Development of a liquid chromatography-mass spectrometry method for monitoring the angiotensin-converting enzyme inhibitor lisinopril in serum

In this study, a sensitive, specific, precise and accurate method for lisonopril quantitative determination in human serum was developed and validated. The method comprises lisinopril isolation from serum by means of solid-phase extraction followed by its quantification by liquid chromatography-mass spectrometry. Chromatographic separation was performed at 55 degrees C on Kromasil C(18) 5 micrometer 250x3.2 mm HPLC column with mobile phase composed of 50 mM ammonium formate buffer (pH 3)-acetonirile-methanol (72:7:21, v/v/v). A Finnigan AQA benchtop mass spectrometer with a pneumatically assisted electrospray (ES) interface and a single quadrupole mass filter was used to detect and quantify lisinopril in column effluent. Ion signals were acquired by selected ion monitoring of the protonated lisinopril ion m/z=406.5 (M+1). The detector response was linear with r>0.9993 in the investigated concentration range 6-150 ng/ml. The mean recovery of lisinopril from serum samples was 88%. The limit of quantitation for lisinopril was 6 ng/ml with a signal-to-noise ratio at this concentration level S/N=34.75+/-3.9 (n=4).     

High temperature-ultra performance liquid chromatography-mass spectrometry for the metabonomic analysis of Zucker rat urine

The applicability and potential of using elevated temperatures and sub 2-microm porous particles in chromatography for metabonomics/metabolomics was investigated using, for the first time, solvent temperatures higher than the boiling point of water (up to 180 degrees C) and thermal gradients to reduce the use of organic solvents. Ultra performance liquid chromatography, combined with mass spectrometry, was investigated for the global metabolite profiling of the plasma and urine of normal and Zucker (fa/fa) obese rats (a well established disease animal model). "Isobaric" high temperature chromatography, where the temperature and flow rate follow a gradient program, was developed and evaluated against a conventional organic solvent gradient. LC-MS data were first examined by established chromatographic criteria in order to evaluate the chromatographic performance and next were treated by special peak picking algorithms to allow the application of multivariate statistics. These studies showed that, for urine (but not plasma), chromatography at elevated temperatures provided better results than conventional reversed-phase LC with higher peak capacity and better peak asymmetry. From a systems biology point of view, better group clustering and separation was obtained with a larger number of variables of high importance when using high temperature-ultra performance liquid chromatography (HT-UPLC) compared to conventional solvent gradients.     

Factors influencing the potential use of Aliquat 336 for the in situ extraction of carboxylic acids from cultures of Pseudomonas putida

The use of extraction techniques to alleviate product inhibition in bioprocesses is one of a number of potential separation methods. However, the intimate contact of an organic phase with the broth implies that the organic components of this phase may be present in the aqueous phase at saturation levels. The quaternary amine Aliquat 336 (trioctyl/decylmethylammonium entity), dissolved in octan-1-ol showed no inhibition on the growth of Pseudomonas putida, at least with respect to molecular toxicity. Nevertheless, it is important to point out two main effects of Aliquat 336 associated with its ion exchange properties. It is able (1) to complex hydroxyl ions and therefore drastically lower the pH of the broth and (2) release its counter ion through these exchanges. Therefore, a strict control of the pH of the cultivation must be conducted, with the constraint that Ps. putida has an optimal pH growth of 7.4-7.5. The pH range tolerated by this strain is, however, between 5.0 and 9.0. In addition, the counter ion of Aliquat 336 needs to be carefully chosen and HSO4- should be preferred to Cl-.     

Effects of organic phase, fermentation media, and operating conditions on lactic Acid extraction

Lactic acid has extensive uses in the food, pharmaceutical, cosmetic and chemical industry. Lately, its use in producing biodegradable polymeric materials (polylactate) makes the production of lactic acid from fermentation broths very important. The major part of the production cost accounts for the cost of separation from very dilute reaction media where productivity is low as a result of the inhibitory nature of lactic acid. The current method of extraction/separation is both expensive and unsustainable. Therefore, there is great scope for development of alternative technology that will offer efficiency, economic, and environmental benefits. One of the promising technologies for recovery of lactic acid from fermentation broth is reactive liquid-liquid extraction. In this paper the extraction and recovery of lactic acid based on reactive processes is examined and the performance of a hydrophobic microporous hollow-fiber membrane module (HFMM) is evaluated. First, equilibrium experiments were conducted using organic solutions consisting of Aliquat 336/trioctylamine (as a carrier) and tri-butyl phosphate (TBP)/sunflower oil (as a solvent) The values of the distribution coefficient were obtained as a function of feed pH, composition of the organic phase (ratio of carrier to solvent), and temperature (range 8-40 degrees C). The optimum extraction was obtained with the organic phase consisting of a mixture of 15 wt % tri-octylamine (TOA) and 15% Aliquat 336 and 70% solvent. The organic phase with TBP performed best but is less suitable because of its damaging properties (toxicity and environmental impact) and cost. Sunflower oil, which performed moderately, can be regarded as a better option as it has many desirable characteristics (nontoxic, environment- and operator-friendly) and it costs much less. The percentage extraction was approximately 33% at pH 6 and at room temperature (can be enhanced by operating at higher temperatures) at a feed flow rate of 15-20 L/h. These results suggest that the hollow-fiber membrane process yields good percentage extraction at the fermentation conditions and its in situ application could improve the process productivity by suppressing the inhibitory effect of lactic acid.     

微小毛霉凝乳酶的分离纯化研究

研究微小毛霉(HL-1)凝乳酶的分离纯化条件及方法。研究酶的最适浸提温度、酶的浸提pH值和最适浸提时间,探讨离子浓度、加水量对浸提效率的影响,利用高速冷冻离心法、有机溶剂沉淀法,膜分离法和层析法等对粗酶液进行了分离。利用光谱法对纯化样品进行检测。酶的最适浸提温度为30℃;最适pH为6.0;浸提10 h活力最高;1%的氯化钠有利于酶的分离,加水比例为15时有利于提取,在10 000 r/min下离心10min澄清效果最好,95%的酒精沉淀效果最好,利用0.2μm的微滤膜可除去发酵液中的菌体,8 000的超滤膜可拦截凝乳酶蛋白,S300的填料可有效分离凝乳酶,纯度达95%以上。     

Quantitative thin-layer chromatographic method for the determination of procainamide and its major metabolite in plasma

A sensitive and accurate spectrodensitometric method was developed for the determination of procainamide and its major metabolite, N-acetylprocainamide, in plasma. The method involves extraction into organic solvent at alkaline pH, separation by thin-layer chromatography and direct measurement of the adsorbance of the compounds on the plate at 275 nm. Quantities as low as 10 ng could be measured and a linear relationship was obtained between peak areas and amounts of the compounds in the spots from 10 to 200 ng. The recovery of both drugs from plasma was from 95.4 to 104.8%. The method is sensitive and specific, and procainamide was well separated from N-acetylprocainamide at all investigated concentrations. The method is recommended for clinical assays and pharmacokinetics studies.     

Investigation on the chemiluminescence reaction of the phenylhydrazine‐luminol–peroxide system

We studied the chemiluminescence (CL) oxidation of phenyl hydrazine–luminol with various organic and inorganic peroxides. Maximum CL intensity for this system was obtained for t‐butylhydroperoxide. The enhancement in CL depended strongly on pH and was greatest at pH 12.5. The solvent drastically enhanced the CL intensity. DMSO was found to increase the CL intensity many‐fold as compared to acetonitrile and water. The effect of temperature on CL intensity has also been studied. The CL spectra revealed a broad peak at 425 nm, which suggests excited 3‐aminophthalate ion as the luminophor. A mechanism to explain the reactions is suggested. Copyright © 2012 John Wiley & Sons, Ltd.     

The solvent dependence of hydrogen exchange kinetics of folded proteins.

The effects of ethanol, ethylene glycol, dioxane, and other organic co-solvents upon the hydrogen exchange rates of randomly coiled oxidized RNase, native RNase, and native trypsin have been measured. The exchange rate of oxidized RNase, the model compound for the proton transfer step in hydrogen exchange, is decreased by all of the co-solvents studied at temperatures in the range 3-20 degrees. This has been ascribed to the combined effects of the disruption of peptide bond solvation due to a reduction in the concentration of water, and of changes in [OH-] ion concentration due to changes in the acid dissociation constant of water, Kw. The solvent dependence for both native RNase and native trypsin is similar in all of the solvents studied. At a low temperature (3-20 degrees), the exchange rates go through a minimum as the solvent concentration is increased. At higher temperatures (20-35 degrees) the exchange rates are increased at all concentrations of the co-solvent. The apparent rate minimum at lower temperatures is due to two opposing effects. Co-solvents decrease the rate of exchange that occurs directly from the folded molecule. At higher concentrations and higer temperature. The decrease in rates for exchange directly from folded protein is primarily due to the effects on the proton transfer step, and not to binding or the solvent effects on protein structure. The solvents used in this study have no apparent effect on conformational processes contributing to the hydrogen exchange process in folded proteins.     

Optimum operational conditions for chiral separation of tryptophan enatiomers using ligand exchange liquid chromatography

A simple and precise method for chiral separation of tryptophan enantiomers using high performance liquid chromatography with aligand exchange mobile phase was developed. Chiral separation was performed on a conventional C₁₈ column, using a mobile phase that consisted of a water-methanol solution (88∶12, v/v) containing 10 mmol/Ll-leucine and 5 mmol/L copper sulfate as a chiral ligand additive at a flow rate of 1.0 mL/min. This method allowed baseline separation of two enantiomers with a resolution of 1.84 in less than 30 min. The effect of various conditions, including concentration, type of ligand, organic modifier, pH, flow rate, and temperature, on enantioseparation were evaluated and chiral recognition mechanisms were investigated. Thermodynamic data (ΔΔH and ΔΔS) obtained by van't Hoff plots revealed that enantioseparation is an enthalpy-controlled process.     

Theory of lipid polymorphism: application to phosphatidylethanolamine and phosphatidylserine

We introduce a microscopic model of a lipid with a charged headgroup and flexible hydrophobic tails, a neutral solvent, and counter ions. Short-ranged interactions between hydrophilic and hydrophobic moieties are included as are the Coulomb interactions between charges. Further, we include a short-ranged interaction between charges and neutral solvent, which mimics the short-ranged, thermally averaged interaction between charges and water dipoles. We show that the model of the uncharged lipid displays the usual lyotropic phases as a function of the relative volume fraction of the headgroup. Choosing model parameters appropriate to dioleoylphosphatidylethanolamine in water, we obtain phase behavior that agrees well with experiment. Finally we choose a solvent concentration and temperature at which the uncharged lipid exhibits an inverted hexagonal phase and turn on the headgroup charge. The lipid system makes a transition from the inverted hexagonal to the lamellar phase, which is related to the increased waters of hydration correlated with the increased headgroup charge via the charge-solvent interaction. The polymorphism displayed upon variation of pH mimics that of the behavior of phosphatidylserine.     

猪肝酯酶催化苯乙二醇环碳酸酯水解最佳反应条件的研究

猪肝酯酶是手性合成中重要的水解酶,在猪肝酯酶的催化下,苯乙二醇环碳酸酯发生水解,生成苯乙二醇。实验围绕影响猪肝酯酶催化反应活性的4个主要因素进行了系统研究,得到了最优的酶浓度（15g/L）、pH值（8.0）、温度（25~30℃）及有机溶剂种类和浓度（二氧六环,65%v/v）,为猪肝酯酶催化苯乙二醇环碳酸酯反应的进一步研究奠定了基础。     

Development of a capillary electrophoresis method for the screening of human urine for multiple drugs of abuse

A new capillary electrophoresis (CE) method was developed and validated for the screening of human urine for nineteen drugs of abuse. In order to achieve sufficient separation, the electrolyte composition was modified using beta-cyclodextrin (beta-CD) and organic solvents. To process each sample, a sequential injection-solid-phase extraction (SI-SPE) system was constructed. Using this device, matrix clean-up, extraction, and preconcentration of analytes were performed onto a C(18) cartridge. Optimal separation and detection were obtained using a background electrolyte consisting of 100mM phosphate adjusted to pH 6.0, with 20 mM beta-CD, 5% acetonitrile and 20% isopropanol. Electrokinetic injection was performed at 5 kV for 10s, separation voltage was 25 kV and column temperature was set to 25 degrees C. The separation was carried out in a 67.0 cm x 50 microm fused-silica capillary with UV detection at 214 nm. The combination of SI-SPE and sample stacking showed significant sensitivity enhancement with limits of detection in the range of 5-30 ng ml(-1). A validation study showed good reproducibility of both migration time (RSD=0.003-0.088%) and peak area (RSD=0.54-4.8%). Overall, this automated and miniaturized SI-SPE system provides a rapid, sensitive, and robust procedure for analysis; as well as minimizes sample and solvent consumption.     

FACTORS INVOLVED IN THE USE OF ORGANIC SOLVENTS AS PRECIPITATING AND DRYING AGENTS OF IMMUNE SERA

1. In concentrations of 70 to 75 per cent the organic solvents methyl, ethyl, and propyl alcohols, and acetone cause complete precipitation of serum proteins and produce maximum loss in solubility. We have referred to this concentration range as the critical concentration. 2. As the concentration of the solvents is increased from about 75 per cent precipitation continues complete but loss in solubility progressively decreases until at all concentrations above about 87 per cent the precipitates formed at room temperature are completely soluble. 3. The degree of resolubility of the precipitates formed even in these high concentrations of the organic solvent decreases as the temperature is raised and as the duration of exposure is increased. 4. At 5°C. the precipitates formed in all concentrations of these organic solvents are completely resoluble. Also these solvents exert maximum precipitating effect at lower temperature. 5. Maximum precipitating effect by these organic solvents occurs at about pH 6.0 precipitation becoming progressively less as the pH value is altered either way from this point. 6. The more concentrated the serum, the greater the proportion of protein present that will be precipitated by any given concentrations of organic solvent. 7. A method for preparing dry immune sera has been given. Such dried sera have been extracted with a number of organic compounds without loss in solubility or antibody activity.     

Determination of some pyridinium aldoxime compounds by means of ion-pair reversed-phase high-performance liquid chromatography: Application in biological material

Two reversed-phase high-performance liquid chromatographic systems are presented for the separation and assay of the pyridinium aldoximes benzyl-P2A, HI-6 and obidoxime in aqueous solutions and biological samples. The systems involve a 5-μm C₁₈ silica gel stationary phase. The eluent consists of methanol, acetic acid buffer (pH 4.80), a counter ion (perchlorate or n-octanesulphonate) and a surfactant. The compounds were detected spectrophotometrically at 304 nm. In the concentration range used, linear plots of concentration versus extinction were obtained, both in blood and in water. Detection limits, even in blood, are satisfactory (0.5–1 μM).Evidence is presented that, at least for HI-6, the addition of counter ions to the system does not lead to the formation of ion pairs to be retained by partition, but rather to a mechanism based on adsorption chromatography.     

Improvements of GC and HPLC analyses in solvent (acetone-butanol-ethanol) fermentation byClostridium saccharobutylicum using a mixture of starch and glycerol as carbon source

A study on the feasibility of using improved computer-controlled HPLC and GC systems was carried out to shorten the time needed for measuring levels of the substrates (glucose, maltose, and glycerol) and products (acetone, butanol ethanol, acetic acid, and butyric acid) produced byClostridium saccharobutylicum DSM 13864 during direct fermentation of sago starch to solvent. The use of HPLC system with a single injection to analyse the composition of culture broth (substrates and products) during solvent fermentation was achieved by raising the column temperature to 80°C. Although good separation of the components in the mixture was achieved, a slight overlap was observed in the peaks for butyric acid and acetone. The shape of the peak obtained and the analysis time of 26.66 min were satisfactory at a fixed flow rate of 0.8 mL/min. An improved GC system was developed, that was able to measure the products of solvent fermentation (acetone, butanol, ethanol, acetic acid, and butyric acid) within 19.28 min. Excellent resolution for each peak was achieved by adjusting the oven temperature to 65°C.     

The Use of Artificial Neural Networks for Optimizing Polydispersity Index (PDI) in Nanoprecipitation Process of Acetaminophen in Microfluidic Devices

Artificial neural networks (ANNs) were used in this study to determine factors that control the polydispersity index (PDI) in an acetaminophen nanosuspension which was prepared using nanoprecipitation in microfluidic devices. The PDI of prepared formulations was measured by dynamic light scattering. Afterwards, the ANNs were applied to model the data. Four independent variables, namely, surfactant concentration, solvent temperature, and flow rate of solvent and antisolvent were considered as input variables, and the PDI of acetaminophen nanosuspension was taken as the output variable. The response surfaces, generated as 3D graphs after modeling, were used to survey the interactions happening between the input variables and the output variable. Comparison of the response surfaces indicated that the antisolvent flow rate and the solvent temperature have reverse effect on the PDI, whereas solvent flow rate has direct relation with PDI. Also, the effect of the concentration of the surfactant on the PDI was found to be indirect and less influential. Overall, it was found that minimum PDI may be obtained at high values of antisolvent flow rate and solvent temperature, while the solvent flow rate should be kept to a minimum.     

The effect of pH and lactose concentration on solvent production from whey permeate using Clostridium acetobutylicum

A study was performed to optimize the production of solvents from whey permeate in batch fermentation using Clostridium acetobutylicum P262. Fermentations performed at relatively low pH values resulted in high solvent yields and productivities, but lactose utilization was incomplete. At higher pH values, lactose utilization was improved but acid production dominated over solvent production. When operating at the higher pH values, an increase in the initial lactose concentration of the whey permeate resulted in lower rates of lactose utilization, and this was accompanied by increased solvent production and decreased acid production. Analysis of data from several experiments revealed a strong inverse relationship between solvent yield and lactose utilization rate. Thus, conditions which minimize the lactose utilization rate, such as low culture pH values or high initial lactose concentrations, favor solventogenesis at the expense of acid production.     

Isolation and purification of protease from human placenta by foam fractionation

The effect of operating parameters like pH, protein concentration, column geometry, and gas flow rate on the separation efficiency of proteolytic enzymes from crude human placental homogenate has been studied in a batch foam column. Purification has been found to be optimum at pH 8.0, close to the isoelectric pH, at which the surface adsorption of the protein on the foam bubbles is maximum. Both purification and recovery varied significantly with total protein concentration. Stable bubble formation was hindered at lower protein concentrations, while extraneous proteins rather than the protease were preferentially adsorbed at higher protein concentrations, decreasing the purification efficiency. Column diameter and column height should be optimized for any specific feed protein concentration and gas flow rate. However, the enrichment ratio was found to decrease with the increase in flow rate. The results indicate that foam fractionation is an effective separation process for recovering valuable biochemicals from biological materials.     

Production of biodiesel using a continuous gas-liquid reactor

A novel continuous reactor process has been developed for the production of biodiesel from fats and oils. The key feature of the process is its ability to operate continuously with a high reaction rate, potentially requiring less post reaction cleaning and product/reactant separation than currently established processes. This was achieved by atomising the heated oil/fat and then spraying it into a reaction chamber filled with methanol vapor in a counter current flow arrangement. This allows the continuous separation of product and the excess methanol stream in the reactor. The overall conversion based on a single cycle of this process has been between 50% and 96% of the feed stock materials. Conversions of 94-96% were achieved while operating with 5-7 g of sodium methoxide/L of methanol at methanol flow rate of 17.2 L/h and oil flow rate of 10 L/h. Additional variations in the reactant stoichiometry (i.e. reactant flow rates), catalyst type/concentration, and reaction temperature on the overall product conversion were investigated.     

The separation of myo-inositol phosphates by ion-pair chromatography

The separation of myo-inositol phosphates by ion pair, reverse-phase high performance liquid chromatography has been investigated. The retention of the inositol phosphates is dependent on both the polarity of the hetaeron utilized and on the pH of the solvent. A method is presented which permits the isocratic separation of multiple forms of inositol phosphates including isomers of myo-inositol trisphosphate. This method appears to be superior to the anion exchange based systems currently employed because of smaller retention volumes, the low ionic strength of the solvent employed, the absence of a requirement for reequilibration, and the ability to perform separations isocratically.