Determination of saccharides by high performance anion-exchange chromatography with pulsed amperometric detection

Summary High performance anion-exchange chromatography (HPAC) coupled with pulsed amperometric detection (PAD) under alkaline conditions (pH 13) separates neutral saccharides based upon their molecular size, saccharide composition, and glycosidic linkages. Carbohydrates were detected by oxidation with a gold-working electrode. HPAC-PAD was compared to high performance liquid chromatography (HPLC) with refractive index (RI) detection in terms of selectivity and sensitivity of saccharides. The results indicate that HPAC-PAD was more precise, two orders of magnitude more sensitive (pmol range) and gives better resolution of saccharides than HPLC-RI. HPAC-PAD required less sample preparation and was not subjected to matrix interferences. The use of HPAC-PAD was applied to the analysis of organic materials (plant residues, animal wastes and sewage sludge) and soil.     

高效阴离子交换色谱-脉冲安培法检测小鼠尿液中的甘露醇、单糖和乳果糖

采用高效阴离子交换色谱-脉冲安培检测法(HPAEC-PAD)建立了小鼠尿液中甘露醇、单糖(包括半乳糖、葡萄糖、甘露糖和果糖)和乳果糖的分析方法。样品经离心沉淀除去蛋白并过分子膜,以CarboPacTM PA1阴离子交换柱为分离柱,采用NaOH梯度淋洗,脉冲安培四电位检测。结果表明,甘露醇、半乳糖、葡萄糖、甘露糖、果糖和乳果糖在0.1～5.0 mg/L内线性良好,线性相关系数r2为0.988～0.999,样品加标回收率为95.5%～104.2%,检出限为0.0013～0.0048 mg/L。此法准确、快速、简便,能同时对6种糖类化合物进行分析,可以跟踪检测整个糖类代谢过程中甘露醇、单糖和乳果糖之间的代谢关系。     

Determination of glycuronic acids by high-performance anion chromatography with pulsed amperometric detection

Summary Acid hydrolysis (0.25M H₂SO₄) coupled with enzyme catalysis (pectolyase and β-D-glucuronidase) were employed to extract galacturonic and glucuronic acids from microbial polysaccharides, plant residues, animal wastes, sewage sludge and soil. The glycuronic acids were separated by high-performance anion chromatography (HPAC) on a strong anion-exchange column using 0.1M sodium hydroxide with 0.25M sodium acetate as the mobile phase and determined by pulsed amperometric detection (PAD). HPAC-PAD was found to be superior to high-performance liquid chromatography with ultra-violet (UV) detection in terms of resolution and sensitivity of glycuronic acids. HPAC-PAD was not subject to interferences present with low UV detection (210 nm) and was highly selective for glycuronic acids. Enzymatic hydrolysis after treatment with mild acid (0.25M H₂SO₄) released galacturonic acids from orange peel and pectin, while glucuronic acid was released from Acacia powder. Large amounts of glycuronic acids were also extracted from plant materials. Low levels of uronic acids were detected in poultry manure, sewage sludge and organic-amended soils.     

阴离子交换色谱-脉冲安培法检测味精中的硫化物

建立了阴离子交换色谱柱分离、脉冲安培检测器测定味精中微量硫化物的方法。味精样品经2 g/L氢氧化钠溶液直接溶解并过滤后直接进样,硫离子与高浓度谷氨酸等阴离子在IonPac AS7阴离子色谱柱(250 mm×4 mm)上可实现较好的分离。以100 mmol/L氢氧化钠-500 mmol/L醋酸钠-0.5%乙二胺为淋洗液等度淋洗,6 min内可完成一次样品测定。脉冲安培检测器检测硫离子的检出限(25 μL进样,信噪比为3)为0.3 μg/L,并具有较宽的线性范围(0.001～1 mg/L),样品加标回收率为94.2%～99.0%。应用该方法检测99%味精、增鲜味精和加盐味精样品中的硫化物,结果表明该方法具有简便快捷、高选择性、高灵敏度等优点。     

Determination of several sugars in serum by high-performance anion-exchange chromatography with pulsed amperometric detection

In this paper, a sensitive, simple and direct method for simultaneous determination of glucose, ribose, isomaltose and maltose in serum sample by high-performance anion-exchange chromatography coupled with integrated pulsed amperometric detection was developed. The four target analytes were easily and completely separated on an anion-exchange column at a flow-rate of 0.25 mL/min by binary step gradient elution in about 16 min and the two eluents were deionized water and 500 mM sodium hydroxide, respectively. The separated four analytes were detected directly by using a gold electrode and quadruple-potential waveform integrated pulsed amperometry without derivatization. Under the optimized conditions, when the injection volume was 25 microL, the detection limits (signal-to-noise ratio equal to 3) for glucose, ribose, isomaltose and maltose were 0.92, 7.50, 12.9 and 10.3 ng/mL, respectively. The calibration graphs of peak area for the four analytes were linear over two to three orders of magnitude with correlation coefficients greater than 0.998. R.S.D. of peak areas of the four analytes for five determinations were no more than 5.6%. The analytical method had been applied to the determination of glucose, ribose, isomaltose and maltose in real serum samples and good results with low relative standard deviation not more than 5.3% were obtained. The accuracy of the proposed method was tested by recovery measurements on spiked samples and good recovery results (98.1-107.9%) were obtained.     

糖的高效阴离子交换色谱-脉冲安培检测法分析

高效阴离子交换色谱-脉冲安培检测法是新近发展的一种分析糖的有效方法。本文讨论了糖的高效阴离子交换色谱分离和脉冲安培法检测的原理,色谱条件的选择,包括分离糖的固定相、流动相以及检测糖的脉冲电位波形,并对方法的应用进行了较为详细的叙述。     

Determination of zidovudine using anion exchange chromatography with integrated pulsed amperometric detection

A rapid and practical method for direct detection of zidovudine in high performance anion exchange chromatography(HPAEC) has been developed with integrated pulsed amperometric detection(IPAD).Dionex AS 18(250 mm×2 mm) and AG 18 (50 mm x 2 mm) columns and 11 mmol/L NaOH solution were used for separation.Multi-step potential waveform parameters were optimized to maximize the signal-to-noise ratio(S/N).Utilizing an optimized waveform,the repeatability(intra-day) precision and intermediate(inter-day) precision are obtained with relative standard deviation(RSD) of 1.88,2.27,respectively.The limit of quantification(LOQ) and limit of detection(LOD) were found to be 9.70,3.0 ng/mL,respectively,with correlation coefficients of 0.9992 over concentration range 0.01-10μg/mL.The present method was successfully applied to the determination of zidovudine in human plasma.The recoveries of plasma sample spiked by 0.7μg/mL,2.7μg/mL obtained were 95.3-101.5%, with relative standard deviation(RSD) of 2.54%,2.21%,respectively.     

Preparation of amino-zirconia bonded phases and their evaluation in hydrophilic interaction chromatography of carbohydrates with pulsed amperometric detection

A series of non-porous, microspherical zirconia-based stationary phases with surface bound amine functionality have been introduced and evaluated in hydrophilic interaction chromatography (HILIC) of underivatized, neutral carbohydrates and anion exchange chromatography of nucleotides using pulsed amperometric detection and ultraviolet detection, respectively. Three aminopropyl alkoxysilane compounds were used in the surface modification of the non-porous zirconia support, namely 3-aminopropyltrimethoxysilane (monoamine), N-(2-aminoethyl)-3-aminopropyltrimethoxysilane (diamine), and trimethoxysilylpropyldiethylenetriamine (triamine). Due to the relatively low specific surface area of the non-porous zirconia support used in this study (ca. 7.3 m²/g), zirconia with surface coating of the triamine type yielded the best results as far as the separations of chitooligosaccharides and maltooligosaccharides are concerned. Since a non-porous zirconia could be readily modified with amine functionality via Zr? O? Si bonds, it is expected that all the three aminopropyl alkoxysilane compounds would yield satisfactory results with porous zirconia microparticles because of their much higher specific surface areas. Although the non-porous zirconia exhibited some limitations, the present study has demonstrated that microspherical zirconia particles are suitable supports for the production of polar sorbents for HILIC of carbohydrates. Another surface modification, which involved the activation of the zirconia surface with aldehyde groups followed by reductive amination with tetraethylenepentamine, was also evaluated. Although this chemistry would in principle yield sorbents with higher concentration in amine groups, the conversion of the majority of the primary amine groups of the tetraethyle-nepetamine molecules to secondary amine functions in the course of the reductive amination reaction have provided a stationary phase that did not afford satisfactory resolution for carbohydrates. However, this same stationary phase behaved as a weak anion exchanger and allowed the high resolution separation of nucleoside-5′-mono-, -di-, and triphosphates. Overall, the results obtained with zirconia-based hydrophilic sorbents paralleled those obtained on amino-silica bonded phases.     

Determination of carnosine in feed and meat by high-performance anion-exchange chromatography with integrated pulsed amperometric detection

Carnosine (beta-alanyl-L-histidine) is a dipeptide regarded as an important molecular marker of the presence of processed animal proteins including meat and bone meal in animal feed. For its identification and quantification a sensitive and selective method by high-performance anion-exchange chromatography coupled with integrated pulsed amperometric detection (HPAEC-IPAD) was developed. The assay is based on isocratic elution with 100 mM NaOH as the mobile phase. Interferences of real matrices were efficiently removed; carnosine could be determined at the concentration ranges 0.1-100 microM with a rather low detection limit of 0.23 ng. Unlike feeds for dogs and cats, no carnosine peak was observed in all examined feeds for ruminants. The good analytical characteristics allowed camosine determination down to 5 microg/g of feed.     

高效阴离子交换色谱-脉冲安培检测法测定乳粉中的低聚果糖

建立了测定乳粉中低聚果糖的高效阴离子交换色谱-脉冲安培检测(HPAEC-PAD)方法。样品经50%(v/v)乙醇水溶液提取,On Guard RP柱除脂,CarboPac PA200阴离子交换色谱柱(250 mm×3 mm)分离。采用水、0.2 mol/L NaOH溶液和0.4 mol/L NaAc溶液三元梯度淋洗,流速为0.4 mL/min,柱温为30 ℃,进样量为25 μL,脉冲安培检测器检测。蔗果三糖、四糖、五糖在0.05~10 mg/L范围内线性关系良好,相关系数(r²)> 0.9993;定量限分别为0.02、0.005和0.02 mg/L。在0.5、1.0、5.0 mg/L添加水平下,蔗果三糖、四糖、五糖的平均回收率为86.0%~114.0%。该方法处理简单、结果准确、灵敏度高,适用于乳粉中低聚果糖的测定。     

高效阴离子交换色谱-脉冲安培检测法定量测定低聚木糖样品中的低聚木糖

建立了低聚木糖样品中的木二糖至木六糖等低聚木糖的高效阴离子交换色谱定量测定方法,并根据低聚木糖的聚合度与色谱保留时间的线性关系,对木七糖和木八糖的保留时间进行预测。采用CarboPacTM PA200阴离子交换柱(3 mm×250 mm),以醋酸钠和氢氧化钠为淋洗液进行二元梯度洗脱,脉冲安培法进行检测。结果表明,木二糖至木六糖在0.804～8.607 mg/L质量浓度范围内的线性关系良好,检出限为0.064～0.111 mg/L,定量限为0.214～0.371 mg/L。将该方法用于低聚木糖产品的检测,3个添加水平的加标回收率为84.29%～118.19%,相对标准偏差(n=3)为0.44%～14.87%。结果表明该方法适用于低聚木糖产品中有效成分的快速、高效分离和定量测定。     

高效阴离子交换色谱-积分脉冲安培检测法同时测定黄酒中的单糖和低聚糖及其指纹图谱的构建

建立了黄酒中葡萄糖、果糖、异麦芽糖、异麦芽三糖、麦芽糖、潘糖、麦芽三糖共7种糖类的高效阴离子交换色谱分离-积分脉冲安培检测的方法。以氢氧化钠和醋酸钠为淋洗液进行梯度洗脱,CarboPac^TM10阴离子交换柱进行分离。该方法可在26 min内实现黄酒中7种糖类的定量分析,在0.5~50 mg/L浓度范围内线性关系良好,样品定量限为0.1 g/L,加标回收率为76.5%~108.4%,精密度(RSD)为3.02%~8.23%。将该法用于不同厂家不同批次的绍兴加饭酒中各种糖类含量的检测,基于所得的检测结果,利用中位数法构建了标准指纹图谱,并采用夹角余弦法评价了各个样品与标准指纹图谱之间的相似度。结果初步显示,不同厂家加饭酒之间的相似度差别较大,该法可作为区别不同厂家酒的依据之一。     

固相萃取-高效阴离子交换色谱-积分脉冲安培法检测人体尿液中的异黄蝶呤

建立了固相萃取-高效阴离子交换色谱-积分脉冲安培法(SPE-HPAEC-IPAD)测定人体尿液中异黄蝶呤的分析方法。尿液经ENVI-18与732型阳离子交换柱串联萃取后,除去了大量干扰物质。采用IonPac AS21分析柱(250 mm×2 mm),以0.025 mol/L NaOH溶液为淋洗液,流速为0.40 mL/min,在优化的安培检测波形条件下,异黄蝶呤的质量浓度在0.005～0.200 mg/L范围内与峰面积呈良好的线性关系,相关系数为0.998 4,检出限为0.003 mg/L。健康人及癌症病人尿液在2 mg/L和5 mg/L两个添加水平的平均回收率在95.4%～96.8%之间,相对标准偏差小于5%。此方法环保、快速、准确,可用于健康人与癌症病人尿液中异黄蝶呤的测定。     

Determination of phenylalanine in blood by high-performance anion-exchange chromatography–pulsed amperometric detection to diagnose phenylketonuria

We have developed a high-performance anion-exchange chromatography with pulsed amperometric detection method for the detection of phenylalanine (Phe) and diagnosis of phenylketonuria (PKU). Sample pretreatment steps were simplified without derivatization. The analyte was separated within 5 min. The detection limit (S/N = 3) for Phe was 50 pg. Linear dynamic range was 1.23–14.43 mg/dL (r² = 0.9999) for a dried blood spot. The mean recoveries of Phe for intra- and inter-day assays were found to be 96.87–104.16%. This method clearly differentiated PKU-positive groups from normal groups, and proved to be a practical procedure for rapid screening and follow-up monitoring of PKU.     

Direct determination of hydrogen cyanide in cigarette mainstream smoke by ion chromatography with pulsed amperometric detection

The determination of hydrogen cyanide in cigarette mainstream smoke has been achieved by ion chromatography (IC) with pulsed amperometric detection (PAD). The proposed method of totally trapping whole cigarette mainstream smoke by Cambridge filters, which are treated with sodium hydroxide/ethanol solution, possesses the advantage of fast analysis time over the widespread used solution absorption method. The possible co-existing interferents are evaluated under the optimized detection conditions and excellent recoveries of cyanide are obtained. The cyanide content of absorption solution can be directly determined by the optimized IC-PAD method without any pretreatments. The linear range is 0.0147-2.45 μg/mL with R2 value of 0.9997. The limit of the detection is 3 μg/L for a 25 μL injection loop. The overall relative standard deviation of the method is less than 5.20% and the recovery range from 94.3% to 101.0%. The results obtained from the developed method are in good agreement with that of continuous flow analyzer (CFA) method.     

微渗析活体取样与液相色谱安培检测法联用测定鼠脑中黄嘌呤和次黄嘌呤

采用微渗析活体取样技术和高效液相色谱安培检测法 ,测定了鼠脑纹状体中的黄嘌呤和次黄嘌呤。安培检测以玻碳电极为工作电极 ,检测电位为 0 .9V。在 5 .0× 1 0 - 7～ 1 .0× 1 0 - 4 mol/L浓度范围内 ,黄嘌呤和次黄嘌呤的浓度分别与峰电流呈良好的线性关系 ,检出限分别为 8 0× 1 0 - 8mol/L和 3 0× 1 0 - 7mol/L。该方法为生命科学的研究提供了一种新的分析手段     

高效阴离子交换色谱-脉冲安培检测法分析啤酒和麦汁中的糖

建立了高效阴离子交换色谱-脉冲安培检测(HPAEC-PAD)同时测定单糖、二糖和多种低聚糖的方法。采用水、0.25 mmol/L NaOH溶液和1 mol/L NaAc溶液三元梯度淋洗,在CarboPac PA-100色谱柱上,11种糖在40 min内达到良好分离;采用积分脉冲安培检测方法,无需对样品进行复杂的前处理或衍生反应便可直接检测。11种糖的检出限(S/N=3)在13～88 pg范围内。将该方法用于啤酒和麦汁样品中单糖、二糖及低聚糖的分析取得了很好的结果,样品中的加标回收率为81%～107%。     

Detection of albiflorin and paeoniflorin in Paeoniae Radix by reversed-phase high-performance liquid chromatography with pulsed amperometric detection

We have developed a reversed-phase high-performance liquid chromatography-pulsed amperometric detection (RP-HPLC-PAD) method for the detection of albiflorin and paeoniflorin in Paeoniae Radix and Wu-ji-san. Albiflorin and paeoniflorin were completely separated using 10% acetonitrile in 5 mM sodium phosphate buffer (pH 3.0) as an eluent and detected by PAD under alkaline conditions after using a post-column delivery system. The limit of detection (S/N = 3) and the limit of quantification (S/N = 10) were 0.10 and 0.35 ng for albiflorin, and 0.20 and 0.50 ng for paeoniflorin, respectively. The coefficients of linear regression were 0.9995 and 0.9999 for concentrations between 0.035 and 100 μg/mL. The intra- and inter-day precision (RSDs) was less than 3.56% in Paeoniae Radix and Wu-ji-san. The average recoveries from Paeoniae Radix and Wu-ji-san were 99.01–100.94% and 99.46–100.64%. This method shows higher selectivity than HPLC–UV method for analyzing albiflorin and paeoniflorin in Chinese medicinal preparation.     

High-performance anion-exchange chromatography of carbohydrates using a new resin and pulsed amperometric detection

Summary This paper reports the results of a study on the use of a new polymer-based, strong anion-exchange, stationary phase for rapid and selective separation of carbohydrates and related compounds by high-pH, anion-exchange chromatography with pulsed amperometric detection. The new adsorbent has been obtained by direct nitration of 2.8 μm, spherical non-porous highly cross-linked, styrene-divinylbenzene copolymer beads, followed by reduction of superficially introduced nitro groups with nascent hydrogen and quaternization of the resultant amino groups with iodomethane. It is reported that by optimizing the ionic strength of the mobile phase, columns packed with the new anion-exchanger can be successfully employed to separate, either in isocratic or gradient elution mode, oligosaccharides, positional isomers of gluco-disaccharides, as well as uronic acids and sugar monophosphates.     

高效阴离子色谱法测定保健食品中的盐酸氨基葡萄糖

建立了保健食品中盐酸氨基葡萄糖的高效阴离子色谱测定方法。在AminoPac PA10离子色谱分离柱(2 mm×250 mm)上,以250 mmol/L NaOH溶液作为流动相,利用Au工作电极、pH参比电极的脉冲安培检测器测定了盐酸氨基葡萄糖。方法的线性范围为0.05～10.0 mg/L,检出限为0.012 mg/L,标准品和样品测定的相对标准偏差分别为0.69%和1.38%。用该法测定了保健食品中盐酸氨基葡萄糖,取得了较为满意的结果,加标回收率为96.6%～105.2%,与国家标准方法的测定结果对照,相对偏差为-1.4%～1.0%,表明所建立的方法具有高的灵敏度和精密度,适合于保健食品中盐酸氨基葡萄糖的分析。