Development and validation of an ion‐exchange chromatography method for heparin and its impurities in heparin products

An anion‐exchange liquid chromatography method for the determination of heparin and its impurities (dermatan sulfate and oversulfated chondroitin sulfate) was developed using chemometric‐assisted optimization, including multivariate experimental design and response surface methodology. The separation of heparin, dermatan sulfate, and oversulfated chondroitin sulfate (R_s above 2.0) was achieved on a Dionex RF IC IonPac AS22 column with a gradient elution of 10–70% of 2.5 M sodium chloride and 20 mM Tris phosphate buffer (pH 2.1) at a flow rate of 0.6 mL/min and UV detection at 215 nm. Method validation shows good linearity (r > 0.99), acceptable precision (%relative standard deviations <11.4%) and trueness (%recovery of 92.3–103.9%) for all analytes. The limits of detection for dermatan sulfate and oversulfated chondroitin sulfate are equivalent to 0.11% w/w (10.5 μg/mL) and 0.07% w/w (7.2 μg/mL), while the limits of quantification are 0.32% w/w (31.5 μg/mL) and 0.22% w/w (22.0 μg/mL) relative to heparin, respectively. The method is specific for heparin, dermatan sulfate, and oversulfated chondroitin sulfate without interference from mobile phase and sample matrices and could be used for accurate quantitation the drug and its impurities in a single run. Applications of the method reveal contents of heparin between 90.3 and 97.8%. Dermatan sulfate and oversulfated chondroitin sulfate were not detected in any of the real‐life samples.     

Gold nanomaterials based pseudostationary phases in capillary electrophoresis: A brand‐new attempt at chondroitin sulfate isomers separation

In this work, a CE method with bare gold nanorods (GNRs) based pseudostationary phase was developed and applied for the separation of chondroitin sulfate (CS) isomers, CS, and dermatan sulfate (DS). The separation efficiency was investigated by varying the experimental parameters such as concentration and pH of the BGE, separation voltage, internal diameter of capillary, different size, and morphology of gold nanomaterials. Results showed that different size and morphology of gold nanomaterials had different effects on the separation of CS and DS. The best separation of CS and DS was achieved in the BGE composed of aqueous 150 mmol/L (mM) ethylenediamine + 20 mM sodium dihydrogen phosphate + 30% v/v GNRs, pH 4.5, at the separation voltage of ?10 kV. Capillary was 59.2 cm in length (effective length 49 cm), 50 μm id capillary thermostated at 25°C. CE with bare GNRs used as pseudostationary phase was shown to be a suitable technique for the separation of CS and DS mixtures with wider peaks. RSD of migration time and peak area of CS and DS were 0.13, 0.14 and 0.86, 1.07%, respectively.     

Online preconcentration and determination of chondroitin sulfate,dermatan sulfate and hyaluronic acid in biological and cosmetic samples using capillary electrophoresis

Capillary electrophoresis with large‐volume sample stacking using an electroosmotic flow pump was developed for the determination of chondroitin sulfate, dermatan sulfate, and hyaluronic acid. Central composite design was used to simultaneously optimize the parameters for capillary electrophoresis separation. The optimized capillary electrophoresis conditions were 200 mM sodium dihydrogen phosphate, 200 mM butylamine, and 0.5% w/v polyethylene glycol as a background electrolyte, pH 4 and ‐16 kV. Exploiting large‐volume sample stacking using an electroosmotic flow pump, the sensitivity of the proposed capillary electrophoresis system coupled with UV detection was significantly improved with limits of detection of 3, 5, 1 mg/L for chondroitin sulfate, dermatan sulfate, and hyaluronic acid, respectively. The developed method was applied to the determination of chondroitin sulfate and hyaluronic acid in cell culture media, cerebrospinal fluid, cosmetic products, and supplementary samples with highly acceptable accuracy and precision. Therefore, the proposed capillary electrophoresis approach was found to be simple, rapid, and reliable for the determination of chondroitin sulfate, dermatan sulfate, and hyaluronic acid in cell culture media, cerebrospinal fluid, cosmetic, and supplementary samples without sample pretreatment.     

Capillary electrophoresis of complex natural polysaccharides

Complex natural polysaccharides, glycosaminoglycans (GAGs), are a class of ubiquitous macromolecules that exhibit a wide range of biological functions and participate and regulate multiple cellular events and (patho)physiological processes. They are generally present either as free chains (hyaluronic acid and bacterial acidic polysaccharides) or as side chains of proteoglycans (PGs; chondroitin/dermatan sulfate, heparin/heparan sulfate, and keratan sulfate) and are most often found in cell membranes and in the extracellular matrix. The recent emergence of modern analytical tools for their study has produced a virtual explosion in the field of glycomics. CE, due to its high resolving power and sensitivity, has been useful in the analysis of intact GAGs and GAG-derived oligosaccharides and disaccharides affording concentration and structural characterization data essential for understanding the biological functions of GAGs. In this review, novel off-line and on-line CE-MS and MS/MS methods for screening of GAG-derived oligosaccharides and disaccharides will be discussed.     

Advances in the separation,sensitive detection,and characterization of heparin and heparan sulfate

Elucidation of the relationship between the structure and biological function of the glycosaminoglycans (GAGs) heparin and heparan sulfate (HS) presents an important analytical challenge mainly due to the difficulty in determining their fine structure. Heparin and HS are responsible for mediation of a wide range of biological actions through specific binding to a variety of proteins including those involved in blood coagulation, cell proliferation, differentiation and adhesion, and host–pathogen interactions. Therefore, there is a growing interest in characterizing the microstructure of heparin and HS and in elucidating the molecular level details of their interaction with peptides and proteins. This review discusses recent developments in the analytical methods used for sensitive separation, detection, and structural characterization of heparin and HS. A brief discussion of the analysis of contaminants in pharmaceutical heparin is also presented.     

硫酸皮肤素的共振瑞利散射法测定

研究了硫酸皮肤素对十六烷基三甲基溴化铵(CTMAB)发生缔合反应的条件、共振瑞利散射特征.结果表明,在pH 5.8的BR缓冲溶液中,硫酸皮肤素能与CTMAB形成离子缔合物,使共振瑞利散射RRS急剧增强并产生新的RRS光谱.硫酸皮肤素浓度在0.04～4.0μg/mL之间与散射强度呈线性关系,方法检出限(3σ)为13 ng/mL,并以CTMAB体系为例研究了共存物质的影响,表明方法选择性好.     

毛细管区带电泳法测定消毒剂中三氯新

建立了消毒剂中三氯新的毛细管电泳分析方法。探讨了缓冲介质和电泳参数对三氯新测定的影响。以15mmol/LNa2HPO4(pH6.0)-乙腈(V(Na2HPO4)∶V(乙腈)=50∶50)为电泳缓冲液,三氯新在12kV电压下电泳,于254nm检测波长处测定,6min可以完成分析。本方法的检出限为0.04mg/L,线性范围0.04～2.00mg/mL(r=0.997),加标回收率在90.9%～108.2%范围内,测定值的相对标准偏差分别为峰高7.7%,迁移时间5.5%。将本法与高效液相色谱法进行比较,样品测定结果的相对误差小于10%。将所建立的方法已用于消毒剂样品中三氯新的测定。     

Quantitative investigation of the interaction between granulocyte-macrophage colony-stimulating factor and heparin by capillary zone electrophoresis

The interactions between granulocyte-macrophage colony-stimulating factor (GM-CSF) and heparin or low-molecular weight heparin (LMWH) were studied by CZE. It was found that GM-CSF could bind to both heparin and LMWH. The binding constants were calculated from Scatchard regression to be (6.5 +/- 0.8) x 10(5)/M and (11.2 +/- 0.7) x 10(5)/M, respectively. The specificity of the interaction between GM-CSF and heparin was also studied by employing another sulfated K carrageenan oligosaccharide as a control. Results showed that K carrageenan oligosaccharide could not interact with GM-CSF, indicating that GM-CSF could specifically interact with heparin.     

硫酸沙丁胺醇的毛细管电泳非接触电导法检测

建立了毛细管电泳-非接触电导检测分离测定硫酸沙丁胺醇的分析方法。分别考察了分离介质、背景电解质及其浓度和pH、分离电压、进样时间、激发电压、激发频率等因素对实验结果的影响。在优化的实验条件(以15 mmol/L乳酸水溶液(pH 2.7)为电泳介质,10 kV下电动进样3 s,分离电压为10 kV,激发电压为60 V,激发频率为120 kHz)下,硫酸沙丁胺醇的检出限(信噪比为3)为1.92 mg/L,在9.60～48.0 mg/L质量浓度范围内有良好的线性关系(r=0.995),迁移时间的相对标准偏差(RSD)为2.7%。将该方法用于硫酸沙丁胺醇片和硫酸沙丁胺醇气雾剂中的硫酸沙丁胺醇含量的测定,加标回收率为90%～113%,检测结果与药厂的标示值相符合,为硫酸沙丁胺醇的检测提供了一种简便、快速、高灵敏的方法。关键词: 毛细管电泳;非接触电导检测法;硫酸沙丁胺醇;硫酸沙丁胺醇片;硫酸沙丁胺醇气雾剂     

Capillary electrophoresis of heparin and other glycosaminoglycans using a polyamine running electrolyte

This study involves the use of polyamines as potential resolving agents for the capillary electrophoresis (CE) of glycosaminoglycans (GAGs), specifically heparin, dermatan sulfate, chondroitin sulfate, over-sulfated chondroitin sulfate (OSCS), and hyaluronan. All of the compounds can be separated from each other with the exception of chondroitin sulfate and hyaluronan. Using optimization software, the final run conditions are found to be 200 mM ethylenediamine and 45.5 mM phosphate as the electrolyte with −14 V applied across a 50 μm ID × 24.5 cm fused silica capillary at 15 °C. The ion migration order, with OSCS as the last instead of the first peak, is in contrast to previous reports using either a high molarity TRIS or lithium phosphate run buffer with narrower bore capillaries. Total analysis time is 12. 5 min and the relative standard deviation of the heparin migration time is about 2.5% (n = 5). The interaction mechanism between selected polyamines and heparin is explored using conductivity measurements in addition to CE experiments to show that an ion-pairing mechanism is likely.     

毛细管区带电泳法测定粉针剂中头孢拉定的含量

用毛细管区带电泳法测定头孢拉定的含量 ,未涂层毛细管柱 (75 μm×48.5cm ,有效长度 40cm) ,电压 2 8kV ,检测波长 2 3 0nm ,温度 2 0℃ ,进样 5×1 0 3Pa× 3s。运行缓冲液为 2 5mmol/L硼砂缓冲液。方法的线性范围 3 1 .2 2μg/mL～ 749.2 8μg/mL ,检测限为 1 .1 7μg/mL。     

Determination of oxytetracycline and some impurities in plasma by non-aqueous capillary electrophoresis using solid-phase extraction

Summary The potential of a non-aqueous, capillary electrophoresis (NACE) system for separating oxytetracycline from three of its impurities—tetracycline, 4-epioxytetracycline and 4-epitetracycline—using UV detection has been studied. The running buffer was: 25 mM sodium acetate, 1 mM EDTA, methanesulfonic acid, pH 4, dissolved in MeOH-ACN (50∶50,v/v). The method was also used to determine these compounds in pig plasma. A solid-phase extraction (SPE) procedure as a clean-up step has also developed. For this we tested Sep Pak C₁₈, LiChrolut EN and OASIS cartridges. OASIS cartridges were best. Recoveries were 90–100% for all compounds except EOTC which had a recovery of 74%.     

Separation, identification, and interaction of heparin oligosaccharides with granulocyte-colony stimulating factor using capillary electrophoresis and mass spectrometry

A capillary electrophoresis (CE) method was developed for the separation of heparin oligosaccharides compatible to study the interactions between the oligosaccharides and granulocyte-colony stimulating factor (G-CSF). Unfractionated heparin was eliminitively degraded to heparin oligosaccharides by an endolytic heparinase. The degraded smaller oligosaccharides (M(r) < 1000) were baseline-separated by CE under a 50 mM phosphate buffer (pH 9.0) in 10 min. Standard heparin disaccharides and larger oligosaccharides (1000 < M(r) < 8000) were all separated under optimized separation conditions. Compared with standard heparin disaccharides, smaller oligosaccharides contained one nonsulfated, two monosulfated, and two disulfated disaccharides, but trisulfated disaccharides were not found. The smaller oligosaccharides were also identified and molecular mass was deduced by electrospray ionization-mass spectrometry (ESI-MS). Furthermore, interactions between G-CSF and the oligosaccharides were studied by using capillary zone electrophoresis (CZE) under the above separation conditions. It was found that larger oligosaccharides could interact with G-CSF while smaller oligosaccharides were not observed to bind to G-CSF under the experimental conditions. In conclusion, the purified heparinase could selectively degrade heparin into oligosaccharides and the interaction between G-CSF and heparin was correlated with the chain length of heparin.     

Linear polyalkylamines as fingerprinting agents in capillary electrophoresis of low-molecular-weight heparins and glycosaminoglycans

Glycosaminoglycan (GAG) analysis represents a challenging frontier despite the advent of many high‐resolution technologies because of their unparalleled structural complexity. We previously developed a resolving agent‐aided capillary electrophoretic approach for fingerprinting low‐molecular‐weight heparins (LMWHs) to profile their microscopic differences and assess batch‐to‐batch variability. In this report, we study the application of this approach for fingerprinting other GAGs and analyze the basis for the fingerprints observed in CE. Although the resolving agents, linear polyalkylamines, could resolve the broad featureless electropherogram of LMWH into a large number of distinct, highly reproducible peaks, longer GAGs such as chondroitin sulfate, dermatan sulfate, and heparin responded in a highly individualistic manner. Full‐length heparin interacted with linear polyalkylamines very strongly followed by dermatan sulfate, whereas chondroitin sulfate remained essentially unaffected. Oversulfated chondroitin sulfate could be easily identified from full‐length heparin. Scatchard analysis of the binding profile of enoxaparin with three linear polyalkylamines displayed a biphasic binding profile suggesting two distinctly different types of interactions. Some LMWH chains were found to interact with linear polyalkylamines with affinities as high as 10 nM, whereas others displayed nearly 5000‐fold weaker affinities. These observations provide fundamental insight into the basis for fingerprinting of LMWHs by linear polyalkylamine‐based resolving agents, which could be utilized in the design of advanced resolving agents for compositional profiling, direct sequencing, and chemoinformatics studies.     

Determination of levodopa by capillary electrophoresis with chemiluminescence detection

A rapid and simple method using capillary electrophoresis (CE) with chemiluminescence (CL) detection was developed for the determination of levodopa. This method was based on enhance effect of levodopa on the CL reaction between luminol and potassium hexacyanoferrate(III) (K₃[Fe(CN)₆]) in alkaline aqueous solution. CL detection employed a lab-built reaction flow cell and a photon counter. The optimized conditions for the CL detection were 1.0 × 10⁻⁵ M luminol added to the CE running buffer and 5.0 × 10⁻⁵ M K₃[Fe(CN)₆] in 0.6 M NaOH solution introduced postcolumn. Under the optimal conditions, a linear range from 5.0 × 10⁻⁸ to 2.5 × 10⁻⁶ M (r = 9991), and a detection limit of 2.0 × 10⁻⁸ M (signal/noise = 3) for levodopa were achieved. The precision (R.S.D.) on peak area (at 5.0 × 10⁻⁷ M of levodopa, n = 11) was 4.1%. The applicability of the method for the analysis of pharmaceutical and human plasma samples was examined.     

Indirect determination of chloride and sulfate ions in alcohol fuel by capillary electrophoresis

A capillary zone electrophoresis method using indirect UV detection for the analysis of chloride and sulfate in alcohol fuel samples was developed. The anions were analyzed in less than 3 min using an electrolyte containing 10 mmol l^–1 chromate and 0.75 mmol l^–1 hexamethonium bromide (HMB) as electroosmotic flow modifier. Coefficients of variation were better than 0.6% for migration time (n=10) and between 2.05 and 2.82% for peak area repeatabilities. Analytical curves of peak area versus concentration in the range of 0.065–0.65 mg kg^–1 for chloride and 0.25–4.0 mg kg^–1 for sulfate were linear with coefficients of correlation higher than 0.9996. The limits of detection for sulfate and chloride were 0.033 and 0.041 mg kg^–1, respectively. Recovery values ranged from 85 to 103%. The method was successfully applied for the quantification of sulfate and chloride in five alcohol fuel samples. The concentration of sulfate varied from 0.45 to 3.12 mg kg^–1. Chloride concentrations were below the methods LOD.     

Studies on interactions of programmed cell death 5 (PDCD5) and its related peptides with heparin by capillary zone electrophoresis

Capillary zone electrophoresis (CZE) was used to investigate interactions between heparin and programmed cell death 5 (PDCD5), and between heparin and PDCD5-related peptides. Samples containing PDCD5, PDCD5-related peptides, and heparin at various ratios were incubated at room temperature and then separated by CZE with tris-acetate buffer at pH 7.2. Both qualitative and quantitative characterizations of the binding of PDCD5 and PDCD5-related peptides to heparin were determined. The changes in the signals of PDCD5 and PDCD5-related peptides were monitored by comparing the electropherograms of the mixtures containing PDCD5 and heparin and PDCD5-related peptides and heparin with that of PDCD5 or PDCD5-related peptides only. The binding constant of the interaction between PDCD5 and heparin was calculated as 4.17 × 10⁴ M⁻¹ by Scatchard analysis. Our investigations show that it is possible to characterize the interaction between PDCD5 and heparin quantitatively and the interaction between PDCD5-related peptides and heparin qualitatively using CZE.     

毛细管电泳安培法检测酚类化合物

使用自行设计组装的毛细管电泳柱端安培检测系统 ,对四个酚类化合物进行了分离检测。研究了工作电极、缓冲液及其 p H值、检测电压和分离电压对分离检测的影响。在优化条件下 ,4个酚在 5× 1 0 -6～ 5× 1 0 -4 mol/L范围内峰高与浓度成良好的线性关系 ,检测下限为 8.5× 1 0 -7mol/L     

毛细管电泳安培法测定脂可平胶囊中的姜黄素

采用毛细管电泳柱端安培检测对脂可平胶囊中的姜黄素进行测定。着重研究了缓冲溶液浓度和酸碱度、检测电位、进样时间和高压对分离测定的影响。以微Pt电极为工作电极,电极电位为 1.0 V,以V(甲醇)∶V(乙醇)∶V(水)=5∶2∶3为非水介质,磷酸二氢钾和硼砂(pH 9.5)为缓冲体系,并用二阶样条小波进行滤波处理,姜黄素在1.0~120 mg/L范围内,峰高与其质量浓度呈良好的线性关系,线性回归方程:Y=20.2 146ρ,检出限为0.02 mg/L。