On-line coupling of size exclusion and capillary zone electrophoresis via a reversed-phase C18 trapping column for the analysis of structurally related enkephalins in cerebrospinal fluid

On-line coupled analytical techniques can be advantageous in the assay of smaller peptides in complex biological matrices such as plasma, cerebrospinal fluid (CSF) and tissues. The present study shows the feasibility of a recently developed system, consisting of a size-exclusion chromatographic (SEC) separation followed by a trapping procedure on an RP18 microcolumn with subsequent elution of the trapped fraction and separation by capillary zone electrophoresis (CZE) for the quantification of structural-related peptides in biological matrices, as demonstrated for a number of enkephalins in CSF. After SEC separation of the enkephalins from large proteins present in CSF a heart-cut of 200 nuL, containing the enkephalin peak, is taken, concentrated on the RP18 microcolumn and, after elution of the enkephalins with 80% acetonitrile, a fraction of the eluate is electrokinetically injected into the CZE system, where stacking and separation is achieved. The degradation of the peptides, caused by endogenous peptidases in the matrix, is sufficiently inhibited with imipramine HCl. The assay has a satisfactory linearity and intraday (9.70-16.3%) precision considering the complexity of this multidimensional separation system. The sensitivity of the method, with a concentration limit of quantification of 2.5 nug/mL, is comparable with other CZE assays for peptides and sufficient for the quantification of peptide drugs in biological matrices.     

Separation of diastereomers of phosphinic pseudopeptides by capillary zone electrophoresis and reverse phase high‐performance liquid chromatography

Capillary zone electrophoresis (CZE) and reverse phase high‐performance liquid chromatography (RP‐HPLC) were used for separation of diastereomers of phosphinic pseudopeptides in achiral separation media. A set of phosphinic pseudopeptides, i. e. peptides with one peptide bond substituted by phosphinic acid moiety ‐PO₂^–‐CH₂‐ derived from the structure N‐Ac‐Val‐AlaB(‐CH₂)Leu‐His‐NH₂ synthesized as a mixture of four diastereomers was used. Separations of diastereomers by CZE were carried out in Tris‐phosphate background electrolytes in the pH range 1.1–3.2 and at least partial separation of the four diastereomers of each pseudopeptide was achieved. A routinely used RP‐HPLC method (C₁₈‐silica column and water/acetonitrile/trifluoroacetic acid mobile phase) was also capable of resolving the diastereomers. In addition, since individual diastereomers of majority of the pseudopeptides were isolated by RP‐HPLC it was possible to check the purity of these RP‐HPLC separated diastereomers and to compare the migration order of the diastereomers in CZE with their elution order in RP‐HPLC. The results obtained by CZE and RP‐HPLC demonstrate a complementarity of both methods in analysis and separation of phosphinic pseudopeptides including their diastereomers.     

Hollow-fiber flow field-flow fractionation: a gentle separation method for mass spectrometry of native proteins

Low-impact ionization sources like electrospray ionization (ESI) and matrix-assisted, laser desorption/ionization (MALDI) equipped with time-of-flight (TOF) mass analyzers provide intact protein analysis over a very wide molar mass range. ESI/TOFMS provides also indications on the higher-order structure of intact proteins and non-covalent protein complexes. However, direct analysis of intact proteins mixtures in real samples shows limited success, mainly because spectra become very complex to interpret. This is also due to sample contaminants, and to the mechanism of competitive ionization in ESI or MALDI. Rapid and efficient sample clean-up and separation methods can significantly enhance the power of TOFMS for intact protein analysis. However, if protein native conditions want to be maintained, the methods should affect neither the three-dimensional structure nor the non-covalent chemistry of the proteins. Reversed-phase (RP) HPLC, size-exclusion chromatography (SEC), and capillary zone electrophoresis (CZE) are on-line or off-line coupled to ESI/TOFMS or MALDI/TOFMS. In fact, these separation methods often show limitations when applied to the analysis of native proteins. Organic modifiers and saline buffers are required in the case of RP HPLC or CZE. They can induce protein degradation or affect ionization when MS is performed after separation. High voltages used in CZE can contribute to alter proteins from their native form. In the case of high molar mass proteins, SEC is scarcely selective, and barely able to detect protein aggregates. Sample entanglement/adsorption on the stationary phase can also occur.     

Application of temperature-controlled micro planar chromatography for separation and quantification of testosterone and its derivatives

Temperature-controlled micro thin-layer chromatography (TLC) was applied for separation and quantification studies of testosterone and its derivatives including methyltestosterone, testosterone propionate, isobutyrate, phenylpropionate, isocaproate, enanthate and caprate. Chromatographic studies were performed on silica-, octadecylsilica- and aluminum-coated plates working inside a small thermostated horizontal chamber unit allowing one-dimensional and two-dimensional developing modes with an elution distance of 45 mm. Retention properties of steroids were investigated across a whole range of binary mixtures such as methanol/water, acetonitrile/water, methanol/dichloromethane and acetone/hexane (0–100% v/v). Moreover, the effect of temperature ranging from −20 to +60 °C under saturated and unsaturated chamber conditions was also investigated. Our results revealed that depending on the mobile phase polarity the separation system based on the low carbon load wettable with water RP18W plates may work as a normal-phase (NP) or reversed-phase (RP) chromatographic system. It has been also demonstrated that micro TLC equipment can be applied as a fast retention screening device as well as simple and robust quantitative tool for determination of testosterone residue containing testosterone derivatives in complex samples. Figure 2D micro TLC separation and quantitative analysis of testosterone and its derivatives Presented at the 11th International Conference on Chemistry and the Environment, 9–12 September 2007, Torun, Poland     

HPLC-ESI-TOFMS检测尿液中的雌二醇

用HPLC—ESI-TOFMS建立了快速灵敏检测尿中雌二醇的方法,尿样中雌二醇经盐酸水解后用OASISHLB小柱萃取．色谱柱为XTerraMS C18反相分离柱（3．5μm,2．1mm×100mm）,流动相为水和乙腈,梯度洗脱,流速为0．2mL／min,紫外检测波长为280nm,外标法定量．质谱采用负离子电离模式．线性方程：y=1．79×10^5x＋2．59×10^4,线性范围：0．0328—32．8mg／L,线性相关系数R^2=0．9979,最小检出浓度0．00328mg／L,萃取回收率101．83％．应用本方法对10例正常人尿样进行测定,结果满意．相比较其它检测尿中雌二醇的方法来说,此法比较简单、快速、准确,为临床的诊断提供有价值的指标．     

Analysis of ginseng root and leaf by multiple columns and detections liquid chromatography

Abstract

A multiple columns and detections liquid chromatography system, including size exclusion chromatography (SEC) and reversed phase liquid chromatography (RPLC), for the analysis of macromolecules and micromolecules in ginseng root and leaf was developed. The columns were connected by two switching valves. Macromolecules were separated on a SEC column (TSK gel SuperMultipore PW-H column, 6?mm× 150?mm, 8?μm) by isocratic elution of 50?mM ammonium acetate aqueous solution, 0.3?mL/min of flow rate and detected by evaporative light scattering detection (ELSD). Micromolecules were analyzed on a Poroshell RP column (Agilent Poroshell 120?SB-Aq column, 4.6?mm × 50?mm, 2.7 µm) with gradient elution of water and acetonitrile, 0.6?mL/min of flow rate and detected by ultraviolet detection (UV). As a result, in the macromolecules chromatogram of ginseng root sample showed two main peaks while only one major peak for ginseng leaf. For micromolecules analysis, 17 compounds (3 nucleosides + 14 saponins) and 17 compounds (3 nucleosides + 1 flavonoid + 13 saponins) were found in ginseng root and leaf, respectively. The developed method is helpful for the quality evaluation of ginseng root and leaf.     

On-column complexation and simultaneous separation of vanadium(IV) and vanadium(V) by capillary electrophoresis with direct UV detection

An on-column complexation method has been developed for the simultaneous determination of V(IV) and V(V). Vanadium species were chelated with aminopolycarboxylic acids to form anionic complexes which were separated by capillary zone electrophoresis (CZE) with direct UV detection. Ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentacetric acid (DTPA), nitrilotriacetic acid (NTA), and N-2-hydroxyethylethlendiaminetriacetric acid (HEDTA) were investigated as both ligand and running electrolyte. Of the ligands studied the complexes of EDTA with V(IV) and V(V) resulted in the highest selectivity and UV response.The conditions used for on-column complexation and separation, including pH, and electrolyte ligand concentration, were examined to achieve reasonable separation selectivity and detection sensitivity. The optimum separation of the anionic forms of V(IV) and V(V) was obtained by use of CZE with UV detection at 185 nm and an electrolyte containing 5 mmol L(-1) EDTA at pH 4.0. Linear calibration plots were obtained in the concentration range10-300 micro mol L(-1); detection limits were 3 micro mol L(-1) for V(IV) and 1 micro mol L(-1) for V(V). The proposed method was demonstrated for the determination of vanadium in groundwater spiked with V(IV) and V(V).     

Separation of selected peptides by capillary electroendoosmotic chromatography using 3 microns reversed-phase bonded silica and mixed-mode phases.

The retention behaviour and selectivity of selected basic, neutral and acidic peptides have been studied by capillary electroendoosmotic chromatography (CEC) with Hypersil C8, C18, Hypersil mixed-mode, and Spherisorb C18/SCX columns, 250 (335) mm x 100 microns, packed with 3 microns particles, and eluted with mobile phases composed of acetonitrile-triethylamine-phosphoric acid (TEAP) at pH 3.0 using a Hewlett-Packard Model HP3DCE capillary electrophoresis system. The selected peptides were desmopressin (D), two analogues (A and B) of desmopressin, oxytocin (O) and carbetocin (C). The peptides eluted either before or after the electroendoosmotic flow (EOF) marker, depending on the concentration of acetonitrile used and the buffer ionic strength. The retention and selectivity of these peptides under CEC conditions were compared to their behaviour in free zone capillary electrophoresis (CZE), where the separation mode was based on the electrophoretic migration of the analytes due to their charge and Stokes radius properties. In addition, their retention behaviour in RP-HPLC was also examined. As a result, it can be concluded that the elution process of this group of synthetic peptides in CEC with a TEAP buffer at pH 3.0 is mediated by a combination of both electrophoretic migration processes and retention mechanisms involving hydrophobic as well as silanophilic interactions. This CEC method when operated with these 3 microns reversed-phase and mixed-mode sorbents with peptides is thus a hybrid of two well-known analytical methods, namely CZE and RP-HPLC. However, the retention behaviour and selectivity of the selected peptides differs significantly in the CEC mode compared to the RP-HPLC or CZE modes. Therefore this CEC method with these peptides represents an orthogonal analytical separation procedure that is complimentary to both of these alternative techniques.     

Determination of Antiviral Drugs and Their Metabolites Using Micro-Solid Phase Extraction and UHPLC-MS/MS in Reversed-Phase and Hydrophilic Interaction Chromatography Modes

Two new ultra-high performance liquid chromatography (UHPLC) methods for analyzing 21 selected antivirals and their metabolites were optimized, including sample preparation step, LC separation conditions, and tandem mass spectrometry detection. Micro-solid phase extraction in pipette tips was used to extract antivirals from the biological material of Hanks balanced salt medium of pH 7.4 and 6.5. These media were used in experiments to evaluate the membrane transport of antiviral drugs. Challenging diversity of physicochemical properties was overcome using combined sorbent composed of C₁₈ and ion exchange moiety, which finally allowed to cover the whole range of tested antivirals. For separation, reversed-phase (RP) chromatography and hydrophilic interaction liquid chromatography (HILIC), were optimized using extensive screening of stationary and mobile phase combinations. Optimized RP-UHPLC separation was carried out using BEH Shield RP18 stationary phase and gradient elution with 25 mmol/L formic acid in acetonitrile and in water. HILIC separation was accomplished with a Cortecs HILIC column and gradient elution with 25 mmol/L ammonium formate pH 3 and acetonitrile. Tandem mass spectrometry (MS/MS) conditions were optimized in both chromatographic modes, but obtained results revealed only a little difference in parameters of capillary voltage and cone voltage. While RP-UHPLC-MS/MS exhibited superior separation selectivity, HILIC-UHPLC-MS/MS has shown substantially higher sensitivity of two orders of magnitude for many compounds. Method validation results indicated that HILIC mode was more suitable for multianalyte methods. Despite better separation selectivity achieved in RP-UHPLC-MS/MS, the matrix effects were noticed while using both chromatographic modes leading to signal enhancement in RP and signal suppression in HILIC.     

Multiple Peaks in HPLC of Proteins: Bovine Serum Albumin Eluted in a Reversed-Phase System

Elution of a commercial sample of bovine serum albumin (BSA) in a reversed-phase (RP) HPLC system at room temperature gives a distorted peak. If a shallow gradient is used during elution a split peak is observed. The nature of the several parts of this multiple peak is studied using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), size-exclusion chromatography (SEC), amino acid analysis, re-elution in RP-HPLC of collected fractions, capillary electrophoresis (CE), and matrix assisted laser desorption ionization-mass spectrometry (MALDI-MS). This study demonstrates that the split peak of BSA observed in these chromatographic conditions is due to the monomer, dimer and other aggregates existing in the commercial sample of the BSA used. Moreover, it is proved that typical RP-chromatographic conditions do not cause aggregation of BSA.     

血管紧张素转化酶活性抑制剂──丝素肽的分离、纯化和结构鉴定

 可溶性丝素粉末经碱性蛋白酶Alcalase水解后 ,其酶解产物对血管紧张素转化酶 (ACE)的活性有很强的抑制作用。采用凝胶过滤色谱SephadexG 15和反相高效液相色谱 (RP HPLC)对水解度为 2 0 %的酶解产物进行分离纯化 ,利用质谱鉴定其中一种ACE抑制剂是肽 ,其结构为Gly Tyr。     

固相萃取-高效液相色谱法同时检测大米中12种磺酰脲类除草剂的残留

建立了固相萃取前处理净化技术-高效液相色谱（HPLC）同时检测大米中12种磺酰脲类除草剂残留的方法。采用ENVITM-18(C18)硅胶柱和ENVI-Carb(GCB)石墨化碳柱对样品进行净化、萃取,采用C8柱,以乙腈和5 mmol/L 冰乙酸混合溶剂为流动相进行梯度洗脱,在240 nm下进行检测。12种磺酰脲类除草剂在0.01~0.50 μg/g添加范围内的回收率为72.2%~106.5%,相对标准偏差为0.6%~6.4%,检出限为0.01~0.02 μg/g。     

Sequential injection-bead injection-lab-on-valve coupled to high-performance liquid chromatography for online renewable micro-solid-phase extraction of carbamate residues in food and environmental samples

A sequential injection‐bead injection‐lab‐on‐valve system was hyphenated to HPLC for online renewable micro‐solid‐phase extraction of carbamate insecticides. The carbamates studied were isoprocarb, methomyl, carbaryl, carbofuran, methiocarb, promecarb, and propoxur. LiChroprep^® RP‐18 beads (25–40 μm) were employed as renewable sorbent packing in a microcolumn situated inside the LOV platform mounted above the multiposition valve of the sequential injection system. The analytes sorbed by the microcolumn were eluted using 80% acetonitrile in 0.1% acetic acid before online introduction to the HPLC system. Separation was performed on an Atlantis C‐18 column (4.6×150 mm, 5 μm) utilizing gradient elution with a flow rate of 1.0 mL/min and a detection wavelength at 270 nm. The sequential injection system offers the means of performing automated handling of sample preconcentration and matrix removal. The enrichment factors ranged between 20 and 125, leading to limits of detection (LODs) in the range of 1–20 μg/L. Good reproducibility was obtained with relative standard deviations of <0.7 and 5.4% for retention time and peak area, respectively. The developed method has been successfully applied to the determination of carbamate residues in fruit, vegetable, and water samples.     

Optimization of a high-performance liquid chromatography method to quantify bilirubin and separate it from its photoproducts

A rapid reversed-phase (RP) high-performance liquid chromatography method for the isolation of bilirubin from its photoproducts (e.g., biliverdin) is reported. The method is based on isocratic elution using methanol:water as the mobile phase. A 2⁴ full-factorial experimental design approach was adopted. For the optimization, the best separation was obtained using a flow rate of 1.50 mL/min, a mobile phase of 99∶1 methanol:water (v/v) at pH 3.60, and a 150×4.6 mm id RP (C₁₈) column containing 5-μm particles. These conditions produced the fastest total retention time of 3.38±0.055 min, and other chromatographic parameters were acceptable. Under the optimum conditions, a linear calibration curve for bilirubin was obtained over the 1.0–40.0 μg/L concentration range studied. The limit of quantification was 0.79 g/L and the limit of detection was 0.24 μg/L. Bilirubin in solution was monitored by ultraviolet detection at 450 nm.     

Accessible silanol sites - beneficial for the RP-HPLC separation of constitutional and diastereomeric azaspirovesamicol isomers

Different RP-HPLC columns (phenyl, conventional ODS, cross-linked C(18) and special end-capped C(8) and C(18) phases) were used to investigate the separation of four basic ionizable isomers. Using ACN/20mM NH(4)OAc aq., a separation was observed exclusively on RP columns with higher silanol activity at unusual high ACN concentration, indicating cation-exchange as main retention mechanism. Using MeOH/20mM NH(4)OAc aq., another separation at low MeOH concentrations was observed on both, RP columns with higher as well as RP columns with lower silanol activity, which is mainly based on hydrophobic interactions. The isomers were also separated on a bare silica column at higher MeOH content using NH(4)OAc. Since cation-exchange governs this retention, the elution order was different compared to the RP phases. A strong retention on the silica column was observed in ACN, which could be attributed to partition processes as additional retention mechanism.     

Silica-based monolithic columns with mixed-mode reversed-phase/weak anion-exchange selectivity principle for high-performance liquid chromatography

This article describes the synthesis, chromatographic characterization, and performance evaluation of analytical (100 x 4.6 mm id) and semipreparative (100 x 10 mm id) monolithic silica columns with mixed-mode RP/weak anion-exchange (RP/WAX) surface modification. The monolithic RP/WAX columns were obtained by immobilization of N-(10-undecenoyl)-3-aminoquinuclidine onto thiol-modified monolithic silica columns (Chromolith) by a radical addition reaction. Their chromatographic characterization by Engelhardt and Tanaka tests revealed slightly lower hydrophobic selectivities than C-8 phases, as well as higher polarity and also improved shape selectivity than RP-18e silica rods. The surface modification enabled separation by both RP and anion-exchange chromatography principles, and thus showed complementary selectivities to the RP-18e monoliths. The mixed-mode monoliths have been tested for the separation of peptides and turned out to be particularly useful for hydrophilic acidic peptides, which are usually insufficiently retained on RP-18e monolithic columns. Compared to a corresponding particulate RP/WAX column (5 microm, 10 nm pore diameter), the analytical RP/WAX monolith caused lower system pressure drops and showed, as expected, higher efficiency (e.g. by a factor of about 2.5 lower C-term for a tetrapeptide). The upscaling from the analytical to semipreparative column dimension was also successful.     

全二维微柱液相色谱-质谱鉴定人胃癌组织中的差异蛋白质

构建了以阳离子交换色谱-反相色谱(SCX-RPLC)为分离模式的新型全二维微柱液相色谱-质谱分离平台.采用了醋酸铵缓冲液梯度洗脱,实现了第一维肽段的分步洗脱,洗脱的肽段经富集除盐后通过接口进入反相色谱微柱,通过线性梯度实现第二维进一步分离,最后进入质谱进行检测.采用此平台分析了人胃癌组织与正常组织提取蛋白质信息,其中正常胃组织鉴定蛋白质数为537个,而癌症组织鉴定蛋白质数目为506个.对胃癌和正常组织两种提取蛋白质酶解产物的蛋白质检索结果进行比较分析,将鉴定的蛋白质按照物理性质进行分布,找出正常组织与癌症组织间蛋白质差异,筛选出一种可能发生变异的癌症特有蛋白.     

三种维生素的毛细管电泳和胶束电动毛细管色谱法分离与安培电化学检测

以自制毛细管电泳－电化学检测系统对ＶＣ,ＶＢ１和ＶＢ６的毛细管区带电泳和胶束电动色谱的分离与检测情况进行了初探。结果表明,在０．０１ｍｏｌ／ＬＮＨ３－ＮＨ４Ｃｌ介质中,检测电势定于５１０～５４０ｍＶ（对ＳＣＥ）时,三种维生素均有较好的ＣＺＥ图,对ＶＣ的分离效率达４６８８００块理论板。使用十二烷基硫酸钠时,分离效果欠佳。     

DNA fragment separations by on‐line combination of capillary isotachophoresis–capillary zone electrophoresis with UV detection

A high‐speed DNA fragment separation system based on an on‐line combination of capillary ITP with CZE (CITP‐CZE) and using UV detection at 260 nm was developed. A novel CITP‐CZE buffer system of pH 6.1 was designed for the separation of ten DNA fragments with sizes ranging from 100 to 1000 bp. An effect of underivatized α‐, β‐ and γ‐cyclodextrins on the resolution of DNA fragments in the CZE step of the CITP‐CZE combination was systematically investigated. Methylhydroxyethylcellulose present in the BGE was used to eliminate the EOF. DNA ladder fragments were separated within 10 min with LODs in the range of 1–5 ng/μL (S/N = 3). The RSDs of the migration time and peak area of individual DNA fragments were in the range of 1–3 and 3–9%, respectively. The developed CITP‐CZE system was further applied to the analysis of digest plasmid DNA samples.     

Fractionation and proteomic analysis of the Walterinnesia aegyptia snake venom using OFFGEL and MALDI‐TOF‐MS techniques

Animal venoms are complex mixtures of more than 100 different compounds, including peptides, proteins, and nonprotein compounds such as lipids, carbohydrates, and metal ions. In addition, the existing compounds show a wide range of molecular weights and concentrations within these venoms, making separation and purification procedures quite tedious. Here, we analyzed for the first time by MS the advantages of using the OFFGEL technique in the separation of the venom components of the Egyptian Elapidae Walterinnesia aegyptia snake compared to two classical methods of separation, SEC and RP‐HPLC. We demonstrate that OFFGEL separates venom components over a larger scale of fractions, preserve respectable resolution with regard to the presence of a given compound in adjacent fractions and allows the identification of a greater number of ions by MS (102 over 134 total ions). We also conclude that applying several separating techniques (SEC and RP‐HPLC in addition to OFFGEL) provides complementary results in terms of ion detection (21 more for SEC and 22 more with RP‐HPLC). As a result, we provide a complete list of 134 ions present in the venom of W. aegyptia by using all these techniques combined.