高效阴离子交换色谱-脉冲安培检测法同时测定生物转化样品中的海藻糖、葡萄糖和麦芽糖

建立了高效阴离子交换色谱-脉冲安培电化学检测法同时测定生物转化样品中海藻糖、葡萄糖和麦芽糖的分析方法。选用CarboPac^TM10色谱柱(250 mm×2 mm)对分离条件进行优化,使用标准样品测定了线性范围和工作曲线,柱温为30 ℃,流速为0.30 mL/min,以氢氧化钠溶液和醋酸钠溶液为流动相进行梯度洗脱,脉冲安培法进行检测。研究结果表明,该方法可在15 min内实现海藻糖生物转化液中3种糖的快速定量分析。海藻糖、葡萄糖和麦芽糖峰面积与质量浓度的线性关系良好,检出限为0.010~0.100 mg/L。将此方法用于酶法制备海藻糖的检测,加标回收率为89.40%~103.2%。在生物转化样品中检测到海藻糖浓度为101.084 g/L,转化率达到了50.5%。该方法灵敏度高,简便快速,可应用于海藻糖制备样品中各种成分的分离和定量检测。     

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.     

A sensitive liquid chromatographic method for the analysis of isovaleric and valeric acids in urine as fluorescent derivatives

A simple and sensitive isocratic liquid chromatographic method was developed for the analysis of isovaleric and valeric acids in human urine as biomarkers in metabolic acidosis. The method is based on the derivatization of isovaleric and valeric acids with a fluorescent reagent 2-(2-naphthoxy)ethyl-2-(piperidino)ethanesulfonate for labeling the analytes with the naphthoxy fluorophore. The resulting fluorescent derivatives of isovaleric and valeric acids were separated on a phenyl-hexyl column, using a mixed solvent of methanol-water-tetrahydrofuran (55:31:14, v/v) as the mobile phase. The separated derivatives were monitored with a fluorimetric detector (excitation at 225 nm and emission at 360 nm). The linear range of the method for the determination of isovaleric acid or valeric acid derivative was over 0.2 approximately 8.0 microM. The detection limit (signal to noise ratio=3 with 10 microl injected) of isovaleric acid or valeric acid was about 0.04 microM. Application of the method to the analysis of isovaleric acid in the urine of a patient with isovaleric acidemia proved feasible.     

Assay of α-glucosidase inhibitory activity using flow-biosensor system

A convenient and continuous method for the assay of α-glucosidase (AGH) inhibitory activity was developed using a continuous-flow/stopped-flow system combined with biosensors. The amount of glucose liberated from maltose by the action of AGH was quantified by an immobilized glucose oxidase (GOD) reactor with a Clark oxygen sensor in the downstream. The immobilized AGH reactor was set in the flow-line. When an inhibitor containing 10 mM maltose substrate was injected and as it reaches the center of the immobilized AGH reactor, the working solution (synthetic intestinal fluid) was stopped for a certain period. After the reaction of inhibitor and/or substrate with AGH, the working solution was propelled again, and the glucose liberated was passed through the immobilized GOD reactor. The inhibition ratios (%) were calculated as the percent inhibition, which were the glucose concentrations in the presence of maltose and inhibitor divided by those in the presence of maltose alone. The multi-channel stopped-flow (MCSF) system was also developed, in which a seven-port, six-positioned rotary valve was inserted and six immobilized AGH reactors were set in a parallel configuration. The IC₅₀ values of acarbose and 1-deoxynojirimycin, a medicinal inhibitor for diabetes, were 0.46±0.062 and 0.23±0.031 μM, respectively, and coincided well with those by our pseudo-in vivo method [Biol. Pharm. Bull. 232 (2000) 1084]. The time to assay the inhibitory activity of one unknown sample was estimated to be 11 min by the 6-channel modified-MCFS system. The proposed MCFS system offers a useful method to evaluate the inhibitory activity for AGH.     

Determination of Plasma Pseudo-Ephedrine by Fluorescence Detection and High Performance Liquid Chromatography

Abstract

A sensitive and specific high-performance liquid chromatographic method has been developed for the determination of pseudoephedrine in plasma. The assay was based on the production of a highly fluorescent derivative of pseudoephedrine using 4-chloro-7-nitrobenzo-2, 1, 3-oxadiazole (NBD-Cl) as the derivatization agent. The fluorescent derivatives were separated using normal phase liquid chromatography after an automated, column-switching, sample clean-up procedure. Pseudoephedrine determination was accurate and precise at concentrations as low as 10 nanograms per milliliter of plasma. The chromatographic step separated derivatives of pseudoephedrine from those of norpseudoephedrine and several other amines. Measurement of pseudoephedrine concentrations in plasma following a single 120 milligram oral dose is illustrated.     

超高效液相色谱法测定卷烟烟丝中7种水溶性糖含量

以ACQUITY UPLC BEH Amide柱为分析柱,乙腈－0．2％三乙胺一水作为流动相,建立了超高效液相色谱－蒸发光散射检测器测定卷烟烟丝中鼠李糖、木糖、果糖、甘露糖、葡萄糖、蔗糖、麦芽糖7种水溶性糖的分析方法。7种水溶性糖回归方程的线性相关系数均大于0．999,检出限为0．56～1．11μg／mL。7种水溶糖的回收率在90．43％106．41％之间,相对标准偏差为1．66％－4．35％仰：6）。该方法灵敏度、准确度高,稳定性好,可用于大批量卷烟烟丝中水溶性糖含量的测定。     

High-performance liquid chromatographic measurement of guanidino compounds of clinical importance in human urine and serum by pre-column fluorescence derivatization using benzoin

High-performance liquid chromatographic microanalyses for guanidino compounds in human physiological fluids have been accomplished by means of a pre-column fluorescence derivatization method using benzoin. The guanidino compounds in urine or deproteinized serum after ultrafiltration are converted to the fluorescent derivatives with benzoin in an alkaline medium, and the derivatives are separated simultaneously within 25 min on a reversed-phase column (mu Bondapak Phenyl) with a linear gradient elution of methanol in aqueous mobile phase (pH 8.5). The method permits the quantitative determination of guanidinosuccinic acid, methylguanidine, taurocyamine and guanidinobutyric acid at concentrations of as low as 8-78 pmol/ml in human urine and serum.     

蜂蜜中外源性γ-淀粉酶残留量的测定

采用液相色谱-同位素质谱联用法(LC-IRMS)建立了测定蜂蜜中外源性γ-淀粉酶残留量的方法。先采用凝胶色谱柱对蜂蜜样品进行预分离,将样品中所含的酶与糖分离开。根据γ-淀粉酶可将底物麦芽糖酶解为葡萄糖的原理,在55 ℃、pH 4.5的0.03 mol/L磷酸盐缓冲液中将γ-淀粉酶与麦芽糖反应48 h后,采用LC分离麦芽糖和葡萄糖,以IRMS测定酶解产物葡萄糖的含量来确定γ-淀粉酶的残留量。本方法的线性范围为5～200 U/kg,定量限为5 U/kg,回收率为89.6%～108.2%,相对标准偏差为3.3%～4.9%。采用本方法对市售蜂蜜和大米糖浆共38个样本进行了考察,γ-淀粉酶的检出率为76.3%。为了进一步验证本方法的检测能力,测定了掺入15%(质量分数)大米糖浆的蜂蜜样品,测得γ-淀粉酶的含量为10.2 U/kg。本方法能够有效地从酶学的角度鉴定蜂蜜中是否含有大米糖浆。     

A manual sequence method of peptides and phosphopeptides using 4-(1'-cyanoisoindolyl)phenylisothiocyanate

A method for sequence analysis and identification of phosphoamino acids in peptides based on high performance liquid chromatography (HPLC) is described. The peptides were derivatized with an Edman type reagent, 4-(1'-cyanoisoindolyl)phenylisothiocyanate (CIPIC) and subsequently cleaved to generate stable and fluorescent 4-(1'-cyanoisoindolyl)phenylthiazolinone (CIP-TZ)-amino acids. Several experimental factors that affected derivatization on membranes were examined. Under the optimized conditions, the CIP-TZ derivatives of Try(p), Thr(p) and Ser(p) were obtained and separated from their parent amino acids with baseline resolution using an isocratic elution system. Up to the 4th residue of phosphorylated pentapeptides was successfully identified, whereas phosphoamino acid residues could not be detected by the conventional procedure using phenylisothiocyanate (PITC). The results demonstrated the potential of CIPIC as a derivatization reagent for peptide sequencing and the applicability of the method for the study and identification of phosphoamino acids in peptides.     

R(-)-4-(3-Isothiocyanatopyrrolidin-1-yl)-7-(N,N-dimethylaminosulfonyl)-2,1,3-benzoxadiazole, a fluorescent chiral tagging reagent: sensitive resolution of chiral amines and amino acids by reversed-phase liquid chromatography

The usefulness of R(-)-4-(3-isothiocyanatopyrrolidin-1-yl)-7-(N,N-dimethylaminosulfonyl)-2,1,3-benzoxadiazole [R(-)-DBD-PyNCS], a fluorescent chiral tagging reagent, for the determination of racemic amines and amino acids, was studied. The reagent reacted with beta-blockers selected as representative secondary amines to produce corresponding fluorescent diastereomers (excitation at 460 nm and emission at 550 nm). The yields of the derivatization reaction were dependent on the stereostructure arround the NH group in beta-blockers. The resulting diastereomers were completely separated with single chromatographic run using linear gradient elutions by reversed-phase chromatography. R(-)-DBD-PyNCS was also applied to the determination of DL-amino acid, considered to be one of the primary amines, in human urine and foodstuffs. DL-amino acids tested equally reacted with the reagent, and the thiocarbamoyl derivatives were separated with an ODS column. The epimerization during the derivatization reaction was negligible judging from the resolution of opposite diastereomers on the chromatogram. The occurence of D-amino acids (D-Ala, D-Ser, D-Asp and/or D-Glu) was identified in the samples tested. The structures and the purities were elucidated with on-line HPLC-MS. The chiral reagent possessing an isothiocyanate group (-NCS) in the structure seems to be applicable to continuous sequential analysis of peptides containing D-amino acids. The thiocarbamoyl derivatives obtained from the reaction with DL-amino acids were converted to thiohydantoins via thiazolinones in acidic medium. The thiohydantoins produced from acidic, basic, neutral, hydroxyl and aromatic amino acids were completely separated with isocratic elutions using acidic mobile phase containing 0.1% TFA. The separations were sufficient for the identification of DL-amino acid in peptide sequences. Although the epimerization during the conversion reaction to thiohydantoins was not avoidable, the descrimination of D- and L-configuration was demonstrated with some commercially available peptides such as beta-lipotropin and [D-Ala2]-deltorphin II. The Edman degaradation method using R(-)-DBD-PyNCS was also adopted to autoanlaysis by gas-phase sequencer. The separation and the detection (UV 254 nm) conditions of the derivatives were used without any change from those for the Edman degradation method using PITC as the tagging reagent. The three DL-amino acid residues (Tyr, Ala and Gly) in [L-Ala2]-leucine-enkephalin and [D-Ala2]-leucine-enkephalin were perfectly identidied with the autoanalysis.     

On the properties of alpha-glucosidase and the binding of glucose to the enzyme.

An alpha-glucosidase was purified from baker's yeast. The molecular weight was approximately 44 000 daltons. SDS-disc gel electrophoresis suggested that the enzyme consisted of four subunits. The isoelectric point was at pH 5.4. The Km values for p-nitrophenyl alpha-D-glucopyranoside and maltose were 2.9 X 10(-4) and 2.5 X 10(-2) M, respectively. Binding of 2-(p-toluidino)naphthalene-6-sulfonate to the alpha-glucosidase was associated with a strong increase in fluorescence. The dissociation constant of the enzyme-TNS complex was 8 X 10(-5) M. The fluorescent probe did not interfere with the binding of glucose to the enzyme although the alpha-glucosidase was inhibited by high concentrations of TNS. The formation of an enzyme-glucose complex was indicated by an increase of fluorescence and by a shift in the wavelength for maximal emission which suggests that the binding process is associated with a change in conformation. The dissociation constant of the glucose--alpha-glucosidase complex KD = 0.57 X 10(-3) M, was calculated from the increase in fluorescence as a function of glucose concentration.     

N-(3,5-二甲基苯基)氨基甲酸酯单糖及二糖作为高效液相色谱 手性固定相研究

广泛使用的环糊精和多糖手性固定相是由吡喃型葡萄糖结构单元构成, 将D-葡萄糖、D-半乳糖、D-甘露糖、D-木糖、麦芽糖、蔗糖、乳糖、纤维二糖的N-(3,5-二甲基苯基)氨基甲酸酯经过3-异氰酸丙基三乙氧基硅烷键合到硅胶上作为高效液相色谱的手性固定相, 结果表明它们也具有好的手性分离能力, 并且它们之间的手性识别能力还具有好的互补性. 该研究表明, 在高效液相色谱中寡糖能成为一类新型的手性固定相.     

Determination of ppb levels of tryptophan derivatives by capillary electrophoresis with homogeneous liquid-liquid extraction and sweeping method

Tryptophan (Trp) derivatives were selectively separated from 24 kinds of amino acid mixture solutions. Capillary electrophoresis (CE) with UV detection using the described pretreatment system was able to separate and determine 4 kinds of Trp derivatives: 5-hydroxy L-tryptophan, 5-methyl L-tryptophan, 1-methyl L-tryptophan and L-tryptophan. The pretreatment system used a combination of a homogeneous liquid-liquid extraction and a sweeping method, the two are different kinds of powerful preconcentration/separation methods. Trp derivatives were thus separated by two different separation systems. First, these derivatives were selectively extracted at 57-100% by a homogeneous liquid-liquid extraction; secondly, they were respectively separated by micellar electrokinetic chromatography (MEKC) with a sweeping effect. The detection limits following complete separation of the Trp derivatives were 10(-8) mol l(-1) levels, respectively. The proposed method provided a more than 10-fold the improvement in sensitivity compared with fluorescence labeling/fluorophotometric analysis; the proposed method was also applied to the component analysis of nutritious medicine.     

Determination of saccharides in sake by high-performance liquid chromatography with polarized photometric detection

A high-performance liquid chromatographic method has been developed for the determination of saccharides in sake, an alcoholic beverage brewed from rice. Saccharides in sake were separated on a normal phase (carbamoyl bonded silica) column using a linear gradient elution of water in acetonitrile. Seven saccharides, glucose, maltose, isomaltose, maltotriose, panose, isomaltotriose and ethyl alpha-D-glucoside, were determined by a polarized photometric detector. Unidentified peaks suggesting saccharides with polymerization degrees over 4 were also observed. The proposed method did not require any sample clean-up treatment. As an application, saccharide compositions in various kinds of sake were compared.     

Assay for enkephalin-degrading peptidases in rat brain tissues by high-performance liquid chromatography with on-line post-column fluorescence detection

The activities of enkephalin-degrading peptidases such as enkephalinases A and B in rat brain tissues were simultaneously assayed by a high-performance liquid chromatographic method with fluorimetric detection with an automatic reaction system. Tyrosine and tyrosine-containing peptides produced enzymatically from the substrate, methionine-enkephaline, were separated by gradient elution on a reversed-phase column (TSK gel ODS-120T), and then converted into fluorescent derivatives for detection by reaction with hydroxylamine, cobalt(II) and borate reagents. The method permits the simple and sensitive detection of N-terminal tyrosine-containing fragments of the enkephalin peptide. The limits of detection are 5-20 pmol per assay tube for the N-terminal tyrosine-containing fragments. The enzyme activities in the regionally separated tissues were 54-191 pmol/min.mg protein for enkephalinase A and 79-153 pmol/min.mg protein for enkephalinase B, which were calculated from the formation of Tyr-Gly-Gly and Tyr-Gly, respectively, during the enzyme reaction.     

A novel self-cleaving phasin tag for purification of recombinant proteins based on hydrophobic polyhydroxyalkanoate nanoparticles

A novel protein purification method was developed using microbial polyhydroxyalkanoates (PHA) granule-associated protein phasin, a pH-inducible self-cleaving intein and PHA nanoparticles. Genes for the target proteins to be produced and purified were fused to genes of intein and phasin, the genes were jointly over-expressed in vivo, such as in E. coli cells in this study. The fused proteins containing target protein, intein and phasin produced by the recombinant E. coli were released together with all other E. coli proteins via a bacterial lysis process. They were then adsorbed in vitro to the surfaces of the hydrophobic polymer nanoparticles incubated with the cell lysates. The nanoparticles attached with the fused proteins were concentrated via centrifugation. Then, the reasonably purified target protein was released by self-cleavage of intein and separated with nanoparticles by a simple centrifugation process. Using this system, enhanced green fluorescent protein (EGFP), maltose binding protein (MBP) and beta-galactosidase were successfully purified in their active forms with reasonable yields, respectively, demonstrating the effectiveness and reliability of this purification system. This method allows the production and purification of high value added proteins in a continuous way with low cost.     

Determination of carbohydrates as their 3-aminophthalhydrazide derivatives by capillary zone electrophoresis with on-line chemiluminescence detection

A method based on pre-capillary derivatization with luminol (3-aminophthalhydrazide) for carbohydrate analysis using capillary electrophoresis with on-line chemiluminescence (CL) detection was developed. The derivatives of seven monosaccharides were separated and detected by using 200 mM borate buffer containing 100 mM hydrogen peroxide at pH 10.0 as separation electrolyte and 25 mM hexacyanoferrate in 3 M sodium hydroxide solution as post-capillary chemiluminescence reagent with separation efficiencies ranging from 160,000 to 231,000 plates per metre. The minimum amount of carbohydrate derivatized was 2 pmol (corresponding to the concentration of 2 microM). The method also provided a linear response for glucose in the concentration range of 0.1-250 microM with a mass detection limit of 420 amol or a concentration detection limit of 0.1 microM. Preliminary work using the CE-CL format to determine glucose in a rat brain microdialysis sample is presented as a typical case.     

Capillary electrophoresis analysis of glucooligosaccharide regioisomers

Complex gluco-oligosaccharide mixtures of two regioisomer series were successfully separated by CE. The gluco-oligosaccharide series were synthesized, employing a dextransucrase from Leuconostoc mesenteroides NRRL B-512F, by successive glucopyranosyl transfers from sucrose to the acceptor glucose or maltose. The glucosyl transfer to both acceptors, occurring through the formation of alpha1-->6 linkages, differed for the two series only in the glucosidic bond to the reducing end namely alpha1-->6 or alpha1-->4 bond for glucose or maltose acceptor, respectively. Thus, the combination of the two series results in mixed pairs of gluco-oligosaccharide regioisomers with different degrees of polymerization (DP). These regioisomer series were first derivatized by reductive amination with 9-aminopyrene-1,4,6-trisulfonate (APTS). Under acidic conditions using triethyl ammonium acetate as electrolyte, the APTS-gluco-oligosaccharides of each series were separated enabling unambiguous size determination by coupling CE to electrospray-mass spectrometry. However, neither these acidic conditions nor alkaline buffer systems could be adapted for the separation of the gluco-oligosaccharide regioisomers arising from the two combined series. By contrast, increased resolution was observed in an alkaline borate buffer, using differential complexation of the regioisomers with the borate anions. Such conditions were also successfully applied to the separation of glucodisaccharide regioisomers composed of alpha1-->2, alpha1-->3, alpha1-->4, and alpha1-->6 linkages commonly synthesized by glucansucrase enzymes.     

Separation and quantification of beer carbohydrates by high-performance liquid chromatography with evaporative light scattering detection

An HPLC method with an evaporative light scattering detector was optimized and validated for quantification of carbohydrates in beer. The chromatographic separation was achieved using a Spherisorb NH2, 5 microm chromatographic column and gradient elution with acetonitrile/water. The determinations were performed in the linear range of 0.05-5.0 g/L for fructose, 0.05-5.0 g/L for glucose, 0.05-15.0 g/L for maltose, 0.05-10.0 g/L for maltotriose, and 0.05-5.0 g/L for maltotetraose. The detection limits were 0.005 g/L for fructose, 0.008 g/L for glucose, and 0.01 g/L for maltose, maltotriose, and maltotetraose. The reliability of the method in terms of precision and accuracy was evaluated in three beer matrices, low alcohol beer, 6% alcohol beer, and beer made with part of adjuncts (4.5% alcohol). Relative standard deviations (RSDs) ranged between 1.59 and 5.95% (n = 10), and recoveries ranged between 94 and 98.4%.