Acids In Complex Samples Research Articles

PHYSIOLOGICAL ROLE OF D-AMINO ACIDS AND BIOANALYTICAL POTENTIAL OF D-AMINO ACID OXIDASES

D-amino acid oxidase (DAAO) plays an important role in the functioning of both prokaryotes and eukaryotes. DAAO is increasingly being used in practice, including for the determination of D-amino acids in complex samples, including human tissues and fl uids. There are generally two types of DAAO in all organisms. The fi rst type is an enzyme highly specifi c for D-aspartate and has its own name D-aspartate oxidase (DASPO). DAAO of the second type is characterized by a wide spectrum of substrate specificity, with preference for one or another D-amino acid varying from source to source. The activity of DAAO with a large number of substrates greatly complicates the selective determination of a particular D-amino acid. The problem is often solved by choosing an enzyme that, under the conditions of analysis, has low or no activity with other D-amino acids present in the sample. For the convenience of selecting a particular enzyme, we have collected and analyzed literature data on the catalytic parameters of known DAAOs with the most important D-amino acids. In addition, similar data are presented for novel recombinant DAAOs from the methylotrophic yeast Ogataea parapolymorpha DL-1. Analysis of the data shows that, with the D-amino acid series, the new OpaDASPO and OpaDAAO have the highest catalytic parameters.

Chiral derivatization-enabled discrimination and on-tissue detection of proteinogenic amino acids by ion mobility mass spectrometry.

The importance of chiral amino acids (AAs) in living organisms has been widely recognized since the discovery of endogenous d-AAs as potential biomarkers in several metabolic disorders. Chiral analysis by ion mobility spectrometry-mass spectrometry (IMS-MS) has the advantages of high speed and sensitivity but is still in its infancy. Here, an N α-(2,4-dinitro-5-fluorophenyl)-l-alaninamide (FDAA) derivatization is combined with trapped ion mobility spectrometry-mass spectrometry (TIMS-MS) for chiral AA analysis. For the first time, we demonstrate the simultaneous separation of 19 pairs of chiral proteinogenic AAs in a single fixed condition TIMS-MS run. The utility of this approach is presented for mouse brain extracts by direct-infusion TIMS-MS. The robust separation ability in complex biological samples was proven in matrix-assisted laser desorption/ionization (MALDI) TIMS mass spectrometry imaging (MSI) as well by directly depositing 19 pairs of chiral AAs on a tissue slide following on-tissue derivatization. In addition, endogenous chiral amino acids were also detected and distinguished. The developed methods show compelling application prospects in biomarker discovery and biological research.

Development of magnetism-assisted in-tube solid phase microextraction of phenolic acids in fruit juices prior to high-performance liquid chromatography quantification.

Magnetism-assisted in-tube solid phase microextraction based on porous monolith mingled with Fe3 O4 nanoparticles was developed for capture of phenolic acids in fruit juices. First, poly (1-allyl-3-methylimidazolium bis [(trifluoro methyl) sulfonyl] imide-co-ethylene dimethacrylate) monolith embedded with Fe3 O4 nanoparticles was facile fabrication in a capillary and employed as microextraction column. Subsequently, a magnetic coil adopted to produce variable magnetic fields during extraction stage was twined on the microextraction column. The analytes contents in eluant were quantified by high performance liquid chromatogram with diode array detector. Various parameters affecting the extraction performance were inspected and optimized in detail. Results revealed that the exertion of magnetic fields in adsorption and desorption steps enhanced the extraction efficiencies of analytes from 44.9-64.0% to 78.6-87.1%. Under the optimal extraction factors, the limits of detection were between 0.012 and 0.061 μg/L, relative standard deviations for precision in terms of intra- and inter-day assay variability ranged from 1.9 to 9.8%. The introduced approach was successfully applied to simultaneously quantify the contents of five analytes in real fruit juices with satisfying fortified recoveries (80.1-116%). The obtained results well demonstrate the promising potential of the developed method in the highly sensitive quantification of trace phenolic acids in complex samples.

Enzyme-Assisted Nucleic Acid Detection for Infectious Disease Diagnostics: Moving toward the Point-of-Care.

Driven by complex and interconnected factors, including population growth, climate change, and geopolitics, infectious diseases represent one of the greatest healthcare challenges of the 21st century. Diagnostic technologies are the first line of defense in the fight against infectious disease, providing critical information to inform epidemiological models, track diseases, decide treatment choices, and ultimately prevent epidemics. The diagnosis of infectious disease at the genomic level using nucleic acid disease biomarkers has proven to be the most effective approach to date. Such methods rely heavily on enzymes to specifically amplify or detect nucleic acids in complex samples, and significant effort has been exerted to harness the power of enzymes for in vitro nucleic acid diagnostics. Unfortunately, significant challenges limit the potential of enzyme-assisted nucleic acid diagnostics, particularly when translating diagnostic technologies from the lab toward the point-of-use or point-of-care. Herein, we discuss the current state of the field and highlight cross-disciplinary efforts to solve the challenges associated with the successful deployment of this important class of diagnostics at or near the point-of-care.

Solid-phase extraction of phenoxyacetic acid herbicides in complex samples with a zirconium(IV)-based metal-organic framework.

A zirconium(IV)-based metal-organic framework material (MOF-808) has been synthesized in a simple way and used for the extraction of phenoxyacetic acids in complex samples. The material has good thermal and chemical stability, large specific surface area (905.36m²/g), and high pore size (22.18Å). Besides, it contains a large amount of Zr-O groups, easy-to-form Zr-O-H bond with carboxyl groups of phenoxyacetic acids, and possesses biphenyl skeleton structure, easy to interact with compounds through π-π and hydrophobic interactions. These characteristics make the material very suitable for the extraction of certain compounds with a high extraction efficiency and excellent selectivity. The extraction conditions were optimized, and then an analytical method was successfully established and applied for analysis of actual samples. The solid-phase extraction method based on prepared material had a wide linear range of 0.2-250μg/L and a low detection limit of 0.1-0.5μg/L for four phenoxyacetic acid compounds including 2,4-dichlorophenoxyacetic acid, 2-(2,4-dichlorophenoxy) propionic acid, 4-chlorophenoxyacetic acid, and dicamba. The relative standard deviations of intra- and interday precision were 1.8-3.8 and 4.3-6.9%, and the recoveries after spiking were between 77.1 and 109.3%. The results showed that the material is a desired substituent for the extraction of compounds with benzene ring structure containing carboxyl groups.

Simple Polydisperse Droplet Emulsion Polymerase Chain Reaction with Statistical Volumetric Correction Compared with Microfluidic Droplet Digital Polymerase Chain Reaction

Nucleic acid amplification technology, such as polymerase chain reaction (PCR), has enabled highly sensitive and specific disease detection and quantification, leading to more accurate diagnosis and treatment regimens. Lab-on-a-chip applications have developed methods to partition single biomolecules, such as DNA and RNA, into picoliter-sized droplets. These individual reaction vessels lead to digitization of PCR enabling improved time to detection and direct quantification of nucleic acids without a standard curve, therefore simplifying assay analysis. Though impactful, these improvements have generally been restricted to centralized laboratories with trained personnel and expensive equipment. To address these limitations and make this technology more applicable for a variety of settings, we have developed a statistical framework to apply to droplet PCR performed in polydisperse droplets prepared without any specialized equipment. The polydisperse droplet system allows for accurate quantification of droplet digital PCR (ddPCR) and reverse transcriptase droplet digital PCR (RT-ddPCR) that is comparable to commercially available systems such as BioRad's ddPCR. Additionally, this approach is compatible with a range of input sample volumes, extending the assay dynamic range beyond that of commercial ddPCR systems. In this work, we show that these ddPCR assays can reduce overall assay time while still providing quantitative results. We also report a multiplexed ddPCR assay and demonstrate proof-of-concept methods for rapid droplet preparation in multiple samples simultaneously. Our simple polydisperse droplet preparation and statistical framework can be extended to a variety of settings for the quantification of nucleic acids in complex samples.

Detection of Nucleic Acids in Complex Samples via Magnetic Microbead-Assisted Catalyzed Hairpin Assembly and "DD-A" FRET.

Nucleic acids, as one kind of significant biomarker, have attracted tremendous attention and exhibited immense values in fundamental studies and clinical applications. In this work, we developed a fluorescent assay for detecting nucleic acids in complex samples based on magnetic microbead (MMB)-assisted catalyzed hairpin assembly (CHA) and a donor donor-acceptor fluorescence resonance energy transfer ("DD-A" FRET) signaling mechanism. Three types of DNA hairpin probes were employed in this system, including Capture, H1 (double FAM-labeled probe as FRET donor), and H2 (TAMRA-labeled probe as FRET acceptor). First, the Captures immobilized on MMBs bound to targets in complex samples, and the sequences in Captures that could trigger catalyzed hairpin assembly (CHA) were exposed. Then, target-enriched MMB complexes were separated and resuspended in the reaction buffer containing H1 and H2. As a result, numerous H1-H2 duplexes were formed during the CHA process, inducing an obvious FRET signal. In contrast, CHA could not be triggered, and the FRET signal was weak, while target was absent. With the aid of magnetic separation and "DD-A" FRET, errors from background interference were effectively eliminated. Importantly, this strategy realized amplified detection in buffer, with detection limits of microRNA as low as 34 pM. Furthermore, this method was successfully applied to detect microRNA-21 in serum and cell culture media. The results showed that our method has the potential for biomedical research and clinical application.

Efficient extraction of perfluorocarboxylic acids in complex samples with a monolithic adsorbent combining fluorophilic and anion-exchange interactions

Efficient extraction of perfluorocarboxylic acids (PFCAs) is a key step for the accurate and sensitive determination of PFCAs due to their low concentration and the complexity of sample matrices. Herein, according to the chemical characteristics of PFCAs, a new adsorbent based on poly (2,2,3,3,4,4,5,5,6,6,7,7-dodecafluoroheptyl acrylate/(vinylbenzyl)trimethylammonium chloride-co-divinylbenzene/ethylenedimethacrylate) monolith was fabricated and utilized as the extraction medium of multiple monolithic fiber solid-phase microextraction (MMF-SPME). Results well indicted that the prepared monolithic adsorbent (MA) could efficient extract PFCAs through fluorophilic and anion-exchange interactions. Under the optimal conditions, the MA/MMF-SPME was combined with HPLC-MS/MS for the sensitive monitoring of ultra-trace PFCAs in water and milks samples. The limits of detection (S/N = 3) of the target PFCAs in water and milk samples were 0.40–4.40 ng/L and 0.9–12.1 ng/L, respectively. Furthermore, the proposed method also exhibited some merits including wide linear dynamic ranges, satisfactory sensitivity, good method precision and low consumption of sample and organic solvent. Isotope internal standard calibration curve method was used to quantify the concentration of PFCAs in real samples, and trace levels of PFCAs in tap water and milk samples had been successfully detected.

Retention Characteristics of a pH Tunable Water Stationary Phase in Supercritical Fluid Chromatography.

The retention characteristics of a novel pH tunable water stationary phase are presented. The method utilizes a change in mobile phase from N2 to CO2 to acidify the water phase in situ and control the ionization and elution of organic acids. With N2 present the phase pH>5.4 and the acids are ionized and strongly retained. Conversely, with CO2 present the pH<3.8 and the acids are neutralized and can elute. This effect is reasonably independent of time. For example, at 80°C hexanoic acid readily elutes from a 10 m column after switching to CO2 at any point over a 1 h period. Beyond this, however, some broadening and peak erosion is noted. Acids are also retained on 10 and 2 m columns similarly, since their elution primarily depends upon the change in stationary phase pH. Altering the CO2 solubility in the water phase alone (i.e., through changing system temperature and pressure without using N2) also produces similar changes in stationary phase acidity. However, this approach yields greater system noise and instability. The N2/CO2 switching mode is used to analyze organic acids in various samples and is found to provide high selectivity for them over other matrix components. Therefore, this approach can potentially simplify the analysis of such acids in complex samples.

Golf ball-assisted electrospray ionization of mass spectrometry for the determination of trace amino acids in complex samples

During the electrospray ionization (ESI) process, ions move through a heated capillary aperture to be detected on arrival at a mass analyzer. However, the ESI process creates an ion plume, which expands into an ion cloud with an area larger than that of the heated capillary aperture, significantly contributing to an ion loss of 50% due to coulombic repulsion. The use of DC and RF fields to focus ions from the ion source into the vacuum chamber has been proposed in the literature, but the improvement of ion transmission efficiency is limited. To improve ion transmission, in this study we propose a novel method using a home-made golf ball positioned between the ion source and the inlet of the mass analyzer to hydrodynamically focus the ions passing through the golf ball. The ion plume produced by the ESI process passes through the golf ball will reduce the size of the ion cloud then be focused and most of them flowed into the mass analyzer. Therefore, the sensitivity will be improved, the aim of this investigation is to study the enhancing of the signal using golf ball-assisted electrospray ionization liquid chromatography tandem mass spectrometry (LC-MS/MS) to determine 20 trace amino acids in complex samples, including tea, urine and serum. The results showed that the analytical performance of the determination of the 20 amino acids in tea, urine and serum samples using the home-made golf ball-assisted ESI source is better than that of a commercial ESI source. The signal intensities of the 20 amino acids were enhanced by factors of 2–2700, 11–2525, and 31–342680 in oolong tea, urine and serum analyses, respectively. The precision of the proposed method ranged from 1–9%, 0.4–9% and 0.4–8% at low, medium and high concentration levels of amino acids, respectively. The home-made golf ball-assisted ESI source effectively increased the signal intensity and enhanced the ion transmission efficiency and is also an easy, convenient and economical device. This technique can be applied to the analysis of trace compounds in complex matrices.

Selective removal of phosphate for analysis of organic acids in complex samples

Accurate quantitation of compounds in samples of biological origin is often hampered by matrix interferences one of which occurs in GC-MS analysis from the presence of highly abundant phosphate. Consequently, high concentrations of phosphate need to be removed before sample analysis. Within this context, we screened 17 anion exchange solid-phase extraction (SPE) materials for selective phosphate removal using different protocols to meet the challenge of simultaneous recovery of six common organic acids in aqueous samples prior to derivatization for GC-MS analysis. Up to 75% recovery was achieved for the most organic acids, only the low pKa tartaric and citric acids were badly recovered. Compared to the traditional approach of phosphate removal by precipitation, SPE had a broader compatibility with common detection methods and performed more selectively among the organic acids under investigation. Based on the results of this study, it is recommended that phosphate removal strategies during the analysis of biologically relevant small molecular weight organic acids consider the respective pKa of the anticipated analytes and the detection method of choice.

Chitosan-modified silver@ruthenium hybrid nanoparticles: evaluation of physico-chemical properties and bio-affinity with sialic acid.

Hybrid nanoparticles (NPs) integrated with dye molecules have attracted interest for biomolecular detection, due to their effortless fabrication, timely operation, controllable specific recognition and low-cost. In this study, hybrid core-shell NPs made of a metal-dye complex (AgNPs@[Ru(bpy)3]2+) core and a chitosan (CS) shell exhibiting a selective fluorescence quenching effect were successfully prepared using a cost-effective wet-chemical approach. The physico-chemical properties of NPs were determined by spectroscopy and light scattering measurements. The bio-affinity of the AgNPs@[Ru(bpy)3]2+/CS was evaluated in aqueous media using sialic acid (SA) as the target molecule in the presence of different monosaccharides and anionic biomolecules as interferents. A significant fluorescence quenching of hybrid NPs was observed in aqueous solutions of SA with interferents, while no significant quenching effect was detected in SA-free interferent solutions. The selective binding of SA to the particles resulted from favorable electrostatic interactions and inter-molecular hydrogen bonding with the functional groups of CS. The hybrid NP system displayed a good sensitivity for SA with a detection limit of 5.1 nM and a concentration dependent fluorescence quenching for SA concentrations ranging from 25 nM to 3.2 μM. This hybrid NP system represents a promising alternative probe for detecting sialic acid in complex samples.

Simple and Rapid Determination of Ferulic Acid Levels in Food and Cosmetic Samples Using Paper-Based Platforms

Ferulic acid is an important phenolic antioxidant found in or added to diet supplements, beverages, and cosmetic creams. Two designs of paper-based platforms for the fast, simple and inexpensive evaluation of ferulic acid contents in food and pharmaceutical cosmetics were evaluated. The first, a paper-based electrochemical device, was developed for ferulic acid detection in uncomplicated matrix samples and was created by the photolithographic method. The second, a paper-based colorimetric device was preceded by thin layer chromatography (TLC) for the separation and detection of ferulic acid in complex samples using a silica plate stationary phase and an 85:15:1 (v/v/v) chloroform: methanol: formic acid mobile phase. After separation, ferulic acid containing section of the TLC plate was attached onto the patterned paper containing the colorimetric reagent and eluted with ethanol. The resulting color change was photographed and quantitatively converted to intensity. Under the optimal conditions, the limit of detection of ferulic acid was found to be 1 ppm and 7 ppm (S/N = 3) for first and second designs, respectively, with good agreement with the standard HPLC-UV detection method. Therefore, these methods can be used for the simple, rapid, inexpensive and sensitive quantification of ferulic acid in a variety of samples.

Investigation of the molecular ion structure for aldononitrile acetate derivatized muramic acid

The muramic acid assay is a powerful tool for detecting both intact bacteria and bacterial debris. Past use of aldononitrile acetate derivatization for determining muramic acid in complex samples by gas chromatography/mass spectrometry met detection needs in many instances; however, questions have been raised regarding the interpretation of the derivative structure and its electron ionization fragments. In this study, we applied different methods and proved that the aldononitrile acetate derivatized muramic acid yields a molecular weight of 398, associated with a lactam structure. We also presented evidence that the structure of aldononitrile acetate derivatized muramic acid is acetylated at four positions, 3 O-acetylations and 1N-acetylation. In practical manner, this communication provides a comprehensive reference to researchers using δ13C value or ion fragments of the muramic acid marker in biogeochemical studies.

Model-based rank annihilation factor analysis for quantitative analysis of mixtures of monoprotic acids using multivariate spectrophotometric acid-base titrations

A new application of rank annihilation factor analysis (RAFA) method is proposed for the quantitative determination of monoprotic acids in complex samples based on spectrophotometric acid-base titrations. The inclusion of a hard-modeling constraint based on the acid-base equilibrium model in the RAFA method allowed for the correct quantitation of the analyte in a sample mixture. An intrinsic difficulty in the analysis of spectrometric titration data is the presence of rank deficiency due to the closure for the mixtures of two or more compounds. Using the proposed approach, accurate quantitation is possible even in the presence of this rank deficiency. The presence of unknown and uncalibrated interferences in the samples does not affect the quantitative determination of the analyte of interest. Extra titration of standard and augmentation with sample matrix and also the matrix effects are avoided in the proposed method. The procedure was successfully applied to the analysis of mixtures of chlorophenols as model compounds.

Two-dimensional strandness-dependent electrophoresis

Two-dimensional strandness-dependent electrophoresis (2D-SDE) separates nucleic acids in complex samples according to strandness, conformation and length. Under the non-denaturing conditions of the first electrophoretic step, single-stranded DNA, double-stranded DNA and RNA.DNA hybrids of similar length migrate at different rates. The second electrophoretic step is performed under denaturing conditions (7 mol l(-1) urea, 55 degrees C) so that all the molecules are single-stranded and separate according to length only. 2D-SDE is useful for revealing important characteristics of complex nucleic acid samples in manipulations such as amplification, renaturation, cDNA synthesis and microarray hybridization. It can also be used to identify mispaired, nicked or damaged fragments in double-stranded DNA. The protocol takes approximately 2 h and requires only basic skills, equipment and reagents.

Analysis of amino acids in complex samples by using voltammetry and multivariate calibration methods

A voltammetric method is proposed for the simultaneous determination of tryptophan, cysteine, and tyrosine using multivariate calibration techniques. Various electrodes and voltammetric techniques were explored to ascertain the optimum measurement strategy. Among them, differential pulse voltammetry (DPV) with a Pt electrode was selected as analytical technique since it provided a suitable compromise between sensitivity and reproducibility while allowing the oxidation peaks of the three compounds to be reasonably discriminated. The sensitivity of DPV with Pt electrode for Trp standards was 8.4×10 −2 A l mol −1, the repeatability 3.7% and the detection limit below 10 −7 M. The lack of full selectivity of the voltammetric data was overcome using multivariate calibration methods on the basis of the differences in the voltammetric waves of each compound. The accuracy of predictions was evaluated preliminarily from the analysis of three-component synthetic mixtures. Subsequently, this method was applied to the analysis of oxidizable amino acids in feed samples. Results obtained were in good concordance with those given by the standard method using an amino acid analyzer.

Quantitative Determination of Erucic Acid in Edible Oils and Fats Containing Marine Oils and/or Hydrogenated Fats

AbstractA method has been developed for the quantitative measurement of erucic acid in complex samples of oils or fats containing marine oils and/or hydrogenated fats. A two step procedure is proposed: in a first step the constituent fatty acids of the transesterified oil or fat are resolved on a silver‐nitrate‐impregnated silicagel thin‐layer in function of the degree of unsaturation and the double bond localization. Positional isomers migrating slowlier on the thin‐layer than erucic acid (e. g. cetoleic acid often present in large amounts in marine oils) are distinctly separated in this way. In a second step components moving ahead of the cetoleic acid (e. g. erucic acid, the added internal standard and geometrical isomers of erucic acid) are scraped off from the thin‐layer plate and the erucic acid is determined by gas chromatography on a packed column with a liquid crystal, operated in the nematic phase temperature region, as the stationary phase. In complex samples recoveries of 90 to 102% are reported with overall relative standard deviations of 2.5 to 3%.

Photometric determination of α-ketoglutaric acid with diazotized sulphanilic acid

A new photometric method for the determination of α-ketoglutaric acid is presented. It is based on the colour reaction of α-ketoglutaric acid in sodium hydroxide solution with diazotized sulphanilic acid in the presence of sodium sulphite and sodium hypophosphite. This method is highly sensitive and fairly selective for α-ketoglutaric acid, and may be suitable for the determination of the acid in complex samples.