反相高效液相色谱法测定小鼠中脑内D-天冬氨酸和D-谷氨酸

建立了测定生物样品中微量D-天冬氨酸、D-谷氨酸的高效液相色谱法。以邻苯二甲醛(OPA)-N-异丁酰基-L-半胱氨酸柱前衍生氨基酸对映体,醋酸钠-甲醇-乙腈混合液为流动相,在Shim-packVP-ODS柱上,采用梯度淋洗技术,2种氨基酸对映体在40min内完全分离。以荧光检测,D-天冬氨酸(D-Asp)、D-谷氨酸(D-Glu)的浓度在1.0×10-8～1.0×10-6mol/L时,其浓度与峰面积呈良好的线性关系。当信噪比为3(S/N=3)时,D-Asp和D-Glu的检出限分别为2.0×10-9mol/L和1.0×10-9mol/L。将方法运用于帕金森模型小鼠中脑中D-Asp和D-Glu含量的测定,对照组与模型组中脑D-Asp含量无显著性差异,D-Glu含量呈现显著性差异。推测在脑组织里,内源性的D-Asp的合成及转运不受神经毒素1-甲基-4-苯基-1,2,3,6-四氢吡啶(MTPT)的毒害仍保持原有的动态平衡;而D-Glu可能具备与L型对映体相似的兴奋毒性,参与帕金森疾病的发病机制。     

D-苯甘氨酸手性固定相的制备及对多种手性化合物的拆分

以D-3,5-二硝基苯甲酰苯甘氨酸和3-氨丙基硅烷化硅胶合成了D-苯甘氨酸衍生手性固定相,并自制了手性高效液相色谱分离柱,用正己烷-异丙醇作流动相,对非衍生化的氨基酸、胺、醇和羧酸类等10种手性化合物进行了高效液相色谱拆分。结果表明:所拆分的10种手性化合物,有7种手性化合物能得到基线分离,最好的分离度Rs=5.56。该文还用苯对柱性能进行了评价,理论塔板数达每米8万块。     

新型紫外衍生试剂用于D-氨基酸的毛细管电泳分离

采用一种新型紫外衍生试剂对乙酰氨基苯磺酰氟(PAABS-F)作为柱前衍生试剂,成功地对19种标准D-氨基酸和甘氨酸进行了衍生和毛细管电泳分离。详细研究了各种分离条件对毛细管电泳分离的影响。实验结果表明:采用20 mmol/L硼砂(pH 9.3),126 mmol/L十二烷基硫酸钠,8 mmol/Lβ-环糊精和20mmol/L NaC l时分离结果最佳,16 m in内实现了20种氨基酸的基线分离。     

配体交换毛细管区带电泳拆分未衍生化氨基酸

以铜(Ⅱ)-L-谷氨酸络合物为手性分离选择剂,对苯丙氨酸、酪氨酸和色氨酸3种非衍生芳香族氨基酸的手性对映体拆分进行了研究,建立了一种快速、简便拆分未衍生化的氨基酸对映体的配体交换毛细管电泳方法.在使用10 mmol/L NH4AC(pH 5.0),5 mmol/L CuSO4和10 mmol/L L-谷氨酸的条件下,成功地拆分了苯丙氨酸、酪氨酸手性对映体;色氨酸手性对映体也得到部分分离;考察了电泳缓冲液组成、pH值等影响分离效果的因素.     

递质氨基酸的毛细管电泳-安培检测

研究了毛细管电泳安培检测氨基酸的方法。氨基酸用萘 -2,3 -二羧醛 (NDA)衍生为电活性物质。碳纤维电极采用1.5V ,30s固定电压电化学预处理 ,检测结果令人满意。作者还对最佳衍生反应条件 ,电极预处理条件对氨基酸衍生物电化学行为的影响进行了研究。该法对谷氨酸、甘氨酸、丙氨酸、γ -氨基丁酸的检出限分别为2.5×10-7、2.5×10-7、5.0×10-8、2.5×10-7 mol/L。     

体内D-酸性氨基酸分析的自动二维手性高效液相色谱法的建立及应用

D-酸性氨基酸的生理功能和疾病标志物作用已逐渐成为代谢组学和创新药物开发的研究热点,本研究利用硅胶基质ODS整体柱为第一维分离色谱柱,乙腈-三氟醋酸-水(9∶0.05∶92,V/V)为流动相;Chiralpak QD-1-AX微径手性色谱柱为第二维手性分离柱,10 mmol/L柠檬酸的甲醇-乙腈(50∶50,V/V)溶液为流动相,4-氟-7-硝基-2,1,3-苯并氧杂恶二唑(NBD)-F衍生化荧光法检测,建立了二维手性高效液相色谱全自动分析系统。本系统实现了对生物样品中酸性氨基酸异构体的高效快速分离(Rs>2.5);高灵敏度检测(LOD=1 fmol)及定量值的确证,酸性氨基酸异构体回收率均在97%~104%之间,日内日间精密度的RSD<5%。应用本系统对免疫功能低下的老年病小鼠动物模型快速老化小鼠亚系1(SAMP1)进行分析,测得SAMP1小鼠胸腺和脾脏中D-天门冬氨酸的含量分别为(206±18)nmol/g和(264±21)nmol/g。首次发现了其胸腺和脾脏中D-天门冬氨酸的含量较对照组同龄抗快速老化小鼠亚系1(SAMR1)有显著升高趋势(p<0.01)。     

表面活性剂对NBD-Cl衍生氨基酸的荧光增敏和芯片电泳分离的影响

研究多种表面活性剂以及溶剂对4-氯-7-硝基苯-2-氧-1,3-二唑(NBD-Cl)衍生氨基酸荧光性质的影响。考察并优化了NBD-Cl与氨基酸的衍生反应条件以及芯片毛细管电泳-激光诱导荧光的分离分析条件。结果表明:在临界胶束浓度以上,阳离子表面活性剂十六烷基三甲基溴化胺(CTMAB)对精氨酸、谷氨酸、天冬氨酸的NBD-Cl衍生物,非离子表面活性剂十二烷基聚氧乙烯醚(Brij-35)对苯丙氨酸、精氨酸的NBD-Cl衍生物有荧光增敏效果。在含有5mmol/LBrij-35和20%乙腈添加剂的15mmol/L硼砂缓冲液(pH9.2)中,精氨酸、苯丙氨酸、丙氨酸、甘氨酸、谷氨酸及天冬氨酸等6种氨基酸的NBD-Cl衍生物可以在70s内实现分离,其中较难分离的丙氨酸、甘氨酸的NBD-Cl衍生物分离度为0.91;6种氨基酸衍生物的检出限为0.15～1.6μmol/L。     

固定化D-氨基酸氧化酶催化DL-氨基酸去消旋化制备非天然L-氨基酸

在过氧化氢酶和氧气存在下,固定化D-氨基酸氧化酶(D-AAO)对映选择性催化DL-氨基酸中的D-对映体氧化脱氨为相应酮酸,L-对映体保留.研究了D-AAO的底物特异性并对反应条件进行了优化.结果表明:D-AAO具有较宽的底物谱,能够催化疏水性α-氨基酸的D-对映体氧化脱氨.在最优反应条件下,D-AAO催化DL-2-氨基丁酸、DL-2-氨基戊酸去消旋化,L-2-氨基丁酸、L-2-氨基戊酸的收率分别为48%和47%,ee分别为99.5%和99.8%.进一步地利用Pd-C/HCOONH4催化氧化脱氨过程中产生的亚氨基酸原位还原,有效提高了L-2-氨基丁酸、L-2-氨基戊酸的收率并保持高的光学纯度.     

D-甘露醇与D-山梨醇在纯水和卤化钠水溶液中的稀释焓

应用等温流动微量热技术测定了298.15 K下D-甘露醇和D-山梨醇在纯水和卤化钠水溶液中的稀释焓,利用M cM illan理论计算了D-甘露醇和D-山梨醇在纯水和卤化钠水溶液中焓相互作用系数.结果表明,D-甘露醇和D-山梨醇在纯水和卤化钠水溶液中的二阶焓相互作用系数均为正值,并随着卤离子半径的变化而相应变化;且同种溶剂中,D-甘露醇的二阶焓相互作用系数要大于D-山梨醇的.结合两分子结构的差异,通过溶质-溶质相互作用和溶剂-溶质相互作用对这一结果进行了讨论.     

衍生化环糊精键合固定相色谱保留和手性识别机理的研究(Ⅰ)

合成了苯基氨基甲酸酯衍生化β-环糊精键合固定相,14个α-氨基膦酸酯类化合物首次在该固定相和商品化的(S)-(+)-萘乙基氨基甲酸酯衍生化β-环糊精固定相上进行液相色谱手性拆分.通过定量结构-对映异构体保留关系对比研究了两种不同的环糊精类固定相上可能的色谱保留和手性识别机理.     

CdS纳米粒子与半胱氨酸相互作用的研究

合成了粒径均匀和分散性好的CdS纳米粒子.通过改变CdS纳米粒子及半胱氨酸的浓度、体系的pH值及CdCl2和CH3CSNH2摩尔比等实验条件跟踪监测了CdS纳米粒子光谱性质的变化,探讨了CdS纳米粒子与半胱氨酸之间的相互作用及化学反应机理.     

Constructing chiral caves and efficiently separating enantiomers of glutamic acid with novel surface-imprinting technique

Dimethylaminoethyl methacrylate (DMAEMA) was first graft-polymerized onto the surfaces of micron-sized silica gel particles in the manner of "grafting from" in a solution polymerization system, obtaining the grafted particles PDMAEMA/SiO(2). Then, the molecular imprinting towards the grafted PDMAEMA was conducted with one enantiomer of glutamic acid (Glu), L-Glu, as template molecule and with 2,2'-dichlorodiethylether (DCEE) as crosslinking agent by adopting the novel surface-molecular imprinting technique established by our research group, and the single enantiomer (L-Glu) molecule-imprinted material MIP-PDMAEMA/SiO(2) was obtained. With another enantiomer of glutamic acid, D-Glu, as the contrast compound, the recognition property of MIP-PDMAEMA/SiO(2) for L-Glu was investigated in depth with both static and dynamic methods, and its ability to separate L-Glu and D-Glu in the racemic solution was examined. The experiment results show that the surface-imprinted material MIP-PDMAEMA/SiO(2) has fine recognition selectivity and binding affinity for l-Glu, whereas its ability to combine D-Glu is poor. The selectivity coefficient of MIP-PDMAEMA/SiO(2) for L-Glu with respect to D-Glu is equal to 3.30, displaying an excellent chiral separation result. It is obvious that in this study, the substance separation at the molecular configuration level has been realized successfully.     

反相-高效液相色谱手性流动相添加剂法测定酸奶及含乳饮料中乳酸对映异构体

选取2,3,6-三甲基-β-环糊精（TM-β-CD）作为流动相手性添加剂,建立了采用反相高效液相色谱法分离酸奶及含乳饮料中乳酸异构体的方法。实验采用hypersil ODS2-C18（250&#215;5．0mm,5μm）色谱柱,以0．5mmol／L PH2．5的TM-β-CD（含4．5mmol／L H2SO4）作为流动相,流速为1．0mL／min,紫外检测波长为210nm。实验考察了不同色谱柱、柱平衡时间、手性流动相添加剂浓度及pH值对分离效果的影响,并进一步研究了方法的线性范围、检出限、精密度及回收率。     

Application of stepwise discriminant analysis to classify commercial orange juices using chiral micellar electrokinetic chromatography-laser induced fluorescence data of amino acids

The use of chiral amino acids content and stepwise discriminant analysis to classify three types of commercial orange juices (i.e., nectars, orange juices reconstituted from concentrates, and pasteurized orange juices not from concentrates) is presented. Micellar electrokinetic chromatography with laser-induced fluorescence (MEKC-LIF) and beta-cyclodextrins are used to determine L- and D-amino acids previously derivatized with fluorescein isothiocyanate (FITC). This chiral MEKC-LIF procedure is easy to implement and provides information about the main amino acids content in orange juices (i.e., L-proline; L-aspartic acid, D-Asp, L-serine, L-asparagine, L-glutamic acid, D-Glu, L-alanine, L-.arginine, D-Arg, and the non-chiral gamma-amino-n-butyric acid (GABA), i.e., gamma-aminobutyric acid). From these results, it is clearly demonstrated that some D-amino acids occur naturally in orange juices. Application of stepwise discriminant analysis to 26 standard samples showed that the amino acids L-Arg, L-Asp and GABA were the most important variables to differentiate the three groups of samples. With these three selected amino acids a 100% correct classification of the samples was obtained either by standard or by leave-one-out cross-validation procedures. These classification functions based on the content in L-Arg, L-Asp and GABA were also applied to nine test samples and provided an adequate classification and/or interesting information on these samples. It is concluded that chiral MEKC-LIF analysis of amino acids and stepwise discriminant analysis can be used as a consistent procedure to classify commercial orange juices providing useful information about their quality and processing. To our knowledge, this is the first report about the combined use of chiral capillary electrophoresis and discriminant techniques to classify foods.     

手性糖基锌卟啉对手性氨基酸甲酯的分子识别

合成了手性四糖基取代锌卟啉(Zn-A)的三种阻转异构体αβαβ-Zn-A、ααββ-Zn-A和αααβ-Zn-A以及一种单糖基取代锌卟啉(Zn-B), 并通过可见光谱滴定法和圆二色(CD)光谱滴定法研究了它们对手性氨基酸甲酯(L/D-LeuOMe, L/D-ThrOMe, L/D-ValOMe和L/D-PheOMe)客体的分子识别行为. 研究发现, 三种锌卟啉对L型氨基酸甲酯的缔合常数均要高于D型, 其中ααββ-Zn-A的对映体选择性(K_L/K_D)最高可达4.75, 可用于L型氨基酸甲酯的选择性识别. 不同的Zn-A 阻转异构体对手性氨基酸甲酯的缔合常数给出相同的顺序:K^θ(LeuOMe) > K^θ(ValOMe) > K^θ(ThrOMe) > K^θ(PheOMe);主体Zn-B对手性氨基酸分子的缔合常数顺序为K^θ(PheOMe) > K^θ(LeuOMe) > K^θ(ValOMe) > K^θ(ThrOMe). 同时, 以咪唑为探针分子研究了非手性分子对Zn-A构象的影响, 发现非手性分子咪唑与手性主体结合后, 也可对主体的构象产生影响. Zn-A的三种阻转异构体与氨基酸甲酯和咪唑类客体的缔合常数关系均为K^θ(ααββ-Zn-A) > K^θ(αβαβ-Zn-A) > K^θ(αααβ-Zn-A).     

以L-天冬氨酸为手性选择剂对手性药物的毛细管电泳拆分

以Ｌ－天冬氨酸为手性选择剂,建立了对１０种手性药物旋光异构体的毛细管电泳快速分离方法,优化了分离条件,并对分离机理作了初步探讨。     

Continuous on-line derivatization and selective separation of D-aspartic acid by a capillary electrophoresis system with a continuous sample introduction interface

A rapid and selective method is described for the separation of D-aspartic acid (D-Asp) using a continuous on-line derivatization system coupled to capillary electrophoresis (CE). D-Asp was derivatized using o-phthaldialdehyde/N-acetyl-L-cysteine (OPA/NAC). By on-line derivatization, amino acid enantiomers were automatically and reproducibly converted to the UV-absorbing diastereomer derivatives which were separated by capillary zone electrophoresis (CZE) in the presence of 10 mmol/L beta-cyclodextrin (beta-CD). Under the investigated separation conditions, D-Asp is resolved from L-aspartic acid (L-Asp) and other amino acids in a standard mixture of amino acids. The separation could be achieved within 4 min and the sample throughput rate can reach up to 16 h(-1). The repeatability (defined as relative standard deviation, RSD) was 3.21%, 3.58% with peak area evaluation and 3.72%, 4.03% with peak height evaluation for L-Asp and D-Asp.     

双水相手性萃取拆分扁桃酸外消旋体

研究了扁桃酸(MA)对映体在含β-环糊精(β-CD)手性选择剂的聚乙二醇(PEG)/(NH_4)_2SO_4双水相体系中的萃取分配行为.考察了pH、PEG和(NH_4)_2SO_4的质量分数、β-CD的浓度、MA的浓度与萃取温度等因素对拆分效果的影响.实验结果表明:双水相手性萃取具有很强的手性分离能力,β-CD对L-MA对映体的识别能力大于对D-MA对映体的识别能力;当温度为30℃、pH=1.0、PEG2000质量分数为30%、(NH_4)_2SO_4质量分数为20%、β-CD的浓度为0.008 mol/L、扁桃酸的浓度为0.05 mol/L时,分离因子(α)达到2.46,上、下相对映体过剩值(e.e.%)分别为42.13%和40.43%.     

Quantification of D-amino acids in the central nervous system of Aplysia californica by liquid chromatography/tandem mass spectrometry

A sensitive, specific and reliable liquid chromatography/tandem mass spectrometry (LC/MS/MS) method has been developed for simultaneous determination of D-amino acids in the central nervous system (CNS) of Aplysia californica. In order to correct for any potential matrix effects on measured signals, deuterium-labeled L-Asp-d3 was used as an internal standard. Pre-column derivatization of the sample with 7-fluoro-4-nitrobenzoxadiazole (NBD-F) allowed both effective in-line pre-concentration and sensitive MS/MS detection of the analytes. An extraction column (50x0.25 mm, 5 microm C18 silica particles) was used to pre-concentrate/stack samples. Enantiomeric separation of amino acid enantiomers was achieved on a chiral column packed with teicoplanin aglycone bonded silica particles (170x0.25 mm, 5 microm) with an MS-friendly mobile phase. The characteristic precursor to product ion transitions, m/z 297-->279 (for NBD-Asp), m/z 269-->223 (For NBD-Ser), m/z 311-->293 (for NBD-Glu) and m/z 300-->282 (for NBD-L-Asp-d3) were monitored for the quantification. Samples from the CNS of A. californica and heart tissues were analyzed. D-Asp was detected at high levels in all the ganglia and nerve tissues, but not in the heart tissue. Further, neither D-Ser nor D-Glu was detected in Aplysia, a widely used neuronal model.     

毛细管电泳测定4个不对称合成化合物的光学纯度

采用毛细管电泳技术,以α-环糊精、β-环糊精和β-环糊精聚合物为手性选择剂,对4种不对称合成物3-(二乙基氨基)-1-苯基-1-丙醇(a)、1-苯基-3-(1-哌啶）-1-丙醇(b)、3-吗啉-1-对甲苯基-1-丙醇(c)和1-（4-氯苯基）-3-吗啉-1-丙醇(d)的消旋体和光学活性异构体进行了手性分离。 结果发现,使用20 kV的分离电压,以浓度为30 g/L的β-环糊精聚合物的80 mmol/L三羟甲基氨基甲烷缓冲溶液（pH=3.2）为背景电解质溶液,4种化合物在40 min内均可得到基线分离。 根据对映异构体峰面积和保留时间的比值确定了4种不对称化合物的光学纯度。 测定的RSD均不大于1.5%。