Analytical procedures for quantification of peptides in pharmaceutical research by liquid chromatography–mass spectrometry

Peptide quantification by liquid chromatography–mass spectrometry (LC–MS) combines the high resolving power of reversed-phase (RP) chromatography with the excellent selectivity and sensitivity of mass spectrometric detection. On the basis of comprehensive practical experience in the analysis of small molecules, pharmaceutical research is developing technologies for analysis of a growing number of peptidic drug candidates. This article is a detailed review of procedures based on LC–MS techniques for quantitative determination of peptides. With the focus on pharmaceutical applications several technologies for sample preparation, various aspects of peptide chromatography, important characteristics of ESI–MS, selectivity of MS-detection modes, the large variability of internal standards, and modern instrumentation are discussed. The demand for reliable, robust, sensitive, and accurate methods is discussed using numerous examples from the literature, complemented by experiments and results from our laboratory.     

Complexes of Click‐Derived Bistriazolylpyridines: Remarkable Electronic Influence of Remote Substituents on Thermodynamic Stability as well as Electronic and Magnetic Properties

2,6‐Bis(1,2,3‐triazol‐4‐yl)pyridine (btp) ligands with substitution patterns ranging from strongly electron‐donating to strongly electron‐accepting groups, readily prepared by means of Cu‐catalyzed 1,3‐dipolar cycloaddition (the “click” reaction), were investigated with regard to their complexation behavior, and the properties of the resulting transition‐metal compounds were compared. Metal–btp complexes of 1:1 stoichiometry, that is, [Ru(btp)Cl₂(dmso)] and [Zn(btp)Br₂], could be isolated and were crystallographically characterized: they display octahedral and trigonal‐bipyramidal coordination geometries, respectively, and exhibit high aggregation tendencies due to efficient π–π stacking leading to low solubilities. Metal–btp complexes of 1:2 stoichiometry, that is, [Fe(btp)₂]²⁺ and [Ru(btp)₂]²⁺, could also be synthesized and their metal centers show the expected octahedral coordination spheres. The iron compounds exhibit quite a complex magnetic behavior in the solid state including spin crossover near room temperature, and hysteresis and locking into high‐spin states on tempering at 400 K, depending on the substituents on the btp ligands. Cyclic voltammetry studies of [Ru(btp)₂]²⁺ reveal strong modulation of the oxidation potentials by more than 0.6 V and a clear linear correlation to the Hammett constant (σ_para) of the substituent at the pyridine core. Isothermal titration calorimetry was used to measure the thermodynamics of the Fe^II–btp complexation process and enabled accurate determination of the complexation enthalpies, which display a linear relationship with the σ_para values for the terminal phenyl substituents. Detailed NMR spectroscopic studies finally revealed that in the case of Fe^II complexation, dynamics are rapid for all investigated btp derivatives in acetonitrile, while replacing Fe^II by Ru^II or changing the solvent to dichloromethane effectively slows down ligand exchange. The results nicely demonstrate the utility of substituent parameters, originally developed for linear free‐energy relationships to explain reactivity in organic reactions, in coordination chemistry, and to illustrate the potential to custom‐design btp ligands and complexes thereof with predictable properties. The fast equilibration of the [Fe(btp)₂]²⁺ complexes together with their tunable stability and interesting magnetic properties should enable the design of dynamic metallosupramolecular materials with advantageous properties.     

Anion recognition by 1,3-disiloxane-1,1,3,3-tetraols in organic solvents

Anion recognition by 1,3-disiloxane-1,1,3,3-tetraols has been elucidated by ¹H NMR titrations and ESI-MS in organic solvents. The association constants of the receptors for halide anions are larger than those of silanediol and 1,3-disiloxane-1,3-diol due to the cooperative hydrogen bonds by four silanol hydroxy groups of 1,3-disiloxane-1,1,3,3-tetraols.     

表面活性单体DMBAC光聚合动力学研究

采用偶氮二异丁脒盐酸盐(AIBA)作为光引发剂,光引发聚合并研究了阳离子表面活性单体——甲基丙烯酰氧乙基二甲基苄基氯化铵(DMBAC)的均聚及其与丙烯酰胺(AM)共聚合反应的动力学行为,聚合反应均在高于DMBAC临界胶束浓度(CMC)的条件下进行.研究结果显示DMBAC均聚合反应速率与引发剂浓度的0.29次方以及单体浓度的0.89次方成正比,均聚合反应的表观活化能约为13.74 kJ/mo1;DMBAC与AM共聚合反应速率与引发剂浓度的0.82次方以及单体总浓度的0.83次方成正比,共聚合反应的表观活化能约为10.97 kJ/mo1;同时测得DMBAC与AM共聚合反应的单体竞聚率为r₁=0.27(AM)、r₂=2.00(DMBAC),说明AM趋向于形成共聚物,而DMBAC更趋向于形成均聚物.     

Co-Cu/SiO2催化剂的表征

用溶胶-凝胶法制备了Co-Cu/SiO2系列的担载型催化剂,采用SBET、TPR、XRD、O2滴定等技术对该催化剂进行了研究,并考察了催化剂在F-T合成反应中的催化性能.结果表明,样品的SBET较大,Co和Cu组分均很好分散;Cu2+的加入使得Co更易于被氢气还原,Co是经由Co3+→Co2+→Co0的还原过程,Cu的还原为Cu2+→Cu0;Cu2+的引入使得样品中钴的还原度增大,对F-T合成的催化性能有了一定的改善.     

基于LabVIEW 8.0的pH电位滴定虚拟仪器

运用离子选择性电极、调理电路、DAQ卡和计算机,构建了基于LabVIEW 8.0的电位滴定虚拟仪器,实现了电位滴定实验数据的自动记录和自动处理.将该虚拟仪器应用到乙酸等的电位滴定实验表明,实验结果相对标准偏差小于0.65%,提高了实验结果的重现性和准确性;且虚拟仪器界面友好、操作简便、实时绘出滴定曲线,自动处理数据及即时得到结果.     

Parallel vector analysis based soft resolution of spectral data from pH‐metric titration using symmetry constraint

In this study, a soft method is proposed to calculate concentration and spectral profiles for the two‐way spectral data from dissociation equilibria of polyprotic acids (H_nA). This method has four main distinct steps: (i) a fixed size moving window evolving factor analysis (FSMWEFA) was used to identify the local rank map, (ii) WFA was applied to calculate the concentration profiles of H_nA and Aⁿ⁻ (selection of the window for application of WFA was performed using EFA), (iii) PVA was used to calculate H_{n − 1}A to HA spectral profiles, and (iv) a symmetry constraint, in addition to the non‐negativity constraint, was utilized to obtain the unique concentration and spectral profiles from different acceptable sets of profiles. In the absence of any selective region in the spectral data, the proposed soft method resulted in unique solution without rotational ambiguity. This study is the first application of symmetry constraint on concentration profiles. The rotational ambiguity drastically decreased on considering the constraint of symmetry of the H_{n − 1}A and HA concentration profiles, in addition to non‐negativity of profiles. Simulated examples were used to confirm these approaches. Effect of closeness of dissociation constants on the estimated values of constants was investigated. The results showed that when the difference between pK_a values is more than 1.2, the obtained errors in the estimation of pK_a values are less than about 6.5%. The considered real data were from pH‐metric titration of fluorescein. The obtained spectral and concentration profiles and the estimated pK_a values for fluorescein were in good agreement with the previously reported data. Copyright © 2009 John Wiley & Sons, Ltd.     

沉淀单点滴定法与双点滴定法研究

导出了沉淀单点滴定法与双点滴定法计算式。以银电极为指示电极 ,Ag 标准溶液为滴定剂 ,对Cl-、Br-、I-及SCN-等离子的测定进行了研究 ;对影响测定结果准确度的因素进行了讨论。实验发现 ,在α =1 .0～ 1 .1的条件下 ,单点滴定法有较高的准确度 ;在α1 ≈ 1 .0～ 1 .1、ΔV =0 .5～ 1 .0mL的条件下 ,双点滴定法有较高的准确度     

微波密闭消解难处理K[Au(CN)2]化合物及其Au的精密电位滴定法测定

首次提出了微波密闭消解难处理K[Au(CN)2]化合物的新方法,优化了试液预处理和电位滴定法测定Au的条件,并与英国标准分析方法进行了对照研究.结果表明两种方法的Au分析结果准确度(相对平均误差≤±0.03%)基本一致,但前者结果精密度好(相对标准偏差≤0.02%),消解快速(30 min)、无污染、无损失、经济、简便,符合绿色分析化学的要求,适宜推广应用.     

Plasma brominated polymer particles as grafting substrate for thiol‐terminated telomers

A combined surface activation and “grafting to” strategy was developed to convert divinylbenzene particles into weak cation exchangers suitable for protein separation. The initial activation step was based on plasma modification with bromoform, which rendered the particles amenable to further reaction with nucleophiles by introducing Br to a surface content of 11.2 atom‐%, as determined by X‐ray photoelectron spectroscopy. Grafting of thiol‐terminated glydicyl methacrylate telomers to freshly plasma activated surfaces was accomplished without the use of added initiator, and the grafting was verified both by reduction in bromine content and the appearance of sulfur‐carbon linkages, showing that the surface grafts were covalently bonded. Following grafting the attached glydicyl methacrylate telomer tentacles were further modified by a two‐step procedure involving hydrolysis to 2,3‐hydroxypropyl groups and conversion of hydroxyl groups to carboxylate functionality by succinic anhydride. The final material was capable of baseline separating four model proteins in 3 min by gradient cation exchange chromatography in a fully aqueous eluent.