亲水作用色谱-电雾式检测用于5种抗病毒药物的定量分析

抗病毒药物具有广泛的应用,对人类和动物的健康有着重要的作用。但抗病毒药物紫外吸收差异大、极性分布广,要实现该类药物的同时分析较为困难。本实验以Click TE-Cys为固定相,结合电雾式检测(CAD),建立了多种抗病毒药物同时分离分析的方法。考察了不同的检测器、色谱模式、色谱柱和流动相对抗病毒药物峰形、分离选择性及检测信号响应的影响。在优化的条件下定量分析了5种抗病毒药物,结果证明该方法具有较好的日内重复性(RSD≤3.06%)和日间重复性(RSD≤5.38%)。同时,该方法还具有较宽的线性范围(0.07~2.28 mg/mL)和较高的灵敏度(LOQ≤0.04 mg/mL),可用于相关药物的定量、定性分析。     

Direct determination of hydrazine,methylhydrazine, and 1,1-dimethylhydrazine by zwitterionic hydrophilic interaction liquid chromatography with amperometric detection

A promising alternative to ion-chromatographic methods currently used for the direct determination of hydrazines is provided by hydrophilic interaction liquid chromatography (HILIC). In this work, we propose a method for the simultaneous determination of hydrazine, methylhydrazine and 1,1-dimethylhydrazine in natural waters and soils based on a combination of chromatographic separation on a zwitterionic sulfobetaine stationary phase (Nucleodur HILIC) in the HILIC mode with amperometric detection.

Effects of different factors on the retention of analytes were studied and the optimum conditions of analysis were found. We recommend a mixture of acetonitrile with an aqueous phosphate buffer solution of pH 2.5 (78:22 v/v) with an ionic strength of 20 mM as a mobile phase. Detection in the direct current mode was performed at a working electrode potential of 1.1 V.

The advantages of the method are the high efficiency of separation, rapidity, high sensitivity and a wide dynamic range of analyte concentrations, covering four orders of magnitude. The attained LOD values for analytes lie in the range 0.07–0.13 μg L^–1, which is two orders of magnitude lower than those in currently used methods of ion chromatography with electrochemical and mass spectrometric detection.

The method was validated on samples of natural waters of different origin using the added–found technique. It was found that the error of analysis did not exceed 10% for river and ground waters and increased to 20–30% for peat bog surface waters.

The possibility of application of the developed method to the analysis of soils was shown on samples of peat bog soils selected at places of impact of the first steps of carrier rockets and polluted by rocket fuel based on 1,1-dimethylhydrazine.     

Simultaneous determination of positive and negative pharmaceutical counterions using mixed-mode chromatography coupled with charged aerosol detector

A mixed-mode chromatography coupled with charged aerosol detector (CAD) method was developed in this work to simultaneously determine pharmaceutical counterions including both inorganic ions and organic ions in the forms of cations and anions. 25 commonly used pharmaceutical ions were studied and simultaneously separated within 20 min by this single method. A silica based mixed-mode column with reversed-phase/cation-exchange/anion-exchange modes was used. It provides reversed-phase, strong cation-exchange and weak anion-exchange properties at the same time. It also provides the HILIC behavior at high percentage of organic solvent. The effects of mobile-phase organic strength, buffer ions, ionic strength, pH and column temperature have been investigated to optimize the method as well as to understand the retention and separation mechanisms. Conventional HPLC system was used and no special chromatography system is needed. The presented method has been employed successfully for screening and quantitative analysis of counterions, unknown ionic impurities and salts in active pharmaceutical ingredients and in process control samples with excellent accuracy, precision and sensitivity. This method provides a simple, fast and generic approach to speed up pharmaceutical research and development process and enhance lab efficiency. The similar methodologies can be applied to other ion analysis.     

亲水作用色谱法测定组织中全基因组DNA甲基化水平

建立了亲水作用色谱(HILIC)测定组织中全基因组DNA甲基化水平的方法。采用苯酚-氯仿提取组织中的DNA,提取的DNA用88%甲酸在140 ℃下裂解,经N2吹干后,加乙腈-水(9:1, v/v)溶解,用Waters BEH HILIC柱进行分离,在277 nm波长下检测胞嘧啶(Cyt)及5-甲基胞嘧啶(5-mCyt)含量。结果表明,以乙腈-10 mmol/L甲酸铵溶液(94:6, v/v)为流动相,流速为0.5 mL/min, Cyt与5-mCyt分离较好,保留时间分别为2.6与3.1 min。胞嘧啶的线性范围为1～900 μmol/L,相关系数为0.9999; 5-甲基胞嘧啶的线性范围为1～64 μmol/L,相关系数为0.9998。胞嘧啶和5-甲基胞嘧啶的检出限为54 nmol/L(柱中为0.54 pmol),定量限为250 nmol/L(柱中为2.5 pmol);在5～900 μmol/L的添加水平下,胞嘧啶和5-甲基胞嘧啶的平均加标回收率为94.7%～100.5%,相对标准偏差小于1.48%。用该方法检测了结肠癌组织中DNA甲基化水平,结果显示该癌组织中全基因组的DNA甲基化均值为4.0%。该方法快速、简单,稳定性好,灵敏度较高,能满足全基因组DNA甲基化的检测要求。     

固相萃取-超高效液相色谱法测定人参中5种原人参二醇型人参皂苷的含量

建立了固相萃取-超高效液相色谱测定人参中5种原人参二醇型人参皂苷的方法。人参药材经粉碎后通过水饱和正丁醇溶液进行超声提取,经过亲水作用固相萃取柱净化后,在ACQUITY UPLC BEH Shield RP18色谱柱(100 mm×2.1 mm, 1.7μm)上分离,利用乙腈/水作为流动相进行梯度洗脱,采用光电二极管阵列检测器检测。结果表明,5种原人参二醇型人参皂苷在5～500μg/mL范围内具有很好的线性关系,相关系数均大于0.999。方法精密度的RSD值在0.95%～2.62%(n=6)之间,22 h内样品稳定性的RSD值在0.90%～2.15%(n=8)之间,日内和日间重复性的RSD值分别为5.35%～6.47%(n=6)和5.56%～6.34%(n=8)。方法的加标回收率在87.16%～101.92%之间,相对标准偏差在1.54%～4.01%(n=6)之间。所建立的方法采用亲水作用色谱模式的固相萃取材料,药材的提取液可直接作为固相萃取的上样溶液进行人参皂苷的富集和净化,并且超高效液相色谱大大缩短了分析时间。该方法简单快速、通量高、重现性好,适用于人参中5种原人参二醇型人参皂苷的...     

应用亲水作用色谱检测鸡蛋和肉中三聚氰胺及三聚氰酸二酰胺

应用亲水作用色谱(HILIC)对从市场上购买的鸡蛋和肉中的三聚氰胺及三聚氰胺降解产物三聚氰酸二酰胺进行了检测。采用的色谱柱为ZIC-HILIC柱,流动相为3 mmol/L磷酸二氢铵溶液(pH 6.9)-乙腈(20:80, v/v),流速为0.8 mL/min,检测波长为220 nm。在该体系下,三聚氰胺和三聚氰酸二酰胺的保留时间适中,与样品中的内源性物质有良好的分离。样品经0.1%磷酸提取,偏磷酸及乙腈沉淀蛋白质和糖类物质,以及P-SCX固相萃取柱净化。三聚氰胺和三聚氰酸二酰胺在0.4～40 mg/L范围内与峰面积呈良好的线性关系,样品定量限(按信噪比(S/N)不小于10计)为2 mg/kg,在2～10 mg/kg添加水平下的平均回收率为80%～105%,相对标准偏差小于10%。该方法具有良好的分离选择性,可用于鸡蛋和肉中三聚氰胺和三聚氰酸二酰胺的同时检测。     

糖类化合物亲水作用色谱保留行为评价

以糖类化合物为研究对象,系统评价了其在亲水模式下的色谱保留行为。分别考察了流动相、固定相和缓冲盐等对糖类化合物保留的影响,建立了糖类化合物在亲水模式下的保留方程。结果表明,糖类化合物随着流动相中乙腈比例的降低,保留时间减小;随着缓冲盐浓度的增加,保留时间增加;同时,糖类化合物的保留行为还会受到有机溶剂种类和固定相类型的影响;其保留行为可使用顶替吸附-液相相互作用模型定量描述。将该模型进一步用于实际样品中糖类化合物保留行为的预测,获得了较好的实验结果,预测保留时间与实测保留时间的相对误差小于0.3%。对糖类化合物亲水模式下的保留行为进行了系统的评价和定量描述,该研究结果将有助于糖类化合物亲水作用色谱分离方法的发展。     

Control of impurities in l-aspartic acid and l-alanine by high-performance liquid chromatography coupled with a corona charged aerosol detector

In this study a reversed phase ion-pair high-performance liquid chromatography (HPLC) method using charged aerosol detection (CAD) was developed and fully validated for the pharmaceutical quality control of l-aspartic acid (Asp). With a slight modification, the method also allows the evaluation of related substances in l-alanine (Ala). The method enables simultaneous control of related amino acids and of possibly occurring organic acids contaminants. A minimum limit of quantification of 0.03% could be achieved for all occurring related substances. Moreover, the detector sensitivity of the CAD was compared with an evaporative light scattering detector (ELSD). Depending on the analyte the CAD was found to be 3.6–42 times more sensitive than the ELSD. The HPLC method was applied to the purity testing of 8 samples of pharmaceutical grade and reagent grade Asp and of 12 samples of Ala supplied by various manufacturers. Both substances were found to be of high purity (greater than 99.8% for Asp and greater than 99.9% for Ala). Malic acid and Ala were the major impurities in Asp. Asp and glutamic acid (Glu) were the only detectable impurities in Ala.     

Separation of monosaccharides by hydrophilic interaction chromatography with evaporative light scattering detection

Hydrophilic interaction liquid chromatography (HILIC) was used to separate monosaccharides that are common in N-linked oligosaccharides in glycoproteins and other compounds. A TSKgel Amide-80 column was eluted with 82% acetonitrile, in 5 mM ammonium formate (pH 5.5). Column temperature was 60 degrees C and evaporative light scattering was used for detection (ELSD). With this method, L-fucose, D-galactose, D-mannose, N-acetyl-D-glucosamine, N-acetylneuraminic acid, and D-glucuronic acid were separated, with detection limits of 0.3-0.5 microg for each monosaccharide, and intermediate precisions were 3-6% RSD (n=6).     

Size exclusion chromatography with Corona charged aerosol detector for the analysis of polyethylene glycol polymer

A technique of using size exclusion chromatography (SEC) with the Corona charged aerosol detector (CAD) was developed and evaluated in comparison with refractive index (RI) and evaporative light scattering detection (ELSD) for fast screening of polyethylene glycol (PEG), a polymer used in preparing pegylated pharmaceutical compounds. These detection techniques were used in the analysis of multiple lots of PEG reagents. CAD was found to provide more accurate impurity and polydispersity profiles of PEG reagents that better differentiate their quality, while RI was not suitable for this application due to its low sensitivity and ELSD led to underestimation of the impurity and polydispersity. The accuracy of polydispersity determination by SEC-CAD was validated against a commercial reference standard of known polydispersity. The SEC-CAD technique and the observed differences between the three detectors can also be applied to polymer analysis in general.     

Determination of atracurium,cisatracurium and mivacurium with their impurities in pharmaceutical preparations by liquid chromatography with charged aerosol detection

The Corona CAD (charged aerosol detection) is a new type of detector introduced for LC applications that has recently become widely applied in pharmaceutical analysis. The Corona CAD measures a physical property of analyte and responds to almost all non-volatile species, independently of their nature and spectral or physicochemical properties. The LC method with charged aerosol detection was developed for the determination of three isomers of atracurium, cisatracurium and also three isomers of mivacurium with their impurities. The limit of quantitation for laudanosine was 1 μg ml⁻¹. The elaborate method for the analysis of those active substances and laudanosine proved to be fast, precise, accurate and sensitive. All other impurities were identified using time-of-flight mass spectrometry with electrospray ionization.     

Exploration of the effects of chloride ions on the analysis of polar compounds at low concentrations by hydrophilic interaction liquid chromatography coupled to a charged aerosol detector: Application to tromethamine

In this study, we discuss the origin of the slightly increased response of the charged aerosol detector when low-concentration polar drugs formulated with sodium chloride are analyzed by hydrophilic interaction liquid chromatography coupled to the charged aerosol detector. In the case of tromethamine mixed with saline solutions, we investigated several levels including the mobile phase, sample matrix, and detection. We show that the analysis of the rich-salted sample results in both interactions with the mobile phase modifiers and the stationary phase during the run time. With 150 mM NaCl as a compounding solution, a slight increase in the tromethamine peak area was observed (<5.5%). Our study suggests that chloride ions in excess sequentially interact firstly with the counterions from the organic modifiers and secondly with the analyte via the stationary phase and the contribution of hydrophilic interaction liquid chromatography retention mechanisms. Because of these effects, the hydrophilic interaction liquid chromatography-charged aerosol detector analysis of drugs in saline solutions requires particular attention, and a correction factor for quantitative purposes that accounts for formulation ions remains appropriate.     

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。     

万古霉素及其杂质的亲水作用色谱分析

目前,万古霉素色谱分析方法主要为反相色谱法,该法分离万古霉素及其杂质时,存在极性选择性不足以及所使用的流动相体系与质谱兼容性差等问题。基于亲水作用色谱(HILIC)对糖肽类物质的色谱保留和极性选择性,本文选取万古霉素及其常见杂质为对象,考察了HILIC固定相、流动相组成比例、缓冲盐添加剂浓度和pH值等色谱条件,对万古霉素及其主要杂质进行了HILIC分离方法研究。确立了以Click XIon色谱柱为固定相,以甲酸铵为流动相添加剂的亲水作用色谱条件,实现了万古霉素及主要杂质的分离,为万古霉素类化合物的分离提供了新的途径。     

亲水/反相二维色谱法制备桔梗中的三萜皂苷

建立了亲水/反相二维色谱用于制备桔梗中三萜皂苷单体的方法。桔梗经水煮醇沉、反相和亲水两种模式的固相萃取后得到三萜皂苷类组分。选定XAmide色谱柱（150 mm×20 mm,5 μm）,以乙腈和水为流动相,在亲水色谱模式下进行组分制备。选择时间触发模式,以1 min为单位进行馏分收集,得到6~25 min之间的20个三萜皂苷精细组分。以第18个馏分（JG23）为例,在反相色谱模式下采用Atlantis Prep T3色谱柱（100 mm×30 mm,5 μm）制备,得到两个单体化合物。通过质谱和核磁共振对其进行定性,确定分别为deapi-platycoside E和platycoside E。实验结果表明,该制备方法具有好的正交选择性,对于复杂样品中三萜皂苷类化合物的分离纯化有一定的借鉴意义。     

Evaluation of different operation modes of high performance liquid chromatography for the analysis of complex mixtures of neutral oligosaccharides

Chromatographic methods based on different HPLC operation modes, reverse phase (RP), high performance anion exchange chromatography (HPAEC), graphitized carbon chromatography (GCC) and hydrophilic interaction liquid chromatography (HILIC), have been developed and compared for the analysis of complex mixtures of neutral oligosaccharides with functional properties. Whereas GCC gave the best chromatographic separation of isomeric oligosaccharides with the same molecular weight (R(s) values in the range 1.0-4.0 and 2.4-5.6 for tetra- and pentasaccharides, respectively), HILIC provided the best results for mixtures including oligosaccharides of different degrees of polymerization (R(s) values of maltooligosaccharides between 3.4 and 6.2). Validation of the HILIC LC-MS method proved its utility for the analysis of oligosaccharide mixtures with functional properties: relative standard deviations lower than 10%, LOD's and LOQ's in the range 12.7-130.2 ng mL(-1) and 39.3-402.2 ng mL(-1), respectively, and linearity up to 10-20 μg mL(-1). Quantitative data for fructooligosaccharides, gentiooligosaccharides and dextransucrase cellobiose acceptor oligosaccharides were obtained by using this method.     

Simple and precise detection of lipid compounds present within liposomal formulations using a charged aerosol detector

In recent decades the use of liposomal preparations as drug delivery systems has become very attractive in pharmaceutical development. Therefore, thorough characterization and quantification of the lipids which form liposomes is wished from both investigators and regulatory authorities when the application in humans is being considered. In this study a new HPLC method for the detection of lipids in liposomal formulations was established using corona charged aerosol detection (CAD) which has the advantage to be independent of the chemical properties of the analytes. The superiority of this method over UV detection was demonstrated. Compared to UV detection no absorption effects of the organic solvent in the mobile phase interfering with the lipid signals were observed with CAD. CAD showed good linearity (R² > 0.990) for all liposomal compounds. The acceptance criteria for precision including repeatability were met. The average recovery for each of the excipients of the liposomal formulation was in the range of 90.0–110%.     

高效液相色谱-蒸发光散射法测定食品中的单糖、双糖、低聚果糖和糖醇

建立了食品中常用的木糖、果糖、葡萄糖、蔗糖、麦芽糖、乳糖、蔗果三糖、蔗果四糖、蔗果五糖、赤藓糖醇、木糖醇、甘露糖醇、麦芽糖醇等13种单糖、双糖、低聚果糖和糖醇的高效液相色谱同时分离检测的方法。该法采用NH₂色谱柱,以乙腈-水为流动相梯度洗脱,蒸发光散射检测器检测;13种糖在0.1~5 g/L内均具有良好的线性关系,检出限均在0.1 g/L以下,精密度(RSD)为2.69%~7.21%,回收率为96.1%~105.2%,结果较为理想。将该法用于实际样品检测,结果显示食品标签明示和实际成分相差较大。     

亲水作用液相色谱脱除人参提取物中农药残留

亲水作用液相色谱法(HILIC)是一种用于改善强极性物质的保留和分离选择性的方法,广泛应用于药物分析、代谢组学、蛋白质组学等领域.该文利用农药分子与皂苷成分在HILIC上的保留行为差异,开发了一种农药残留脱除方法.以市售高纯人参提取物为例,该文评价了农药分子和人参皂苷在亲水色谱柱上的保留行为,并考察了上样量、淋洗体积、...     

Determination of glutathione content in grape juice and wine by high-performance liquid chromatography with fluorescence detection

A modified preparation of sample was developed for the determination of glutathione content in grape juice and wine by high-performance liquid chromatography with fluorescence detection, using on-line pre-column derivatization. Ice-cold deoxygenated methanol was used to deactivate the oxidation enzymes in juices or wines and keep the glutathione stable. The optimum recovery of glutathione content in grape juice and wine was obtained when either the sample of grape juice or wine was mixed in ice-cold deoxygenated methanol in the ratio 10:90 (v:v) and further diluted in sodium acetate buffer in the ratio 1:1 (v:v). The optimized method was validated for linearity, limit of detection, limit of quantification, precision and uncertainty. According to the validation data the method is appropriate for the determination of glutathione content in grape juice and wine. Glutathione contents in grape juices made from White Muscat grapes and Sauvignon Blanc wines were analysed. The average glutathione content in 28 young Sauvignon Blanc wines was 12.5 mg L⁻¹.