Detection of single nucleotide polymorphisms by the specific interaction between transition metal ions and mismatched base pairs in duplex DNA

Single nucleotide polymorphisms (SNPs) are base differences in the human genome. These differences are favorable markers for genetic factors including those associated with risks of complex diseases and individual responses to drugs. When two duplex DNAs with different types of SNPs are mixed and reannealed, the two novel heteroduplexes containing mismatched base pairs are formed in addition to the two initial perfectly matched homoduplexes. Heteroduplex analysis recognizing the newly formed mismatched base pairs is useful for SNP detection. Various strategies to detect the mismatched base pairs were devised due to the potential applications of SNPs. However, they were not always convenient and accurate. Here, we propose a novel strategy to detect the mismatched base pairs by the specific interaction between the Hg²⁺ ion and a T:T mismatched base pair and that between the Ag⁺ ion and a C:C mismatched base pair. UV melting indicated that the melting temperature of only the heteroduplexes with the T:T and C:C mismatched base pair specifically increased on adding the Hg²⁺ and Ag⁺ ion, respectively. Fluorescence resonance energy transfer analyses indicated that the intensity of fluorophore emission of only the fluorophore and quencher-labeled heteroduplexes with the T:T and C:C mismatched base pair specifically decreased on adding the Hg²⁺ and Ag⁺ ion, respectively. We propose that the addition of the metal ion could be a convenient and accurate strategy to detect the mismatched base pair in the heteroduplex. This novel strategy might make the heteroduplex analysis easy and eventually lead to better SNP detection.     

反相硅胶整体柱离子对色谱法快速测定吡啶离子液体阳离子

建立了整体柱离子对色谱-紫外检测法梯度淋洗快速分离测定4种吡啶离子液体阳离子的方法。分离采用C18反相硅胶整体柱,以离子对试剂(用柠檬酸调节pH值)-乙腈为淋洗液,并采用多级梯度洗脱程序。实验考察了色谱柱、离子对试剂、乙腈浓度、色谱柱温度及流速对吡啶阳离子保留的影响,并讨论了其保留规律。咪唑阳离子的保留符合碳数规律。最佳色谱条件是:在流速3.0 mL/min,柱温30℃下,以1.0 mmol/L庚烷磺酸钠(pH 4.0)(A)+乙腈(B)为淋洗液进行梯度洗脱。淋洗梯度为0～2.0 min,10%B;2.0～2.5 min,10%～15%B;2.5～4.0 min,15%B;4.0～4.5 min,15%～20%B;4.5～10.0 min,20%B。在此条件下,4种吡啶阳离子可在7 min内基线分离。所测阳离子的检出限(S/N=3)为0.05～0.17 mg/L;峰面积的相对标准偏差(n=5)小于0.6%。将本方法用于实验室合成的离子液体样品和污水样品的分析,加标回收率在95.7%～99.0%之间。本方法准确、快速,具有较好的实用性。     

梯度洗脱高效液相色谱法测定红花玉兰中4种植物激素

建立了梯度洗脱高效液相色谱法测定红花玉兰中赤霉素(GA3)、生长素(IAA)、脱落酸(ABA)和玉米素(ZT)等4种植物激素的方法。采用Agilent ZORBAX SB-C18柱和紫外检测器,以甲醇和0.1 mol/L乙酸作为流动相进行梯度洗脱,流速1 mL/min,进样量10μL。GA3,IAA和ABA的检测波长为254 nm,柱温35℃;ZT的检测波长为270 nm,柱温40℃。采用外标法进行定量测定,4种植物激素的相关系数均大于0.9990。4种激素的回收率为98.1%~125.2%,相对标准偏差为0.31%~0.92%,日内和日间精密度RSD均<10%。方法可适用于红花玉兰多种组织的植物激素测定,为红花玉兰生长发育特性的研究奠定了基础。     

离子对色谱法同时分离测定吡啶及咪唑离子液体阳离子

建立了用紫外检测的反相离子对色谱梯度淋洗同时分离测定4种吡啶离子液体阳离子和5种咪唑离子液体阳离子的方法。实验采用ZORBAX Eclipse XDB-C18反相色谱柱,以离子对试剂水溶液(用柠檬酸调节pH值)+乙腈为流动相,考察了离子对试剂种类和浓度、乙腈浓度和色谱柱温度对保留的影响,探讨了相关保留规律,确定最佳色谱条件为:流速1.0 mL/min、柱温30℃,以1.0 mmol/L庚烷磺酸钠水溶液(pH 4.0)-乙腈为淋洗液进行梯度洗脱。在此条件下,4种吡啶阳离子和5种咪唑阳离子在15 min内达到基线分离。检出限(S/N=3)为0.31～0.54 mg/L,峰面积的相对标准偏差为0.10%～0.75%。将该方法用于实验室合成的离子液体样品分析,加标回收率为94%～98%。该方法准确、可靠,具有较好的实用性。     

Determination of Pollutant Phenols by Capillary High‐Performance Liquid Chromatography with UV Detection

This paper describes a liquid chromatographic method using a reversed phase capillary column coupled to an UV detector for the quantitation of thirteen pollutant phenols. Chromatographic separation was carried out with gradient elution at 25.0 ± 0.1°C. The two major anisocratic elution modes (gradient elution and temperature programming) were evaluated. The detection limit range was 10–81 pg (100 nL injected). The chromatographic method combined with liquid‐liquid extraction was applied to analysis of these compounds in river water. Recoveries of 75–103% were achieved for most of them.     

Rapid and Complete Separation of Inorganic Polyphosphates from Monomer to 35-Mer by Hplc

Abstract

The on-line coupling system of high-performance liquid chromatography (HPLC) and flow injection analysis (FIA) was developed for the rapid separation and sensitive detection of inorganic polyphosphates. Effects of gradient elution conditions on the chromatographic behavior were studied. The column temperature as well as a chloride concentration gradient were found to be very effective for the improvement of resolution(1). To minimize analysis time and to maximize resolution, the gradient elution conditions were optimized by use of a computer-assisted retention prediction system(2). More than 35 kinds of inorganic polyphosphates could be separated completely within 200 min under the optimum conditions as shown in Fig. 1.     

离子色谱法测定氯膦酸二钠及其制剂的含量和有关物质

采用抑制型电导检测-离子色谱法同时测定氯膦酸二钠及其制剂的含量和有关物质.色谱条件为阴离子交换色谱柱(IonPac AS11-HC);检测方式为抑制电导检测;柱温30 ℃;流速1.2 mL/min;以KOH溶液为淋洗液,含量测定采用等度洗脱,有关物质检查采用梯度洗脱.氯膦酸二钠在0.0568～0.1895 g/L范围内线性关系良好(r＝0.9999); 注射液和胶囊的平均回收率分别为100.1%(RSD=0.7%)和98.9%(RSD=0.6%);检出限为0.3 ng.各杂质与主成分色谱峰能完全分离.     

离子色谱-积分脉冲安培检测法同时测定酱油中20种氨基酸和6种糖

建立了梯度淋洗-离子色谱-积分脉冲安培检测法同时测定酱油中20种氨基酸和6种糖的方法，通过对色谱柱温度、pH值、放置时间等实验影响因素的考察，探索出了适合26种组分测定的多级梯度淋洗条件。结果表明，当流速为0.25 mL/min，pH值在5.2~6.7，柱温在35℃时，26种组分在各自的浓度范围内具有良好的线性关系（r² ≥ 0.995）。除了谷氨酰胺、亮氨酸、异亮氨酸、蛋氨酸外，其他22种组分的检出限均小于0.03 mg/L；在0.20、0.50、2.00 mg/L 3个添加水平下，回收率为84.2%~109%，RSD为2.7%~7.8%。该方法高效、简便、灵敏、准确，可用于酱油中多种氨基酸和糖的同时测定以及掺假辨别技术研究。     

Detection of low-abundance impurities in synthetic thyroid hormones by stationary phase optimized liquid chromatography–mass spectrometry

The transfer of a gradient method to an isocratic or multistep gradient method employing stationary phase optimized liquid chromatography facilitated a reduction in analysis time by 50% and significantly improved the mass spectrometric detectability of impurities in synthetic thyroid hormones. Four column segments packed with different stationary phases were combined into a single chromatographic column, which allowed the separation and photometric as well as mass spectrometric detection of thyroid compounds in less than 30 min under isocratic- or step gradient elution conditions with 0.10% acetic acid/acetonitrile. Signal instability and baseline drift during detection by negative electrospray ionization time-of-flight mass spectrometry were minimized by optimizing the spray parameters for each individual elution step. This resulted in improved detectabilities and higher mass spectral quality, especially for low-abundance components in the sample mixture. The method was applied to the separation and detection of the low-abundance impurities formed upon the thermal stressing of a sample of synthetic levothyroxine.     

高效液相色谱法分析逆转录聚合酶链反应产物

建立了分离逆转录聚合酶链反应（ＲＴ ＰＣＲ）产物的高效液相色谱方法,反应液直接进样,用ＴＳＫｇｅｌＤＥＡＥ ＮＰＲ柱分离,Ｔｒｉｓ ＨＣｌ缓冲溶液（ｐＨ９０） 氯化钠线性梯度洗脱,于２６０ｎｍ处检测。用所建立的方法分析了大鼠肠缺血／再灌注损伤后外周血中性粒细胞（ＰＭＮ）磷脂酶Ａ２ｍＲＮＡ的表达。     

Some factors affecting separation and detection of amino acids by high-performance anion-exchange chromatography with integrated pulsed amperometric detection

Some factors influencing the separation and detection of amino acids by high-performance anion-exchange chromatography with integrated pulsed amperometric detection were investigated. These factors include eluent concentration, column temperature, and detection waveform. The selectivity changes in weakly retained amino acids are slight with changing sodium hydroxide eluent concentration. When sodium acetate eluent concentration is changed, the selectivity variations between strongly retained amino acids containing two carboxyl groups and containing only one carboxyl group are obviously different. Significant but slight selectivity changes in weakly retained amino acids can be achieved through changing the column temperature. Sodium hydroxide and sodium acetate eluent concentration affect the detection of amino acids. Detection sensitivity of amino acids can be improved by increasing the concentration of sodium hydroxide and sodium acetate in a certain concentration range. The detections of amino acids at two different detection waveforms were compared. The hydroxyl amino acids can be selectively detected by choosing a modified detection waveform. The optimized gradient elution condition and column temperature for analyzing 19 amino acids were obtained. The time for the gradient elution program was 60 min. The column temperature was 35 degrees C. Under the optimized conditions, detection limits for 19 amino acids were 0.15-4.52 pmol. The calibration graphs of peak area for all the analytes were linear for about three orders of magnitude. The RSDs (n=5) of peak area were 0.6-5.6%. The determination of trace amino acid impurities in valine product is shown as an application example.     

Theoretical background of short chromatographic layers. Optimization of gradient elution in short columns

Although linear salt gradient elution ion-exchange chromatography (IEC) of proteins is commonly carried out with relatively short columns, it is still not clear how the column length affects the separation performance and the economics of the process. The separation performance can be adjusted by changing a combination of the column length, the gradient slope and the flow velocity. The same resolution can be obtained with a given column length with different combinations of the gradient slope and the flow velocity. This results in different separation time and elution volume at the same resolution. Based on our previous model, a method for determining the separation time and the elution volume relationship for the same resolution (iso-resolution curve) was developed. The effect of the column length and the mass transfer rate on the iso-resolution curve was examined. A long column and/or high mass transfer rate results in lesser elution volume. The resolution data with porous bead packed columns and monolithic columns were in good agreement with the calculated iso-resolution curves. Although the elution volume can be reduced with increasing column length, the pressure drop limits govern the optimum conditions.     

Determination of Antibiotics in Surface Water by Solid-Phase Extraction and High-Performance Liquid Chromatography with Diode Array and Mass Spectrometry Detection

A high-performance liquid chromatography method is reported for the determination of antibiotics in water. The antibiotics were simultaneously preconcentrated by solid-phase extraction. High-performance liquid chromatography was performed on a C18 modified column with gradient elution in 25?min at 40°C. The separation was performed using gradient elution with 90:10 acetonitrile:water and 0.1% aqueous formic acid. The antibiotics were identified by diode array detection and mass spectrometry. The established method was suitable for the determination of antibiotics in surface water.     

Column-switching high-performance liquid chromatographic detection of pholcodine and its metabolites in urine with fluorescence and electrochemical detection.

A sensitive and selective method for the detection of pholcodine and its metabolite morphine in urine using high-performance liquid chromatography is described. It involves on-line clean-up of urine on a trace enrichment column packed with a polymeric strong cation-exchange material. Pholcodine and its metabolites were separated on two analytical columns with different selectivities. Pholcodine was detected by a fluorescence detector and morphine was detected electrochemically. One system, based on reversed-phase chromatography, applied a polystyrene-divinylbenzene column and gradient elution. The other system was based on normal-phase chromatography with a silica column and isocratic elution. Morphine was confirmed to be a metabolite of pholcodine by reversed-phase chromatography and electrochemical detection. Two unidentified metabolites of pholcodine were separated from pholcodine by normal-phase chromatography and detected by fluorescence detection.     

高效液相色谱分析鱼毒性藻类海洋卡盾藻溶血毒素

采用高效液相色谱技术(HPLC)分离和分析了海洋卡盾藻(香港株)溶血毒素的甲醇粗提物,通过对比不同流动相、检测波长、等度和梯度洗脱方式,初步建立了分析其溶血毒素的HPLC方法.结果表明,采用梯度洗脱的方法,海洋卡盾藻溶血毒素的粗提物在50 min内取得了较好地分离.色谱条件为C8硅胶柱(4.6 mm× 150 mm,5 μm),流动相为乙腈和水,梯度洗脱(0～10 min,60％→80％乙腈;10～40 min,80％ →90％乙腈;40～50 min,90％乙腈);紫外检测器检测波长为205和448nm;流速为0.8 mL/min;柱温为25℃.用兔血红细胞法对各个主要色谱峰进行溶血活性检测显示,海洋卡盾藻(香港株)的溶血毒素至少含有5种成分.光谱分析显示一种成分的紫外吸收高峰在448 nm,一种未完全分离的混合组分的紫外吸收高峰为440和446nm,另外3种成分的紫外吸收高峰为205 nm.     

天然酚酸类化合物的反相高效液相分析

 采用反相高效液相法梯度洗脱 ,对 12种天然酚酸进行定性定量分析研究。通过条件的优化 ,确定了最佳的分离条件 ;同时探讨了流动相的酸度对分离结果的影响 ,以及酚酸结构与保留行为之间的关系。实验中以确定的最佳分离条件对 12种天然酚酸进行了定量分析 ,测定了各个化合物的回归方程、相关系数、线性范围、检测限和相对标准偏差。实验结果显示 ,各个化合物回归方程的相关系数r为 0 9980～ 0 9999,检测限为 0 96ng～ 4 10ng ,相对标准偏差RSD≤ 2 6 4 % ,满足定量分析要求。     

葡萄酒中7种酚酸的色谱电化学分析

建立了同时测定葡萄酒中没食子酸、原儿茶酸、丁香酸、p-香豆酸、咖啡酸、绿原酸和阿魏酸等7种生物活性酚酸的反相高效液相色谱电化学分析新方法,并测定了5种国产不同品牌的葡萄酒.采用HypersilODS色谱柱(250mm×4.0mm,5.0μm),流动相为甲醇-4%醋酸,梯度洗脱,流速为0.8mL/min,工作电压为0.7V,柱温为30℃.实验结果表明,电化学法的检出限比紫外法的检出限低4～600倍.     

Gradient elution in normal-phase high-performance liquid chromatographic systems

Gradient elution is widely used for separation of complex samples in reversed-phase HPLC systems, but is less frequently applied in normal-phase HPLC, where it has a notoriously bad reputation for poor reproducibility and unpredictable retention. This behaviour is caused by preferential adsorption of polar solvents used in mixed mobile phases, which may cause significant deviations of the actual gradient profile from the pre-set program. Another important source of irreproducible retention behaviour is gradual deactivation of the adsorbent by adsorption of even traces of water during normal-phase gradient elution. To avoid this phenomenon, carefully dried solvents should be used. Finally, column temperature should be carefully controlled during normal-phase gradient elution if reproducible results are to be obtained. Working with dry solvents at a controlled constant temperature and using a sophisticated gradient-elution chromatograph, reproducibility of the retention data in normal-phase gradient elution better than 2% may be achieved even over several months of column use. The retention data in gradient elution can be calculated accurately if appropriate corrections are adopted for the gradient dwell volume and for the preferential adsorption of the polar solvents using experimental adsorption isotherms. The average error of prediction for the corrected calculated gradient retention data was lower than 2% for a silica gel column and lower than 3% for a bonded nitrile column, which may be suitable for the optimization of separation. Further, a simple approach is suggested for rapid estimation of changes in the retention induced by a change in the gradient profile in normal-phase HPLC. For such a rough estimation, it is not necessary to know the parameters of the dependence of the solute retention factors on the composition of the mobile phase.     

Determination of phenols in wines by liquid chromatography with photodiode array and fluorescence detection

A reversed-phase LC method, optimised for the separation of trans- and cis-resveratrol, catechin, epicatechin, quercetin and rutin, is reported. Analyses were performed on a reversed-phase column by gradient elution. Detection was carried out by photodiode array, although the use of a fluorimetric detector considerably lowered the detection limits for catechin, epicatechin and both resveratrol isomers. Identification by the two different detection systems was based on retention characteristics, UV spectra and peak purity index were compared with commercial standards. The procedures were applied to the determination of the phenolic compounds in different types of wines and musts.     

Simultaneous determination of geniposide and its metabolites genipin and genipinine in culture of Aspergillus niger by HPLC

When cultivated with Aspergillus niger, geniposide, an important drug, is transformed into genipin and genipinine. A simple and rapid HPLC method for simultaneous determination of geniposide and its two metabolites in broth of A. niger is described. The chromatographic separation was achieved on a C18 ODS column (250 x 4.6 mm) by gradient elution with 0.1% formic acid in water and 0.1% formic acid in acetonitrile as the gradient mixtures. The flow rate was 1 mL/min, the detection wavelength was 238 nm and the column temperature was kept at 28 degrees C. The retention times of geniposide, genipin and genipinine were 10.9, 13.8 and 21.5 min, respectively. The mean absolute recoveries of three analysts were over 98%. Quantification limits were 0.01 microg/mL for geniposide and 0.02 microg/mL for the two metabolites. The method was applied for the quantification of geniposide, genipin and genipinine during fermentation and the evaluation of the bioavailabilities of these three compounds in Caco-2 monolayer.