Preparation Of Calibration Curves Research Articles

Looking into the Quantification of Forensic Samples with Real-Time PCR.

The quantification of human DNA extracts from forensic samples plays a key role in the forensic genetics process, ensuring maximum efficiency and avoiding repeated analyses, over-amplified samples, or unnecessary examinations. In our laboratory, we use the Quantifiler® Trio system to quantify DNA extracts from a wide range of samples extracted from traces (bloodstains, saliva, semen, tissues, etc.), including swabs from touched objects, which are very numerous in the forensic context. This method has been extensively used continuously for nine years, following an initial validation process, and is part of the ISO/IEC 17025 accredited method. In routine practice, based on the quantitative values determined from the extracts of each trace, we use a standard method or a low-copy-number method that involves repeating the amplification with the generation of a consensus genetic profile. Nowadays, when the quantification results are less than 0.003 ng/μL in the minimum extraction volume (40 μL), we do not proceed with the DNA extract analysis. By verifying the limits of the method, we make a conscious cost-benefit choice, in particular by using the least amount of DNA needed to obtain sufficiently robust genetic profiles appropriate for submission to the Italian DNA Forensic Database. In this work, we present a critical re-evaluation of this phase of the method, which is based on the use of standard curves obtained from the average values of the control DNA analysed in duplicate. Considering the various contributions to uncertainty that are difficult to measure, such as manual pipetting or analytical phases carried out by different operators, we have decided to thoroughly investigate the contribution of variability in the preparation of calibration curves to the final results. Thus, 757 samples from 20 independent experiments were re-evaluated using two different standards for the construction of curves, determining the quantitative differences between the two methods. The experiments also determined the parameters of the slope, Y-intercept, R2, and the values of the synthetic control probe to verify how these parameters can provide information on the final outcome of each analysis. The outcome of this revalidation demonstrated that it is preferable to use quantification ranges rather than exact quantitative limits before deciding how to analyse the extracts via PCR or forgoing the determination of profiles. Additionally, we present some preliminary data related to the analysis of samples that would not have been analysed based on the initial validation, from which genetic profiles were obtained after applying a concentration method to the extracts. Our goal is to improve the accredited analytical method, with a careful risk assessment as indicated by accreditation standards, ensuring that no source of evidence is lost in the reconstruction of a criminal event.

GC-MS and HPTLC bioautography-based phytochemical profiling and evaluation of biological activity Neptunia prostrata Linn whole plant and leaves

This work used experimental methods to evaluate the chemical composition of GC-MS, HPTLC-based bioautographaphy, antioxidant, anti-diabetic, and protein denaturation properties of the Neptunia prostrata Linn (family: Mimosaceae) whole plant and leaves. Three methods, namely DPPH, metal chelating, and reducing power assays, were used to estimate the antioxidant effects. In addition, α-amylase, a carbohydrate-hydrolyzing enzyme, was targeted to test the anti-diabetic activity. The protein denaturation assay was used to evaluate the anti-inflammatory activity of the plant. GC-MS, an important analytical tool, was performed to identify bioactive compounds of medicinal plants used in pharmaceutical industries. Biochemical derivatization HPTLC-based bioautography with DPPH was executed to identify antioxidant agents from N. prostrata. The validated HPTLC method was used to quantify identified antioxidant agents. The stability of compounds, preparation of calibration curves, linearity, range, precision, limit of detection, limit of quantification, robustness, and accuracy are examined to develop a robust method. Higher total phenolic content (TPC) content was observed in N. prostrata hydroalcoholic extract of the whole plant (81.12 ± 4.19 mg GAE/g) and methanolic extract of leaves (105.16 ± 3.11 mg GAE/g). Methanolic extract of N. prostrata leaves showed significantly high antioxidant potential for all DPPH (IC50:28.05 ± 1.78 µg/mL), metal chelating (IC50:1598 ± 23.51 µg/mL), and reducing power assay compared to standard ascorbic acid (DPPH assay: IC50:24.14 ± 2.34 µg/mL and metal chelating: IC50:1301.16 ± 6.14 µg/mL). N. prostrata aqueous extract of the whole plant (IC50:179.47 ± 10.59 µg/mL) and leaves (IC50:130.86 ± 3.58 µg/mL) showed significantly high α-amylase inhibitory activity as compared to standard acarbose (IC50:148.62 ± 7.84 µg/mL). Methanolic extract of whole plants (IC50:131.46 ± 11.81 µg/mL) and chloroform extract of leaves (IC50:122.61 ± 8.19 µg/mL) exhibited strong anti-inflammatory activity as compared to standard acetylsalicylic acid (IC50:117.77 ± 4.95 µg/mL). GC-MS analysis revealed the presence of several bioactive compounds, where 2-cyclopenten-1-one, 2-hydroxy showed the most abundant bioactive compounds in the plant with 17.01%. HPTLC-bioautography identified three phenolic acids (chlorogenic acid, gallic acid, and 4-hydroxybenzoic acid) and one flavonoid (naringenin), further quantified with the validated HPTLC method. These findings propose that N. prostrata has therapeutic potential for treating diseases related to oxidative damage, diabetes, and inflammation, which may be due to the presence of bioactive compounds.

Korean Proficiency Tests for Pesticide Residues in Rice: Comparison of Various Proficiency Testing Evaluation Methods and Identification of Critical Factors for Multiresidue Analysis.

Establishing pesticide safety management for agricultural products necessitates accurate pesticide analysis at analytical laboratories. Proficiency testing is regarded an effective method for quality control. Herein, proficiency tests were carried out for residual pesticide analysis in laboratories. All samples satisfied the homogeneity and stability criteria of the ISO 13528 standard. The obtained results were analyzed using the ISO 17043 z-score evaluation. Both individual pesticide and multiresidue proficiency evaluations were performed, and the proportion of z-scores within the ±2 range ("Satisfactory" rating) obtained for seven pesticides ranged 79-97%. Of the laboratories, 83% were classified as Category A using the category A/B method, and these also received AAA ratings in the triple-A evaluations. Furthermore, 66-74% of the laboratories were rated "Good" via five evaluation methods based on their z-scores. The sum of weighted z-scores and scaled sum of squared z-scores were considered as the most suitable evaluation techniques, as they compensated for the drawbacks of good results and corrected the poor results. To identify the main factors affecting laboratory analysis, the experience of the analyst, sample weight, calibration curve preparation method, and cleanup status were considered. A dispersive solid phase extraction cleanup significantly improved the results (p < 0.01).

Strategies for mapping protein hydrolysate profiles and pharmacokinetics based on non-targeted proteomics combining skyline-aided quantitative techniques

Numerous works have been focused on the bioactivities of protein hydrolysates (PHs) and their application in food or drug formulations, but their composition and pharmacokinetics have never been addressed due to their complex constitutes, short half-life, extremely low concentrations and lack of authentic standards. The present study aims to develop systematic analytical strategy and technical platform with optimized sample preparation, separation and detection protocols for PHs. Lineal peptides (LPs), extraction of the spleen of healthy pigs or calves, were used as cases. First, solvents with polarity gradients were used to globally extract peptides of LP from biological matrix. Non-targeted proteomics based on a high-resolution MS system was used to establish a reliable qualitative analysis workflow for PHs. Based on the developed approach, 247 unique peptides were identified using NanoLC-Orbitrap-MS/MS, and then further verified on the MicroLC-Q-TOF/MS system. In the quantitative analysis workflow, Skyline software was used to predict and optimize the LC-MS/MS detection parameters of LPs followed by investigating the linearity and precision of the developed analytical assay. Note worthily, we innovatively prepared calibration curves by sequential dilution of LP solution to overcome the bottleneck of lacking authentic standards and complex PH composition. All the peptides exhibited good linearity and precision in biological matrix. The established qualitative and quantitative assays were successfully applied to study the distribution characteristics of LPs in mice, and would be conductive to systematically map the profile and pharmacokinetics of peptides in various PHs in vivo and in vitro.

Synthesis and Acidity Study of Mixed MFI-MORD Type Zeolite

Zeolites of various nature are widely used in the chemical industry, the fuel and energy sector of the economy as sorbents, catalysts and materials for the creation of inorganic membranes for various purposes. At the same time, it is possible to change the acid properties of the surface of zeolites both by varying the ratio of silicon to aluminum or silicon to phosphorus, and by joint synthesis of zeolites of various types with different acidic properties. The presented article provides a method for the sequential production of a zeolite of a mixed structure type MFI and mordenite. Synthesis of the original MFI type zeolite was carried out using seed grains by the hydrothermal method for 72 hours, followed by washing and drying of the zeolite. To obtain a layer of mordenite on the surface of the MFI type zeolite, the initial zeolite was pretreated with alkali and then treated with n-butylamine. In this way, nine samples of zeolite with different acidic surface properties were obtained. Determination of the acidic properties of the surface was carried out by the method of ammonia chemisorption followed by its desorption from the surface of the zeolite. For this purpose, the test sample was loaded into a quartz cuvette, purged with argon at a temperature of 800°С, after which the temperature dropped to 150°С, and the surface of the zeolite was treated with ammonia. Subsequently, the test sample was heated up to 800°С with registration of desorption curves. The amount of adsorbed ammonia was carried out according to previously prepared calibration curves. The synthesized samples of zeolites had different acidity from 0.48 to 0.72 mmol(NH3)/g(sample). In this case, the total acidity of the samples correlated with the ratio of silicon to aluminum in the zeolite samples. Also, depending on the ratio of the MFI and mordenite structures in the zeolite sample, it is possible to vary not only the number, but also the strength of the formed acid sites. So, an increase in the content of mordenite contributes to an increase in the strength of acid centers. The developed method for the synthesis of mixed structure zeolites of the MFI type mordenite made it possible to control the surface acidity of the synthesized samples.

Effect of Long Term Fertilization Practices on Tembotrione Movement in Soil: Assessment by Plant Bioassay

The herbicide behavior in soils is executed nowadays via the chemical extraction and instrumental analysis. However, the biological availability and the dynamics of herbicide in soil could be studied effectively through bioassay in the absence of sophisticated techniques and also in situ conditions. Tembotrione is one of the recently registered triketone herbicide and its movement behavior was not studied much in India. Hence the present study was executed to understand the influence of long term fertilization practices on its transport and movement using the bioassay technique involving Randomized Block Design (RBD). The plot wise soil samples were collected from the 50 years old long term fertilization experiment, processed and used to conduct the leaching column (made of PVC) experiment under controlled condition for 30 days. Soil samples were dissected depth wise and residue of tembotrione in each depth was obtained by conducting bioassay using green gram and sunflower as test species. For calibration curve preparation, a separate bioassay was conducted by fortifying tembotrione in control soil at different concentrations. The recorded plant parameters viz., germination percent and biomass after 15th day of both the test species predicted the tembotrione transport efficiently and the results of both the test species were comparable. The tembotrione has shown movement upto 30 cm soil depth and the concentration of residue decreased with increase in soil depth. The long term fertilization practices with the balanced nutrients supply viz., 100% NPK with hand weeding, Zn, FYM showed significant effect on the transport of tembotrione in soil at various depths.&#x0D; The study concludes that the plant bioassay technique using green gram and sunflower could be very well used to investigate the transport behavior of Tembotrione in soil. Based on data, tembotrione was identified as moderately mobile compound in sandy clay loam and its movement has been altered by the long term fertilization practices.

Hydrogen detection during photocatalytic water splitting: A tutorial

The increasing interest in the usage of clean technologies pointed toward the photocatalytic evolution of hydrogen. Consequently, an appropriate analytical procedure becomes mandatory to detect and quantify the produced hydrogen in different photocatalytic systems so that the quantification, as well as the photocatalytic experiment is reliable and repeatable. Herein, this work represents a first-of-the-kind approach proposing guidelines for a robust detection and quantification of hydrogen evolved from photocatalytic reaction systems together with standard operation procedures to consider when performing photocatalytic hydrogen evolution reactions. Gas chromatography with thermal conductivity detector (GC-TCD) was chosen as the analytical tool for the detection and quantification through the preparation of calibration curves carried out emulating the reaction conditions i.e. introducing hydrogen into a reactor containing electrolyte solution and sealed with a silicon septum for gas tight assurance. Validation parameters were calculated and linearity of the calibration curves obtained with the proposed methodology was proved.

A novel calibration strategy based on isotopic distribution for high-throughput quantitative analysis of pesticides and veterinary drugs using LC-HRMS

Preparation of calibration curves is a critical step for large-scale quantification. However, this procedure is time-consuming, labor intensive. Herein, a novel isotopologue multipoint calibration (IMC) strategy, was proposed and demonstrated for the simultaneous quantitation of 120 pesticides and 83 veterinary drugs in surface water samples using Liquid Chromatography-High Resolution Mass Spectrometry (LC-HRMS). In this strategy, the natural isotopic distribution was used to generate external calibration curves, eliminating the need of analyst’s adjustment and many sets of chemical standard solutions required in external calibration curves. Additionally, this strategy was comprehensively validated, and the results indicated this strategy had better performance in both accuracy and precision, fully meeting the requirements for the quantitative analysis. Interestingly, for the samples with high concentration beyond the upper limit of quantitation, the IMC strategy could avoid samples dilution by monitoring the less abundant isotopic channels. Furthermore, the IMC method was successfully applied in the surface water samples collected from Anhui province, China. Among which, sulfamethoxazole and imidacoprid were the main contributors. In conclusion, we present a promising LC-HRMS strategy for the accurate quantitation of small molecules, which has a potential application in food and environmental analysis.

Pyrolysis-GC–MS analysis of ingested polystyrene microsphere content in individual Daphnia magna

Microplastic (MP) pollution in the aquatic environment is a cause for increasing concern. However, analyzing MPs ingested by small organisms, such as zooplankton, is difficult because of the low content and small size of the ingested MPs. We attempted to determine the content of ingested MPs in individual zooplankton using pyrolysis–gas chromatography–mass spectrometry (Py-GC–MS). To establish zooplankton model of MP ingestion, individual Daphnia magna were cultivated separately in microplate cells with polystyrene (PS) microspheres (10 μm in diameter, 245,000 particles, 135 μg) under different conditions. To prepare calibration curves for determining ingested PS content, approximately 100–150 μg of commercially available Daphnia-based powdered fish food, roughly corresponding to the weight of a single D. magna organism, was mixed with PS microspheres (0.005–26 μg) and analyzed using Py-GC–MS at 600 °C. In the resulting pyrograms, peaks of the styrene monomer and trimer from PS were detected, and linear relationships were obtained between the relative peak area and the amount of added PS. Finally, the cultivated zooplankton were individually subjected to Py-GC–MS analysis, and the ingested PS content in each zooplankton was successfully determined. Individual zooplankton cultured with PS in the absence of food ingested 2.3–7.9 μg of PS particles, whereas that in the presence of food (Chlorella vulgaris) ingested only 0.1–0.2 μg of PS particles. This result suggests that zooplankton might preferentially ingest ordinary food when both food and MPs are present, although further systematic studies are necessary to validate this observation.

Internal calibration as an emerging approach for endogenous analyte quantification: Application to steroids

The use of mass spectrometry methods with triple quadrupole instruments is well established for quantification. However, the preparation of calibration curves can be time-consuming and prone to analytical errors. In this study, an innovative internal calibration (IC) approach using a one-standard calibration with a stable isotope-labeled (SIL) standard version of the endogenous compound was developed. To ensure optimal quantitative performance, the following parameters were evaluated: the stability of the analyte-to-SIL response factor (RF), the chemical and isotopic purities of the SIL, and the instrumental reproducibility. Using six clinically important endogenous steroids and their respective SIL standards, we demonstrated that RFs obtained on different LC-MS platforms were consistent. The quantitative performance of the proposed approach was determined using quality control samples prepared in depleted serum, and showed both satisfactory precision (1.3%–12.4%) and trueness (77.5%–107.0%, with only 3 values outside ±30%). The developed method was then applied to human serum samples, and the results were similar to those obtained with the conventional quantification approach based on external calibration: the Passing-Bablok regression showed a proportional bias of 6.8% and a mean difference of −5.9% between the two methodologies. Finally, we showed that the naturally occurring isotopes of the SIL can be used to provide additional calibration points and increase the accuracy for analytes with low concentrations.

Validated Quantitative 1H NMR Method for Simultaneous Quantification of Indole Alkaloids in Uncaria rhynchophylla.

Uncariae Ramulus Cum Uncis, known as “Gou-Teng” in Chinese, is derived mainly from the dried hook-bearing stems of Uncaria rhynchophylla. Quantitative determination of monoterpenoid indole alkaloids is critical for controlling its quality. In the present study, a rapid, accurate, and precise method was developed for the simultaneous quantitation of four characteristic components, namely, rhynchophylline (1), isorhynchophylline (2), corynoxeine (3), and isocorynoxeine (4), through 1H NMR spectrometry techniques. This method was performed on a 600 MHz NMR spectrometer with optimized acquisition parameters for performing quantitative experiments within 14 min. The highly deshielded signal of NH was at δH 10–11 in the aprotic solvent DMSO-d6, which enables satisfactory separation of the signals to be integrated. Validation of the quantitative method was also performed in terms of specificity, linearity, sensitivity, accuracy, and precision. The method is linear in the concentration range of 25–400 μg/mL. The lower limit of quantification is 25 μg/mL. The intra- and interday relative standard deviation across three validation runs over the entire concentration range is less than 2.51%. The accuracy determined at three concentrations was within ±4.4% in terms of relative error. The proposed qNMR method was demonstrated to be a powerful tool for quantifying the alkaloids in traditional Chinese medicines (TCMs) due to its unique advantages of high precision, rapid analysis, and nonrequirement of standard compounds for calibration curve preparation. Moreover, qNMR represents a feasible alternative to high-performance liquid chromatography-based methods for the quality control of TCMs.

Development and validation of a HILIC-MS/MS method for the quantitation of fructose in human urine in support of clinical programs

In a previous publication [1], a 20-minute UPLC®-MS/MS method, employing a surrogate analyte approach, was developed and validated to measure fructose and sorbitol, as mechanistic biomarkers, in human plasma to support first-in-human (FIH) studies. Different from plasma which maintains its homeostasis, urine has no such homeostasis mechanisms [2], therefore it is expected to be able to accommodate more changes. Here we describe the development and validation of a LC-MS/MS method for the quantiation of fructose in human urine to support clinical trials. A hydrophilic interaction chromatography (HILIC) method using an Asahipak NH2P-50 column (Shodex, 4.6 × 250 mm, 5 µm) was developed. Acetone precipitation was utilized to extract fructose from urine. For validation, stable isotope-labeled 13C6-fructose was used as the surrogate analyte for fructose in the preparation of calibration curves. QCs were prepared using both the surrogate analyte (13C6-fructose) and the authentic analyte (fructose). Difficulties were encountered for post-extraction stability experiments especially for authentic fructose QCs at low concentrations. Extensive troubleshooting revealed that fructose’s chromatography improved as the column aged. As a result, the response factor of fructose increased over time for low concentration samples, leading to failed post-extraction stability experiments. A column cleaning procedure was implemented to ensure consistency in chromatography performance. The HILIC-MS/MS method was successfully validated and applied to analyze clinical samples with a 91% overall run passing rate.

Comparison of allergen quantification strategies for egg, milk, and peanut in food using targeted LC-MS/MS.

Methods for the detection and quantification of food allergens in complex matrices are necessary to ensure compliance with labeling regulations and assess the effectiveness of food allergen preventive controls. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) has emerged as an orthogonal technique in complement to immunochemical-based assays. However, the absence of established guidelines for MS-based quantification of allergens in food has limited harmonization among the method development community. In this study, different quantification strategies were evaluated using a previously developed multiplexed LC-MS/MS method for the detection of egg, milk, and peanut. Peptide performance criteria (retention time, signal-to-noise ratio, and ion ratio tolerance) were established and quantification approaches using varying calibrants, internal standards, background matrices, and calibration curve preparation schemes were systematically evaluated to refine the previous method for routine laboratory use. A matrix-matched calibration curve using allergen ingredients as calibrants and stable isotope-labeled peptides as internal standards provided the most accurate quantitative results. The strategy was further verified with commercially available reference materials and allowed for the confident detection and quantification of food allergens. This work highlights the need for transparency in calibration strategy and peptide performance requirements for effective evaluation of mass spectrometric methods for the quantification of food allergens.

Calibration-Curve-Locking Database for Semi-Quantitative Metabolomics by Gas Chromatography/Mass Spectrometry.

Calibration-Curve-Locking Databases (CCLDs) have been constructed for automatic compound search and semi-quantitative screening by gas chromatography/mass spectrometry (GC/MS) in several fields. CCLD felicitates the semi-quantification of target compounds without calibration curve preparation because it contains the retention time (RT), calibration curves, and electron ionization (EI) mass spectra, which are obtained under stable apparatus conditions. Despite its usefulness, there is no CCLD for metabolomics. Herein, we developed a novel CCLD and semi-quantification framework for GC/MS-based metabolomics. All analytes were subjected to GC/MS after derivatization under stable apparatus conditions using (1) target tuning, (2) RT locking technique, and (3) automatic derivatization and injection by a robotic platform. The RTs and EI mass spectra were obtained from an existing authorized database. A quantifier ion and one or two qualifier ions were selected for each target metabolite. The calibration curves were obtained as plots of the peak area ratio of the target compounds to an internal standard versus the target compound concentration. These data were registered in a database as a novel CCLD. We examined the applicability of CCLD for analyzing human plasma, resulting in time-saving and labor-saving semi-qualitative screening without the need for standard substances.

Methodology of controlling amino compounds and latex in drilling muds

In industrial practice, rock hydration inhibitors, classified as amine compounds, are being increasingly used in drilling mud compositions. There is no methodology for determining this type of compounds content in the drilling mud, and proper servicing of this type of muds requires constant monitoring of their content to ensure appropriate inhibitory properties. During drilling, amine compounds adsorb on the surface of rocks and their concentration decreases during drilling and reduces the inhibitory properties of the mud. A common additive used in the composition of drilling muds is latex, the determination of which is necessary to ensure the mud parameters stability. Compounds of this type ensure the stability of the borehole wall, while their content also decreases during drilling. The results obtained from the research presented in the article allowed to propose methods for the determination of amine compounds and latexes. These methods can be used in the field laboratory when servicing drilling muds. The article presents the methodology for determining the content of new types of chemicals used in drilling muds, i.e. rock hydration inhibitors classified as amine compounds and latexes. The proposed methods enable proper servicing of muds with the addition of the agents that ensure the maintenance of appropriate mud properties. The method for the determination of amine compounds consists in determining the total nitrogen in the drilling mud filtrate. Dilution of the filtrate provides the results within the measuring range of the method and reduces the concentration of chloride ions, the amount of which may not exceed 10 000 mg/dm3. The first method of latex determination is weight determination of the dry residue after separation of the latex from the filtrate with acetone. The second one is based on nephelometric measurements of a tenfold diluted filtrate. The measurement result is the NTU (nephelometric turbidity unit) value depending on the latex content in the filtrate. All of the developed methods require the preparation of calibration curves, which are the basis for calculating the content of a given agent in the mud.

A robust multiplexed assay to quantify C1-inhibitor, C1q, and C4 proteins for in vitro diagnosis of hereditary angioedema from dried blood spot

Hereditary angioedema (HAE) is a rare genetic disease caused by deficiency or dysfunction of C1 esterase inhibitor (C1-INH). Plasma C1-INH activity and concentrations of C1-INH and complement components 1q and 4 (C1q, C4) are critical to the HAE diagnosis. We describe a novel multiplexed assay to simultaneously measure C1-INH, C1q, and C4 levels in dried blood spot (DBS) of HAE patients. The blood proteins were extracted from 3 mm punches of DBS samples and were subsequently digested by trypsin. The signature peptide derived from each protein was quantified by liquid chromatography-tandem mass spectrometry (LC–MS/MS). Analyte-depleted blood was generated as a surrogate matrix for the preparation of calibration curves to overcome the interference of endogenous proteins, and the assay reproducibility was further monitored by assessing the signal of plasma transferrin as a house-keeping protein. The assay was fully validated following regulatory guideline, with a quantification range of 12.5–800 μg/mL for C1-INH and C4 and 3.13–200 μg/mL for C1q. The precision and accuracy ranged from 3.3%–9.8% and -8.2%–12.6%, respectively. All the patient samples exhibited C1-INH levels lower than normal range except the Type II patient and the C4 and C1q concentrations were as expected. Results from the DBS-based LC–MS assay were highly correlated with the ELISA data measured in plasma of the same subjects. The method described here offers unique advantages such as less invasive sampling, minimal blood processing, and easy transportation and sample storage, allowing, for the first time, C1-INH, C4, and C1q levels to be simultaneously determined in a drop of dried blood.

Cell size influences inorganic carbon acquisition in artificially selected phytoplankton.

Cell size influences the rate at which phytoplankton assimilate dissolved inorganic carbon (DIC), but it is unclear whether volume-specific carbon uptake should be greater in smaller or larger cells. On the one hand, Fick's Law predicts smaller cells to have a superior diffusive CO2 supply. On the other, larger cells may have greater scope to invest metabolic energy to upregulate active transport per unit area through CO2 -concentrating mechanisms (CCMs). Previous studies have focused on among-species comparisons, which complicates disentangling the role of cell size from other covarying traits. In this study, we investigated the DIC assimilation of the green alga Dunaliella tertiolecta after using artificial selection to evolve a 9.3-fold difference in cell volume. We compared CO2 affinity, external carbonic anhydrase (CAext ), isotopic signatures (δ13 C) and growth among size-selected lineages. Evolving cells to larger sizes led to an upregulation of CCMs that improved the DIC uptake of this species, with higher CO2 affinity, higher CAext and higher δ13 C. Larger cells also achieved faster growth and higher maximum biovolume densities. We showed that evolutionary shifts in cell size can alter the efficiency of DIC uptake systems to influence the fitness of a phytoplankton species.

Simple Measurement of Carbon Films on Copper Tubes and Their Effects on Corrosion

It has been reported that pitting corrosion in copper tubes occurs due to the effect of a carbon film produced by the influence of undergoing an oil and heat treatment. As a quantitative method for determining the residual carbon amount, it has been reported that the inner surface of a copper tube can be dissolved with a mixed acid to collect and analyze the adhering carbon; however, this method is dangerous and difficult. Therefore, two methods were examined as a simple quantitative method for obtaining the residual carbon amount using copper tubes with known residual carbon amounts. One method utilizes X-ray photoelectron spectroscopy (XPS), and the other method utilizes the potential difference between the carbon film-adhered surface and carbon film-removed surface. In regard to XPS measurement, a linear correlation was found between the spectral intensity of C and the residual carbon amount; therefore, XPS measurements were considered to be effective as a simple measurement method for the carbon film on the inner surface of a copper tube. In the evaluation method by measuring the corrosion potential, a correlation was observed between the potential difference ΔE and the residual carbon amount of the inner surface of the tube and the outer surface of the polished tube. It is considered possible to estimate the residual carbon amount from the prepared calibration curve. Through these studies, it is suggested that the carbon film was non-uniformly present on the surface of the copper tube. Therefore, the galvanic current was measured, and the effect of a non-uniform carbon film on corrosion behavior was investigated. As a result, in the measurement of galvanic current, the current flowed from the copper tube with a large amount of residual carbon (cathode) to the copper tube with a small amount of residual carbon (anode). In addition, the higher the area ratio of the carbon film was, the larger the galvanic current tended to be.

PSVII-31 Impact of Total Dissolved Solids (TDS) in Drinking Water on Nursery Pig Performance

Abstract High concentrations of total dissolved solids (TDS) in water have been reported to increase the incidence of diarrhea and reduce nursery pig growth performance. The particular minerals of concern for pigs are typically intakes of magnesium and sodium sulfate salts. Therefore, the objective of this study was to investigate the effects of drinking water with added concentrations of TDS from sulfate salts on animal performance and feed and water disappearance. Newly weaned pigs (n = 1,144; 20 d old PIC) were stocked at 26 pigs per pen of equal gender in 44 pens in a wean-to-finish commercial research barn. Each pen was randomly assigned to receive one of four water treatments into two cup waters per pen: 1) combination of CaSO4, MgSO4, and NaSO4; 2) CaSO4; 3) MgSO4; 4) NaSO4. The drinking water treatments were derived from stock solutions delivered at 1:128 to relevant pens using a separate medicator per water line (Table 1). Pigs were provided free access to four phase nursery diets and water throughout the trial. Pen weights were measured on d 0, 7, 21, 35, and 42 using a scale that weighs the entire pen of pigs. Feed remaining on weigh days was calculated according to a prepared calibration curve by measuring the distance from the top of the feeder to the top of the feed. Water meters at each pen were read on weigh days to determine the water disappearance per pen. Water treatment did not affect (P &amp;gt; 0.07) ADG nor ADFI of nursery pigs. Water disappearance tended to be greater (P = 0.10) in pens receiving the CaSO4 water compared to the combination treatment from d 21 to 35. The TDS concentrations from sulfate complexes used in this experiment did not impact the growth performance or feed or water disappearance of newly weaned nursery pigs.

LC-MS/MS assays to quantify sulfatides and lysosulfatide in cerebrospinal fluid of metachromatic leukodystrophy patients.

Aim: Two separate LC-MS/MS assays were developed to quantitate sulfatides and lysosulfatide in human cerebrospinal fluid (CSF). Materials & methods: Lysosulfatide and the 15 most abundant sulfatide species were quantitated by LC-MS/MS using artificial CSF as surrogate matrix to prepare calibration curves. Results: Validation criteria were met (linear range: 0.02-1.00μg/ml sulfatides [0.02-1.00ng/ml lysosulfatide]); accuracy/precision were within ±15%. CSF from 21 children with metachromatic leukodystrophyhad significantly higher sulfatide and lysosulfatide concentrations than CSF from 60 healthy children (p<0.0001). Worse motor function correlated with higher CSF sulfatide (p=0.0087) and lysosulfatide (p=0.0034) levels. Conclusion: These assays, validated in patients with metachromatic leukodystrophy, may aid the clinical assessment of therapeutic responses.