胰岛素反相制备色谱的方法开发

以胰岛素反相制备色谱方法的开发和优化为目标,通过考察色谱保留参数、峰展宽及样品流出曲线的浓度分布等色谱参数,对流动相梯度、色谱填料、载样量等色谱条件进行了优化,并建立了胰岛素制备色谱峰参数的描述方法。结果表明,所建立的方法可快速筛选出最适于胰岛素分离的色谱条件(包括流动相梯度及分离填料),即流动相中的强洗脱溶剂(有机相)需采取缓梯度窄区间的变化条件,筛选出的分离填料需具备峰向两侧展宽且展宽程度较小、样品最高浓度居中分布的特点。将方法用于实际胰岛素粗品的纯化制备,获得了杂质去除效果好、胰岛素纯度高的产品。该法为胰岛素反相色谱纯化制备方法的快速建立提供了指导,具有较强的实用价值,同时为发展大分子化合物的制备色谱方法提供了参考。     

Comparative molecular field analysis of chromatographic hydrophobicity indices for some coumarin analogs

The chromatographic hydrophobicity index (CHI) is an HPLC‐based parameter that provides reliable guidance in optimization of pharmacological efficiency and adsorption, distribution, metabolism and exertion (ADME) profile of drug candidates. In the present work, classical and three‐dimensional quantitative structure–property relationship (QSPR) models were developed for prediction of CHI values of some 4‐hydroxycoumarin analogs on immobilized artificial membrane column. Comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA) as 3D–QSPR methods were performed to gain insight into the key structural factors affecting on the chromatographic hydrophobicity of interested chemicals. The calculated parameters of Q ², R ² and standard error were 0.545, 0.996 and 0.773 for CoMFA model and 0.815, 0.986 and 1.44 for CoMSIA model, respectively. The contour maps for steric fields of the CoMFA model illustrate that the hydrophobicity of chemicals will be higher when the positions of R6, R7 and R8 in the 4‐hydroxycuomarin ring are substituted by alkyl groups. Moreover, by the analysis of the plots of electrostatic fields, it was concluded that the CHI value greatly increases if one hydrogen on coumarin ring is substituted by the F, Cl, Br, OH or OCH₃ group.     

二维反相液相色谱制备五味子中的木脂素

应用自主研发的具有分离-富集模式的制备色谱工厂,建立了分离制备五味子木脂素有效部位及其单体化合物的方法。该方法首先以C₁₈（250 mm×4.6 mm,5 μm）为色谱柱,水和甲醇为流动相,分离度和保留时间为考察指标,采集4个不同梯度的色谱信息,通过XTool色谱专家系统软件模拟,确定五味子木脂素第一、二维色谱条件;然后采用线性放大的方法,以C₁₈（250 mm×30 mm,10 μm）为第一、二维分离柱,C₁₈（80 mm×30 mm,10 μm）为富集柱,水为富集稀释液,对五味子木脂素进行二维色谱分离纯化;最后第一维分离得到9个可重复组分,第二维分离得到20个高纯度化合物,其中有6个单体化合物。结果表明该法重现性良好,可以实现五味子木脂素的系统性分离,对五味子化学成分的研究具有重要意义。     

Prediction of peptides retention behavior in reversed-phase liquid chromatography based on their hydrophobicity

Hydrophobicity is an important physicochemical property of peptides and proteins. It is responsible for their conformational changes, stability, as well as various chemical intramolecular and intermolecular interactions. Enormous efforts have been invested to study the extent of hydrophobicity and how it could influence various biological processes, in addition to its crucial role in the separation and purification endeavor as well. Here, we have reviewed various studies that were carried out to determine the hydrophobicity starting from (i) simple amino acids solubility behavior, (ii) experimental approach that was undertaken in the reversed-phase liquid chromatography mode, and ending with (iii) some examples of more advanced computational and machine learning models.     

反相高效液相色谱法同时测定罗氏海盘车中的7种核苷化合物

建立了罗氏海盘车中7种核苷化合物的反相高效液相色谱分析测定方法。采用超声波辅助提取,选用两根不同的C18色谱柱串联,以甲醇和0.2%(体积分数)乙酸/水溶液为流动相梯度洗脱分离。优化的色谱条件为: 柱温为室温,检测波长为260 nm,流速为0.8 mL/min,进样量为20 μL。结果表明,7种核苷化合物在一定的浓度范围内线性关系良好,次黄嘌呤和胸苷的线性范围为0.65～40 mg/L,尿苷、黄嘌呤和肌苷的线性范围为0.80～40 mg/L,胸腺嘧啶的线性范围为1.15～40 mg/L,鸟苷的线性范围为0.50～40 mg/L。样品中7种核苷化合物的加标回收率为90.00%～105.00%,相对标准偏差为0.72%～3.23%。该方法操作简便、灵敏度高、重复性好,回收率高,适用于罗氏海盘车中7种核苷类成分的同时分析,也可用于罗氏海盘车的质量控制和综合评价。     

环孢素A在反相液相色谱中的吸附行为及分离纯化北大核心CSCD

环孢素A(cyclosporine A,CsA)是由11个氨基酸组成的中性环状多肽,是临床拮抗器官和组织移植后排异反应的首选药物。高效液相色谱法广泛应用于CsA的分离分析,开展CsA色谱行为的研究是使用制备高效液相色谱纯化CsA的关键。该文首先在C18固定相上比较了CsA在甲醇-水和乙腈-水两种流动相体系中的保留行为,结果表明其保留时间对有机相比例变化比较敏感。控制甲醇比例在84%~88%,或者乙腈比例在75%~85%,CsA的保留因子(k)在3~7范围内。考察了上样量对CsA峰形的影响。随着上样量增加,在两种流动相体系中,CsA的峰形都由对称开始变得拖尾,保留时间前移。因此在进行CsA纯化时,需要特别注意前杂的去除情况。然后采用吸附等温线描述CsA的保留行为,当流动相中CsA的质量浓度较低时,有机相比例对溶质在固定相上的吸附量影响并不明显。随着溶质的质量浓度增加至0.5 g/L以上,有机相比例降低有助于提高CsA在固定相上的吸附量。和甲醇-水体系相比,在乙腈-水体系中固定相对溶质有更大的吸附容量。用模型对CsA的等温吸附曲线拟合,在甲醇-水体系中符合Langmuir模型,在乙腈-水体系中为Moreau模型。由模型参数可知在两种体系下,CsA在C18固定相上均为单层吸附,区别在于乙腈-水体系中CsA会产生较大的分子间作用力。最后,实验采用0~60 min 65%~75%乙腈、60~80 min 75%乙腈的条件开展了环孢素A纯化的探索实验,可将CsA的杂质控制在0.2%以下。本研究结果对采用制备高效液相色谱纯化CsA具有指导意义。     

胆固醇基键合相-反相高效液相色谱法测定正辛醇/水分配系数

通过反相高效液相色谱法系统地考察了中性和弱酸性化合物在新型胆固醇基键合相色谱柱（Cholester柱）上的保留行为。以甲醇和乙腈为有机调节剂,建立了保留因子（k）与有机调节剂比例（φ）间的关系,并外推获取100%水相为流动相时的log k_w;同时进一步建立并验证了不同流动相下正辛醇/水分配系数的对数（log K_ow）和log k_φ（log k_w）间的模型,并预测了部分化合物的log K_ow。结果表明,使用Cholester柱测定log K_ow时,甲醇比乙腈更适合作为有机调节剂;对中性化合物和中性状态的酸性化合物,可以用任意甲醇比例下获取的log k_φ预测log K_ow;对存在离解的酸性化合物,依然用外推方式获取的log k_w预测log K_ow。将采用Cholester柱与文献中采用C₁₈柱、C₈柱建立的log K_ow-log k_φ模型进行对比,结果表明化合物与胆固醇基键合相存在特别的作用。     

反相液相制备色谱法结合超临界流体制备色谱法分离纯化海风藤中的化合物

建立了基于反相液相制备色谱和超临界流体制备色谱的组合方法,用于分离纯化醇提水沉后石油醚层中的海风藤。首先以甲醇作为改性剂,采用醇提水沉法去除海风藤甲醇提取物中的叶绿素,加入硅藻土后用石油醚回流富集目标成分。选用反相C18制备色谱柱将其分为18个组分,然后将组分在SFC模式下进行制备。选用酰胺色谱柱,以甲醇为改性剂,在柱温30℃、背压15.0 MPa的条件下进行分离。基于反相色谱和超临界流体色谱不同的分离选择性,最后分离得到6个高纯度化合物。该法展示了反相制备色谱和超临界流体制备色谱在海风藤分离纯化方面的优势,特别是超临界流体色谱在天然产物的分析和制备方面的巨大潜力。     

反相高效液相色谱中同系物组分的线性S－logk＇w相关分析

研究了反相高效液相色谱中不同冲洗剂体系中同系物组分的线性Ｓ－ｌｏｇｋ＇ｗ相关分析（ＬＳＬＣ）。ＬＳＬＣ分析可以用来表征反相高效液相色谱的柱系统，在ＬＳＬＣ方程Ｓ＝ｐ·ｌｏｇｋ＇ｗ＋ｑ中，ｑ／ｐ主要表征了柱相比的对数（ｌｏｇΦ），而且对给定冲洗剂系统是一个常数；由ＬＳＬＣ所获得的相比并不显著的随同系物的种类的变化而变化，它是随Ｇ＿１８键含量的增加而增加的。     

反相高效液相色谱法定量分析木质素的主要降解产物

建立了反相高效液相色谱定量分析玉米秸秆蒸汽爆破预处理过程中产生的主要木质素降解产物的方法。采用C18色谱柱,柱温30 ℃,乙腈-水(含1.5%的醋酸)为流动相,梯度洗脱,流速为0.8 mL/min, 254 nm和280 nm波长下紫外检测,可实现4-羟基苯甲酸、香草酸、紫丁香酸、4-羟基苯甲醛、香草醛和紫丁香醛的有效分离。6种主要木质素降解产物线性回归方程相关系数为0.9999～1.0000,加标回收率均在96%以上,相对标准偏差低于2.5%(n=6),满足定量分析要求。     

亲水/反相二维制备液相色谱法分离纯化络石藤中的化学成分

建立了亲水/反相二维制备液相色谱(Pre-2D-HILIC/RPLC)分离纯化络石藤中化学成分的分析方法。络石藤药材经醇提、活性炭脱色后用反相固相萃取柱除去色素和强极性物质,最终得到干燥的浅黄色粉末。一维亲水色谱选择Click XIon色谱柱(250 mm×20 mm,10μm)作为固定相,水和乙腈作为流动相,进行梯度洗脱,以紫外触发模式收集馏分,共得到15个组分。二维反相色谱选择C18色谱柱(250 mm×20 mm,5μm)作为固定相,水和乙腈作为流动相,进行梯度洗脱,最终得到14个高纯度化合物,并通过质谱和核磁共振对其进行确认。实验结果表明,该法具有良好的正交选择性,可以有效提高分离度和峰容量,对于分离络石藤等复杂样品具有重要意义。     

Simultaneous determination of cefuroxime axetil and ornidazole in tablet dosage form using reversed-phase high performance liquid chromatography

 A simple, accurate and sensitive reversed-phase high performance liquid chromatographic (RP-HPLC) method for the simultaneous determination of cefuroxime axetil and ornidazole in combined tablet dosage form has been developed. The method was performed with a HiQ-SiL C18 column (250 mm×4.6 mm) and photodiode array (PDA) detector, using 0.01 mol/L potassium dihydrogen orthophosphate-methanol (56∶44, v/v) as the mobile phase and tinidazole as the internal standard. Beer’s law obeys in the concentration ranges of 5-25 μg/mL and 10-50 μg/mL for cefuroxime axetil and ornidazole, respectively. The method has been successfully validated statistically and applied for the analysis of the drugs in pharmaceutical formulation.     

Evaluation of solute hydrophobicity by reversed-phase high performance liquid chromatography using aqueous binary mobile phases

Summary The dependence of the capacity factor (k′) on the concentration of the organic modifier (D) in the aqueous binary mobile phase in reversed-phase high-performance liquid chromatography has been investigated to evaluate the hydrophobicity of the solute molecule. The r-values, defined as the slope of log k′ vs. log(1/D) plots, were measured for various solutes and related to the non-polar surface area and the partition coefficients. The r-value was found to be a good indication of solute hydrophobicity. Detailed investigation of the results allowed to consider statistically the molecular posture of the solute adsorbed onto the stationary alkyl ligand.     

反相高效液相色谱法同时测定化妆品中7种萘二酚类物质

建立了反相高效液相色谱((RP-HPLC))同时测定化妆品中7种萘二酚类物质的分析方法。膏霜类、乳液类和水类样品用95%(v/v)乙醇提取,粉类样品用95%乙醇-0.1%乙酸(3:2, v/v)溶液提取,经离心、过滤后,用C18柱,以甲醇-0.1%乙酸溶液为流动相梯度洗脱分离,使用二极管阵列检测器检测,以保留时间定性,并以紫外吸收光谱图辅助定性,外标法定量。结果表明,萘二酚类物质在线性范围内线性关系良好,相关系数均不低于0.999 0,方法的定量限(以信噪比为10计)为0.5～1.2 mg/kg,添加水平为5.0～50 mg/kg时回收率为84.0%～102%,相对标准偏差(n=6)为1.3%～5.7%。该法前处理简单、回收率高、精密度好,适用于非蜡基类化妆品中萘二酚类物质的测定。     

非水反相高效液相色谱法测定海藻中3种叶黄素类化合物

建立了非水反相高效液相色谱测定不同种类海藻中3种叶黄素类化合物的方法。采用超声波辅助提取海藻中的叶黄素类化合物,选用Alltima C18色谱柱分离,以甲醇-乙腈-丙酮为流动相系统,梯度洗脱分离,检测波长445 nm。结果表明,在选定的色谱条件下,待分析的3种叶黄素类化合物与其他化合物分离良好,在一定浓度范围内线性关系良好(紫黄素:6.24～624μg/L,环氧玉米黄素:4.56～456μg/L,玉米黄素:5.60～560μg/L)。该方法适用于不同海洋藻类中3种叶黄素类化合物的定量分析。     

Comprehensive characterization of Stevia Rebaudiana using two-dimensional reversed-phase liquid chromatography/hydrophilic interaction liquid chromatography

Two-dimensional reversed-phase liquid chromatography/hydrophilic interaction liquid chromatography (2D-RPLC/HILIC) system was successfully applied for comprehensive characterization of steviol glycosides from Stevia rebaudiana. The experiments were performed in offline mode using an XCharge C18 column in first dimension and an XAmide column in second dimension. In first dimension, preliminary separation of Stevia aqueous extract was accomplished and 30 fractions were collected. Then fractions 1-20 were selected for further purification and 13 compounds with high purity were obtained in second dimension. Comprehensive characterization of these compounds was completed by determination of their retention time, accurate molecular weight, diagnostic fragmentation ions, and nuclear magnetic resonance spectroscopy. As a result, all nine known steviol glycosides, as well as other four steviol glycosides were fully purified. The result demonstrated that this procedure is an effective approach for the preparative separation and comprehensive characterization of steviol glycosides in Stevia. This 2D-RPLC/HILIC method will be a promising tool for the purification of low-abundance compounds from natural products.     

Analysis of commercial ceramides by non-aqueous reversed-phase liquid chromatography with evaporative light-scattering detection

Summary This paper describes the development of a chromatographic system for analysis of commercial ceramides structurally similar to those found in the stratum corneum. The ceramides used in this study contain different amine based (phytosphingosine, sphingosine and dihydrosphingosine) and fatty acids of different chain lengths and with different functional groups (hydroxylated and unsaturated). Non-aqueous reversed-phase (NARP) liquid chromatography with evaporative light-scattering detection (ELSD) were the techniques chosen in accordance with the nature of the ceramides. The eluent strength and the potential selectivity of different organic solvents were investigated. On a C₁₈-bonded silica, the most promising chromatographic conditions employed a gradient from ACN-THF, 95∶5, to ACN-THF-PrOH, 35∶5∶60, in 15 min with a constant concentration of TEA (10 mM) and a stoichiometric amount of formic acid.     

Quantitative structure chromatography relationships in reversed-phase high performance liquid chromatography: Prediction of retention behaviour using theoretically derived molecular properties

Summary The use of theoretically calculated molecular properties as predictors for retention in reversed-phase HPLC has been explored. HPLC retention times have been measured for a series of 47 substituted aromatic molecules in three solvent mixtures and steric and electronic properties of these compounds have been derived using semi-empirical molecular orbital and empirical theoretical methods. A subset of the experimental data (a training set) was used to derive property-retention time relationships and the remaining data were then used to test the predictive capability of the methods.Good retention time prediction was possible using derived regression equations for individual solvents and after including solvent parameters it was possible to predict retention for all solvents using a single equation. This method showed that the most useful properties were calculated log P and the calculated dipole moment of the solutes, and the calculated solvent polarisability. In addition, 90% of the data were used to train an artificial neural network and the remaining 10% of the data used to test the network; excellent prediction was obtained, the neural network approach being as successful as the regression analysis.