg。该方法快速简便、灵敏度和分离度好,适用于藜芦药材中活性生物碱的测定。     

离子色谱-抑制电导检测法同时测定草甘膦母液中的含磷副产物及无机阴离子

建立了一种抑制电导检测-离子色谱(IC)同时测定草甘膦生产工艺中母液里的草甘膦及其副产物、无机阴离子的方法。样品经过滤后直接进样,色谱条件: IonPac AS11-HC分离柱(250 mm×4 mm)和IonPac AG11-HC保护柱(50 mm×4 mm),在线淋洗液发生器KOH梯度淋洗,流速1.0 mL/min,采用抑制电导检测。草甘膦、甲基草甘膦、六甲基磷酰三胺(HMPA)、增甘膦、亚磷酸、磷酸、Cl~和SO2~4的线性范围分别为0.1～20 mg/L、0.1～20 mg/L、0.1～50 mg/L、0.25～50 mg/L、0.05～20 mg/L、0.2～50 mg/L、0.02～20 mg/L和0.05～50 mg/L,相关系数分别为0.9995、0.9993、0.9999、0.9998、0.9999、0.9985、0.9999和0.9980,加标回收率为93.7%～104.0%,相对标准偏差均小于2.5% (n=7),检出限(以信噪比(S/N)=3计)为0.002～0.025 mg/L。该方法用于草甘膦生产工艺中母液里草甘膦及其含磷副产物和无机阴离子的测定,结果令人满意。     

快速高分离度液相色谱法测定贝类中软骨藻酸残留量

介绍了用快速高分离度液相色谱(RRIC)测定贝类中记忆丧失性贝类毒素软骨藻酸的方法.样品以V(甲醇):V(水)=1:1的溶液提取,经LC-SAX强阴离子柱固相萃取净化,以ZORBAX SB-C18柱(1.8μm,2.1×50mm)分离,乙腈+0.1%三氟乙酸水溶液等度洗脱,1 min内完成检测.用RRIC外标法定量分析.在10,15,20,40 mg/kg 4个添加水平下进行了验证试验,方法在0～18mg/L范围内有良好的线性关系,相关系数大于0.999,回收率为79.3%～106%.     

柱前衍生化气相色谱法分析豌豆根尖粘液中的多糖

采用三氟乙酸水解豌豆根尖粘液中的多糖,衍生化后用气相色谱分离测定6种单糖.实验表明,根尖粘液用三氟乙酸水解并乙酰化,采用OV-17石英毛细管色谱柱(35 m×0.32 mm, 0.33 μm),利用气相色谱法对6种单糖进行分离测定,6种糖全部达到基线分离,总分析时间为42 min.用保留时间定性,外标法定量.方法线性范围为0.0025～2.5 g/L,各标准曲线相关系数r>0.9991; 检出限0 0216～0.1578 mg/L,加样回收率为96.7%～107.4%.实验结果表明,本方法可有效测定逆境胁迫下植物根尖粘液中的糖类物质.     

高效液相色谱法定量测定中药千层塔提取物中的石杉碱甲

建立了高效液相色谱快速定量测定中药千层塔提取物中石杉碱甲含量的分析方法。千层塔提取物经甲醇/水/甲酸(10/90/0.2, v/v/v)提取并定容后,过滤膜后直接分析。色谱分离选用XCharge C18色谱柱(150 mm×4.6 mm, 5 μm),以水(含0.1%三氟乙酸)和乙腈(含0.09%三氟乙酸)为流动相进行梯度洗脱,流速为2 mL/min,于310 nm波长下检测,可在10 min内完成石杉碱甲的快速分离分析。结果表明,石杉碱甲在2.12～106 mg/L范围内线性关系良好(相关系数为0.9999);平均加标回收率为102.34%,相对标准偏差(RSD)为0.46%;日内及日间精密度均小于2%,满足定量要求。该方法简便、快速,结果可靠,重现性好,可作为千层塔提取物质量评价的依据。     

同位素内标超高效液相色谱-串联质谱法检测大鼠血清中丙烯酰胺和环氧丙酰胺

建立了检测大鼠血清中丙烯酰胺和环氧丙酰胺的超高效液相色谱-串联质谱法(UPLC-MS/MS).血清样品中加入乙腈沉淀蛋白质,超高速离心取上清液,离心浓缩后,采用ACQUITY UPLC T3 C18色谱柱(50 mm×2.1 mm, 1.7 μm),以0.1%甲酸溶液和甲醇为流动相进行梯度洗脱,经UPLC分离,电喷雾离子化四级杆串联质谱以正离子多反应监测方式(MRM)检测,用同位素内标法定量.丙烯酰胺和环氧丙酰胺在0.5～20.0 mg/L浓度范围内线性良好,相关系数r均大于0.999;检出限均为0.1 mg/L;在3个浓度水平(2.0, 8.0, 和15.0 mg/L)进行添加实验,样品的加标回收率分别为98.0%～108.3%和99.6%～106.7%;日内精密度(RSD)为0.54%～5.8%和1.1%～5.8%;每个样品测试时间仅为6 min.本方法测定大鼠血清中的丙烯酰胺和环氧丙酰胺操作简单快速, 重现性好, 灵敏度高.     

高效液相色谱－蒸发光散射法测定液态乳中乳果糖含量

液态乳中乳糖和乳果糖是同分异构体,因保留特性相似,成为色谱法分离检测乳果糖的难点。目前,虽有多种液相色谱检测乳果糖的报道,但方法在前处理的简便性、色谱柱的适用性及分离效果的稳定性等方面存在不足。该研究建立了高效液相色谱－蒸发光散射（HPLC－ELSD）检测液态乳中乳果糖的方法。采用0.4 mol/L醋酸铵（pH 3.8）沉降蛋白,氨基色谱柱分离,蒸发光散射检测器检测。液态乳中乳果糖与乳糖得到了良好的分离,在1.0～100 mg/L范围内线性关系良好（r2 = 0.998 0）,检出限为15 mg/L,定量下限为50 mg/L,回收率为85.1%～110%,相对标准偏差（RSD）不大于7.5%。该方法操作简单,可在8 min内完成检测,结果重复性好、准确度高,满足液态乳中乳果糖的日常检测要求。     

微乳液相色谱法同时测定山楂叶提取物中4种黄酮成分

建立了一种新的微乳体系,用于微乳液相色谱同时分析山楂叶提取物中牡荆素鼠李糖苷、芦丁、牡荆素及金丝桃苷4种黄酮成分.通过对影响分离选择性的主要因素进行考察,得到最佳微乳体系的组成为: 1.0%(w/w) 聚氧乙烯月桂醇(Brij35)-1.1%(w/w)正丁醇-0.1%(w/w)正辛醇-0.3%三乙胺(V/V)(H_3PO_4调节至pH 2.5).在此微乳体系中,表面活性剂类型和浓度、油相种类、添加剂三乙胺、流动相的pH值对4种黄酮成分的分离起到了重要的作用.选择Venusil ASB C_(18)色谱柱(150 mm×4.6 mm, 5μm),流速为0.8 mL/min,检测波长为360 nm,柱温为35 ℃.结果显示,4种黄酮成分在27 min内达到基线分离,在0.95～140.8 mg/L范围内,4个黄酮成分的线性相关系数r≥0.9995,平均回收率98.6%～101.6%.本方法可应用于山楂叶提取物中4种主要黄酮成分的质量分析.     

离子色谱法紫外检测器测定印染废水中的染料酸性红26

采用离子色谱法测定印染废水中的酸性红26,以100 mmol/L高氯酸钠–乙腈(体积比1∶1)为淋洗液,流速为1.0 mL/min,经IonPacAS16(250 mm×4 mm)分析柱分离,采用紫外–可见检测器于540 nm波长处检测,用外标法定量。酸性红26的线性范围为0.1～20 mg/L,相关系数为0.9999,加标回收率为98.4%～101.0%,以信噪比为3计算检出限,酸性红26的检出限为0.03 mg/L。色谱保留时间、色谱峰面积和色谱峰高测定结果的相对标准偏差分别为0.10%,0.50%,0.15%(n=10)。在淋洗液中添加乙腈以增强洗脱能力和改善峰形。使用紫外检测法可以避免印染废水中大量阴离子的干扰。     

气相色谱-质谱法测定轨道交通塑料产品中18种有害物质

建立气相色谱–质谱联用检测轨道交通塑料产品中的18种有毒有害物质的方法。用甲苯超声萃取60 min,选用DB–5HT(15 m×0.25 mm, 0.1μm)色谱柱,气相色谱–质谱法对样品进行检测。在优化的分析条件下,18种待测物质于20 min内获得对称的色谱峰,分离效果良好。待测物质的线性范围为0.5～4.5 mg/L,线性相关系数均在0.999以上,检出限达到0.5 mg/L,测定结果的相对标准偏差小于7%(n=6),样品加标回收率为85.3%～107.9%。该测试方法方便快速,测试结果准确、可靠,可以满足轨道交通塑料产品中多种有害物质的测定要求。     

Thermal stability of whey proteins studied by differential scanning calorimetry

The thermal stability of the bovine whey proteins.; β-lactoglobulin (β-1g), α-lactalbumin (α-1a) and serum albumin (BSA) was studied individually and in mixtures in the temperature range 25–140°C by differential scanning calorimetry. The thermal denaturation temperature (T_D) and the transition enthalpies (ΔH_app) were determined at different pH-values (3.0–10.0) in simulated milk ultrafil-trate (SMUF).β-Lg was, except at pH 9.0 and 10.0, the most thermostable protein at all pH-values. At acidic pH-values BSA was the least thermostable. At alkaline pH-values, however, α-la had lower thermal stability than BSA. α-La exhibited double peak behaviour at acidic pH-values and ΔH_app was dependent on Ca-content. Mixtures of the proteins were studied at pH 4.0, 5.0 and 6.6. In general, when mixed, the proteins seemed to denaturate independently of each other.     

Separation and quantitation of milk whey proteins of close isoelectric points by on-line capillary isoelectric focusing--electrospray ionization mass spectrometry in glycerol-water media

On-line coupling between CIEF and ESI/MS based on the use of bare fused-silica capillaries and glycerol-water media, recently developed in our laboratory, has been investigated for the separation of milk whey proteins that present close pI values. First, a new rinsing procedure, compatible with MS detection, has been developed to desorb these rather hydrophobic proteins (α-casein (α-CN), bovine serum albumin (BSA), lactoferrin (LF)) from the inner capillary wall and to avoid capillary blockages. Common hydrochloric acid washing solution was replaced by a multi-step sequence based on the use of TFA, ammonia and ethanol. To achieve the separation of major whey proteins (β-lactoglobulin A (β-LG A), β-lactoglobulin B (β-LG B), α-lactalbumin (α-LA) and BSA, which possess close pI values (4.5-5.35), CIEF parameters i.e. carrier ampholyte nature, capillary partial filling length with ampholyte/protein mixture and focusing time, have been optimized with respect to total analysis time, sensitivity and precision on pI determination. After optimization of sheath liquid composition (80:20 (v/v) methanol-water+1% HCOOH), quantitation of β-LG A, β-LG B, α-LA and BSA was performed. The limits of detection obtained from extracted ion current (EIC) and single ion monitoring (SIM) modes were in the 57-136 nM and 11-68 nM range, respectively. Finally, first results obtained from biological samples demonstrated the suitability of CIEF-MS as a potential alternative methodology to 2D-PAGE to diagnose milk protein allergies.     

Quantitative Characterization of Bovine Serum Albumin, α-Lactalbumin and β-Lactoglobulin in Commercial Whey Sample by RP-LC

A simple, sensitive and precise reverse phase liquid chromatographic method has been developed and validated for quantification of bovine serum albumin (BSA), α-lactalbumin (α-La) and β–lactoglobulin (β-Lg) that are removed from whey waste by foam fractionation method. The data is reproducible over a wide concentration range. This optimized method allowed analysis of BSA, α-La, β-Lg in a mixture within 5 min and could be applied to the analysis of a variety of commercial and laboratory whey products within a short time.     

反相高效液相色谱法测定牛奶中的主要蛋白质

建立了测定牛奶中的主要蛋白质的反相高效液相色谱法,对前处理方法进行了优化,采用Agilent Zorbax 300SB-C8(250 mm×4.6 mm,5 μm)色谱柱,三氟乙酸-水-乙腈流动相梯度洗脱,214 nm检测,柱温45 ℃,外标法定量.测定κ-CN, αs2-CN, αs1-CN, β-CN, Whey和Igg的线性关系良好,相关系数均大于0 999,加标回收率在74.8%～132.5%之间.大豆蛋白质不影响分离与检测.采用本方法分析了9种牛奶样品中上述蛋白质的含量与总量,结果表明,本方法测定结果准确可靠.不同样品中4种酪蛋白与乳清蛋白的比例基本接近,但是不同品牌之间存在一定差异.     

原料奶、婴儿配方奶粉及酸奶中蛋白去除效果的研究

建立了毛细管电泳法分析α-乳白蛋白、β-乳球蛋白A及β-乳球蛋白B的方法,考察了不同浓度冰乙酸(HAc)及三氯乙酸(TCA)对原料奶、婴儿配方奶粉A和B及酸奶中蛋白的去除效果。结果表明,TCA去除蛋白效果优于HAc,不同样品所需TCA浓度不同:2g原料奶需3mL100g/LTCA;0.5g婴儿配方奶粉A和B分别需5mL10g/L及20g/LTCA;2g酸奶则需3mL20g/LTCA才能将蛋白完全沉淀。为原料奶、婴儿配方奶粉及酸奶等乳制品的样品前处理提供了有价值的参考。     

Proteomic characterization of donkey milk “caseome”

At present, compared with bovine milk, the characterization of donkey milk caseins is at a relatively early stage progress, and only limited data are related to its genetic polymorphism. In this work, the heterogeneity of donkey caseome was investigated using a proteomic approach, based on one- (PAGE, UTLIEF) and two-dimensional (PAGE → UTLIEF) electrophoresis, stained with either Coomassie Brilliant Blue or specific polyclonal antibodies, and structural MS analysis. These combined methodologies allowed the contemporary identification of donkey α_s1, α_s2, β and κ-CN with their related heterogeneity due to phosphorylation (α_s1, α_s2 and β-CN), glycosylation (κ-CN) and incorrect splicing of RNA in mRNA (deleted forms of α_s1-CN and β-CN). The results achieved showed 11 components for κ-CN, six phosphorylated components for β and α_s1-CN and three main phosphorylated components for α_s2-CN, each accounting for 10, 11 and 12 P/mole. At this regard, for the first time, the primary structure of the expressed protein corresponding to the only available donkey α_s2-CN cDNA sequence was determined. Furthermore β-CN was found in homozygous and heterozygous state for the occurrence of a genetic β-CN variant having a MW value 28 mass units higher than the common β-CN phenotype.     

Structural rearrangement of β-lactoglobulin at different oil-water interfaces and its effect on emulsion stability

Understanding the factors that control protein structure and stability at the oil-water interface continues to be a major focus to optimize the formulation of protein-stabilized emulsions. In this study, a combination of synchrotron radiation circular dichroism spectroscopy, front-face fluorescence spectroscopy, and dual polarization interferometry (DPI) was used to characterize the conformation and geometric structure of β-lactoglobulin (β-Lg) upon adsorption to two oil-water interfaces: a hexadecane-water interface and a tricaprylin-water interface. The results show that, upon adsorption to both oil-water interfaces, β-Lg went through a β-sheet to α-helix transition with a corresponding loss of its globular tertiary structure. The degree of conformational change was also a function of the oil phase polarity. The hexadecane oil induced a much higher degree of non-native α-helix compared to the tricaprylin oil. In contrast to the β-Lg conformation in solution, the non-native α-helical-rich conformation of β-Lg at the interface was resistant to further conformational change upon heating. DPI measurements suggest that β-Lg formed a thin dense layer at emulsion droplet surfaces. The effects of high temperature and the presence of salt on these β-Lg emulsions were then investigated by monitoring changes in the ζ-potential and particle size. In the absence of salt, high electrostatic repulsion meant β-Lg-stabilized emulsions were resistant to heating to 90 °C. Adding salt (120 mM NaCl) before or after heating led to emulsion flocculation due to the screening of the electrostatic repulsion between colloidal particles. This study has provided insight into the structural properties of proteins adsorbed at the oil-water interface and has implications in the formulation and production of emulsions stabilized by globular proteins.     

The "dark side" of β-lactoglobulin: unedited structural features suggest unexpected functions

The in-depth characterization of water buffalo (WB) whey proteins based on chromatographic and mass spectrometric techniques revealed unexpected structural co- and post-translational modifications for β-lactoglobulin (β-Lg). The residues Lys⁴⁷ and Lys⁶⁹ of β-Lg were found to be lactosylated early, at the time of milking. Thiol groups of β-Lg underwent a dynamic sulfhydryl/disulfide exchange that is probably essential in accomplishing specific physiological requirements in which proteins may alternatively act either as a trigger or as a target. In this sense, the free sulfhydryl group of β-Lg established a glutathionylation/deglutathionylation equilibrium, which could be functional in conveying and delivering glutathione. Furthermore, the N-lauroylated β-Lg occurring exclusively in WB milk has been characterized for the first time. N-acylation could be an evolutionary remnant of ancestral lipocalins. Combined with the known aptitude of β-Lg to interact with phospholipid bilayers, this suggests that the protein could also be involved in the membrane translocation of small molecules, in addition to targeting, trafficking or the maintenance of membrane integrity. This structural characterization of β-Lg adds to the currently existing data and expands our understanding of the possible biological roles of this enigmatic protein.     

Real time observation of proteolysis with Fourier transform infrared (FT-IR) and UV-circular dichroism spectroscopy: watching a protease eat a protein

Fourier transform infrared (FT-IR)- and UV-circular dichroism (UV-CD) spectroscopy have been used to study real-time proteolytic digestion of β-lactoglobulin (β-LG) and β-casein (β-CN) by trypsin at various substrate/enzyme ratios in D(2)O-buffer at 37°C. Both techniques confirm that protein substrate looses its secondary structure upon conversion to the peptide fragments. This perturbation alters the backbone of the protein chain resulting in conformational changes and degrading of the intact protein. Precisely, the most significant spectral changes which arise from digestion take place in the amide I and amide II regions. The FT-IR spectra for the degraded β-LG show a decrease around 1634 cm(-1), suggesting a decrease of β-sheet structure in the course of hydrolysis. Similarly, the intensity around the 1654 cm(-1) band decreases for β-CN digested by trypsin, indicating a reduction in the α-helical part. On the other hand, the intensity around ～1594 cm(-1) and ～1406 cm(-1) increases upon enzymatic breakdown of both substrates, suggesting an increase in the antisymmetric and symmetric stretching modes of free carboxylates, respectively, as released digestion products. Observation of further H/D exchange in the course of digestion manifests the structural opening of the buried groups and accessibility to the core of the substrate. On the basis of the UV-CD spectra recorded for β-LG and β-CN digested by trypsin, the unordered structure increases concomitant with a decrease in the remaining structure, thus, revealing breakdown of the intact protein into smaller fragments. This model study in a closed reaction system may serve as a basis for the much more complex digestion processes in an open reaction system such as the stomach.     

H/α-CN versus H/β-CN rate ratios in the solvolysis of sulfonate esters in unconstrained systems. Additional evidence for conjugative stabilization of attached carbocations by the cyano moiety

H/α-CN and H/β-CN rate ratios have been measured for the solvolysis of sulfonate esters of relatively simple, noncyclic aliphatic alcohols. The β-CN function was found to be far more rate retarding than the α-CN function.