Quantitative Detection of Trace Explosive Vapors by Programmed Temperature Desorption Gas Chromatography-Electron Capture Detector

The direct liquid deposition of solution standards onto sorbent-filled thermal desorption tubes is used for the quantitative analysis of trace explosive vapor samples. The direct liquid deposition method yields a higher fidelity between the analysis of vapor samples and the analysis of solution standards than using separate injection methods for vapors and solutions, i.e., samples collected on vapor collection tubes and standards prepared in solution vials. Additionally, the method can account for instrumentation losses, which makes it ideal for minimizing variability and quantitative trace chemical detection. Gas chromatography with an electron capture detector is an instrumentation configuration sensitive to nitro-energetics, such as TNT and RDX, due to their relatively high electron affinity. However, vapor quantitation of these compounds is difficult without viable vapor standards. Thus, we eliminate the requirement for vapor standards by combining the sensitivity of the instrumentation with a direct liquid deposition protocol to analyze trace explosive vapor samples.     

Analysis and quantitative determination of group B saponins in processed soybean products

Pure analytical standards for the major saponins present in processed soy products, the group B saponins (soyasaponins I, II, III and IV), were isolated in mg quantities by a combination of processing, precipitation/re-solubilisation, TLC and preparative HPLC. These standards were determined to be pure by LC-ESI/MS analysis and NMR. The standards were used to perfect a facile analytical HPLC method using spectrometric detection to determine the percent composition of the group B soyasaponins in various products from processing of soybean.     

Improved assays of alpha-lactalbumin and galactosyltransferase

The development and evaluation of a method for the determination of galactosyltransferase and alpha-lactalbumin activities using the addition of Dowex resin to the sample to separate substrate from products are described. For both assays galactosyltransferase activity was optimized by the addition of detergent, and relevant control incubations were included. The assay conditions were optimized for epididymal tissue and standards, and the assays were validated for accuracy and specificity with authentic bovine proteins and lactating rat mammary gland homogenates. Galactosyltransferase and alpha-lactalbumin activities in tissues were dependent on the extraction procedure used. Epididymal and testicular homogenates reduced the slopes of internal standards of galactosyltransferase but only testicular homogenates depressed slopes of internal standards of alpha-lactalbumin, necessitating the use of internal standards in the validation of the assays.     

JEV-DNA实时荧光定量标准品的构建

利用TaqMan荧光定量PCR技术,建立JEV-DNA定量标准品的制备方法。通过处理JEV减毒活疫苗提取病毒RNA,进行RT-PCR扩增目的片段,与T载体连接,转化感受态细胞,T-A克隆,测序鉴定后定量。获得预期的重组质粒,建立的标准曲线有较大的线性范围。此法制备的重组质粒标准品可用于对病毒载量进行测定。     

Simplified, Accurate Method for Antibiotic Assay of Clinical Specimens

Large glass plates are used for this modified agar-well diffusion assay method, allowing up to 81 replications on a single plate. With a specially designed agar punch, it is possible to prepare the small agar wells very quickly. The saving in serum resulting from fewer replications of standards with the large plates, and the small volume of the agar wells, makes it economically feasible to use pooled human serum for the standard antibiotic solutions. Methods are described for preparing the standard solutions, and for providing controls for the deterioration of standards and unknowns. Procedures for preparing and maintaining the commonly used assay organisms are presented. Serum specimens are tested directly rather than diluting them to a narrow range of antibiotic concentrations. This is possible because of a procedure for calculations that recognizes the curvilinear relationship between zone sizes and antibiotic concentrations. Adaptation of this method to a number of the commonly used antibiotics is described. With this method, it has been possible to test large numbers of clinical specimens in a minimal time, and with accuracy consistently better than 10%.     

Synthesis of β-Peptide Standards for Use in Model Prebiotic Reactions

A one-pot method was developed for the preparation of a series of β-alanine standards of moderate size (2 to ≥12 residues) for studies concerning the prebiotic origins of peptides. The one-pot synthesis involved two sequential reactions: (1) dry-down self-condensation of β-alanine methyl ester, yielding β-alanine peptide methyl ester oligomers, and (2) subsequent hydrolysis of β-alanine peptide methyl ester oligomers, producing a series of β-alanine peptide standards. These standards were then spiked into a model prebiotic product mixture to confirm by HPLC the formation of β-alanine peptides under plausible reaction conditions. The simplicity of this approach suggests it can be used to prepare a variety of β-peptide standards for investigating differences between α- and β-peptides in the context of prebiotic chemistry.     

Toward Système International d'Unité-traceable protein quantification: from amino acids to proteins

Here we present a demonstration of the proof of principle that absolute concentration of a protein within a mixture of other proteins can be measured with SI traceability. The method used was based on tryptic digestion of a protein followed by quantification using double exact matching isotope dilution mass spectrometry (IDMS) of the peptides released. To provide full SI traceability to measurements of protein concentration we demonstrated a method of SI traceable peptide quantification in which the peptide standards used were quantified by an amino acid analysis method that incorporated double exact matching IDMS and amino acid standards of known purity. The concentration of the protein was therefore determined based upon the concentration of tryptic peptides, which in turn had been quantified based upon amino acid standards. This allowed fully SI-traceable measurements of protein concentration to be made. Important caveats in the implementation of this approach are also discussed and examples of how these can have detrimental effects on the measurements are shown.     

Specific assay for radiolabelled digoxin and its known apolar metabolites in biological fluids. I.

A specific assay method for radiolabelled digoxin and its known apolar metabolites in plasma, urine and saliva was developed. The assay permits the delineation of the pharmacokinetics of digoxin and its metabolites after single-dose administration of the drug to humans. Column chromatographic and solvent extraction procedures were used for the separation of apolar and polar compounds. Thin-layer chromatography was applied for the individual and specific assessment of digoxin and its apolar metabolites. Apolar and polar standards were used for quantitative assessments of all the procedures used. Accuracy and precision of the assay developed were evaluated in plasma, urine and saliva using biological samples spiked with known amounts of standards and by measuring replicates of biological samples obtained from pharmacokinetic studies with digoxin administration to humans.     

Pellicle associated adherence film above incubation broth surface - an inexpensive adjunct to recognizing Candida krusei in the laboratory

Background

Targeting Induced Local Lesions in Genomes (TILLING) is a high throughput reverse genetics tool which detects mismatches (single point mutations or small indels) in large number of individuals of mutagenized populations. Currently, TILLING is intensively used for genomics assisted molecular breeding of several crop plants for desired traits. Most commonly used platform for mutation detection is Li-COR DNA Analyzer, where PCR amplified products treated with single strand mismatch specific nuclease are resolved on denaturing gels. The molecular size of any cut product can be easily estimated by comparing with IR dye labeled markers of known sizes. Similar fluorescent dye labeled size markers are also used for several genotyping experiments. Currently, commercially available size standards are expensive and are restricted up to only 700 bp which renders estimation of products of sizes greater than 700 bases inaccurate.

Findings

A simple protocol was developed for labeling 5' end of multiple DNA size markers with fluorescent dyes. This method involves cloning a pool of different size markers of DNA in a plasmid vector. PCR amplification of plasmid using IR dye labeled universal primers generates 5' fluorescent labeled products of various sizes. The size of products constituting the ladder can be customized as per the need. The generated size markers can be used without any further purification and were found to be stable up to one year at -20°C.

Conclusions

A simple method was developed for generating fluorescent dye labeled size standards. This method can be customized to generate different size standards as per experimental needs. The protocol described can also be adapted for developing labeled size standards for detection on platforms other than Li-COR i.e. other than infra red range of the spectrum.     

Partial synthesis of ganglioside and lysoganglioside lipoforms as internal standards for MS quantification

Within recent years, ganglioside patterns have been increasingly analyzed by MS. However, internal standards for calibration are only available for gangliosides GM1, GM2, and GM3. For this reason, we prepared homologous internal standards bearing nonnatural fatty acids of the major mammalian brain gangliosides GM1, GD1a, GD1b, GT1b, and GQ1b, and of the tumor-associated gangliosides GM2 and GD2. The fatty acid moieties were incorporated after selective chemical or enzymatic deacylation of bovine brain gangliosides. For modification of the sphingoid bases, we developed a new synthetic method based on olefin cross metathesis. This method was used for the preparation of a lyso-GM1 and a lyso-GM2 standard. The total yield of this method was 8.7% for the synthesis of d17:1-lyso-GM1 from d20:1/18:0-GM1 in four steps. The title compounds are currently used as calibration substances for MS quantification and are also suitable for functional studies.     

汉坦病毒S基因的克隆及体外转录

目的:通过基因克隆和体外转录,获得汉坦病毒汉滩型76118株及汉城型R22株S基因的RNA全长cRNA,为汉坦病毒病原学检测提供阳性定量标准品。方法:设计汉滩型76118株和汉城型R22株S基因克隆引物,PCR获得相应片段,分别克隆至含双启动子的PCRⅡ载体中,测序鉴定无误后,重组质粒分别经内切酶SpeⅠ、SacⅠ线性化,用T7 RNA聚合酶进行体外转录,产物经DNase处理、纯化后测定浓度,经RT-PCR验证。结果:获得汉滩型76118株及汉城型R22株S基因的cRNA片段,并可准确定量其拷贝数,76118株和R22株的质量浓度分别为80、17.58 ng/μL。结论:获得的cRNA样品可作为汉坦病毒核酸快速检测方法的阳性定量标准品。     

A sensitive and specific stable isotope assay for warfarin and its metabolites

A capillary gas chromatographic mass spectrometric method for the quantification of warfarin and its known metabolites from microsomal incubations is described. Deuterium labelled 4', 6-, 7- and 8-hydroxy warfarins are used as internal standards and the method has detection limits of 1 ng ml-1 with 20 ng ml-1 being the lower limit for accurate quantification.     

Comparison of product carbon footprint standards with a case study on poinsettia (Euphorbia pulcherrima)

Purpose

A method to quantify the climate impact of products called product carbon footprint (PCF) has been gaining popularity in recent years. However, variations of this method have resulted in several competing standards to guide the carbon calculation process. The aim of the current paper was to compare PCF results when calculated according to the different standards.

Methods

The three leading PCF standards are Publicly Available Specification (PAS) 2050:2011, ISO.DIN 2 14067 and Product Life Cycle Accounting and Reporting Standard (PARS) 2011. These standards were compared conceptually, and a case study was performed in which the PCF of a poinsettia plant produced in Germany was calculated according to all three standards.

Results and discussion

The PCF results were 0.45–0.50, 0.53–0.58 and 0.53–0.59 kg carbon dioxide equivalent according to PAS 2050:2011, ISO.DIN 2 14067 and PARS 2011, respectively. According to all standards, the life cycle stage contributing the most greenhouse gases (GHGs) was the production of the poinsettia plant, and the single process with the highest emissions was the electricity use in the production. It was found that if nonrenewable fuels were used for heating instead of wood chips, then heating would be the highest GHG contributor—accounting for over 80 % of emissions of the total PCF.

Conclusions

A key finding was that both the production system used and the decisions taken by the person carrying out the PCF calculation result in greater differences in the PCF result than the use of different standards. Differences among the three standards could be harmonised by more specific cut-off rules and exclusion criteria with the publication of ISO.DIN 2 14067, as well as the development and use of product category rules.     

A quantitative system of age analysis for the dog-whelk, Nucella lapillus

Past attempts to age classify dog-whelks are revised briefly. The most satisfactory method is a system which relies upon familarity of morphological characteristics with which to separate population samples into five different classes. An attempt has been made to quantitate this system. Definite standards have been suggested as the criteria with which to separate the five age classes, and a series of simple methods have been demonstrated which, when followed give finite values for each of these standards. The standards suggested are: (i) the lower limits for shell-length in mature individuals; (ii) the lip-thickness coinciding with maturity; (iii) the upper limits for shell-length in juvenile crevice dwellers. The methods of estimate for values for these standards are illustrated in practice with population samples from Blackrock, Sussex. The values thus obtained and the methods used in each estimation are discussed with view to their validity. It is reasonable to suspect that the quantitative age classification system can be used to classify dog-whelks from any shore provided large enough population samples are collected to estimate the values for each standard. Use of the system is further demonstrated by classification of animals from the shore at Martin's Haven, Pembrokeshire.     

Growth charts of head length and breadth for regional areas? A study in Sardinia (Italy)

There exist few standards of head length and breadth from childhood to adulthood in Europoid populations. Moreover, such standards are based on samples that cannot be used as references for all populations since they were taken from different ethnic groups and from different periods. The aims of this study were: (1) to test whether standards derived from North Americans of European extraction can be used to assess the Sardinian population; and (2) to produce growth charts for head length and breadth for Sardinian males and females from 3 to 22 years of age.The cross sectional sample consisted of 9,721 subjects of Sardinian origin (4,884 males and 4,837 females), aged 3–22 years, measured from 1998 to 2008. Growth percentiles were produced with the LMS method. The mean values for each sex in each age class (3–18 years) are almost always significantly lower for both head length and breadth than the corresponding North American values. The exceptions are the head length of boys of 14 years and girls of 16–18 years where values for Sardinians are lower, but not significantly so. The results show that the North American standards are not appropriate for the assessment of Sardinian children. For the Sardinian population, specific regional growth charts should be used to correctly evaluate the normal range and the cut-off points of the extreme percentiles.     

Telomere length measurement in mouse chromosomes by a modified Q-FISH method

Telomeres are physical ends of mammalian chromosomes that dynamically change during the lifetime of a cell or organism. In order to understand mechanisms responsible for telomere dynamics, it is necessary to develop methods for accurate telomere length measurement. The most sensitive method for measuring telomere length in mouse chromosomes is quantitative fluorescence in situ hybridization (Q-FISH). The usual protocol for Q-FISH requires plasmids with variable numbers of telomeric repeats and fluorescence beads as calibration standards. Here, we describe a Q-FISH protocol in which two mouse lymphoma cell lines with well-defined telomere lengths are used as calibration standards. Using this protocol we demonstrate that reproducible results can be obtained in a set of four different mouse cell lines. This method can be adapted so that any pair of mammalian cell lines can serve as an internal calibration standard.     

Fibrinolysis standards: A review of the current status

Biological standards are used to calibrate measurements of components of the fibrinolytic system, either for assigning potency values to therapeutic products, or to determine levels in human plasma as an indicator of thrombotic risk. Traditionally WHO International Standards are calibrated in International Units based on consensus values from collaborative studies. The International Unit is defined by the response activity of a given amount of the standard in a bioassay, independent of the method used. Assay validity is based on the assumption that both standard and test preparation contain the same analyte, and the response in an assay is a true function of this analyte. This principle is reflected in the diversity of source materials used to prepare fibrinolysis standards, which has depended on the contemporary preparations they were employed to measure. With advancing recombinant technology, and improved analytical techniques, a reference system based on reference materials and associated reference methods has been recommended for future fibrinolysis standards. Careful consideration and scientific judgement must however be applied when deciding on an approach to develop a new standard, with decisions based on the suitability of a standard to serve its purpose, and not just to satisfy a metrological ideal.