Thermally induced conformation transition of triple-helical lentinan in NaCl aqueous solution

Lentinan, a beta-(1-->3)-D-glucan, was isolated from Lentinus edodes by using an improved extraction and purification method to show good water solubility and high yield. The results from 13C NMR, size-exclusion chromatography combined with multiangle laser light scattering (SEC-MALLS), dynamic light scattering (DLS), and optical rotation revealed that lentinan existed in a triple-helical conformation in the aqueous solution at 25 degrees C, whereas the thermally induced conformation transition from triple helix to single flexible chains occurred at elevated temperatures. The dependences of the weight-average molecular weight (Mw), radius of gyration (z1/2), hydrodynamic radius (Rh), intrinsic viscosity ([eta]), and specific optical rotation of lentinan on temperature in 0.9% NaCl aqueous solution showed an abrupt drop at 130-145 degrees C. It was confirmed that the conformation transitions from triple strand to single chain and from extended chains to winding chains for lentinan were completed rapidly at 130-145 degrees C, as a result of the simultaneous destruction of the intra- and intermolecular hydrogen bonds in lentinan. The thermally induced conformational transition was irreversible. The results from atomic force microscopy (AFM) and DLS demonstrated the existence of intrachain entanglement for the triple-helical chains, leading to the wormlike linear, circular, and crossover species for lentinan having high Mw (1.71x10(6)) in aqueous solution at 25 degrees C.     

杜氏盐藻多糖的高效体积排阻色谱的保留特性及其分析方法的研究

通过对从杜氏盐藻中提取出的不同多糖级分在高效体积排阻色谱柱(Waters Ultrahydragel Linear,7.8 mm i.d.×300 mm,2根串联)上的保留特性的考察及其分离分析条件的优化,建立了高效体积排阻色谱分析盐藻多糖平均相对分子质量及其分布的方法。结果表明:流动相中盐的种类及其浓度、pH值对3种酸性多糖级分(特别是硫酸化多糖级分PD4a)的保留行为有显著影响;在柱温为45 ℃,流速为0.9 mL/min条件下,使用0.1 mol/L的NaAc水溶液作流动相基本上能消除非特异性吸附作用及分子间缔合等因素的干扰,使各多糖级分基本以非缔合状态按立体排除机制保留和分离。在优化的色谱条件下,测得的盐藻多糖5个级分的重均相对分子质量(Mw)分别为1548000,33000,67000,424000,10000;测得的硫酸化多糖级分PD4a的Mw和峰面积的相对标准偏差分别为1.7%和 0.88%(n=5)。     

Xylooligosaccharides Production from Alkali-Pretreated Sugarcane Bagasse Using Xylanases from Thermoascus aurantiacus

Sugarcane bagasse hemicellulose was isolated in a one-step chemical extraction using hydrogen peroxide in alkaline media. The polysaccharide containing 80.9% xylose and small amounts of l-arabinose, 4-O-methyl-d-glucuronic acid and glucose, was hydrolyzed by crude enzymatic extracts from Thermoascus aurantiacus at 50?°C. Conditions of enzymatic hydrolysis leading to the best yields of xylose and xylooligosaccharides (DP 2-5) were investigated using substrate concentration in the range 0.5–3.5% (w/v), enzyme load 40–80 U/g of the substrate, and reaction time from 3 to 96 h, applying a 2² factorial design. The maximum conversion to xylooligosaccharides (37.1%) was obtained with 2.6% of substrate and xylanase load of 60 U/g. The predicted maximum yield of xylobiose by a polynomial model was 41.6%. Crude enzymatic extract of T. aurantiacus generate from sugarcane bagasse hemicellulose 39% of xylose, 59% of xylobiose, and 2% of other xylooligosaccharides.     

Tailoring Wet Explosion Process Parameters for the Pretreatment of Cocksfoot Grass for High Sugar Yields

The pretreatment of lignocellulosic biomass is crucial for efficient subsequent enzymatic hydrolysis and ethanol fermentation. In this study, wet explosion (WEx) pretreatment was applied to cocksfoot grass and pretreatment conditions were tailored for maximizing the sugar yields using response surface methodology. The WEx process parameters studied were temperature (160–210 °C), retention time (5–20 min), and dilute sulfuric acid concentration (0.2–0.5 %). The pretreatment parameter set E, applying 210 °C for 5 min and 0.5 % dilute sulfuric acid, was found most suitable for achieving a high glucose release with low formation of by-products. Under these conditions, the cellulose and hemicellulose sugar recovery was 94 % and 70 %, respectively. The efficiency of the enzymatic hydrolysis of cellulose under these conditions was 91 %. On the other hand, the release of pentose sugars was higher when applying less severe pretreatment conditions C (160 °C, 5 min, 0.2 % dilute sulfuric acid). Therefore, the choice of the most suitable pretreatment conditions is depending on the main target product, i.e., hexose or pentose sugars.     

Polarographic behaviour and determination of acrivastine in capsules and human urine

Dibenzo-18-crown-6 (DB18C6) is used for the liquid-liquid extraction, and recovery of titanium from paints, pigment, paper and pulp industries. The extraction mechanism of titanium(IV) from pH 4 medium with DB18C6 in dichloromethane was investigated. The DB18C6 concentration in organic phase, the concentration of titanium, the effect of pH and interference ions such as Mo(6+), V(5+), Nb(5+), Ta(5+), Zr(4+), Fe(3+), etc. in the aqueous phase and the temperature on the distribution coefficient for the Ti(IV) have been examined. The titanium was determined by spectrophotometric and inductively coupled plasma atomic emission spectroscopic (ICP-AES) method. Titanium forms a colourless complex with DB18C6 at pH 4.0 which is extracted with dichloromethane having molar absorptivity 1.53x10(4) lmol(-1)cm(-1) at 285 nm. It obeys Beer's law in the range of 0.16-3.84 ppm of titanium. The colour was developed with thiocyanate which has molar absorptivity 1.50x10(3) lmol(-1)cm(-1) at 425 nm. The extract is directly inserted in the plasma for ICP-AES measurement, which enhance the sensitivity several folds and the limits for estimation are 0.5-30 ngml(-1). The overall formation (logbeta(2)K'e) and extraction (K(ex)) constants calculated are 18.61+/-0.02 and 1.03+/-0.03x10(-10), respectively. The transportation of titanium has been discussed. The titanium is preconcentrated and determined in standard and environmental samples.     

牡励中脂肪酸在储藏过程中的稳定性

用超临界流体萃取及ＧＣ－ＭＳ分析了新冷冻干燥及保存１５ｄ,３０ｄ,４５ｄ,６０ｄ,７５ｄ,９０ｄ后的鲜牡蛎粉中的２３种脂肪酸组分的质量分数。发现在存放过程中牡蛎脂肪酸的稳定性与其不饱和度有关;不饱和度越高,脂肪酸越易被氧化,其中多不饱和记酸的氧化是逐渐进行的,没有特定的稳定期。     

Application of pressurized liquid extraction technology to pharmaceutical solid dosage form analysis

The technique of pressurized liquid extraction has been evaluated for the extraction of active ingredients from pharmaceutical dosage forms using montelukast sodium oral chewable tablets as a model. The extraction method was optimized for the number of extraction cycles, extraction time, extraction solvent composition and temperature. Samples were extracted using two cycles of water for 2 min with a cell temperature of 40 degrees C and a pressure of 1.0 x 10(4) kPa, to disintegrate the tablet, followed by three cycles of methanol for 3 min at 70 degrees C and 1.0 x 10(4) kPa, to solubilize montelukast sodium. The method demonstrated an extraction efficiency of 98.2% of label claim and an RSD of 1.3% (n=10), as compared to 97.6% and an RSD of 0.9% obtained using a validated mechanical extraction method.     

Multiresidue determination of organophosphorus pesticides in ginkgo leaves by accelerated solvent extraction and gas chromatography with flame photometric detection

A rapid and simple method using accelerated solvent extraction and solid-phase extraction cleanup was developed and validated for the determination of 15 organophosphorus pesticides in ginkgo leaves by capillary gas chromatography with flame photometric detection. The pesticides were extracted at 100 degrees C under 1500 psi pressure in <20 min. The average recovery from 10 g ginkgo leaves, fortified at 3 levels ranging from 0.05 to 1.00 mg/kg, was 95.2% with a relative standard deviation of 4.6%. The limits of detection ranged from 1.11 x 10(-3) mg/kg (dimethoate) to 4.44 x 10(-3) mg/kg (dichlorvos). The proposed method showed acceptable accuracy and precision while minimizing environmental concerns, time, and labor. Furthermore, the method could be easily applied to the monitoring of these 15 organophosphorus pesticides in ginkgo leaves.     

Influence of extraction methodologies on the analysis of five major volatile aromatic compounds of citronella grass (Cymbopogon nardus) and lemongrass (Cymbopogon citratus) grown in Thailand

This paper deals with the systematic comparison of extraction of major volatile aromatic compounds (VACs) of citronella grass and lemongrass by classical microhydrodistillation (MHD), as well as modern accelerated solvent extraction (ASE). Sixteen VACs were identified by GC/MS. GC-flame ionization detection was used for the quantification of five VACs (citronellal, citronellol, geraniol, citral, and eugenol) to compare the extraction efficiency of the two different methods. Linear range, LOD, and LOQ were calculated for the five VACs. Intraday and interday precisions for the analysis of VACs were determined for each sample. The extraction recovery, as calculated by a spiking experiment with known standards of VACs, by ASE and MHD ranged from 64.9 to 91.2% and 74.3 to 95.2%, respectively. The extraction efficiency of the VACs was compared for three solvents of varying polarities (hexane, dichloromethane, and methanol), seven different temperatures (ranging from 40 to 160 degrees C, with a gradual increment of 20 degrees C), five time periods (from 1 to 10 min), and three cycles (1, 2, and 3 repeated extractions). Optimum extraction yields of VACs were obtained when extractions were carried out for 7 min with dichloromethane and two extraction cycles at 120 degrees C. The results showed that the ASE technique is more efficient than MHD, as it results in improved yields and significant reduction in extraction time with automated extraction capabilities.     

High-efficiency protein extraction from polyacrylamide gels for molecular mass measurement by matrix-assisted laser desorption/ionization-time of flight-mass spectrometry

A simple and fast method of protein extraction from Coomassie Brilliant Blue (CBB)-stained polyacrylamide gels suited for molecular mass measurement of proteins by matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS) is reported. Proteins in CBB-stained gel pieces were extracted by a 10-min soaking in 0.1 M NaOH at 25 degrees C. The recovery of this one-step extraction method was 34-73% for proteins <67 kDa. CBB adduction to proteins during mass spectrometric analysis was avoided by a destaining step before the alkaline extraction. The molecular mass values of the extracted proteins coincided with those of purified proteins within +/-0.01-0.10% deviation for all the proteins <36 kDa. Because of the high extraction recovery, mass measurement was possible for the proteins extracted from CBB-stained gels with loaded protein quantities as little as 34 ng for cytochrome c, alpha-lactalbumin, myoglobin, beta-lactoglobulin, trypsinogen, and carbonic anhydrase (12.4-29.0 kDa), 340 ng for glyceraldehyde-3-phosphate dehydrogenase (35.6 kDa) and albumin (66.3 kDa). This method provides a highly efficient approach to utilize CBB-stained one- or two-dimensional gels for whole protein analysis using MALDI-TOF-MS.     

Binding of adenosine to pendant phenylboronate groups of thermoresponsive copolymer: a quantitative study

Binding of adenosine to the thermosensitive copolymer of N-isopropylacrylamide and 3-(acrylamido)aminophenylboronic acid (82:18, Mn = 47,000 g . mol(-1)) was studied by equilibrium dialysis at 22 degrees C and 37 degrees C, in a 0.1 M glycine buffer containing 0.1 M NaCl at pH 9.2. The copolymer exhibited a the phase transition temperature (T(p)) of 26.5 degrees C under the above conditions. At 22 degrees C the binding of adenosine to the water-soluble copolymer was well described by a Langmuir model, accounting for preferential ionisation of the boronate-nucleoside complexes and, therefore, restricted reactivity of the rest of boronates. At saturation, the copolymer contained 38% of its phenylboronic acid groups in the form of complexes, whereas the association constant was 1,400 M(-1). At 37 degrees C no binding of adenosine to thermally precipitated copolymer was found, presumably owing to interaction of the phenylboronates with hydrophobic segments of polyNIPAM. At high loading of the copolymer by the reversibly bound adenosine the T(p) steeply increases with increasing fraction of the phenylboronate-adenosine complexes in the chains. The increase of the T(p) observed above the saturating adenosine concentration (>1 x 10(-3) M, 22 degrees C) very probably testifies to competition of the nucleoside with hydrophobic polyNIPAM segments for binding to the pendant phenylboronates.     

SPE and large-volume sample stacking in MEKC for determination of doxycycline in biological fluids: comparison of direct injection to SPE-MEKC

A novel and simple method has been developed for the determination of doxycycline (DOX) in biological fluids. The method is based on SPE, large-volume sample stacking (LVSS) and MEKC with UV-DAD detection. Six SPE cartridges have been used in investigation for sample clean up and pre-concentration (Supelco LC-8, LC-18, LC-SCX, and LC-WCX, as well as Strata-X and X-C). DOX was determined on a 56 cm (effective length 50 cm) x 50 microm id fused-silica capillary. The BGE was 20 mM borate buffer, pH 9.3, containing 80 mM SDS and 7.5% v/v of methanol (30 sx50 mbar), and the temperature and voltage were 25 degrees C and 30 kV, respectively. The analytical wavelength was set at 210 nm. Under optimized conditions it is possible to determine DOX in human serum, urine, semen, tears and saliva with recovery of 97.5% (RSD 2.5%). The method was shown to be sensitive (LOD is 1 microg/L) and precise (intra-day RSD 0.2 and 2.4%; inter-days 0.4 and 3.5% for migration time and peak area, respectively). Results for developed SPE-LVSS-MEKC were compared with LVSS-MEKC method with direct sample injection. The new LVSS-MEKC method is presented as a useful technique for rapid determination without extraction procedure of DOX in human urine and serum, using 80 mM of SDS, 10% v/v of methanol and 40 mM borate buffer (pH 9.3; 30 s x 50 mbar; 25 degrees C; 30 kV; 350 nm), but not for the other biological fluids, according to lower sensitivity of the method and because of the sample composition.     

High-performance liquid chromatographic method for the simultaneous measurement of remacemide (a novel anticonvulsant and cerebroprotectant) and an active metabolite in human plasma.

A high-performance liquid chromatographic (HPLC) method was developed and validated for the determination of both remacemide (a novel anticonvulsant and cerebroprotectant) and an active, major metabolite in human plasma. After the addition of an internal standard, the analytes were extracted from the plasma by ion-exchange solid-phase extraction and measured by an isocratic HPLC system with ultraviolet detection at 210 nm. The recovery of the analytes was > 90%. The standard curves were linear over the range of quantitation of approximately 10-500 ng/ml for remacemide itself and 15-250 ng/ml for the metabolite. Both intra-day and inter-day accuracy and precision data were excellent. Remacemide and its metabolite were shown to be stable in human plasma for at least a year when stored at -20 degrees C.     

Application of high-performance liquid chromatography--inductively coupled plasma mass spectrometry to the investigation of cadmium speciation in pig kidney following cooking and in vitro gastro-intestinal digestion

The speciation of cadmium in retail pig kidney has been examined by size-exclusion chromatography (SEC) coupled directly to inductively coupled plasma mass spectrometry (ICP-MS). Approximately 35% of the cadmium from uncooked kidney was soluble after aqueous extraction at pH 8 and SEC - ICP-MS revealed three discrete peaks whose retention times corresponded to estimated relative molecular masses of 1.2 x 10(6), 7.0 x 10(4) and 6 x 10(3)-9 x 10(3). In the cooked kidney, 35% of the Cd was soluble and was all associated with a peak of a relative molecular mass (Mr) of 6 x 10(3)-9 x 10(3). After simulated gastric digestion of cooked pig kidney at pH 2.5, 60% of the cadmium was solubilised and associated with a species of Mr less than 1 x 10(3). When the digest was also subjected to simulated intestinal digestion at pH 6.8, a single peak, which corresponded to 20% of the original cadmium, was eluted. This peak co-eluted with the single peak extracted at pH 8.0 from the cooked kidney. It was also of similar estimated Mr to the single broad peak observed after simulated gastro-intestinal digestion of equine renal metallothionein (Mr = 1.1 x 10(4]. The results suggest that the majority of soluble cadmium in retail pig kidney is associated with a metallothionein-like protein that survives both cooking and simulated in vitro gastro-intestinal digestion.     

固相萃取-气相色谱-质谱法测定利多卡因代谢物单乙基甘氨酰二甲苯胺的血药浓度

建立了固相萃取-气相色谱-质谱(GC-MS)测定利多卡因代谢物单乙基甘氨酰二甲苯胺(MEGX)血药浓度的方法。血清中的MEGX采用固相萃取小柱萃取、GC-MS测定。色谱条件为：HP-5MS毛细管柱(15 m×0.25 mm×0.1 μm),初始柱温100 ℃,保持1 min后以40 ℃/min速率升温至200 ℃,保持0.5 min;进样口温度250 ℃;分流进样,分流比1∶1,进样量2 μL;载气为氦气,流量为1.0 mL/min。质谱条件为:离子源温度230 ℃,电子轰击电离,电子能量70 eV,选择离子检测（m/z 58(MEGX)、 m/z 86(普鲁卡因,内标)）。结果表明,MEGX在血清中的浓度在1.562～25 ng/mL范围内的线性关系良好,相关系数0.9981,最低检测限为0.5 ng/mL,不同浓度MEGX的萃取回收率在80.1%～85.7%之间。实验证明该方法快速、准确,选择性好,灵敏度高,适合用于血清中微量MEGX的测定。     

Counter-current chromatographic estimation of hydrophobicity of Z-(cis) and E-(trans) enalapril and kinetics of cis/trans isomerization

The kinetics of Z-(cis)/E-(trans) isomerization of enalapril was investigated by reversed phase high-performance liquid chromatography (RP-HPLC) using a monolith ODS column under a series of different temperature and pH conditions. At a neutral pH 7, the rate (k(obs)) of Z-(cis)/E-(trans) isomerization of enalapril at 4 degrees C (9.4 x 10(-3)min(-1)) is much lower than at 23 degrees C (1.8 x 10(-1)min(-1)), while the fractional concentration of Z-(cis) isomer is always higher than that of E-(trans) isomer in the pH range 2-7. The fractional concentration of the E-(trans) isomer becomes a maximum (about 40%) in the pH range 3-6, where enalapril exists as a zwitterion. The hydrophobicity (logP(O/W)) of both isomers was estimated by high-speed counter-current chromatography (HSCCC). Normal phase HSCCC separation using a tert-butyl methyl ether-acetonitrile-20mM potassium phosphate buffer (pH 5) two-phase solvent system (2:2:3, v/v/v) at 4 degrees C was effective in partially separating the isomers, and the partition coefficient (K) of each isomer was directly calculated from the retention volume (V(R)). The logP(O/W) values of Z-(cis) and E-(trans) isomers were -0.46 and -0.65, respectively.     

Chestnut Shell as Unexploited Source of Fermentable Sugars: Effect of Different Pretreatment Methods on Enzymatic Saccharification

Chestnut shell (CS) is an agronomic residue mainly used for extraction of antioxidants or as adsorbent of metal ions. It also contains some polysaccharide that has not been considered as potential source of fermentable sugars for biofuel production until now. In this study, the effect of different pretreatment methods on CS was evaluated in order to obtain the greatest conversion of cellulose and xylan into fermentable sugars. Hot acid impregnation, steam explosion (acid-catalysed or not), and aqueous ammonia soaking (AAS) were selected as pretreatments. The pretreated biomass was subjected to saccharification with two enzyme cocktails prepared from commercial preparations, and evaluation of the best pretreatment and enzyme cocktail was based on the yield of fermentable sugars produced. As AAS provided the best result after preliminary experiments, enhancement of sugar production was attempted by changing the concentrations of ammonium hydroxide, enzymes, and CS. The optimal pretreatment condition was 10 % ammonium hydroxide, 70 °C, 22 h with CS at 5 % solid loading. After saccharification of the pretreated CS for 72 h at 50 °C and pH 5.0 with a cocktail containing cellulase (Accellerase 1500), beta-glucosidase (Accellerase BG), and xylanase (Accellerase XY), glucose and xylose yields were 67.8 and 92.7 %, respectively.     

Determination of 1-(2-methoxyphenyl)piperazine derivatives of isocyanates at low concentrations by temperature-programmed miniaturized liquid chromatography

A temperature-programmed packed capillary LC method with large-volume injection on-column focusing has been developed for screening and determination of 1-(2-methoxyphenyl)piperazine derivatives of airborne toluene-2,4-diisocyanate, toluene-2,6-diisocyanate, hexamethylenediisocyanate and methylenebisphenyl-4,4-diisocyanate, based on sampling methods described in MDHS 25/3. Injection volumes up to 100 microl were successfully loaded onto the 250x0.32 mm I.D. capillary column packed with 3 microm Hypersil ODS particles. The isocyanate derivatives were loaded at 10 degrees C and eluted by a three-step temperature program starting at 10 degrees C for 10 min, followed by a temperature ramp of 2.5 degrees C min(-1) to 45 degrees C and then 9.9 degrees C min(-1) to 90 degrees C. The mobile phase consisted of acetonitrile-acetate buffer (3% triethylamine, pH 4.5) (45:55, v/v). The isocyanate derivatives were dissolved in acetonitrile-acetate buffer (3% triethylamine, pH 4.5) (30:70, v/v) to achieve sufficient focusing. The concentration limit of detection of the individual derivatives utilizing an "U" shaped flow cell with a 8.0 mm light path and an injection volume of 100 microl was 44, 87, 43 and 210 pg ml(-1) for toluene-2,6-diisocyanate, hexamethylenediisocyanate, toluene-2,4-diisocyanate and methylenebisphenyl-4,4-diisocyanate, respectively. Within the investigated concentration range, 10-500 ng ml(-1), the linear calibration curves gave correlation coefficients ranging from 0.994 to 0.998. The repeatability of the method with regard to retention time and peak height ranged from 0.3 to 1.1% and 1.1 to 2.3% (n=9) relative standard deviation, respectively. The average recovery of the method, with regard to toluene-2,4-diisocyanate, was 97.7+/-1.6% (n=9).     

Quantitative determination of rocuronium in human plasma by liquid chromatography-electrospray ionization mass spectrometry

Liquid chromatography-electrospray ionization mass spectrometry (LC-ESI-MS) was used for the quantification of the neuromuscular blocking agent rocuronium in human plasma. Verapamil was used as internal standard. The samples were subjected to a dichloromethane liquid-liquid extraction after ion pairing of the positively charged ammonium compound with iodide prior to LC-MS. Optimized conditions involved separation on a Symmetry Shield RP-18 column (50 x 2.1 mm, 3.5 microm) using a 15-min gradient from 10 to 90% acetonitrile in water containing 0.1% trifluoroacetic acid at 250 microl/min. Linear detector responses for standards were observed from 25 to 2,000 ng/ml. The extraction recovery averaged 59% for rocuronium and 83% for the internal standard. The limit of quantification (LOQ), using 500 microl of plasma, was 25 ng/ml. Precision ranged from 1.3 to 19% (LOQ), and accuracy was between 92 and 112%. In plasma samples, at 20 and 4 degrees C, rocuronium was stable at physiological pH for 4 h; frozen at -30 degrees C it was stable for at least 75 days. The method was found suitable for the analysis of samples collected during pharmacokinetic investigations in humans.