Liquid chromatographic analysis of glucosamine in commercial dietary supplements using indirect fluorescence detection

A method of using indirect fluorescence detection is evaluated for the analysis of glucosamine in commercial dietary supplements following chromatographic separation. In this method, the eluting analyte, glucosamine, was detected by monitoring an increase in the fluorescence signal for L-tryptophan (L-Trp) or DL-5-methoxytryptophan (5-MTP) after glucosamine complexed with a copper(II) ion and released either L-Trp or 5-MTP from a copper(II) complex, which is introduced postcolumn. The fluorescence of L-Trp and 5-MTP are quenched when complexed with the copper(II) ion. The results obtained using indirect fluorescence detection are compared with the results obtained for precolumn derivatization with phenylisothiocyanate. Statistical analysis is performed to compare the results obtained for the two postcolumn interaction components, Cu(L-Trp)2 and Cu(5-MTP)2, as well as the results obtained using the indirect fluorescence detection method and a precolumn derivatization method. The indirect fluorescence detection method provided an alternative to precolumn derivatization for determining the concentration of glucosamine in commercial dietary supplements. The stability of the glucosamine-o-phthalaldehyde-3-mercaptopropionic acid derivative is also evaluated to investigate the applicability of the popular precolumn derivatization reagent, o-phthalaldehyde-3-mercaptopropionic acid, for this analysis.     

Biochemical characteristics, metabolism, and antitumor activity of several acetylated hexosamines

We have synthesized several potential inhibitors and/or modifiers of the carbohydrate portion of plasma membrane glycoconjugates. These include fluorinated and actylated analogs of D-glucosamine, D-galactosamine, and D-mannosamine. These compounds have been tested to determine their effects on both[14C] glucosamine and [3H] leucine incorporation into glycoconjugate and on cell growth and viability using P-288 murine lymphoma cells maintained in tissue culture. The most cytotoxic agent tested was 2-acetamido-2-deoxy-1,3,4,6-tetra-O-acetyl-beta-D-glucopyranose or simply beta-pentaacetylglucosamine which prevented cell growth at 10(-4)-10(-3) M. beta-Pentaacetylglucosamine cytotoxicity was correlated with its high lipid solubility, having an octanol/water partition coefficient of 0.424 as compared with 0.278 for the alpha-anomer and 0.017 for N-acetylglucosamine. In vitro metabolism studies with [4C]- and/or [3H]-labeled pentaacetylglucosamine have indicated intracellular de-O-acetylation leading to the biosynthesis of UDP-N-acetylglucosamine, followed by the incorporation of this sugar into cellular glycoprotein. Concomitant with the formation of increased amounts of this nucleotide sugar, intracellular UTP and CTP pools fell to one third normal within 3 h after the administration of 1 mM pentaacetylglucosamine. At present it is unclear whether the cytotoxicity of beta-pentaacetylglucosamine or other similar agents is due to alterations in nucleotide and nucleotide-sugar pools causing a decrease in energy charge and polynucleotide biosynthesis or is due to a direct effect on membrane glycoconjugate biosynthesis.     

A gas chromatographic/mass spectrometric method for tracing the microbial conversion of glucose into amino sugars in soil

Amino sugars in soils are heterogeneous and have been used as microbial residue biomarkers to investigate the microbial contribution to soil organic matter. However, it is not clear what the available carbon source is and how glucose is utilized for the synthesis of soil amino sugars. This paper presents a new gas chromatography/mass spectrometry (GC/MS) approach for the identification of 13C incorporation into three amino sugars, D-glucosamine, D-galactosamine, and muramic acid, in soil incubated with U-13C-glucose. Method evaluation showed that the chemical ionization (CI) mode was suitable for all these amino sugars, but that electron impact (EI) mode was applicable only to glucosamine and galactosamine. The 13C conversion rate was estimated based on the abundance ratio of the ions corresponding to the masses of the ions F+n and F (where n is the skeleton carbon number in the fragment ions F of the amino sugars) and calculated as atom percentage excess. The reproducibility of the method was excellent and clearly adequate for the present purpose. In addition, the new approach is highly accurate as tested with mixtures of U-13C-glucose and natural glucose.     

Liquid chromatography and postcolumn indirect detection of glyphosate.

Glyphosate [N-(phosphonomethyl)glycine] and its metabolite aminomethylphosphonic acid (AMPA) were separated and detected by a postcolumn indirect detection strategy. Separation can be done on a cation-exchange column, where glyphosate elutes before AMPA, or on an anion-exchange column, where the elution order is reversed. Detection was achieved by using a fluorescent Al(3+)-morin postcolumn reagent. When the postcolumn reagent combines with the column effluent in a mixing tee, the fluorescence decreases in the presence of both analytes. Variables affecting the postcolumn indirect fluorescence detection were established and optimized; the major factors were postcolumn pH and volume and temperature of the postcolumn reaction coil. Detection limits, defined as three times the background noise, for glyphosate and AMPA separated on an anion-exchange column were 14 and 40 ng, respectively.     

Postcolumn fluorescence as an alternative to evaporative light scattering detection for ceramide analysis with gradient elution in non-aqueous reversed-phase liquid chromatography

Ceramide analysis was developed with gradient elution in non-aqueous reversed-phase liquid chromatography with evaporative light scattering detection (ELSD) or postcolumn fluorescence detection. Fluorescence detection (excitation, 360 nm; emission, 425 nm) after postcolumn formation of mixed assemblies between eluted ceramides and 1,6-diphenyl-1,3,5-hexatriene was developed. In comparison with ELSD, fluorescence detection allows a better detection of the minor species ceramide from ceramide type III (commercial mixture of non-hydroxy fatty acid-sphingosine) and appears to be more sensitive for quantitation of ceramides at low concentrations. The fluorescence response is linear over a wide range of injected amount of ceramide III (expressed as stearoyl-phytosphingosine): 10 ng to 1000 ng. The response of ELSD is non linear but can be linearized in double logarithmic coordinates for calculations over a narrow range, e.g. between 10 to 350 ng ceramide III injected. The lower quantitation limits of these two detectors are similar: 5 ng ceramide III was injected.     

Capillary liquid chromatography fraction collection and postcolumn reaction using segmented flow microfluidics

A challenge for capillary LC (cLC) is fraction collection and the manipulation of fractions from microscale columns. An emerging approach is the use of segmented flow or droplet technology to perform such tasks. In this work, a fraction collection and postcolumn reaction system based on segmented flow was developed for the gradient cLC of proteins. In the system, column effluent and immiscible oil are pumped into separate arms of a tee resulting in regular fractions of effluent segmented by oil. Fractions were generated at 1 Hz corresponding to 5 nL volumes. The fraction collection rate was high enough to generate over 30 fractions per peak and preserve chromatographic resolution achieved for a five‐protein test mixture. The resulting fractions could be stored and subsequently derivatized for fluorescence detection by pumping them into a second tee where naphthalene dicarboxyaldehyde, a fluorogenic reagent, was pumped into a second arm and added to each fraction. Proteins were derivatized within the droplets enabling postcolumn fluorescence detection of the proteins. The experiments demonstrate that fraction collection from cLC by segmented flow can be extended to proteins. Further, they illustrate a potential workflow for protein analysis based on postcolumn derivatization for fluorescence detection.     

色氨酸与功能单体自组装体系的分子间非共价相互作用

利用荧光猝灭分析、 三维荧光光谱和计算机模拟等方法分别研究色氨酸(L-Trp)与己二酸二酰肼(ADH)、 双丙酮丙烯酰胺(DAAM)和丙烯酸(AA)3种功能单体间的非共价相互作用. 考察了荧光猝灭机理、 色氨酸-功能单体的相互作用强弱以及作用力类型. 研究结果表明, 功能单体对L-Trp的荧光猝灭过程是由于形成不发射荧光的复合物而引起的静态猝灭; L-Trp与功能单体的结合常数较大, 所形成的复合物较稳定, 非共价作用中以氢键和静电作用贡献最大; L-Trp主要通过羧基与功能单体产生相互作用, 芳杂环与功能单体间的相互作用相对较弱; L-Trp与功能单体相互作用的强弱顺序为L-Trp-ADH > L-Trp-DAAM > L-Trp-AA.     

Rapid analysis of amino sugars by microchip electrophoresis with laser-induced fluorescence detection.

Microchip electrophoresis for the short-time analysis of amino sugars is described. D-Glucosamine, D-galactosamine and their reduced forms were labeled with 4-nitro-2,1,3-benzoxadiazole 7-fluoride (NBD-F) at pH 6.0 and the fluorescent derivatives were purified on an octadecyl silica (ODS) gel plate. The derivatives were analyzed by electrophoresis on a microfabricated chip with a 33 mm long separation channel with argon laser-induced fluorescence detection. Under the established conditions, these amino sugarderivatives were well separated from each other within 60 s. Amino sugars of as small an amount as 0.5 fmol could be detected with a signal-to-noise (S/N) ratio of 3, and peak response showed good linearity between at least 0.8 and 8 fmol of samples with a relative standard deviation (RSD) of ca. 4%. This method was also applied to the analysis of amino sugar composition of O-linked glycans released from bovine submaxillary mucin with alkali in the presence of borohydride. The result of amino sugar composition analysis for individual O-glycans fractionated by high-performance liquid chromatography was quite useful for their identification.     

还原性负离子的间接荧光检测离子色谱法

采用４个铈柱后反应和３价铈荧光检测的离子色谱法分离维生素Ｃ,亚硝酸根,硫代硫酸根,亚硫酸根,草酸根和碘离子６种还原性负离子,同时也给出了使用这种方法的一些最佳条件。     

HPLC analysis of an amino bisphosphonate in pharmaceutical formulations using postcolumn derivatization and fluorescence detection

Monosodium 4-amino-1-hydroxybutane-1, 1-diphosphonic acid (MK-217) is a bone resorption inhibitor implicated in the treatment of malignant hypercalcemia. This compound is very water soluble and has five ionizable groups with pKa values over the entire pH range. As a result, it is difficult to maintain a single species in solution for chromatographic separation. Since there is no chromophore in the molecular structure, UV detection is ineffective. The compound and its potential degradation products are separated by ion-pair chromatography using 0.01 M cetyltrimethylammonium bromide as the ion-pairing agent and a polymeric stationary phase. Detection is by fluorescence detection after postcolumn derivatization of the primary amine with ophthalaldehyde and mercaptoethanol (OPA-MERC). Optimization of the chromatographic separation and the postcolumn reaction has been carried out, and the method has been applied to the analysis of MK-217 in intravenous solutions and tablet formulations.     

Postcolumn derivatization of amino acids using reaction flow chromatography columns with fluorescence detection: A fast new approach to selective derivatization techniques

Reaction flow (RF) chromatography with fluorescamine reagent and fluorescence detection (FLD) was used for the analysis of amino acids. The performance of RF chromatography was tested against several optimized conventional postcolumn derivatization (PCD) methods. RF columns achieved greater sensitivity compared to conventional PCD methods, without the need for reaction loops, which resulted in more efficient separations. The RF-PCD method also achieved limits of detection (LOD) from the low picomole to subnanomole range. The calibration data of the RF-PCD technique yielded R²?≥?0.99 and % relative standard deviation in peak areas ranging from 0.34% to 5%. Through reaction flow chromatography, multiplexed detection was also achieved allowing the monitoring and analysis of derivatized and nonderivatized flow streams simultaneously.     

Separation of pectin methylesterase isoenzymes from tomato fruits using short monolithic columns

One of the main forms of tomato pectin methylesterase (PME; EC 3.1.1.1.1) that is applicable to the food industry was isolated from fresh tomato fruit. The extraction of the PME isoenzymes involved washing the fresh tomato flesh with water in order to remove sugars and than solubilizing the enzymes with a diluted HCl solution at pH 1.6. The extract was then neutralized to pH 7.4 using buffer solution. After filtration, the solution was directly fractioned using Convective Interaction Media (CIM) short monolithic disk column bearing sulfonyl (SO3) groups and using a linear gradient from 0 to 700 mM NaCl. The injection volume was 3 ml and the diameter of the column was 12 mm and length 3 mm. The isolated fractions were monitored for protein content and PME activity. The fraction with the targeted enzyme, which showed NaCl independent activity, was further purified and concentrated by ultrafiltration and finally purified by a second semi-preparative cation-exchange chromatography step using a CIM carboxymethyl (CM) disk monolithic column consisting of two disks and applying a step gradient. From 1 kg of fresh tomato fruits, 7.5 mg of purified PME with molecular mass estimated to be 26 000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was obtained. A fraction with mixed PME and polygalacturonase activity was also obtained. Compared to the published procedures for the isolation and purification of PME from plant materials, this new procedure is much faster and more efficient. The potential application of CIM disk short monolithic columns in the analysis and semi-preparative extraction and isolation of the PME isoenzyme is presented.     

Sodium N-Chlorobenzenesulfonamide as a Selective Oxidant for Hexosamines in Alkaline Medium: A Kinetic and Mechanistic Study

Oxidation of D-mannosamine (1), D-glucosamine (2), and D-galctosamine (3) by sodium N-chlorobenzenesulfonamide or chloramine-B (CAB) at 313 K is followed by a shortening of carbon chain and obeys the rate law, rate = k[CAB][sugar][HO(-)](x)(), where x is less than unity. The products are arabinonic acid, ribonic acid, and erythronic acid for 1 and 2 with smaller amounts of glyceric and hexonic acids, while lyxonic and threonic acids are predominant in the oxidation of 3 with smaller amounts of glyceric and hexonic acids. Proton inventory studies made in a H(2)O-D(2)O mixture point toward a single transition state. In the proposed mechanism the alkoxy anion (S(-)) of the hexosamine formed in a base-catalyzed reaction at C-1 carbon is subjected to an electrophilic rate-limiting attack by Cl(+) of the oxidant. The hexonic acid formed is decarboxylated with loss of ammonia to form the respective pentose, which is further converted into the corresponding pentonic acid. The breaking of the bond between C-1 and C-2 carbons in pentose yields tetronic acids. The thermodynamic parameters for sugar alkoxy anion formation and activation parameters for the rate-limiting step have been evaluated.     

Metal Ion Chromatography with Fluorescence Detection

Abstract

Nonpolar, agglomerated anion exchanger, and surface-sulfonated cation exchanger stationary phases have been used in conjunction with 8-hydroxyquinoline-5-sulfonic acid (HQS) in the eluent or as a postcolumn reagent for the separation and detection of a number of metals that form fluorescent HQS complexes. Several metals, notably those classified as transition metals, form nonfluorescent HQS chelates and quenches the fluorescence of other metal-HQS metal chelates. Such transition metals have been detected by introducing the fluorescent Al-HQS chelate postcolumn. Cation exchange stationary phases are the most useful for chromatographic applications involving HQS and are able to provide a variety of useful separations by tailoring elution conditions. Although not sensitive to Ba, the approach may be particularly good for the determination of the other alkaline earth metals. Fluorescence quenching resulting from Fe and Ni leaching from stainless steel chromatographic systems present a problem for trace analysis and accentuate the need for nonmetallic hardware. Subpicomole detection limits are attainable for Cd, Mg and Zn.     

Development and performance of an automated HPLC-analyzer for catecholamines

Summary An automated, dual-column HPLC method has been developed that can be used routinely to quantify the parent catecholamines in body fluids. The method is based on a new, laboratory-prepared column material with size exclusion and high performance affinity chromatography properties. A microprocessor controlled column-switching technique is used with an optional integrated reaction-system for postcolumn derivatization for fluorescence detection. Natural fluorescence can be used for higher concentration levels. The commercially available analyzer allows, for the first time, the direct injection (up to 0.5 ml) of an appropriate biological fluid. In its integrated sample-processing mode, it exhibits, chemoselectivity and -specificity, with a detection limit of 2 pg, high precision and speed of analysis.     

Determination of thiamine by high-performance liquid chromatography

High-performance liquid chromatographic methods for the determination of thiamine (vitamin B1) in foodstuffs or biological tissues and fluids are outlined and discussed. The methods are often similar and interchangeable, sample extraction and clean up procedures being the major difference. Most of the methods use either ultraviolet or fluorescence detection. Fluorescence detection requires either precolumn or postcolumn oxidation of thiamine to thiochrome. A number of methods are recommended and problems with standardization are emphasized.     

Postcolumn derivatization of proteins in capillary sieving electrophoresis/laser‐induced fluorescence detection

The separation methods for proteins with high resolution and sensitivity are absolutely important in the field of biological sciences. Capillary sieving electrophoresis (CSE) is an excellent separation technique for DNA and proteins with high resolution, while LIF permits the most sensitive detection in CSE. Therefore, proteins have to be labeled with fluorescent or fluorogenic reagent to produce fluorescent derivatives. Both precolumn and oncolumn derivatization have been employed for the labeling of proteins in CSE. However, there is no report on the postcolumn derivatization due to the limitation in the use of a standard migration buffer, despite it being a promising method for sensitive detection of proteins. Here, we show a novel postcolumn derivatization method for protein separation by CSE, using a tertiary amine as a buffer component in the running buffer. Tris, which is commonly used as a base in CSE separation buffers, was substituted by tertiary amines, 2‐(diethylamino)ethanol and triethanolamine. A buffer solution containing 2‐(diethylamino)ethanol or triethanolamine can be used for the CSE separation followed by the postcolumn derivatization of proteins, since both reagents are unreactive toward a fluorogenic labeling reagent, naphthalene‐2,3‐dicarbaldehyde. Thus, LIF detection using the postcolumn derivatization permits significant reduction in the LOD (by a factor of 2.4–28) of proteins, compared with conventional absorbance detection.     

L-色氨酸分子印迹传感器敏感膜的制备与性能

以L-色氨酸(L-Trp)为模板分子, 邻苯二胺(o-PD)为功能单体, 在金电极表面原位合成了分子印迹聚合物敏感膜; 通过循环伏安法(CV)、 差示脉冲伏安法(DPV)和电化学阻抗谱法(EIS)考察了该电极的性能. DPV测试结果表明, 在1×10^-8~2×10^-7 mol/L范围内, 峰电流与L-Trp的浓度呈线性关系, 检出限为0.3×10^-8 mol/L; 选择识别性实验结果表明, L-Trp印迹敏感膜的印迹因子达到3.72, 相对于干扰物的选择因子均大于1, 对与L-Trp结构相似的L-酪氨酸(L-Tyr)的选择因子也达到2.30, 说明该印迹膜对L-Trp具有良好的选择性; 识别过程动力学研究结果表明, 印迹膜对L-Trp的识别是一个两步连续发生的过程, 即快结合过程和慢吸附过程.     

High-performance liquid chromatographic determination of peptides in biological fluids by automated pre-column fluorescence derivatization with fluorescamine

Peptides containing a free alpha- or epsilon-amino group react with fluorescamine under mild alkaline conditions to generate a highly fluorescent but unstable reaction product and, consequently, practical high-performance liquid chromatographic (HPLC) approaches to analysis have typically involved the use of postcolumn derivatization. An automated precolumn approach is reported in which peptides are reacted with fluorescamine just prior to HPLC analysis by a commercially available autoinjector with derivatization capabilities. The autoinjector added base and fluorescamine reagent solutions to a sample vial containing peptide analytes, and the derivatization reaction was allowed to proceed for 5 min at room temperature prior to injection into the HPLC system. The derivatized peptides were analyzed by reversed-phase HPLC with fluorescence detection (excitation at 390 nm; emission 470-nm cut-off filter) on an octylsilica column. Optimization of the precolumn reaction conditions and the use of narrower HPLC columns (2 mm I.D.) resulted in a typical on-column detection limit of 30-50 fmol of peptide, which was substantially lower than that in previously reported post-column methods. This approach was applied to the HPLC of several naturally occurring and synthetic peptides containing alpha- and epsilon-amino groups. In combination with solid-phase extraction, prior to automated precolumn fluorescence derivatization and chromatographic analysis, the methodology was used for the determination of a synthetic growth hormone-releasing peptide in plasma samples.     

Simultaneous determination of inorganic nitrogen species by microcolumn ion chromatography

Inorganic nitrogen species (nitrate, nitrite and ammonium ions) were simultaneously determined by microcolumn ion chromatography. Nitrate and nitrite were determined by UV detection at 206 nm, whereas ammonium ion was determined by fluorescence detection at excitation 410 nm and emission 470 nm. The latter fluorescence detection is based on the postcolumn reaction of ammonium ion with o-phthalaldehyde in the presence of 2-mercaptoethanol. Effects of the reagent concentration, pH, and other reaction conditions on the signal intensity were examined, and the optimum condition was explored. The present method allowed simultaneous determination of nitrate, nitrite and ammonium ions in river water.