A high performance liquid chromatographic method for the quantitation of flecainide in plasma.

A routine high performance liquid chromatographic method for the rapid determination of fleca?nide (Flecaine), using a novel internal standard, N-methylfleca?nide, has been developed. After deproteinization of spiked samples, fleca?nide was totally recovered at neutral pH. Fleca?nide and the internal standard were separated on a reversed phase XL 3 microns ODS column using 10 mM phosphate buffer, pH 3.0: acetonitrile (70:30) as mobile phase, in less than 10 min. With spectrofluorometric detection, the limit of quantitation for fleca?nide was 10 ng/mL. Intra- and inter-assay precision variations were 0.24% and 1.4%.     

Solid phase extraction and simultaneous high performance liquid chromatographic determination of antipyrine and its major metabolites in urine.

A reversed phase gradient high performance liquid chromatographic method utilizing solid phase extraction has been described for the simultaneous determination of antipyrine (AP), 4-hydroxyantipyrine (4-OHAP), norantipyrine (NorAP) and 3-hydroxymethylantipyrine (3-OHMAP) in human urine after hydrolysis with beta-glucuronidase. The C-18 sorbent cartridges were conditioned and urine samples were applied, washed with 1 x 4 mL of phosphate buffer and eluted with 3 x 100 microL of 20% v/v of acetonitrile in methylene chloride. The eluent was evaporated to dryness, reconstituted in 100 microL phosphate buffer and injected. The calibration ranges were 2.0-250 micrograms/mL (AP), 2.5-250 micrograms/mL (NorAP), 2.0-250 micrograms/mL (3-OHMAP) and 5.0-500 micrograms/mL (4-OHAP) with regression coefficients of 0.998 or greater. Specificity was indicated by the absence of interferences in chromatogram of blank urine from normal as well as cirrhotic patients. The average recovery was 86.7% for AP, 90.5% for NorAP, 85.2% for 4-OHAP and 74.2% for 3-OHMAP. The within-assay precision as indicated by the reproducibility of the assayed spiked urine was less than 9% in all cases and the between-assay precision was less than 12%. The method was applied to studies on antipyrine metabolism in stable cirrhotic patients. Following administration of a single oral dose of about 1000 mg to nine stable cirrhotic patients and eight age-matched healthy volunteers, the cumulative account excreted in the urine up to 48 h for AP and the three metabolites was comparable to other literature reports.     

原料奶的高效液相色谱指纹图谱的建立

采用高效液相色谱-质谱联用(HPLC-MS)技术,在负离子扫描模式下建立了原料奶的乙腈提取成分的指纹图谱。采用乙腈和0.1%的乙酸水溶液为流动相进行二元梯度洗脱,柱温30℃,分析时间为85min。确定了11个共有峰,以其中的7号峰为参照物,通过相对峰面积和相对保留时间进行了方法学考察。结果表明,本方法具有良好的重现性,各指纹峰相对保留时间的相对标准偏差(RSD)小于0.79%,相对峰面积的RSD小于2.84%。在原料奶指纹图谱基础上,选取有代表性的危害物如防腐剂苯甲酸等进行了添加,建立了危害物添加图谱,对沈阳地区超市的50个纯牛奶样品进行了筛查,取得初步的应用结果。     

酪蛋白多肽的制备和色谱分离方法

为了得到低成本的多肽,本文利用胰蛋白酶对酪蛋白进行了充分的酶解。采用分析级反相高效液相色谱-电喷雾质谱联用技术(RP-HPLC/ESI-MS)分析了酶解产物各组分的组成,并通过改变流动相的梯度洗脱程序,优化了分析级色谱条件以充分分离相对含量较高的多肽组分;将优化的分析级色谱条件直接放大到制备级RP-HPLC中,在程序控制下通过紫外吸收信号结合ESI-MS信号共同引导实现了多肽的全自动化分离和收集。整个过程方便快捷,经过这样一个单一的分离步骤,得到了多个纯度较高的多肽。除此之外,本文还考察了流动相的酸碱性、柱上样量等因素对该体系制备级分离的影响,并对一次分离中分辨率不好的亲水性多肽混合物进行了二次分离,得到了多个新的多肽。本文建立的多肽制备方法为多肽和多肽材料的广泛应用提供了一种选择。     

High-efficiency high performance liquid chromatographic analysis of red wine anthocyanins

The analysis of anthocyanins in natural products is of significant relevance in recent times due to the recognised health benefits associated with their consumption. In red grapes and wines in particular, anthocyanins are known to contribute important properties to the sensory (colour and taste), anti-oxidant- and ageing characteristics. However, the detailed investigation of the alteration of these compounds during wine ageing is hampered by the challenges associated with the separation of grape-derived anthocyanins and their derived products. High performance liquid chromatography (HPLC) is primarily used for this purpose, often in combination with mass spectrometric (MS) detection, although conventional HPLC methods provide incomplete resolution. We have previously demonstrated how on-column inter-conversion reactions are responsible for poor chromatographic efficiency in the HPLC analysis of anthocyanins, and how an increase in temperature and decrease in particle size may improve the chromatographic performance. In the current contribution an experimental configuration for the high efficiency analysis of anthocyanins is derived using the kinetic plot method (KPM). Further, it is shown how analysis under optimal conditions, in combination with MS detection, delivers much improved separation and identification of red wine anthocyanins and their derived products. This improved analytical performance holds promise for the in-depth investigation of these influential compounds in wine during ageing.     

北五味子的液相色谱指纹图谱的建立

利用反相高效液相色谱法建立中药材北五味子的指纹图谱,为科学评价及有效控制北五味子质量提供了新方法。实验分析了10个不同产地的北五味子样品,采用国家食品药品监督管理局推荐的“中药色谱指纹图谱相似度评价系统(2004 A版)”计算处理,建立了由26个指纹峰和19个共有峰组成的北五味子指纹图谱,确定了5个主要的指纹峰。通过夹角余弦法和相关系数法计算了北五味子10个样本与指纹图谱间的相似度,得到了满意的结果。所建立的指纹图谱可以用来区别不同产地北五味子药材的优劣,为进一步控制北五味子的质量提供依据。     

Improved high performance liquid chromatographic determination of amanitins with electrochemical detection

Summary The determination of amanitins, the main toxins of the poisonous fungus Amanita Phalloides, through an improved HPLC method with electrochemical detection is presented. It uses an improved reverse phase separation on a wide pore butyl column and amperometric detection in oxidizing mode at + 0.6 V. Compared to the previously reported methods employing narrow pore C18 packings and UV detectors, the described method gives a much lower detection limit (40, 80 and 70 pg ca. for alpha-, beta- and gamma-amanitin, respectively), and a better chromatographic efficiency. Coupled with a sample preparation procedure using disposable cartridges packed with C18 and underivatized silica (recovery of alpha-amanitin =65 %, RSD 4.1 at 20 ng/ml), the method permits the quantitative determination of alphaamanitin in serum at low concentrations (10–20 ng/ml). A tentative immuno-extraction of urine samples, by means of a solid phase antiserum, bound to nylon nets, allowing the detection of few nanograms of toxin per milliliter is also briefly described.     

反相液相色谱法测定α-甲氧基萘的含量

用反相液相色谱法分析了α-甲氧基萘的含量和其中的有害杂质。色谱条件：色谱柱Nucleosil－C18;流动相V（甲醇）：V（水）=90：10;紫外检测器,面积归一化法定量。方法的变异系数为0．29％。     

非线性电泳

电泳大家当已耳熟能详了,它发现于18世纪初期,有悠久的历史.电泳因Tiselius而进入分离领域,为蛋白质科学的建立和发展立下了汗马功劳.其贡献至今不息,一直是生物学家的掌中宝.     

剖析液相色谱柱技术的复杂状况(英文)

 Column packings continue to evolve as the needs of users for high efficiency, high resolution and highly sensitive high performance liquid chromatographic (HPLC) analysis drive further developments. In comparing and contrasting modern HPLC columns technologies, diameters of column packings and particle materials are covered. Some products and applications of modern HPLC columns are provided. Future directions in packing developments are predicted in this introductory article.     

虎纹捕鸟蛛毒素-Ⅴ去折叠过程的色谱分析

 将纯化的天然虎纹捕鸟蛛毒素-Ⅴ经盐酸胍变性30 min后,在pH 3.0、反应温度为37 ℃的条件下与三羧甲基磷酸(TCEP)反应12 min,用反相高效液相色谱分离得到其全部去折叠中间体,通过基体辅助激光解吸电离飞行时间质谱(MALDI-TOF MS)进行鉴定,并利用烷基化反应对这些去折叠中间体予以进一步确证。 根据其保留时间,分析虎纹毒素-Ⅴ各去折叠中间体的色谱行为,初步探讨了多肽或蛋白质构象异构体反相色谱行为的多样性。     

Normal-phase high performance liquid chromatographic separation and characterization of short- and long-chain triacylglycerols

Summary Short- and long-chain triacylglycerols (SLCT) are a family of lipids prepared by chemical or enzymatic interesterification of triacetin, tripropionin and/or tributyrin, and long-chain (C_16!18) hydrogenated vegetable oils. In this study, a normal-phase cyanopropyl high-performance liquid chromatographic (HPLC) method was developed for the separation and quantification of SLCT. The method is capable of separating SLCT mixtures, free fatty acids and the neutral lipid classes of saturated long-chain triacylglycerols, diacylglycerols and monoacylglycerols. To characterize the specific SLCT classes, a normal-phase HPLC procedure using a non-modified silica column was developed to separate the SLCT into individual isomers based on total carbon number and position of fatty acids on the glycerol backbone. Online coupling with a mass detector (LC/MS) allowed the identification of the individual triacylglycerol structures.     

Thermospray high performance liquid chromatographic/mass spectrometric analysis of some fusarium mycotoxins

Thermospray high performance liquid chromatography/mass spectrometry (TSP HPLC/MS) was used to analyze five Fusarium mycotoxins in porcine plasma and urine. Four cytotoxic trichothecene mycotoxins, T-2 toxin, HT-2 toxin, diacetoxyscirpenol (DAS), deoxynivalenol (DON), T2 tetraol, and the fungal estrogen zearalenone (F-2 toxin) were analyzed. The thermospray mass spectrum contained molecular weight information with few, if any, fragment signals. Detection limits ranging from 1 to 10 ng of mycotoxin injected onto the HPLC column were obtained using selected ion monitoring (SIM) HPLC/MS. Neither the plasma nor the urine matrix interfered with TSP HPLC/MS analysis of these mycotoxins and no sample derivatization was necessary for the analysis. The TSP HPLC/MS technique appears to be ideal for very sensitive analysis of mycotoxins in biological samples.     

免疫法制备液相色谱生物样品的研究——血中沙丁胺醇的分析

 用动物免疫法制备了免疫亲和柱纯化水溶性的沙丁胺醇血浆样品。琥珀酸酐交联沙丁胺醇和牛血清白蛋白获得抗原免疫家兔抗沙丁胺醇抗体——免疫球蛋白。琼脂糖Ｓｅｐｈａｒｏｓｅ４Ｂ与抗体交联制成免疫球蛋白亲和柱。对高效液相色谱法测定中的一般提取方法和固相小柱提取法作了比较，后者具有内源性杂质干扰少的优点，是生物样品预处理的一种有效的方法。     

Solid phase extraction and high performance liquid chromatographic determination of doxophylline in plasma

A sensitive and selective high performance liquid chromatographic doxophylline assay with ultraviolet detection has been developed for plasma samples. The drug is isolated from biological samples with a reversed phase C18 disposable extraction column. Plasma standard curves are linear for concentrations of doxophylline from 0.03 to 10 mg/L. At the therapeutic range concentrations, the recoveries are better than 96.9%. The coefficients of variation for the procedure are 4.1% and 2.7% for the concentrations 0.03 mg/L and 10 mg/L, respectively. By this method, pharmacokinetic profiles are obtained for six adult volunteers.     

Two high-performance liquid chromatographic methods for quantitation of theophylline in plasma

Two high-performance liquid chromatographic methods are described for the assay of theophylline in plasma. Both allowed the separation of theophylline from the caffeine metabolites, theobromine and 1,7-dimethylxanthine. Method A, using 8-chlorotheophylline as internal standard, involved back extraction of theophylline from organic extract with 0.1 M sodium hydroxide. Method B used generally accepted solvent extraction followed by evaporation and beta-hydroxyethyltheophylline as internal standard. High-performance liquid chromatographic analyses were performed on reversed-phase phenyl columns (25 X 0.46 and 25 X 0.41 cm) using 20% methanol in 20 mM phosphate buffer at pH 5.6 for Method A and 2% acetonitrile and 8% methanol in 20 mM phosphate buffer for Method B. The column effluent was monitored at UV 273 nm. Standard curves for both Methods A and B were fitted by linear regression (r greater than 0.999) in the concentration range of 0.05-50 micrograms/ml. Either method was selective, accurate and reproducible over the concentration range 0.08-26 micrograms/ml. However, compared with Method B, Method A provided significant advantages in terms of simplicity, speed and efficiency.     

丁香酚键合硅胶液相色谱固定相的制备和色谱性能

采用固液相表面连续反应法,先将偶联剂γ-[(2,3)-环氧丙氧]丙基三甲氧基硅烷(KH-560)键合到球形硅胶表面,然后再将植物有效成分丁香酚与硅胶上的KH-560活性基团反应,合成了丁香酚键合硅胶液相色谱固定相(EGSP)。采用元素分析、热重分析和红外光谱对该固定相的结构进行了表征。以萘作为溶质探针,乙腈-水(35:65, v/v)为流动相,流速为0.8 mL/min,测得EGSP柱的柱效。以一系列的中性、碱性和酸性化合物为溶质探针,C₁₈柱和苯基柱作参比,对该固定相的色谱性能及保留机理进行了研究。结果表明,硅胶表面成功键合上了丁香酚配体,键合量为0.28 mmol/g, EGSP柱理论塔板数约为24707 N/m。该固定相不仅具有良好的反相色谱性能,同时由于配体结构中含有芳环、烯基和甲氧基,还能与溶质发生π-π电荷转移、偶极-偶极和氢键作用。与传统的反相C₁₈柱和苯基柱相比,EGSP在极性芳香族化合物的快速、简便分离中占优势。