Gradient Procedure Research Articles

Fast gradient high performance liquid chromatography method with UV detection for simultaneous determination of seven angiotensin converting enzyme inhibitors together with hydrochlorothiazide in pharmaceutical dosage forms and spiked human plasma and urine

The development of a reversed phase liquid chromatographic method for the simultaneous determination of seven angiotensin converting enzyme (ACE) inhibitors; five drugs namely benazepril HCl (BZL), enalapril maleate (ENL), fosinopril sodium (FSP), lisinopril (LSP) and ramipril (RMP) and two metabolites captopril disulfide (CPD) and enalaprilat (ENT) together with hydrochlorothiazide (HCT) is described. The method can serve as a substitute for many published papers for the analysis of the targeted compounds with or without hydrochloothiazide in pharmaceutical formulations as well as in spiked human plasma and urine samples. The method utilizes a simple gradient procedure for the separation in a 11min run time using acetonitrile aqueous ammonia buffer (pH 9) solution and an Extend RP-C18 (25μm particle size, 4.6mm×250mm, Agilent) HPLC column. The effluent was monitored on a UV detector at 215nm. The effect of pH, solvent strength and analysis time on the peak shape and quantification were carefully studied in order to optimize the method. Adopting the proposed procedure, the analytes produce well-shaped peaks with good linear relationship over the investigated concentration ranges. The limits of detection (LOD) and limits of quantification (LOQ) from standard drug solutions lie in the range of 17–64 and 56–212ngmL−1, respectively. Correlation coefficient values (r) higher than 0.997 were obtained for all the studied drugs in spiked human plasma and urine samples. The intra-day and inter-day precision of the method was evaluated with relative standard deviation values being satisfactory for their purposed analysis. The method was validated with respect to specificity, recovery, accuracy, precision and linearity.

Functional optimization technique for numerical solution of the radiative transfer equation

A numerical method for solving the time-independent radiative transfer problem in a flat layer with given properties and temperature distribution is proposed. This method avoids the numerical diffusion; rather, it is based on a gradient procedure for the functional minimization of the residual of the radiative transfer integral equation. Means for suppressing computational instabilities are proposed that reduce requirements for the approximation of the operators in the optimization problem but do not change the problem objective functional.

Búsqueda exhaustiva por entornos aplicada al diseño económico de bóvedas de hormigón armado

This paper presents two gradient algorithms applied to the economic optimization of reinforced concrete vaults, typically used in the construction of underpasses. The algorithms are gradient multi start neighbourhood exhaustive search procedures. The vault is defined by 45 design variables and the objective function is an economic one. Both methods have been applied to a vault of 12.40 m of diameter and 3.00 m of lateral walls. Design variables have been coded in base 2 for algorithms GB1 and in Gray coding for GB2. The neighbourhood is defined by the set of solutions that differ in one bit. The Gray coding solves the lack of proximity between two solutions typical of the ordinary binary coding. The positive effect of the Gray coding is proven in the present paper, where the average cost of 3,000 runs of GB2 improves by 3.81% a similar run by algorithm GB1. In addition, GB2 attains the best cost solution. The paper includes a stop criterion for the algorithm based on the stability of the statistics of the multi start results. The algorithms are simple and can be applied to other structural problems. The structure of best cost has a high slenderness and a span to depth ratio of 40. The study reports savings of 6% when compared to a design by an experienced practitioner office.

F101 BDD前処理の統一的構築方法についての一考察(F1.低炭素社会向けシミュレーション技術,フォーラム)

An iterative domain decomposition method is applied to numerical analysis of 3-Dimensional (3D) linear magnetostatic problems taking the magnetic vector potential as an unknown function. The iterative domain decomposition method is combined with the Preconditioned Conjugate Gradient (PCG) procedure and the Hierarchical Domain Decomposition Method (HDDM) which is adopted in parallel computing. Our previously employed preconditioner was the Neumann-Neumann preconditioner. Numerical results showed that the method was only effective for smaller problems. In this paper, we consider its improvement with the Balancing Domain Decomposition (BDD) preconditioner. Also, we discuss a unified approach for the construction of BDD preconditioners in various applications including elastic problems, heat transfer problems and incompressible viscous flow problems besides magnetostatic problems.

Identification of internal control genes for quantitative expression analysis by real-time PCR in bovine peripheral lymphocytes

Gene expression studies in blood cells, particularly lymphocytes, are useful for monitoring potential exposure to toxicants or environmental pollutants in humans and livestock species. Quantitative PCR is the method of choice for obtaining accurate quantification of mRNA transcripts although variations in the amount of starting material, enzymatic efficiency, and the presence of inhibitors can lead to evaluation errors. As a result, normalization of data is of crucial importance. The most common approach is the use of endogenous reference genes as an internal control, whose expression should ideally not vary among individuals and under different experimental conditions. The accurate selection of reference genes is therefore an important step in interpreting quantitative PCR studies. Since no systematic investigation in bovine lymphocytes has been performed, the aim of the present study was to assess the expression stability of seven candidate reference genes in circulating lymphocytes collected from 15 dairy cows. Following the characterization by flow cytometric analysis of the cell populations obtained from blood through a density gradient procedure, three popular softwares were used to evaluate the gene expression data. The results showed that two genes are sufficient for normalization of quantitative PCR studies in cattle lymphocytes and that YWAHZ, S24 and PPIA are the most stable genes.

Rosa multiflora Thunberg fruit 추출물로부터 Helicobacter pylori 억제물질의 분리 및 동정

The antimicrobial activity of 70% ethanol extracts from Rosa multiflora Thunberg fruit against Helicobacter pylori was examined. The inhibitory activity of Rosa multiflora Thunberg fruit extracts against H. pylori was determined to clear a zone of 14 mm with 70% ethanol extracts. Purification of inhibitory compounds was carried on Sephadex LH-20 and cartridge column chromatography using a gradient procedure, with increasing ethanol () in . The chemical structure of the purified inhibitory compounds on H. pylori was identified to be protocatechuic acid, chlorogenic acid and quercetin by FAB-MS, NMR and IR spectrum.

Ohr (Organic Hydroperoxide Resistance Protein) Possesses a Previously Undescribed Activity, Lipoyl-dependent Peroxidase

The Ohr (organic hydroperoxide resistance) family of 15-kDa Cys-based, thiol-dependent peroxidases is central to the bacterial response to stress induced by organic hydroperoxides but not by hydrogen peroxide. Ohr has a unique three-dimensional structure and requires dithiols, but not monothiols, to support its activity. However, the physiological reducing system of Ohr has not yet been identified. Here we show that lipoylated enzymes present in the bacterial extracts of Xylella fastidiosa interacted physically and functionally with this Cys-based peroxidase, whereas thioredoxin and glutathione systems failed to support Ohr peroxidase activity. Furthermore, we could reconstitute in vitro three lipoyl-dependent systems as the Ohr physiological reducing systems. We also showed that OsmC from Escherichia coli, an orthologue of Ohr from Xylella fastidiosa, is specifically reduced by lipoyl-dependent systems. These results represent the first description of a Cys-based peroxidase that is directly reduced by lipoylated enzymes.

Variational approach to the solution of linear multiparameter eigenvalue problems

We associate a multiparameter spectral problem in a real Euclidean space with a variational problem of finding a minimum of a certain functional. We establish the equivalence of the spectralproblem and the variational problem. On the basis of the gradient procedure, we propose a numerical algorithm for the determination of its eigenvalues and eigenvectors. The local convergence of the algorithm is proved.

진달래꽃(Rhododendron mucronulatum Flos.) 추출물로부터 Helicobacter pylori 억제 효과를 가지는 phenol성 물질의 정제 및 동정

진달래꽃을 60% ethanol로 추출하여 추출물의 H. pylori에 대한 저해활성의 측정과 저해물질의 분리 및 동정을 시행하였다. 또한 H. pylori균에 대한 항균활성은 60% ethanol 추출물에서 phenol 화합물을 200<TEX>${\mu}g/ml$</TEX> 이상 주입하였을 때부터 22 mm이상의 clear zone이 관찰되어 H. pylori균에 대한 높은 항균력을 나타내었다. 저해물질의 분리는 Sephadex LH-20 column을 이용하여 분리한 결과 6개의 fraction으로 분리되었으며, H. pylori 저해활성을 나타낸 fraction D를 MCI-gel CHP-20 column을 이용하여 normal phase type인 ethanol 100%에서 0%로 농도를 감소시키며 용출한 결과 5개의 단일 물질 분획을 얻을 수 있었다. 그 중 세 가지의 물질에 대해 H. pylori저해활성이 나타나 FAB-MS, <TEX>$^1H$</TEX>와 <TEX>$^{13}C$</TEX> NMR 및 IR spectrum을 사용하여 구조 동정한 결과 quercitrin (quercetin-3-O-rhamnopyranoside), myricitrin (myricetin-3-O-rhamnopyranoside) 및 quercetin인 것으로 확인되었다. The antimicrobial activities of Helicobacter pylori as a functional food source with water and 60% ethanol extracts from Rododendron mucronulatum Turcz. flowers were examined. The total phenol content of 60% ethanol extracts (30.6<TEX>${\pm}$</TEX>0.14 mg/g) from Rododendron mucronulatum Turcz. flowers was higher than that of water extracts (23.2<TEX>${\pm}$</TEX>0.21 mg/g). The inhibitory activities of Rododendron mucronulatum Turcz. extracts on H. pylori was determined to clear zone of 15 mm in 80% ethanol extracts. Purification of inhibitory compounds was carried out in Sephadex LH-20 and MCI-gel CHP-20 column chromatography using a gradient procedure, with increasing ethanol(0<TEX>${\rightarrow}$</TEX>100%) in <TEX>$H_2O$</TEX>. The chemical structure of the purified inhibitory compounds of H. pylori was identified to be quercitrin (quercetin-3-O- rhamnopyranoside), myricitrin (myricetin-3-O-rhamnopyranoside), quercetin by FAB-MS, NMR and IR spectra.

Quantitation of Irinotecan and its two major metabolites using a liquid chromatography–electrospray ionization tandem mass spectrometric

A sensitive and selective liquid chromatographic–tandem mass spectrometric (LC–MS/MS) method for the determination of Irinotecan (CPT-11) and its metabolites in human plasma has been developed. Samples were prepared after protein precipitation and analyzed on a C 18 column interfaced with a Q-Trap tandem mass spectrometer. Positive electrospray ionization was employed as the ionization source. The mobile phase consisted of acetonitrile–water (0.05% formic acid), using gradient procedure. The analytes and internal standard camptothecin were both detected by use of multiple reaction monitoring mode. The method was linear in the concentration range of 10.0–2000.0 ng/ml for CPT-11 and 0.5–200.0 ng/ml for 7-ethyl-10-hydroxycamptothecin (SN-38), respectively. The lower limit of quantification (LLOQ) was 10 ng/ml for CPT-11 and 0.5 ng/ml for SN-38. The intra- and inter-day relative standard deviation across three validation runs over the entire concentration range was less than 10.6%. The accuracy determined at three concentrations was within ±11.4% in terms of relative error. Due to the unavailability of standard for 7-ethyl-10- O-glucuronyl-camptothecin (SN-38G) and the importance of knowing the concentration of this metabolite, we developed a method for analysis SN-38G by taking advantage of the quantitive conversion of SN-38G to SN-38 using glucuronidase. This enzymatic method of identification and quantitation of gluconated compound can be widely used when the standard for phase II glucuronide metabolites are not available.

청목노상 뽕잎으로부터 Helicobacter pylori 억제물질의 정제 및 동정

본 연구에서는 청목노상 추출물로부터 Helicobacter pylori를 억제하는 물질을 정제 및 동정을 실시하였다. 80% 에탄올 추출물의 페놀성 물질의 함량은 16.1 mg/g으로 측정되었으며, H. pylori 저해활성은 clear zone이 15 mm로 나타났다. 저해물질의 분리는 <TEX>$C_{18}$</TEX> column을 이용하여 분리한 결과 9개의 fraction으로 분리되었으며, H. pylori 저해활성을 나타낸 fraction 7과 8을 MCI-gel CHP-20 column을 이용하여 normal phase type인 ethanol 100%에서 0%로 농도를 감소시키며 10 mL/min의 유속으로 용출한 결과 6개의 단일 물질 분획을 얻을 수 있었다. 그 중 세 가지의 물질이 H. pylori저해활성을 보여 FAB-Mass, <TEX>$^1H$</TEX>와 <TEX>$^{13}C$</TEX> NMR과 IR spectrum을 사용하여 구조 동정한 결과 chlorogenic acid, caffeic acid 및 rosmarinic acid인 것으로 확인되었다. In this study, we tried to find the subject to inhibit H. pylori from Cheongmoknosang mulberry leaves extracts and to purify and identify them. Total phenolic compounds of hot water and 80% ethanol extracts are 17.6 and 16.1 mg/g. The activity of H. pylori inhibition at 80% ethanol extracts was determined as 15mm clear zone. The purification of inhibitory compounds were carried on <TEX>$C_{18}$</TEX> column and MCI-gel CHP-20 column chromatography which were used a gradient procedure as increasing ethanol in <TEX>$H_2O$</TEX>. The chemical structure of purified inhibitory compounds on H. pylori were identified chlorogenic acid, caffeic acid, and rosmarinic acid by FAB-MS, <TEX>$^1H-NMR$</TEX>, <TEX>$^{13}C-NMR$</TEX> and IR spectrum.

Interaction of Mint2 with TrkA Is Involved in Regulation of Nerve Growth Factor-induced Neurite Outgrowth

TrkA receptor signaling is essential for nerve growth factor (NGF)-induced survival and differentiation of sensory neurons. To identify possible effectors or regulators of TrkA signaling, yeast two-hybrid screening was performed using the intracellular domain of TrkA as bait. We identified muc18-1-interacting protein 2 (Mint2) as a novel TrkA-binding protein and found that the phosphotyrosine binding domain of Mint2 interacted with TrkA in a phosphorylation- and ligand-independent fashion. Coimmunoprecipitation assays showed that endogenous TrkA interacted with Mint2 in rat tissue homogenates, and immunohistochemical evidence revealed that Mint2 and TrkA colocalized in rat dorsal root ganglion neurons. Furthermore, Mint2 overexpression inhibited NGF-induced neurite outgrowth in both PC12 and cultured dorsal root ganglion neurons, whereas inhibition of Mint2 expression by RNA interference facilitated NGF-induced neurite outgrowth. Moreover, Mint2 was found to promote the retention of TrkA in the Golgi apparatus and inhibit its surface sorting. Taken together, our data provide evidence that Mint2 is a novel TrkA-regulating protein that affects NGF-induced neurite outgrowth, possibly through a mechanism involving retention of TrkA in the Golgi apparatus.

Quantification of plasmid DNA copies in the nucleus after lipoplex and polyplex transfection

Nuclear uptake of plasmid DNA is one of the many cellular barriers that limit the efficiency of non-viral gene delivery systems. We have determined the number of plasmids that reach the nucleus of a transfected cell using an internally standardized quantitative PCR (qPCR) assay. We isolated nuclei using two different protocols: a density gradient technique and a detergent-based method. The density gradient procedure yielded nuclei with substantially less adhering plasmids on the outside of the nuclei. Using the density gradient protocol we determined that cells transfected with Lipofectamine™ lipoplexes or polyethylenimine polyplexes contained between 75 and 50,000 plasmids/nucleus, depending on the applied plasmid dose. Any increase above 3000 plasmids/nucleus resulted in only marginal increases in transgene expression. Furthermore, lipoplex-delivered plasmids were more efficiently expressed, on the basis of protein expression per plasmid number in the nucleus, than polyplex-delivered plasmids. This indicates that polymer may remain bound to some plasmids in the nucleus. Lastly, by sorting transfected cells into high- and low-expressing sub-populations, we observe that a sub-population of cells contain 3× greater plasmids/nucleus but express nearly 100× more transgene than other cells within a single transfection reaction. Taken together these results suggest the importance of considering the processes downstream from nuclear entry for strategies to improve the efficiency of gene transfer reagents.

Numerical optimization for the calculus of variations by gradients on non-Hilbert Sobolev spaces using conjugate gradients and normalized differential equations of steepest descent

Numerical optimization for the calculus of variations by gradients on non-Hilbert Sobolev spaces using conjugate gradients and normalized differential equations of steepest descent

A rapid Percoll gradient procedure for preparation of synaptosomes

Homogenization of fresh brain tissue in isotonic medium shears plasma membranes causing nerve terminals to become separated from their axons and postsynaptic connections. The nerve terminal membranes then reseal to form synaptosomes. The discontinuous Percoll gradient procedure described here is designed to isolate synaptosomes from brain homogenates in the minimum time to allow functional experiments to be performed. Synaptosomes are isolated using a medium-speed centrifuge, while maintaining isotonic conditions and minimizing mechanically damaging resuspension steps. This protocol has advantages over other procedures in terms of speed and by producing relatively homogeneous synaptosomes, minimizing the presence of synaptic and glial plasma membranes and extrasynaptosomal mitochondria. The purified synaptosomes are viable and take up and release neurotransmitters very efficiently. A typical yield of synaptosomes is between 2.5 and 4 mg of synaptosomal protein per gram rat brain. The procedure takes approximately 1 h from homogenization of the brain until collection of the synaptosomal suspension from the Percoll gradient.

LC and Principal Component Analysis in the Lipophilicity Study of Seven Angiotensin II-AT1 Receptor Antagonists (Sartans)

Seven sartans have been chromatographed with acetonitrile-buffer and methanol–buffer in different proportions as mobile phases. The retention values, log k or R M were extrapolated to zero organic modifier content to obtain the log k w or R MW values. Calibration equations were obtained for standards of known lipophilicity. A simple method employing a gradient procedure of 10–100% acetonitrile or methanol in 60 min and standards of the extreme lipophilicity was also elaborated. Chromatographic log P values were compared to those calculated by use of different software products. Finally, principal component analysis was performed to explore and visualize similarities and differences among the drugs and among the methods.

A reduced-order model of the low-voltage cascade electroosmotic micropump

In the present investigation, we have derived an efficient reduced-order model of the low-voltage cascade electroosmotic micropump. This model can be combined with the equivalent circuit model of straight microchannels to construct a complete model for a microfluidic device, which can be employed to implement modern control schemes. To demonstrate the efficiency of the reduced-order model we employ it to estimate the zeta potentials of many subchannels in the micropump cascade using velocity measurements, which is a preliminary step to the implementation of modern control schemes. It is found that a conjugate gradient procedure employing the reduced-order model estimates accurately the zeta potential variation in the subchannels, which may be caused by adhesion of biomolecules, even with noisy velocity measurements.

Lipoprotein separation in a novel iodixanol density gradient, for composition, density, and phenotype analysis

Separation of lipoproteins by traditional sequential salt density floatation is a prolonged process ( approximately 72 h) with variable recovery, whereas iodixanol-based, self-generating density gradients provide a rapid ( approximately 4 h) alternative. A novel, three-layered iodixanol gradient was evaluated for its ability to separate lipoprotein fractions in 63 subjects with varying degrees of dyslipidemia. Lipoprotein cholesterol, triglycerides, and apolipoproteins were measured in 21 successive iodixanol density fractions. Iodixanol fractionation was compared with sequential floatation ultracentrifugation. Iodixanol gradient formation showed a coefficient of variation of 0.29% and total lipid recovery from the gradient of 95.4% for cholesterol and 84.7% for triglyceride. Recoveries for VLDL-, LDL-, and HDL-cholesterol, triglycerides, and apolipoproteins were approximately 10% higher with iodixanol compared with sequential floatation. The iodixanol gradient effectively discriminated classic lipoproteins and their subfractions, and there was evidence for improved resolution of lipoproteins with the iodixanol gradient. LDL particles subfractionated by the gradient showed good correlation between density and particle size with small, dense LDL (<25.5 nm) separated in fractions with density >1.028 g/dl. The new iodixanol density gradient enabled rapid separation with improved resolution and recovery of all lipoproteins and their subfractions, providing important information with regard to LDL phenotype from a single centrifugation step with minimal in-vitro modification of lipoproteins.

A hybrid KA‐MoM algorithm for computation of scattering from a 3‐D PEC target above a dielectric rough surface

A new hybrid analytical‐numerical iterative algorithm, which combines the Kirchhoff approximation (KA) and the Method of Moment (MoM), is developed for computing the electromagnetic scattering from a three‐dimensional (3‐D) perfect electric conducting (PEC) target above a 2‐D infinite randomly rough dielectric surface. The equations of difference scattering due to the target presence above the rough surface are derived. The induced difference fields on the rough surface due to the interactions between the target and the rough surface are calculated by using the KA method. The excitation term on the right‐hand‐side (RHS) of target's surface integral equation (SIE), which contains the difference scattering of the rough surface, is then updated for calculating new target currents with the Conjugate Gradient (CG) procedure. Then the target currents are used to compute the difference field induced on the rough surface with the KA method. Multiple iterations take account of the multiorder interactions between the target and the underlying rough surface. Numerical quadrature upon the rough surface is performed only once to compute the coupling scattering field from the rough surface to the target, and it takes N steps (N is the discretized mesh number of rough surface). By using this hybrid KA‐MoM algorithm, the requirements of memory and CPU time can be reduced significantly. Moreover, the validity conditions and the convergence performance of this hybrid algorithm are also discussed. With Monte‐Carlo generations of randomly rough surfaces, bistatic scattering from different‐shaped targets above a Gaussian rough surface is numerically simulated. Finally, dependence of bistatic scattering pattern on the surface dielectric property and the target geometry is discussed.

Validation of an ultra‐fast UPLC‐UV method for the separation of antituberculosis tablets

A simple method using ultra performance LC (UPLC) coupled with UV detection was developed and validated for the determination of antituberculosis drugs in combined dosage form, i. e. isoniazid (ISN), pyrazinamide (PYR) and rifampicin (RIF). Drugs were separated on a short column (2.1 mm x 50 mm) packed with 1.7 mum particles, using an elution gradient procedure. At 30 degrees C, less than 2 min was necessary for the complete separation of the three antituberculosis drugs, while the original USP method was performed in 15 min. Further improvements were obtained with the combination of UPLC and high temperature (up to 90 degrees C), namely HT-UPLC, which allows the application of higher mobile phase flow rates. Therefore, the separation of ISN, PYR and RIF was performed in less than 1 min. After validation (selectivity, trueness, precision and accuracy), both methods (UPLC and HT-UPLC) have proven suitable for the routine quality control analysis of antituberculosis drugs in combined dosage form. Additionally, a large number of samples per day can be analysed due to the short analysis times.