Efficient Method For Quantification Research Articles

Determination of plasma lysophosphatidylethanolamines (lyso-PE) by LC-MS/MS revealed a possible relation between obesity and lyso-PE in Japanese preadolescent children: The Hokkaido study.

Lysophosphatidylethanolamines (lyso-PEs) are the partial hydrolysis products of phosphatidylethanolamine. Although lyso-PEs are important biomarkers in various diseases, their determination is limited by the lack of simple and efficient quantification methods. This study aims to develop an improved quantitative method for the determination of lyso-PEs and its application to an epidemiological study. Single reaction monitoring channels by collision-induced dissociation for seven lyso-PEs were established using liquid chromatography-tandem mass spectrometry. Plasma lyso-PEs were extracted with a single-phase method using an isotopically labelled internal standard for quantification. The proposed method was adopted to define lyso-PEs in plasma samples of children aged 9-12years living in Sapporo, Japan. The limit of detection and limit of quantification for each lyso-PE ranged between 0.001-0.015 and 0.002-0.031pmol/μL, respectively. Recoveries were found to be > 91% for all the species. The analysis results of children's plasma showed that the total lyso-PE concentrations in boys (n = 181) and girls (n = 161) were 11.53 and 11.00pmol/μL (median), respectively. Participants were further classified by the percentage of overweight and subgrouped as underweight (n = 12), normal range (n = 292), or overweight (n = 38). Interestingly, the reduction of lyso-PE 16:0 and increased lyso-PE 22:6 were observed in overweight children compared with normal range (Fold change: 0.909 and 1.174, respectively). This study successfully established a simple quantitative method to determine lyso-PE concentrations. Furthermore, our method revealed the possible relation between plasma lyso-PEs and overweight status.

Enhancing 2-Pyrone Synthase Efficiency by High-Throughput Mass-Spectrometric Quantification and In Vitro/In Vivo Catalytic Performance Correlation.

Engineering efficient biocatalysts is essential for metabolic engineering to produce valuable bioproducts from renewable resources. However, due to the complexity of cellular metabolic networks, it is challenging to translate success in vitro into high performance in cells. To meet such a challenge, an accurate and efficient quantification method is necessary to screen a large set of mutants from complex cell culture and a careful correlation between the catalysis parameters in vitro and performance in cells is required. In this study, we employed a mass-spectrometry based high-throughput quantitative method to screen new mutants of 2-pyrone synthase (2PS) for triacetic acid lactone (TAL) biosynthesis through directed evolution in E. coli. From the process, we discovered two mutants with the highest improvement (46 fold) in titer and the fastest kcat (44 fold) over the wild type 2PS, respectively, among those reported in the literature. A careful examination of the correlation between intracellular substrate concentration, Michaelis-Menten parameters and TAL titer for these two mutants reveals that a fast reaction rate under limiting intracellular substrate concentrations is important for in-cell biocatalysis. Such properties can be tuned by protein engineering and synthetic biology to adopt these engineered proteins for the maximum activities in different intracellular environments.

Life history of oysters influences Vibrio parahaemolyticus accumulation in Pacific oysters (Crassostrea gigas).

Vibrio parahaemolyticus infection in humans is associated with raw oyster consumption. Evaluation of V. parahaemolyticus presence in oysters is of most interest because of the economic and public health issues that it represents. To explore V. parahaemolyticus accumulation and depuration in adult Crassostrea gigas, we developed a GFP-tagged V. parahaemolyticus strain (IFVp201-gfp+ ), as well as a rapid and efficient quantification method in C. gigas oysters haemolymph by flow cytometry. Impact of the life history of C. gigas on accumulation and depuration of V. parahaemolyticus IFVp201 was subsequently investigated. We found that naive oysters, i.e. grown in controlled facilities with UV treated seawater, accumulated significantly more IFVp201 than environmental oysters, i.e. grown in intertidal environment. We hypothesized that environmental oysters could have been immune primed, thus could limit V. parahaemolyticus accumulation. Meanwhile, both naive and environmental oysters had similar depuration rates.

Simple and rapid analysis of Linagliptin in dried blood spot using an ionic liquid based vortex‐assisted dispersive liquid–liquid microextraction coupled with liquid chromatography–electrospray ionization–tandem mass spectrometry: Application to pharmacokinetic studies

AbstractA simple, rapid, and convenient ionic liquid based vortex‐assisted dispersive liquid–liquid micro extraction method for the determination of Linagliptin was developed. The main novelty of the present work deals with the analysis of Linagliptin in dried blood spot with significant advantages with regard to invasive sampling, volume of blood used (< 20 μL), storage and transport of biological materials and requirements for special biohazard arrangements. The extraction sample was assayed using liquid chromatography–tandem mass spectrometry using electropray ionization techniques. The effects of significant factors in extracting and disperser solvent, and salt contents were investigated. An efficient quantification method was developed for bioassay of Linagliptin using Sitagliptin as internal standard through Inertsil ODS‐3V (250 mm × 4.6 mm, 5 μm) column using 10 mM ammonium acetate buffer (pH adjusted to 4.6 with 0.1% Trifluoroacetic acid): Acetonitrile [10:90 (v/v)] as mobile phase. Tandem mass spectrometry detection was performed to monitor precursor → product ion transitions m/z 473.3 → 364.2 for Linagliptin, m/z 408.2 → 235.1 for internal standard. The assay was linear from 6 to 1500 ng/mL and the mean recoveries are more than 90%. The peak dried blood spot concentration (Cmax) after 1.5 h was determined to be 1391.2 ng/ml for Linagliptin.

Quantification of Actaea racemosa L. (black cohosh) from some of its potential adulterants using qPCR and dPCR methods

The demand for popular natural health products (NHPs) such as Black Cohosh is increasing considerably, which in turn challenges quality assurance (QA) throughout the supply chain. To detect and quantify the target species present in a given NHP, DNA-based molecular techniques such as Real-time quantitative PCR (qPCR) and digital PCR (dPCR) are standard tools in the food and pathogen testing industries. There is a gap in the literature concerning validated quantitative PCR methods for botanicals that can be utilized for QA and good manufacturing practices. The objective of this study is to develop an efficient quantification method using qPCR and dPCR techniques for the detection and quantification of Actaea racemosa (Black cohosh) NHPs from its potential adulterants. These developed methods are validated for applicability on commercial NHPs. Species-specific hydrolysis probe assays were designed to analyze the black cohosh NHPs using qPCR and dPCR techniques. The results confirmed that the developed qPCR and dPCR methods are highly precise for identifying and quantifying black cohosh NHPs, indicating their potential applicability in future routine industrial and laboratory testing. This enables a single qPCR test to determine not only the presence of a specific botanical, but also the amount when mixed with an adulterant.

Application of Image Compression Ratio Analysis as a Method for Quantifying Complexity of Dental Enamel Microstructure.

It is well recognized that enamel microanatomy in mammals reflects biomechanical demands placed upon teeth, as determined by mechanical properties of species' diets, use of teeth as weapons, and so forth. However, there are limited options for researchers wishing to perform large-scale comparisons of enamel microstructure with adaptive questions in mind. This is because to date there has been no efficient method for quantification and statistical analysis of enamel complexity. Our study proposes to apply a method previously developed for quantification of 3D tooth cusp morphology to the problem of quantifying microstructural enamel complexity. Here, we use image compression ratio (ICR) as a proxy variable for enamel complexity in 2D enamel photomicrographs taken using circularly polarized transmitted light microscopy. ICR describes the relationship between a digital image captured in an uncompressed file format and the identical image that has had its file size compressed using computer algorithms; more complex images receive less compression. In our analyses, ICR analysis is able to distinguish between images of teeth with simple, radial enamel and teeth with complex decussating enamel. Moreover, our results show a significant correlation between ICR and enamel complexity ranks assigned via visual assessment. Therefore, our results demonstrate that ICR analysis provides a viable methodology for efficient comparison of overall enamel complexity among dental samples. Anat Rec, 302:2279-2286, 2019. © 2019 American Association for Anatomy.

Comparison of Methods for Determination of Carbon in Calcareous Soils

ABSTRACTThe global cycle of carbon (C) has raised attention in recent decades due to the great increase in carbon dioxide levels (CO2) levels in the atmosphere and its influence on climate change. Calcareous soils represent a significant fraction of the areas with potential for agriculture and have differential attributes, such as high calcium contents, magnesium, carbonates, and pH values. These attributes have been ignored in analytical procedures despite these characteristics, resulting in an overestimation or underestimation of the soil carbon. Several methods have been proposed for determining the soil carbon contents, however, studies evaluating the analytical procedures of C quantification methods, considering the soil characteristics, such as the calcareous soils, are needed, in order to improve their accuracy. Therefore, the objective of this work was to evaluate and compare methods for C determination and to propose adjustments in the methodology for calcareous soil analysis. The Yeomans and Bremner (YB) was the most efficient method for quantification of organic C among the wet oxidation methods. On the other hand, the Donagemma (WB) method underestimated the organic carbon contents. The results showed that the samples must be macerated and pretreated with a hydrochloric acid solution for the use of CHNS-O, in order to eliminate carbonates in the form of nodules and concretions.

Application of ethidium bromide monoazide for quantification of viable and dead cells of Salmonella enterica by real-time loop-mediated isothermal amplification

A rapid and efficient method for quantification and discrimination of Salmonella enterica ser. Enteritidis between viable and dead cells killed by heat was developed using ethidium bromide monoazide (EMA) in combination with a real-time loop amplified (Rti-LAMP) DNA assay. The use of 8.0μg/ml or less of EMA did not inhibit DNA amplification in Rti-LAMP assays derived from viable cells. However, 8.0μg/ml of EMA notably inhibited DNA amplification and significantly increased the Tp values with dead cells. When the DNA from 2000 viable CFU was subjected to EMA-Rti-LAMP the resulting Tp value was 13min. In contrast, the DNA from 2000CFU completely heat destroyed CFU still yielded a Tp value, which was greatly increased to 33.1min. When the DNA from viable plus heat killed CFU at a ratio of 7:1993 was subjected to EMA-Rti-LAMP, the resulting Tp value was 19.3min, which was statistically identical (P<0.05) to the Tp value of 19.9min. obtained with the DNA from only 7 viable CFU. These results indicate that even though 2000 dead cells yielded a Tp value of 33.1min., low numbers of viable cells in the presence of much higher numbers of dead cells still yielded a linear plot for enumerating viable CFU from Tp values. In addition, propidium monoazide (PMA) was found to be ineffective in distinguishing between low numbers of viable and heat killed cells of S. enterica.

Eco-balance analysis of six agricultural land uses in the Ikushunbetsu watershed

This study quantified and evaluated the greenhouse gas emissions of farmland soils at a watershed scale using parameters available at a regional scale. The estimation was based on field monitoring data in the Ikushunbetsu River Watershed, Hokkaido, Japan. Simple regression models were created for carbon dioxide, nitrous oxide and methane emissions associated with six major agricultural land uses, and forest as an alternative land use to farmland. An eco-balance method involves conducting an analysis on the basis of farmland surplus nitrogen (N) and global warming potential (GWP). Uncertainties associating the estimations were estimated using Mote Carlo simulation. The eco-balance is the analysis of the relationship between production and environmental load. In this study, production and environmental load were compared by changing each of the land use combinations by 10%. Farmland surplus N was lowest for soybean with 8.2 kg N ha−1 year−1, followed by paddy rice. The highest value was recorded for vegetables with 99.8 kg N ha−1 year−1. The weighted mean of total farmland based on the land use proportion was 44 ± 33.8 kg N ha−1 year−1. The calculated GWP was 7.3 Mg CO2 eq ha−1 year−1 for paddy rice and 0.1 to 6.8 Mg CO2 eq ha−1 year−1 for upland crops. The weighted mean of the total farmland area was 4.0 ± 3.4 Mg CO2 eq ha−1 year−1. The eco-balance analysis showed that there were 59 combinations out of 8008 combinations able to reduce GWP more than 6%, but less than 7%, than the value in 2005. Among them, in 30 combinations, farmland surplus N became less than the value in 2005; production was reduced compared with the value in 2005 in 27 combinations. Soybean occupied 20–80% in the seven combinations where production was increased compared with 2005, while keeping farmland surplus N below the value in 2005. The estimation of greenhouse gases in this study exhibited high uncertainty because of variability in management and errors in evaluation procedures. Quantification of the data variability is set at maximum, which comprises the measured values in this area. Based on the quantification of the uncertainty, more efficient quantification methods can be established to clarify mitigation potential. This type of quantification and comparison between production and emission enables decision makers to set some threshold values that allow a compromise between production and environmental load.

Quantification of perfume compounds in shampoo using solid‐phase microextraction

AbstractMethods for the quantitative analysis of perfume compounds in shampoo were developed using SPME (solid‐phase microextraction). The method development started with the extraction of the perfume compounds from water, and subsequently from shampoo aqueous dispersion. It was observed that the addition of salts decreased the extraction efficiencies with the concentration of shampoo in water as low as 0.1% in contrast to the effect of the salt in water. Step‐by‐step optimization to increase extraction efficiency and minimize matrix effect was investigated. Dilution of shampoo with water was found to be an efficient method for quantification. It decreased the viscosity of the shampoo and increased the concentration of free analytes. Largely diluted shampoos enabled an efficient SPME extraction and allowed the use of a unified calibration applicable to different types of shampoo. The exhaustive SPME extraction of small amounts of sample, e.g. 50 µl, provided another fast quantitative method. This unprecedented work presents quantitative methods for the analysis of perfume compounds in shampoo. This research extends the knowledge of perception and reconstitution of perfume compounds in shampoo, and facilitates development of new detergent products. Copyright © 2006 John Wiley &amp; Sons, Ltd.