LC System Research Articles

Extraction of intrinsic column peak profiles of narrow-bore and microbore columns by peak deconvolution methods

The intrinsic peak profiles (free from the delay and dispersion caused by state-of-the art UHPLC systems) generated by narrow-bore and microbore chromatographic columns used in liquid chromatography-mass spectrometry (LC-MS) proteomic analyses are extracted from two different deconvolution methods. The first method is based on the classical discrete Fourier transform (DFT) while the second method refers to the Taylor expansion of the continuous Fourier transform (FT). The two numerical methods are compared regarding the accurate determination of the intrinsic peak profiles of the non-retained compound (toluene) expected on a narrow-bore 2.1 mm × 100 mm column packed with 1.6 μm CORTECS-C18 superficially porous particles and installed on three different LC systems (ACQUITY i-class UPLC, ACQUITY H-class UPLC, and Arc LC systems). The DFT-based method is most relevant when the low-frequency band of the chromatographic peak does not overlap with the high-frequency bands related to the experimental baseline noise (pump/detector). The Taylor expansion-based method is successful for the extraction of the intrinsic peak profiles of narrow-bore 2.1 mm i.d. columns packed with sub-2 μm particles installed on standard UHPLC systems. When the LC system dispersion significantly exceeds that of the column, the DFT-based method is preferred over the Taylor expansion-based method and is successfully applied to extract the intrinsic peak profiles generated by a microbore 1.0 mm × 100 mm column packed with 1.8 μm HSS-C18 fully porous particles (volume variance ∼ 0.15 μL2 for the non-retained compound toluene) run on the low-dispersion ACQUITY i-class UPLC system (∼ 1 μL2 volume variance). This result opens up promising avenues for the development, quality control, and LC-MS analyses of microbore 1 mm i.d. columns using the state-of-the-art UHPLC instruments at flow rates larger than 0.1 mL/min.

Preparation of cholesteric polymer networks with broadband reflection memory effect

ABSTRACT In this study, polymer network is prepared by broadband reflective cholesteric liquid crystal (CLC) film with washing out method. By the method of photoinduced molecular diffusion, the CLC film is prepared by adding different concentrations of ultraviolet (UV) absorption dyes into the LC system and irradiated with a series of UV light intensities for different times. The selective reflection property and broadband reflection memory effect of the polymer network are proved, and the response mechanism is given. By refilling the polymer network with different LCs, reflective bands with different central wavelengths and bandwidths can be obtained. It is confirmed that different fillers can recurrence the initial reflection bandwidth of CLCs in different degrees. These results may provide a new idea for total reflection CLC films.

Re-Examining the Impact of Minimal Scans in Liquid Chromatography-Mass Spectrometry Analysis.

Liquid chromatography-mass spectrometry (LC-MS) is one of the most widely used analytical tools. High analysis volumes and sample complexity often demand more informative LC-MS acquisition schemes to improve efficiency and throughput without compromising data quality, and such a demand has been always hindered by the prerequisite that a minimum of 13-20 MS scans (data points) across an analyte peak are required for accurate quantitation. The current study systematically re-evaluated and compared the impact of different scan numbers on quantitation analysis using both triple quadrupoles mass spectrometry (TQMS) and high-resolution mass spectrometry (HRMS). Contrary to the 13-20 minimal scan prerequisite, the data obtained from a group of eight commercial drugs in the absence and presence of biological matrices suggest that 6 scans per analyte peak are sufficient to achieve highly comparable quantitation results compared to that obtained using 10 and 20 scans, respectively. The fewer minimal scan prerequisite is presumably attributed to an improved LC system and advanced column technology, better MS detector, and more intelligent peak detection and integration algorithms leading to a more symmetric peak shape and smaller peak standard deviation. As a result, more informative acquisition schemes can be broadly set up for higher throughput and more data-rich LC-MS/MS analysis as demonstrated in a hepatocyte clearance assay in which fewer MS scans executed on HRMS led to broader metabolite coverage without compromising data quality in hepatic clearance assessment. The demonstrated acquisition scheme would substantially increase the throughput, robustness, and richness of the nonregulatory analysis, which can be broadly applied in diverse fields including pharmaceutical, environmental, forensic, toxicological, and biotechnological.

Identification of 7 000-9 000 Proteins from Cell Lines and Tissues by Single-Shot Microflow LC-MS/MS.

A current trend in proteomics is to acquire data in a "single-shot" by LC-MS/MS because it simplifies workflows and promises better throughput and quantitative accuracy than schemes that involve extensive sample fractionation. However, single-shot approaches can suffer from limited proteome coverage when performed by data dependent acquisition (ssDDA) on nanoflow LC systems. For applications where sample quantities are not scarce, this study shows that high proteome coverage can be obtained using a microflow LC-MS/MS system operating a 1 mm i.d. × 150 mm column, at a flow-rate of 50 μL/min and coupled to an Orbitrap HF-X mass spectrometer. The results demonstrate the identification of ∼9 000 proteins from 50 μg of protein digest from Arabidopsis roots, 7 500 from mouse thymus, and 7 300 from human breast cancer cells in 3 h of analysis time in a single run. The dynamic range of protein quantification measured by the iBAQ approach spanned 5 orders of magnitude and replicate analysis showed that the median coefficient of variation was below 20%. Together, this study shows that ssDDA by μLC-MS/MS is a robust method for comprehensive and large-scale proteome analysis and which may be further extended to more rapid chromatography and data independent acquisition approaches in the future.̀.

Improving LC/MS/MS-based bioanalytical method performance and sensitivity via a hybrid surface barrier to mitigate analyte – Metal surface interactions

The accurate determination of the pharmacokinetics (PK) of a candidate drug molecule is critical in both drug discovery and development. Over the last 30 years, the sensitivity and selectivity of LC/MS has resulted in it being established as the technology of choice for these studies. However, unwanted chemical interactions between analyte(s) and the metal components in a chromatography system can result in poor peak shape and reduction in signal response, which can adversely affect the analysis of low concentrations of drugs and their metabolites in biological samples. This study evaluated the benefits of employing an inert hybrid surface technology (HST) applied to the metallic components in the LC flow path, column frits and column wall to mitigate these interactions. The results obtained were compared with that of an identical conventional LC for the bioanalysis of two steroid phosphate drugs (dexamethasone phosphate and hydrocortisone phosphate) and an epidermal growth factor receptor (EGFR) inhibitor (gefitinib) in human plasma. The results showed that for the two steroid phosphates, the peak width was reduced by 20%, peak tailing factors reduced by up to 30% and the assay sensitivity improved by factors of 7.5 and 10. This resulted in a significant improvement in the limit of detection. The new LC system also improved the reproducibility of peak integration for gefitinib, thereby reducing assay coefficients of variation (%CV) from greater than 10% to less than 5% at the lower limit of quantification.

Functionalized graphene oxide tablets for sample preparation of drugs in biological fluids: Extraction of ritonavir, a HIV protease inhibitor, from human saliva and plasma using LC–MS/MS

In this work, graphene oxide based tablets (GO-Tabs) were prepared by applying functionalized GO as a thin layer on a polyethylene substrate. The graphene oxide was functionalized with amine groups (-NH2 ) by Poly (ethylene glycol) bis(3-aminopropyl) terminated (GO-NH2 -PEG-NH2 ). The functionalized GO-Tabs were used for extraction of ritonavir in human saliva samples. The analysis of ritonavir in plasma and saliva samples was performed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Gradient LC system with MS/MS in the positive ion mode (ESI+) was used. The transitions m/z 721→269.0 and m/z 614→421 were used for the ritonavir and the internal standard indinavir, respectively. In this study, the determination of the HIV protease inhibitor ritonavir in human saliva samples was carried out using functionalized GO-Tab and LC-MS/MS and the method was validated. Standard calibration curve for plasma and saliva samples was constructed from 5.0 to 2000 nmol L-1 . The limit of detection was 0.1 nmol L-1 and the limit of quantification was 5.0 nmol L-1 in both plasma and saliva matrices. The intra- and inter-assay precision values were found to be between 1.5 and 5.8%, and the accuracy values ranged from 88.0% to 108% utilizing saliva and plasma samples. The extraction recovery was over 80%, and the presented functionalized GO-Tabs could be re-used for more than ten extractions without deterioration in recovery.

Electronic games classification in the library of congress and Dewey classification schemes: a comparative study

PurposeThe study aims to explore the classification of electronic games in Dewey decimal classification (DDC) and The Library of Congress classification (LCC) schemes.Design/methodology/approachThe study adopted a comparative analytical method to explore the topic in both the DDC and the LCC schemes by comparing its processing method in both schemes. The study measures the extent to which both schemes succeed in allocating notations covering the topic’s literature.FindingsThe study reached several results, the most important of which are: the difference between the two main cognitive sections, to which they belong to the topic, namely, arts and recreation (700) in the DDC scheme and the geography section (G) in the LCC scheme, while they were found to share the same sub-section scheme. The two schemes do not allocate notations to address the subject of electronic games as literature and other notations that have not been embodied for electronic games themselves or in the form of a compact disc or other media.Originality/valueAs far as we know, this is the first paper that compares the treatment of video games in DDC and Library of Congress classification schemes. The study allows for understanding the difference in the treatment of topics in both schemes, which would help in the decision of the adoption of a particular classification scheme.

HPLC method for levothyroxine quantification in long-acting drug delivery systems. Validation and evaluation of bovine serum albumin as levothyroxine stabilizer

Deficiency of thyroid hormones (hypothyroidism) is treated with oral levothyroxine (LEVO). However, the effectiveness of oral administration is highly dependent on the co-administration of food and other drugs. This factor, in combination with the chronic nature of this condition, mean that there are concerns with patient compliance. Development of long acting formulations to treat hypothyroidism could potentially solve this problem. However, LEVO instability in solution could be problematic. In order to develop long acting LEVO delivery systems in vitro drug release experiments should be carried out. However, short term LEVO stability in aqueous solution will prevent this. BSA was used as a stabiliser for LEVO; extending the stability of the drug in aqueous solutions from a few hours to 2 weeks. In order to achieve this, the required concentration of the protein was 0.1% w/v. Subsequently, an HPLC method capable of separating LEVO from the protein was developed and validated following ICH guidelines. The analysis was carried out using a reverse phase HPLC method on an Agilent 1220 Infinity II LC system. The column used to achieve separation was a Zorbax Eclipse plus C18 (95 Å pore size, 250 mm length x 4.6 mm internal diameter; 5 μm particle size). The mobile phase used was composed of acetonitrile and 0.1% trifluoroacetic acid at a ratio of 50:50% v/v. UV detection of LEVO sodium was carried out at 225 nm. The retention time for the drug was 6.6 minutes. The method showed a limit of detection of 0.03 μg/mL and a limit of quantification of 0.09 μg/mL. Finally, this method was used to evaluate the release from implants containing 20% w/w of LEVO. These devices were prepared using a solvent casting method with poly(caprolactone) and LEVO. These devices showed an initial burst release over the first 3 days. Subsequently, they were capable of providing a linear release rate over the following 25 days.

Chemoinformatics based comprehensive two-dimensional liquid chromatography-quadrupole time-of-flight mass spectrometry approach to chemically distinguish Chrysanthemum species

An effective comprehensive two-dimensional liquid chromatography (LC × LC) coupled with quadrupole time-of-flight mass spectrometry (Q-TOF/MS) method was developed for quantitative determination of 19 active constituents in chrysanthemum species. Separation was achieved on a LC × LC system, with Zorbax Eclipse Plus C18 column (2.1 × 100 mm, 3.5 μm) used in the first dimension (1D), Poroshell 120 Bonus-RP column (3.0 × 50 mm, 2.7 μm) in the second dimension (2D). An optimal flow rate of 0.1 mL/min in 1D combined with 2.0 mL/min in 2D was utilized to improve the resolution and peak shape of the analytes. A synchronized gradient elution program was optimized to make for the 2D chromatographic resolution and the final peak capacity. The established method was then applied for the contents determination of the 19 analytes in all the chrysanthemum samples collected. Based on the content data set of these components, principal component analysis (PCA), partial least squares discriminant analysis (PLS-DA), and hierarchical cluster analysis (HCA) were performed to distinguish the chrysanthemum of different varieties. 29 selected chrysanthemum products were classified into five clusters, with Chuju, Jinzhanju, Taiju and Wanshouju distinguished from each other. However, Hangbaiju, Huangju, Gongju, and Xueju clustered into a group due to their similarities in the analytes content. This study shows that LC × LC/Q-TOF MS in combination with PCA, PLS-DA and HCA can be applied successfully as an effective method for the classification and quality control of chrysanthemum.

Effects of Chronic Secondhand Smoke (SHS) Exposure on Cognitive Performance and Metabolic Pathways in the Hippocampus of Wild-Type and Human Tau Mice.

Background:Exposure to secondhand smoke (SHS) is a risk factor for developing sporadic forms of sporadic dementia. A human tau (htau) mouse model is available that exhibits age-dependent tau dysregulation, neurofibrillary tangles, neuronal loss, neuroinflammation, and oxidative stress starting at an early age (3–4 months) and in which tau dysregulation and neuronal loss correlate with synaptic dysfunction and cognitive decline.Objective:The goal of this study was to assess the effects of chronic SHS exposure (10 months’ exposure to ) on behavioral and cognitive function, metabolism, and neuropathology in mice.Methods:Wild-type (WT) and htau female and male mice were exposed to SHS (90% side stream, 10% main stream) using the SCIREQ® inExpose™ system or air control for 168 min per day, for 312 d, 7 d per week. The exposures continued during the days of behavioral and cognitive testing. In addition to behavioral and cognitive performance and neuropathology, the lungs of mice were examined for pathology and alterations in gene expression.Results:Mice exposed to chronic SHS exposure showed the following genotype-dependent responses: a) lower body weights in WT, but not htau, mice; b) less spontaneous alternation in WT, but not htau, mice in the Y maze; c) faster swim speeds of WT, but not htau, mice in the water maze; d) lower activity levels of WT and htau mice in the open field; e) lower expression of brain PHF1, TTCM1, , and HSP90 protein levels in WT male, but not female, mice; and f) more profound effects on hippocampal metabolic pathways in WT male than female mice and more profound effects in WT than htau mice.Discussion:The brain of WT mice, in particular WT male mice, might be especially susceptible to the effects of chronic SHS exposure. In WT males, independent pathways involving ascorbate, flavin adenine dinucleotide, or palmitoleic acid might contribute to the hippocampal injury following chronic SHS exposure. https://doi.org/10.1289/EHP8428

Inter‐individual variability and differential tissue abundance of mitochondrial amidoxime reducing component 1 (mARC1) enzyme in human

Mitochondrial amidoxime-reducing component (mARC) enzymes are the recently discovered molybdenum-containing enzymes that metabolize a multitude of endobiotic and xenobiotics, e.g., N-hydroxylated nucleobases, N-oxides, N-hydroxy compounds. While two isoforms of mARC enzymes (mARC1 and mARC2) have been identified in humans, little is currently known about their inter-individual variability and differential tissue abundance. To fill this knowledge gap, the specific aims of this study were to i) develop a novel microflow liquid-chromatography tandem mass spectrometry (µLC-MS/MS) method for mARC1 quantification in human tissues, ii) quantify mARC1 abundance in five human tissues (liver, intestine, kidney, lung, and, heart), and iii) quantify inter-individual variability in mARC1 abundance by determining the effect of age and sex. Tissue samples were digested by trypsin using an optimized protocol and peptide separation was performed on an Ionkey column using an M-class microflow LC system coupled to Xevo TQ-XS MS instrument (Waters). Surrogate peptides of mARC1 were selected and quantified in multiple reaction monitoring (MRM) mode. The best two surrogate peptides of mARC1 for the targeted analysis were: DLLPIK and VGDPVYLLGQ. Using recombinant protein as a calibrator, the absolute levels of mARC1 in the liver was 41 ± 4 pmol/mg liver protein, which is comparable to the microsomal abundance of major cytochrome P450 enzymes, CYP3A4, CYP2E1, and CYP2C9. The average mARC1 abundance in the human kidney was 8-fold lower than the liver, but the abundance was independent of age in both the organs. In particular, mARC1 abundance in the liver was 35 ± 8, 41 ± 11, 39 ± 13, 42 ± 11, and 41 ± 11 pmol/mg liver protein in infants (0 to 1 year), early childhood (1 to 6 years), middle childhood (6 to 12 years), adolescence (12 to 18 years), and adults (>18 years). Similarly, the abundance was not associated with sex, i.e., mARC1 abundance in men and women was 39 ± 11 and 35 ± 10 pmol/mg protein, respectively. mARC1 was also expressed in other tissues, but its abundance (mean) in the human intestine, lung, and heart was 6-, 3-, and 28-fold lower than the liver. In summary, mARC1 abundance in humans is higher in the liver than in other studied tissues. In addition, the enzyme did not show age or sex-dependent changes in the abundance, which signifies its important physiological role. The mARC1 abundance data presented here can be used to develop physiologically-based pharmacokinetic models for the prediction of in vivo pharmacokinetics of mARC substrates.

Towards a universal automated and miniaturized sample preparation approach

Nowadays, analytical chemistry plays a fundamental role in developing strategies for monitoring residues and contaminants in complex matrices (e.g., biological, environmental, and food samples). Considering the significant number of interfering substances present, combined with the lower concentration levels in which the target analytes are commonly found, sample preparation is becoming a crucial step in the analytical workflow. For these reasons, automation and miniaturization of this process represent a current trend since by these means, several principles of Green Chemistry can be meet all together, including: (i) lower volumes of reagents and samples required; (ii) reduced waste generation; and (iii) high-throughput with lesser operators' handling. The employment of online sample preparation based on LC systems (e.g., online SPE and In-Tube SPME) can be considered promising tools to perform sample preparation in a more sustainable way than conventional approaches. For these reasons, herein, we present a review discussing the essential characteristics of miniaturized, automated systems, including column-switching modes and modern approaches based on chip-LC. These methodologies deserve attention mainly due to their greener character derived from automation and miniaturization of the process. Also, selected applications, mostly covering the last five years, are presented. A brief comparison between them and conventional LC-based approaches is carried out to bring an adequately discussed review to this journal audience.

Publishing classification systems on the Internet: representation and use

Classification systems are published primarily in the book form, for various purposes and in different versions. They are identified by the number or year of publication. The authors propose another variant of maintaining classification systems, i.e. in the card format.The authors discuss the issues of presenting classification schedules of popular classification systems on the Internet with special focus on their applications. They explain why privately owned classification systems, like Dewey Decimal Classification (DDC) and Universal Decimal Classification (UDC), are not published on the Internet, while the systems supported by government organizations (e.g. national libraries) are traditionally published openly. The authors provide the example of the Library of Congress Classification (LCC), which is the United States’ National Classification System and which is published on open access. The project of the open presentation of the Library-Bibliographical Classification (LBC) in several versions supplemented with indices on the website of the Russian State Library (RSL) is described in more detail. The possibilities for their use are analyzed.

One Domain Divided in Twain: Ontological Perspectives of Design Expressed via Classification

Abstract Epistemological and ontological perspectives on design have changed dramatically in the past century, and yet library classification systems have not been updated accordingly. This article examines where design is—and is not—classified in the two dominant library classification systems in America, Dewey Decimal Classification and Library of Congress Classification, and what this expresses regarding design as a domain. This article demonstrates how such classification relegates design theory as subordinate to the product and can lead to information siloes, diminish creativity, and interdisciplinary study.

Revisit the Vertical Structure of the Eddies and Eddy‐Induced Transport in the Leeuwin Current System

AbstractThe vertical structure of eddies in the Leeuwin Current system affects the eddy volume, heat, and salt transport, even the ecosystem. However, the understanding of eddy vertical structure and eddy‐induced transport here are still very limited. In this study, satellite observed sea surface heights were combined with decade‐long in situ measurements of Argo floats to study the vertical structure of mesoscale eddies in the LC system and their volume, heat, and salt transport. A novel eddy reconstruction method, which considers the influences of both the eddy and background flow, is devised to study the three‐dimensional structure of eddies. Result shows that, in LC system, anticyclonic eddies (AEs) are usually surface‐intensified, with the geostrophic velocity decreasing sharply below the mixed layer, while cyclonic eddies (CEs) are subsurface‐intensified, with a maximum speed at 240 m. The density anomaly core of the average AE (CE) is at a depth of 130 m (650 m) with a density anomaly of −0.51 (0.24) kg/m3. The volume‐integrated eddy kinetic energy and available potential energy of the average CE are much larger than those of the average AE. The average lifespan of CEs is significantly longer than that of AEs, which can be explained by the deeper vertical scale of CEs. The offshore volume transport by eddy drift across the coastal (107°E) section is 9.05 Sv (12.5 Sv). The heat and salt onshore transport by eddy drift across the coastal (107°E) section are, respectively, 10.6 Tw and 143.1 ton/s (17.1 Tw and 241.0 ton/s).

Total Retention Liquid Chromatography-Mass Spectrometry to Achieve Maximum Protein Sequence Coverage.

Peptide identification by liquid chromatography-mass spectrometry (LC-MS) requires retention and elution of peptides from the LC column. Although medium and hydrophobic peptides are readily retained by the C18 columns that are commonly used in proteomics, short and hydrophilic peptides are not retained nor measured by MS due to their elution in the void volume after sample injection. These nonretained peptides can possess important post-translational modifications, such as glycosylation or phosphorylation. We describe a total retention LC-MS method that employs a reverse phase C18 column and porous graphitic carbon (PGC) column to retain both hydrophobic and hydrophilic peptides for LC-MS analysis. Our setup uses a single valve with a trapping column and two LC pumps run at low microliter/minute flow rates to deliver separate gradients to parallel capillary C18 and PGC columns. Our capillary LC system balances the need for high sensitivity with ease of implementation as compared to other 2D LC systems that use nanocolumns with multiple trapping columns and multiport valves. We demonstrate the utility of the method identifying hydrophilic peptides that went undetected when only a C18 nanocolumn was used. These missed hydrophilic peptides include tripeptides and N-glycosylated species.

Library Classification Systems in the U.S.: Basic Ideas and Examples

This article offers a basic introduction to classification in the context of librarianship in the United States with an aim toward filling gaps in formal education and practical experience. The article defines the concept of classification and discusses the goals and purposes of classification, both functional and intellectual. Overviews of two common classification systems frequently used in U.S. libraries are presented: Dewey Decimal Classification (DDC), Library of Congress Classification (LCC), as well as an introduction to a group of classifications known as “reader-interest classifications.”

Associations among total p-cresylsulfate, indoxyl sulfate and hippuric acid levels with hemodialysis quality indicators in maintenance hemodialysis patients

BackgroundTotal p-cresylsulfate (PCS), indoxyl sulfate (IS) and hippuric acid (HA) are harmful uremic toxins known to be elevated in patients with uremia. Serum total PCS, IS and HA levels have been associated with coronary atherosclerosis, left ventricular hypertrophy, metabolic acidosis, neurological symptoms, and accelerated renal damage associated with chronic kidney disease; however, no study has examined the effect of total PCS, IS and HA on hemodialysis (HD) quality indicators. The aim of this study was to examine associations among total PCS, IS and HA with HD quality indicators in patients undergoing HD treatment. MethodsThis study included 264 consecutive patients at a single HD center who assessed using previously demonstrated HD quality indicators including anemia, bone-mineral metabolism, dialysis dose, cardiovascular risk, and middle molecule removal area. Serum HA was measured using a capillary electrophoresis method. Serum total PCS and IS concentrations were measured using an Ultra Performance LC System. ResultsMultiple regression analysis showed that sex, potassium, systolic blood pressure (SBP), average BP, β2-microglobulin, and creatinine were independently positively associated with IS level, and that age, total cholesterol, and estimated glomerular filtration rate (eGFR) was independently negatively associated with IS level. In addition, β2-microglobulin was independently positively associated with total PCS. Moreover, potassium, diastolic blood pressure, average BP, β2-microglobulin, dialysis vintage, and albumin were independently positively associated with HA level, and age, transferrin saturation, fasting glucose, and eGFR were independently negatively associated with HA level. When the patients were stratified by age and sex, serum IS and HA levels were still independently associated with some hemodialysis quality indicators. In addition, canonical correlation analysis also confirmed the relationship between uremic toxins (IS and HA) and HD quality indicators (potassium, β2-microglobulin, average BP, creatinine, and eGFR). ConclusionThis study demonstrated that uremic toxins (IS and HA) and HD quality indicators (potassium, β2-microglobulin, average BP, creatinine, and eGFR) constructs were correlated with each other, and that there were sex and age differences in these associations among maintenance HD patients.

Proteomic profile of bone "collagen" extracted for stable isotopes: Implications for bulk and single amino acid analyses.

Protein studies in archaeology and paleontology have been dominated by stable isotope studies to understand diet and trophic levels, but recent applications of proteomic techniques have resulted in a more complete understanding of protein diagenesis than stable isotopes alone. In stable isotope analyses, samples are retained or discarded based on their properties. Proteomics can directly determine what proteins are present within the sample and may be able to allow previously discarded samples to be analyzed. Protein samples that had been previously analyzed for stable isotopes, including those with marginal and poor sample quality, were characterized by liquid chromatography/mass spectrometry using an LTQ Orbitrap Velos mass spectrometer after separation on a Dionex Ultimate 3000 LC system. Data were analyzed using MetaMorpheus and custom R scripts. We found a variety of proteins in addition to collagen, although collagen I was found in the majority of the samples (most samples >80%). We also found a positive correlation between total deamidation and wt% N, suggesting that deamidation may impact the overall nitrogen signal in bulk analyses. The amino acid profiles of samples, including those of marginal or poor stable isotope quality, reflect the expected collagen I percentages, allowing their use in single amino acid stable isotope analyses. All the samples regardless of quality were found to have high concentrations of collagen I, making interpretations of dietary routing based on collagen I reasonably valid. The amino acid profiles on the marginal and poor samples reflect an expected collagen I profile and allow these samples to be recovered for single amino acid analyses.

Extraction, Radical Scavenging Activities, and Chemical Composition Identification of Flavonoids from Sunflower (Helianthus annuus L.) Receptacles.

This study was focused on extraction, radical scavenging activities, and chemical composition identification of total flavonoids in sunflower (Helianthus annuus L.) receptacles (TFSR). We investigated the optimal extract parameters of TFSR using response surface methodology. The highest yield of TFSR was 1.04% with the ethanol concentration 58%, the material-to-liquid ratio 1:20 (v/w), the extraction time 2.6 h, and the extraction temperature 67 °C. The results of radical scavenging activities showed that ethyl acetate fraction (EAF) was the strongest by using 2-diphenyl-1-picrylhydrazyl (DPPH), 2, 2’-azino-bis (3-ethylbenzo thiazoline-6-sulfonic acid) (ABTS) and iron ion reducing analysis. The EAF had the highest flavonoids contents. Four fractions A, B, C and D were enrichment from EAF by polyamide resin. Fraction B had the highest flavonoids content. Thirteen chemical components of flavonoids in fraction B were first identified by Ultimate 3000 Nano LC System coupled to a Q Exactive HF benchtop Orbitrap mass spectrometer (UHPLC-HRMS/MS). Among of the thirteen chemical components, isoquercetin and daidzein were identified accurately by comparing with standard samples. Radical scavenging analysis showed that isoquercetin and EAF had strong activities. Therefore, sunflower receptacles can be used as a source of natural flavonoids. TFSR as a natural radical scavenger has potential applications in pharmaceutical industry.