High-throughput SNP detection by using DNA pooling and denaturing high performance liquid chromatography (DHPLC)

One of the critical steps in the positional cloning of a complex disease gene involves association analysis between a phenotype and a set of densely spaced diallelic markers, typically single nucleotide repeats (SNPs), covering the region of interest. However, the effort and cost of detecting sufficient numbers of SNPs across relatively large physical distances represents a significant rate-limiting step. We have explored DNA pooling, in conjunction with denaturing high performance liquid chromatography (DHPLC), as a possible strategy for augmenting the efficiency, economy, and throughput of SNP detection. DHPLC is traditionally used to detect variants in polymerase chain reaction products containing both allelic forms of a polymorphism (e.g., heterozygotes or a 1:1 mix of both alleles) via heteroduplex separation and thereby requires separate analyses of multiple individual test samples. We have adapted this technology to identify variants in pooled DNA. To evaluate the utility and sensitivity of this approach, we constructed DNA pools comprised of 20 previously genotyped individuals with a frequency representation of 0%-50% for the variant allele. Mutation detection was performed by using temperature-modulated heteroduplex formation/DHPLC and dye-terminator sequencing. Using DHPLC, we could consistently detect SNPs at lower than 5% frequency, corresponding to the detection of one variant allele in a pool of 20 alleles. In contrast, fluorescent sequencing detected variants in the same pools only if the frequency of the less common allele was at least 10%. We conclude that DNA pooling of samples for DHPLC analysis is an effective way to increase throughput efficiency of SNP detection.     

Simultaneous analysis of the non-canonical amino acids norleucine and norvaline in biopharmaceutical-related fermentation processes by a new ultra-high performance liquid chromatography approach

In this study, a precise and reliable ultra-high performance liquid chromatography (UHPLC) method for the simultaneous determination of non-canonical (norvaline and norleucine) and standard amino acids (aspartic acid, glutamic acid, serine, histidine, glycine, threonine, arginine, tyrosine, methionine, valine, phenylalanine, isoleucine, leucine) in biopharmaceutical-related fermentation processes was established. After pre-column derivatization with ortho-phthaldialdehyde and 2-mercaptoethanol, the derivatives were separated on a sub-2 μm particle C₁₈ reverse-phase column. Identification and quantification of amino acids were carried out by fluorescence detection. To test method feasibility on standard HPLC instruments, the assay was properly transferred to a core–shell particle C₁₈ reverse-phase column. The limits of detection showed excellent sensitivity by values from 0.06 to 0.17 pmol per injection and limits of quantification between 0.19 and 0.89 pmol. In the present study, the newly established UHPLC method was applied to a recombinant antibody Escherichia coli fermentation process for the analysis of total free amino acids. We were able to specifically detect and quantify the unfavorable amino acids in such complex samples. Since we observed trace amounts of norvaline and norleucine during all fermentation phases, an obligatory process monitoring should be considered to improve quality of recombinant protein drugs in future.     

High performance liquid chromatography analysis of 2-mercaptoethylamine (cysteamine) in biological samples by derivatization with N-(1-pyrenyl) maleimide (NPM) using fluorescence detection

2-Mercaptoethylamine (cysteamine) is an aminothiol compound used as a drug for the treatment of cystinosis, an autosomal recessive lysosomal storage disorder. Because of cysteamine's important role in clinical settings, its analysis by sensitive techniques has become pivotal. Unfortunately, the available methods are either complex or labor intensive. Therefore, we have developed a new rapid, sensitive, and simple method for determining cysteamine in biological samples (brain, kidney, liver, and plasma), using N-(1-pyrenyl) maleimide (NPM) as the derivatizing agent and reversed-phase high performance liquid chromatography (HPLC) with a fluorescence detection method (lambda(ex)=330 nm, lambda(em)=376 nm). The mobile phase was acetonitrile and water (70:30) with acetic acid and o-phosphoric acid (1 mL/L). The calibration curve for cysteamine in serine borate buffer (SBB) was found to be linear over a range of 0-1200 nM (r(2)=0.9993), and in plasma and liver matrix, the r(2) values were 0.9968 and 0.9965, respectively. The coefficients of the variation for the within-run and between-run precisions ranged from 0.68 to 9.90% and 0.63 to 4.17%, respectively. The percentage of relative recovery ranged from 94.1 to 98.6%.     

A rapid method for analysis of fermentatively produced D-xylonate using ultra-high performance liquid chromatography and evaporative light scattering detection

An ultra-high performance liquid chromatography (UHPLC) based method for the analysis of d-xylonate was developed using an amide column in combination with an evaporative light scattering (ELS) detector. Separation of d-xylonate from other components of the fermentation medium was achieved. The dynamic range of the method was 0.2–7.0 g/L.     

Separation and quantitation of bile acids using an isocratic solvent system for high performance liquid chromatography coupled to an evaporative light scattering detector.

We developed a quantitative method for the analysis of bile acids using a high performance liquid chromatograph coupled to an evaporative light scattering detector. An isocratic solvent system was used to resolve in a single run conjugated and unconjugated bile acid species relevant in human and rodent physiology. The detection of various bile acids was linear over a range of 0.08 to 10 nmol of injected molecules. The developed system is a convenient and cost-effective method for the routine analysis of a wide variety of bile acids.     

Drug binding to human serum albumin: abridged review of results obtained with high-performance liquid chromatography and circular dichroism

The drug binding to plasma and tissue proteins are fundamental factors in determining the overall pharmacological activity of a drug. Human serum albumin (HSA), together with alpha1-acid glycoprotein (AGP), are the most important plasma proteins, which act as drug carriers, with drug pharmacokinetic implications, resulting in important clinical impacts for drugs that have a relatively narrow therapeutic index. This review focuses on the combination of biochromatography and circular dichroism as an effective approach for the characterization of albumin binding sites and their enantioselectivity. Furthermore, their applications to the study of changes in the binding properties of the protein arising by the reversible or covalent binding of drugs are discussed, and examples of physiological relevance reported. Perspectives of these studies reside in supporting the development of new drugs, which require miniaturization to facilitate the screening of classes of compounds for their binding to the target protein, and a deeper characterization of the mechanisms involved in the molecular recognition processes.     

Test of circular dichroism (CD) methods for crambin and CD-assisted secondary structure prediction of its homologous toxins

Methods that analyze protein circular dichroism (CD) spectra for fractions of secondary structure are evaluated for the plant protein crambin, which has a known high-resolution crystal structure. In addition, a two-step secondary structure prediction scheme is presented and used for the toxins homologous to crambin, shown by others to have secondary structures similar to crambin. The test of CD spectral analysis methods with the protein crambin employed two computer programs and several CD basis sets. Crambin's crystal structure, known to 0.945A resolution (Hendrickson, W.A., Teeter, M.M. Nature 290:107-113, 1981), allows accurate evaluation of results. Analysis with the protein spectra basis sets (Provencher, S.W., Gl?ckner, J. Biochemistry 20:33-37, 1981) as modified (Manavalan, P., Johnson, W.C., Jr. Anal. Biochem. 167:76-85, 1987) agreed most closely with crambin's crystal structure. This method was then applied to the CD spectra of the membrane-active toxins homologous to crambin (alpha 1- and beta-purothionin, phoratoxin A and B, and viscotoxin A3 and B). The new program SEQ (pronounced "seek") was developed to assign the secondary structure along the protein chain in a hierarchical fashion and applied to the plant toxins. The method constrained the secondary structure fractions to those from CD analysis and combined standard statistical methods with amphipathic helix location. Both CD-arrived secondary structure percentages and sequence assignment indicate that the viscotoxins are structurally most similar to crambin. Purothionin's secondary structure was predicted to be fundamentally similar to crambin's with a difference at the start of the first helix. This assignment agreed with Raman and NMR analyses of purothionin and lends validity to the method presented here. Differences from the NMR in the CD secondary structure fraction analysis for phoratoxin suggest interference in the CD from tryptophan residues.     

猪苓甾酮类化合物的HPLC含量测定及指纹图谱研究

收集中国12个省区35个猪苓样品,利用高效液相色谱技术,测定5个甾酮类化合物含量,获得指纹图谱,利用相似度评价对图谱数据进行分析。共有28个样品能获得定量分析结果,其中25个样品中猪苓酮A和B的相对含量大于70%;经相关分析,猪苓酮A和B的含量之间呈极显著正相关(r=0.955,P0.01)。陕西省样品平均总甾酮含量以及猪苓酮A和B相对含量均最高,分别为182.70μg/g(C.V=82.4%)和92.3%(C.V=3.1%)。35个样品之间指纹图谱相似度0.412–0.943,标定了8个共有峰,指认了其中的3个色谱峰。11个陕西省样品之间指纹图谱相似度0.812–0.989,标定了17个共有峰,指认了其中的9个色谱峰。以上结果说明,猪苓酮A和B是猪苓主要甾酮类成分,对猪苓药材的质量控制有重要意义;陕西省的猪苓质量好且稳定。     

Fluorometric determination of sialic acids using malononitrile in weakly alkaline media and its application to postcolumn labeling in high-performance liquid chromatography

A sensitive method for determination of sialic acids by monitoring the fluorescence produced with malononitrile in borate buffer has been established. Measurement of the fluorescence intensity of the reaction mixture at 430 nm with irradiation at 360 nm allowed determination of 3-60 nmol of sialic acids with high reproducibility. A few amino sugars and deoxy sugars, as well as catecholamines reacted with this reagent; however other carbohydrates, amino acids, amines, aldehydes, and carboxylic acids including alpha-keto acids, etc., showed little reactivity. This method was successfully applied to postcolumn fluorescence labeling of sialic acids in high-performance liquid chromatography.     

Analysis of lichen substances including triterpenoids by high performance liquid chromatography with a differential refractive index detector and a photodiode array detector

A new method for analysis of lichen triterpenoids was established using high performance liquid chromatography with the combination of a differential refractive index detector (RID) and a photodiode array detector (PDA). It is proved that this method was convenient to detect and identify aromatic and aliphatic lichen substances; it enabled quantitative analysis of substances having no or less absorption of ultraviolet rays such as triterpenoids. In addition, they can be measured in high accuracy compared with the TLC method.     

Determination of tramadol in human plasma and urine samples using liquid phase microextraction with back extraction combined with high performance liquid chromatography

Liquid phase microextraction by back extraction (LPME-BE) combined with high performance liquid chromatography (HPLC)-fluorescence detection was developed for the determination of tramadol in human plasma. Tramadol was extracted from 2 mL of basic sample solution (donor phase) with pH 11.5 through a micro liter-size organic solvent phase (100 microL n-octane) for 25 min and finally into a 3.5 microL acidic aqueous acceptor microdrop with pH 2.5 suspended in the organic phase from the tip of a HPLC microsyringe needle for 15 min with the stirring rate of 1250 rpm. After extraction for a period of time, the microdrop was taken back into the syringe and injected into HPLC. Effected the experimental parameters such as the nature of the extracting solvent and its volume, sample temperature, stirring rate, volume of the acceptor phase, pH and extraction time on LPME-BE efficiency was investigated. At the optimized condition, enrichment factor of 366 and detection limit (LOD) of 0.12 microg L(-1) were obtained. The calibration curve was linear (r=0.999) in the concentration range of 0.3-130 microg L(-1). Within-day relative standard deviation RSD (S/N=3) and between-day RSD were 3.16% and 6.29%, respectively. The method was successfully applied to determine the concentration of tramadol in the plasma and urine samples and satisfactory results were obtained.     

Poly(acrylic acid)-block-poly(L-valine): evaluation of beta-sheet formation and its stability using circular dichroism technique

Secondary structure formation in four novel hybrid poly(acrylic acid)-b-poly(L-valine) (PAA-b-PLVAL) block copolymers, that is, PAA(40)-PLVAL(100), PAA(80)-PLVAL(100), PAA(80)-PLVAL(80), and PAA(80)-PLVAL(60), was investigated by circular dichroism. The formation of stable and well-defined beta-sheet structure in the PLVAL hydrophobic domains was observed for all the copolymers. At pH 5, PAA(80)-PLVAL(60) with the lowest PLVAL/PAA molar ratio possessed the lowest beta-sheet content of 12%, and it increased to 62% for PAA(40)-PLVAL(100) system. The beta-sheet formation in the block copolymers was controlled by both random PAA-PLVAL hydrogen bonds at low pH and electrostatic repulsive forces on the PAA segment at high pH; hence, the beta-sheet structure was most stable at intermediate pH. The length of PAA segments was critical in the beta-sheet solubilization and in providing sufficient shielding of the hydrophobic core from denaturing agents such as urea.     

Development of a liquid chromatography method for the determination of linezolid and its application to in vitro and human microdialysis samples

Linezolid is a new, promising antibacterial agent to treat severe infections. A rapid HPLC assay using UV detection for the determination in microdialysate and human plasma was developed. After sample preparation, using acetonitrile for plasma and water for microdialysate, 20 microl was injected and separated on a RP-18 column. Overall, the assay exhibited good precision and accuracy. The diffusion properties of linezolid investigated in in vitro microdialysis experiments revealed a mean relative recovery of 77.5% (CV: 5.4%; delivery and recovery experiments). Following characterization of linezolid in in vitro microdialysis, the setting is suitable for application in clinical studies.     

Analysis of human breath samples with a multi-bed sorption trap and comprehensive two-dimensional gas chromatography (GCxGC)

A multi-bed sorption trap designed to quantitatively collect volatile organic compounds from large-volume vapor samples and inject them into a gas chromatograph is combined with a comprehensive two-dimensional gas chromatograph (GCxGC) for the analysis of organic compounds in human breath samples. The first-column effluent of the GCxGC is modulated by a single-stage, resistively-heated and air-cooled segment of 0.18-mm i.d. stainless steel column using the same stationary phase as the first column. Cooling gas is provided by a two-stage conventional refrigeration system, and thus no consumables other than carrier gas and electric power are required. The sorption trap uses four discreet beds, three containing different grades of graphitized carbon and one containing a carbon molecular sieve. The ordering of the beds in the trap tube is from the weakest to strongest adsorbent during sample collection. Breath samples are collected in gas sampling bags, and samples are passed through the trap at a flow rate of about 50 cm3/min. After sample collection, hydrogen carrier gas flow is initiated in the direction opposite to the sample collection flow, and the metal trap tube is resistively heated to inject a sample plug into the GCxGC. Performance data for the combined GCxGC/sorption-trap instrument is described, and human breath-sample chromatograms are presented.     

Determination of glucosamine sulfate in human plasma by precolumn derivatization using high performance liquid chromatography with fluorescence detection: its application to a bioequivalence study

A simple, rapid, selective and specific high-performance liquid chromatography (HPLC) method with fluorescence detection was developed for determination of glucosamine sulfate in human plasma and application to a bioequivalence in healthy volunteers. Precipitation of plasma was accomplished with acetonitrile to separate interfering endogenous products from the compound of interest. After vortex mixing and centrifugation, the supernatant was transferred and derivatized with 9-fluorenylmethoxycarbonyl chloride-acetonitrile solution in borate buffer (pH=8.0) at 30 degrees C for 30 min. The chromatographic separation was performed on a Diamonsil C18 column (150.0 mmx4.6 mm, 5 microm) with a mobile phase gradient consisting of water and acetonitrile at a flow rate of 1 mL/min. The method was linear in the range of 0.1-10.0 microg/mL with a correlation coefficient (r) of 0.9996. The limit of detection was 15 ng/mL. Inter- and intra-day precisions were 相似文献   

Detection of rutin by high-performance liquid chromatography and its application to taxonomic studies ofCalamagrostis (Poaceae)

A rapid and reproducible microanalysis technique for detecting rutin was established using reversed phase high-performance liquid chromatography. The usefulness of its application to studies in complicated internal structures of some species or species complexes ofCalamagrostis was discussed.     

Quantification of a novel natural antioxidant (UP302) in rat plasma using ultra-high performance liquid chromatography-tandem mass spectrometry

UP302 is a novel natural antioxidant isolated from Dianella ensifolia (Liliaceae). In the investigation, a specific and sensitive ultra-high performance liquid chromatography-tandem mass spectrometry for quantitative determination of UP302 in rat plasma was developed and validated. UP302 and the internal standard daidzein were extracted from 100 μL aliquots of rat plasma using methanol. Detection of UP302 and IS was done by tandem mass spectrometry, operating in negative ion and selected reaction monitoring acquisition mode. The precursor-product ion transitions monitored for UP302 and daidzein were m/z 301.1→135.2 and 252.9→132.0, respectively. The linearity of the method was observed within the concentration range of 5-2000 ng/mL. Intra- and inter-day assay variations were less than 15%, and the accuracy values were between 99.2% and 107.3%. The method was successfully applied to stability investigation of UP302 incubated in rat plasma at 37°C and measurement of UP302 in plasma after intravenous administration of UP302 to rats at a single dose of 5 mg/kg. Incubation stability revealed that within first one hour, UP302 was rapidly declined approximately 35% and remained stable after 4 h. Pharmacokinetic values of half-life, volume of distribution, systemic clearance and mean residence time were 0.87 ± 0.58 h, 6.90 ± 3.35 L/kg, 5.89 ± 1.21 L/h kg and 0.34 ± 0.13 h, respectively.     

Evaluation of cefquinome's efficacy in controlling avian colibacillosis and detection of its residues using high performance liquid chromatography (HPLC)

This study aimed to evaluate the efficacy of cefquinome in treatment and controlling of Escherichia coli experimentally infected broiler chickens, in addition of detection of its residues using High performance liquid chromatography (HPLC). In this study, 150 one-day old Cobb broiler chicks were used. On the 14^th day chicks experimentally infected and divided into 3 equal groups (50 each); control group (G1) non-infected, non-treated, (G2) infected with E. coli O₇₈ non treated, (G3) infected with E. coli O₇₈, cefquinome treated. Cefquinome was administrated 5^th day post infection, intramuscularly by a dose of (2 mg/ kg b w.t) for 3 consecutive days. Experimental E. coli infection in broilers induced weakness, loss of appetite, depression, cough and watery diarrhea in addition to a recorded mortality (30%) with reduction in growth performance, erythrogram, total proteins, albumin, antioxidants and haemagglutination inhibition (HI) titers. In addition, a significant increase in feed conversion rate (FCR), leukocytic count, liver enzymes, kidney functions, total globulins, malondialdehyde, nitric oxide and lysozyme activity. Treatment with cefquinome led to decreased mortality rate, improvement in clinical signs, growth performance and modulated most of these altered parameters. Cefquinome's residues was not detected in breast muscles 3^rd day and liver and kidneys 7^th days post treatment. Therefore, it's recommended that cefquinome is a good choice for controlling of colibacillosis in broilers and its withdrawal time 3 days in breast muscles and 7 days in liver and kidney post treatment.     

Conformation of antibiotic X-537A (lasalocid-A) and its calcium complex in acetonitrile solvent using circular dichroism spectroscopy

The calcium binding characteristics of antibiotic X-537A (lasalocid-A) in a lipophilic solvent, acetonitrile (CH3CN), have been studied using circular dichroism (CD) spectroscopy. The analysis of the data indicated that in this medium polar solvent, X-537A forms predominantly the charged complexes of stoichiometries 2:1 and 1:1, the relative amounts of the two being dependent on [Ca2+]. The conformations of the complexes, arrived at on the basis of the data, seem to indicate a rigid part encompassing Ca2+, liganded to 3 oxygens of the molecule, viz., the carbonyl, the substituted tetrahydrofuran ring and the substituted pyran ring oxygens (apart from, possibly, the liganding provided by nitrogen atoms of the solvent molecules), and a flexible part consisting of the salicylic acid group of the molecule.