Quantification Of Small Molecules Research Articles

Metabolomic Applications in Radiation Biodosimetry.

Metabolomics, the collective assessment and quantification of small molecules in a given biofluid or tissue sample, has provided new ways in evaluating an individual's exposure level to ionizing radiation or other genotoxic stressors. Protocols that are routinely utilized for the preparation of samples from rodents to patients are presented here in order to be analyzed by high-throughput liquid chromatography-mass spectrometry techniques. These protocols are based on established methods in our laboratory that have been used extensively in radiation biodosimetry through metabolomics. These protocols are focused on general profiling of samples and therefore do not concentrate on extraction of specific classes on metabolites (e.g., eicosanoids).

Determination of copper and lead in tequila by conventional matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and partial least squares regression.

Quantification of small molecules by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) is challenging yet attractive, due to micro-scale procedural simplicity, high throughput and lack of memory effects. Since these features are important while analyzing trace elements in quality control schemes, MALDI-TOFMS was used for the determination of copper (Cu) and lead (Pb) in tequila with quantification carried out by partial least squares regression (PLS2) and by univariate calibration (UC). In the proposed procedure, Bi(III) was added as internal standard (IS), diethyldithiocarbamate complexes were formed (pH 7.4) and extracted into chloroform; after solvent evaporation and re-constitution in acetonitrile, the sample was co-crystallized with α-cyano-4-hydroxycinnamic acid on a steel target. From the acquired mass spectra, UC was performed using IS-normalized signals of the monoisotopic ions of analytes, and the m/z range 350-513 was used for PLS2. Accuracy was tested by recovery experiments and by inductively coupled plasma (ICP)-MS analysis. When compared with direct analyte signal measurements, application of IS yielded enhanced analytical performance using either UC or PLS2; the method quantification limits were: 11.1 μg L-1 , 23.4 μg L-1 for Cu and 89.8 μg L-1 , 97.1 μg L-1 for Pb, respectively. In tequila, MALDI-TOFMS and ICP-MS provided consistent results for Cu (165-2599 μg L-1 ); Pb was not detected in any sample by MALDI-TOFMS, yet recoveries obtained after standard addition were indicative of acceptable accuracy (400 μg L-1 Pb added; recoveries: 91.2-108% for UC and 98.8-120% for PLS2). New experimental evidence has been provided supporting the inclusion of trace metals quantification within a range of MALDI-TOFMS applications. Slightly better results were obtained for UC as compared with PLS2 yet both methods can be recommended for testing the compliance of Cu and Pb levels with Official Mexican Norm. Of note, while using PLS2, there is no need for signal integration nor for IS normalization.

Recognition-Enhanced Metastably Shielded Aptamer for Digital Quantification of Small Molecules.

Aptamers are recognized as competitive affinity reagents; their application, however, often suffers from their relatively low target binding affinity, especially for small molecules. We herein introduce the concept of a recognition-enhanced metastably shielded aptamer probe (RMSApt) and explore its performance for digital quantification of low-affinity small molecules. The RMSApt design employs the idea of constructing an allosteric aptamer probe conferring a minor energy gap in the recognition switch process to facilitate target binding and probe response, in turn significantly improving the recognition efficiency for low-affinity targets. The probe design strategy boosts the application of aptamers for precisely quantifying targets with a dissociation constant Kd ranging from 10-4 to 10-9 M, which would cover most of the small-molecule species that exist binding aptamers. Thus, RMSApt would facilitate the translation of aptamers for medical diagnosis, food safety, and environmental screening.

Quantification of Methotrexate in Human Serum and Plasma by Liquid Chromatography Tandem Mass Spectrometry.

Mass spectrometry (MS) is a highly specific and sensitive technique that is used for the detection of many different analytes with diverse chemical characteristics. It has been adopted by clinical laboratories for the quantification of small molecules and, by extension, has been widely used for therapeutic drug monitoring. It is an attractive alternative to immunoassay methods, because it is not subject to the same interferences. A limitation of MS (relative to immunoassays) is the turnaround time. However, this can be addressed by workflow parallelization with other assays. Herein we describe a tandem LC-MS/MS method for the detection and quantification of methotrexate in human plasma with a lower limit of quantification of 0.01μM and within-assay and between-assay coefficients of variation of less than 15%. This method lacks interference from high-abundance metabolites and utilizes kindred chromatography to improve turnaround time in the therapeutic drug monitoring laboratory.

Rapid Detection of Drugs in Human Plasma Using a Small-Molecule-Linked Hybridization Chain Reaction.

Rapid detection and quantification of pharmaceutical drugs directly in human plasma is of major importance for the development of relevant point-of-care testing devices. Here, we report a method for detection and quantification of small molecules in human plasma. An assay employing a small molecule-linked hybridization chain reaction (HCR) has been devised for the detection of the pharmaceutically relevant drugs digoxin (Dig) and methotrexate (MTX). Double modification by small molecule ligands on the initiator strand act as sites to control the rate of the HCR. Upon protein binding to the modified initiator strand, the HCR is greatly inhibited. If the protein is preincubated with a sample containing the small molecule analyte, the protein binding site is occupied by the analyte and the initiator strand will initiate the HCR. This enables efficient detection and quantification of small-molecule analytes in nanomolar concentration even in 50% human plasma within 4 min. Thus, the rapidity and simplicity of this assay has potential for point-of-care testing.

Multifunctional solid-state electrochemiluminescent chemosensors and aptasensor with free-standing active sites based on task-specific pyrene-terminated polymers via RAFT polymerization

Based on the flexible molecular engineering technique of reversible addition-fragmentation chain transfer (RAFT) polymerization, various polymers carrying positive or negative charges and different terminal groups such as pyrene or tertiary amine were synthesized for fabricating multifunctional solid-state electrochemiluminescent (ECL) sensors. Accordingly, the chemosensors immobilizing the ECL probe or co-immobilizing the ECL probe and the coreactant were realized for the quantification of small molecules (e.g., tripropylamine, tetracycline), and an aptasensor was developed for the specific and sensitive lysozyme assay (limit of detection: 0.1 ng/mL). All of the sensors were realized via a simple design exploiting the π−π stacking and electrostatic interactions. It was confirmed that the proposed strategy is simple but universal for the fabrication of versatile ECL sensors that showed simplicity, cost-effectiveness, high sensitivity, long-term stability, and excellent reproducibility.

A targeted metabolomics approach for clinical diagnosis of inborn errors of metabolism

Metabolome, the ultimate functional product of the genome, can be studied through identification and quantification of small molecules. The global metabolome influences the individual phenotype through clinical and environmental interventions. Metabolomics has become an integral part of clinical research and allowed for another dimension of better understanding of disease pathophysiology and mechanism. More than 95% of the clinical biochemistry laboratory routine workload is based on small molecular identification, which can potentially be analyzed through metabolomics. However, multiple challenges in clinical metabolomics impact the entire workflow and data quality, thus the biological interpretation needs to be standardized for a reproducible outcome. Herein, we introduce the establishment of a comprehensive targeted metabolomics method for a panel of 220 clinically relevant metabolites using Liquid chromatography-tandem mass spectrometry (LC-MS/MS) standardized for clinical research. The sensitivity, reproducibility and molecular stability of each targeted metabolite (amino acids, organic acids, acylcarnitines, sugars, bile acids, neurotransmitters, polyamines, and hormones) were assessed under multiple experimental conditions. The metabolic tissue distribution was determined in various rat organs. Furthermore, the method was validated in dry blood spot (DBS) samples collected from patients known to have various inborn errors of metabolism (IEMs).Using this approach, our panel appears to be sensitive and robust as it demonstrated differential and unique metabolic profiles in various rat tissues. Also, as a prospective screening method, this panel of diverse metabolites has the ability to identify patients with a wide range of IEMs who otherwise may need multiple, time-consuming and expensive biochemical assays causing a delay in clinical management.

Panoply of Fluorescence Polarization/Anisotropy Signaling Mechanisms for Functional Nucleic Acid-Based Sensing Platforms.

Fluorescence polarization/anisotropy is a very popular technique that is widely used in homogeneous-phase immunoassays for the small molecule quantification. In the present Feature, we discuss how the potential of this signaling approach considerably expanded during the last 2 decades through the implementation of a myriad of original transducing strategies that use functional nucleic acid recognition elements as a promising alternative to antibodies.

Microprobe Capillary Electrophoresis Mass Spectrometry for Single-cell Metabolomics in Live Frog (Xenopus laevis) Embryos.

The quantification of small molecules in single cells raises new potentials for better understanding the basic processes that underlie embryonic development. To enable single-cell investigations directly in live embryos, new analytical approaches are needed, particularly those that are sensitive, selective, quantitative, robust, and scalable to different cell sizes. Here, we present a protocol that enables the in situ analysis of metabolism in single cells in freely developing embryos of the South African clawed frog (Xenopus laevis), a powerful model in cell and developmental biology. This approach uses a capillary microprobe to aspirate a defined portion from single identified cells in the embryo, leaving neighboring cells intact for subsequent analysis. The collected cell content is analyzed by a microscale capillary electrophoresis electrospray ionization (CE-ESI) interface coupled to a high-resolution tandem mass spectrometer. This approach is scalable to various cell sizes and compatible with the complex three-dimensional structure of the developing embryo. As an example, we demonstrate that microprobe single-cell CE-ESI-MS enables the elucidation of metabolic cell heterogeneity that unfolds as a progenitor cell gives rise to descendants during development of the embryo. Besides cell and developmental biology, the single-cell analysis protocols described here are amenable to other cell sizes, cell types, or animal models.

Microprobe Capillary Electrophoresis Mass Spectrometry for Single-cell Metabolomics in Live Frog (<em>Xenopus laevis</em>) Embryos

The quantification of small molecules in single cells raises new potentials for better understanding the basic processes that underlie embryonic development. To enable single-cell investigations directly in live embryos, new analytical approaches are needed, particularly those that are sensitive, selective, quantitative, robust, and scalable to different cell sizes. Here, we present a protocol that enables the in situ analysis of metabolism in single cells in freely developing embryos of the South African clawed frog (Xenopus laevis), a powerful model in cell and developmental biology. This approach uses a capillary microprobe to aspirate a defined portion from single identified cells in the embryo, leaving neighboring cells intact for subsequent analysis. The collected cell content is analyzed by a microscale capillary electrophoresis electrospray ionization (CE-ESI) interface coupled to a high-resolution tandem mass spectrometer. This approach is scalable to various cell sizes and compatible with the complex three-dimensional structure of the developing embryo. As an example, we demonstrate that microprobe single-cell CE-ESI-MS enables the elucidation of metabolic cell heterogeneity that unfolds as a progenitor cell gives rise to descendants during development of the embryo. Besides cell and developmental biology, the single-cell analysis protocols described here are amenable to other cell sizes, cell types, or animal models.

Improving Visualization and Interpretation of Metabolome-Wide Association Studies: An Application in a Population-Based Cohort Using Untargeted 1H NMR Metabolic Profiling.

1H NMR spectroscopy of biofluids generates reproducible data allowing detection and quantification of small molecules in large population cohorts. Statistical models to analyze such data are now well-established, and the use of univariate metabolome wide association studies (MWAS) investigating the spectral features separately has emerged as a computationally efficient and interpretable alternative to multivariate models. The MWAS rely on the accurate estimation of a metabolome wide significance level (MWSL) to be applied to control the family wise error rate. Subsequent interpretation requires efficient visualization and formal feature annotation, which, in-turn, call for efficient prioritization of spectral variables of interest. Using human serum 1H NMR spectroscopic profiles from 3948 participants from the Multi-Ethnic Study of Atherosclerosis (MESA), we have performed a series of MWAS for serum levels of glucose. We first propose an extension of the conventional MWSL that yields stable estimates of the MWSL across the different model parameterizations and distributional features of the outcome. We propose both efficient visualization methods and a strategy based on subsampling and internal validation to prioritize the associations. Our work proposes and illustrates practical and scalable solutions to facilitate the implementation of the MWAS approach and improve interpretation in large cohort studies.

Aptamer-based detection of adenosine triphosphate via qPCR

Sensitive and specific detection and quantification of small molecules often remain challenging. We developed a novel magnetic bead-based aptamer-assisted real-time PCR (Apta-qPCR) assay to provide a versatile platform for quantification of small molecules. The assay has been realized for the detection of ATP as a model system. The assay relies on a combination of qPCR with the target-induced dissociation (TID) of ATP aptamer from an oligonucleotide, complementary to the ATP binding site of the aptamer. The complementary oligonucleotide was immobilized on deoxythymidine (dT)-modified magnetic beads (dT-beads) and hybridized with the aptamer. The presence of ATP resulted in dissociation of the aptamer from the dT-beads and the dissociated aptamer was quantified using qPCR. The Apta-qPCR assay was able to detect 17nM ATP with a broad dynamic range from 50nM to 5mM. The assay is label-free, and real-time PCR-based detection of aptamer facilitates high sensitivity. The presented method is highly versatile and can be applied to various aptamer-target pairs to allow detection of a broad range of target analytes.

Quantification of Small Molecule–Protein Interactions using FRET between Tryptophan and the Pacific Blue Fluorophore

We report a new method to quantify the affinity of small molecules for proteins. This method is based on Förster resonance energy transfer (FRET) between endogenous tryptophan (Trp) residues and the coumarin-derived fluorophore Pacific Blue (PB). Tryptophan residues are frequently found in proteins near ligand-binding sites, making this approach potentially applicable to a wide range of systems. To improve access to PB, we developed a scalable multigram synthesis of this fluorophore, starting with inexpensive 2,3,4,5-tetrafluorobenzoic acid. This route was used to synthesize fluorescent derivatives of biotin, as well as lower affinity thiobiotin, iminobiotin, and imidazolidinethione analogues that bind the protein streptavidin. Compared with previously published FRET acceptors for tryptophan, PB proved to be superior in both sensitivity and efficiency. These unique properties of PB enabled direct quantification of dissociation constants (Kd) as well as competitive inhibition constants (Ki) in the micromolar to nanomolar range. In comparison to analogous binding studies using fluorescence polarization, fluorescence quenching, or fluorescence enhancement, affinities determined using Trp-FRET were more precise and accurate as validated using independent isothermal titration calorimetry studies. FRET between tryptophan and PB represents a new tool for the characterization of protein–ligand complexes.

Ultrasensitive quantitative LC-MS/MS of an inhibitor of apoptosis protein's antagonist in plasma using protein target affinity extraction.

A target protein-based affinity extraction LC-MS/MS method was developed to enable plasma level determination following ultralow dosing (0.1-3 µg/kg) of an inhibitor of apoptosis proteins molecule. Methodology & results: Affinity extraction (AE) utilizing immobilized target protein BIR2/BIR3 was used to selectively capture the inhibitor of apoptosis proteins molecule from dog plasma and enable removal of background matrix components. Pretreatment of plasma samples using protein precipitation was found to provide an additional sensitivity gain. A LLOQ of 7.8 pM was achieved by combining protein precipitation with AE. The method was used to support an ultralow dose dog toxicity study. AE-LC-MS/MS, utilizing target protein, is a highly sensitive methodology for small molecule quantification with potential for broader applicability.

Quantitative online sheath-flow surface enhanced Raman spectroscopy detection for liquid chromatography.

Sheath-flow surface-enhanced Raman spectroscopy (SERS) was used for online detection and quantification of small molecules separated by liquid chromatography. A mixture of model metabolites (thiamine, folic acid, and riboflavin) was separated and characterized by UV-Vis and SERS detectors connected in series. Acetonitrile in the mobile phase provided an internal standard enabling quantitative detection across SERS experiments.

Metabolomics approaches for resolving and harnessing chemical diversity in grapes, yeast and wine

Metabolomics is the scientific study of the unique chemical fingerprints that result from specific biochemical processes. It enables the comprehensive detection and quantification of small molecules that are intermediates or end-products of metabolism in a biological sample, such as a grape or yeast. The metabolomics approach also provides for a holistic characterisation of complex chemical transformations and oxidation reactions during winemaking, ageing and transport. The development of metabolomics has made major advances in the last two decades. Improved chromatographic techniques for separation of polar metabolites and enhanced sensitivity and selectivity of nuclear mass resonance and mass spectrometric instrumentation now enable detection and quantification of hundreds of metabolites in a single analysis. New bioinformatics tools and statistics capabilities are available which are key to efficiently analysing and annotating comprehensive datasets. This review provides an overview of analytical methods for untargeted profiling, biomarker discovery and targeted analysis of non-volatile and volatile metabolites. It summarises bioinformatics tools for data processing, data mining and metabolite identification and explores metabolomics approaches which have been successfully employed to comprehensively analyse, annotate and understand the broad chemical diversity in grapes, yeast and wine. It also describes opportunities for the application of metabolomics in grape and wine science and provides an outlook about further development needs and emerging trends.

Accurate and reliable quantification of 25-hydroxy-vitamin D species by liquid chromatography high-resolution tandem mass spectrometry

In general, mass spectrometric quantification of small molecules in routine laboratory testing utilizes liquid chromatography coupled to low mass resolution triple-quadrupole mass spectrometers (QQQs). Here we introduce high-resolution tandem mass spectrometry (quadrupole-Orbitrap) for the quantification of 25-hydroxy-vitamin D [25(OH)D], a marker of the vitamin D status, because the specificity of 25(OH)D immunoassays is still questionable and mass spectrometric quantification is becoming increasingly important. Liquid chromatography coupled to high-resolution tandem mass spectrometry (LC-MS/HR-MS) was used to quantify 25-hydroxy-cholecalciferol [25(OH)D3], 25-hydroxy-ergocalciferol [25(OH)D2], and their C3-epimers 3-epi-25(OH)D3 and 3-epi-25(OH)D2. The method has a run time of 5 min and was validated according to the US Food and Drug Administration and the European Medicines Agency guidelines. High mass resolution was advantageously applied to separate a quasi-isobaric interference of the internal standard D6-25(OH)D2 with 3-epi-25(OH)D3. All analytes showed an imprecision of below 10% coefficient of variation (CV), trueness between 90% and 110%, and limits of quantification below 10 nM. Concentrations measured by LC-MS/HR-MS are in good agreement with those of the National Institute of Standards and Technology reference methods using LC-MS/MS (QQQ). In conclusion, quantification of 25(OH)D by LC-MS/HR-MS is applicable for routine testing and also holds promise for highly specific quantification of other small molecules.

MS-DIAL: data-independent MS/MS deconvolution for comprehensive metabolome analysis.

Data-independent acquisition (DIA) in liquid chromatography tandem mass spectrometry (LC-MS/MS) provides more comprehensive untargeted acquisition of molecular data. Here we provide an open-source software pipeline, MS-DIAL, to demonstrate how DIA improves simultaneous identification and quantification of small molecules by mass spectral deconvolution. For reversed phase LC-MS/MS, our program with an enriched LipidBlast library identified total 1,023 lipid compounds from nine algal strains to highlight their chemotaxonomic relationships.

The application of high resolution diffusion NMR for the characterisation and quantification of small molecules in saliva/dentifrice slurries

The application of DOSY NMR as a technique for the virtual separation of toothpaste adjuvants in model saliva is reported for the first time.

Quantitative performance of liquid chromatography coupled to Q-Exactive high resolution mass spectrometry (HRMS) for the analysis of tetracyclines in a complex matrix

The presence of antibiotics in the environment is of increased interest and, as modern mass spectrometers become more efficient, we are increasingly aware of traces of pharmaceuticals appearing in a wide range of environmental and biological matrices. The Q-Exactive mass spectrometer is part of these innovative hybrid high-resolution mass spectrometers (HRMS) which is often associated with peptide sequencing or metabolomics but with a limited number of studies focusing on its application to the quantification of small molecules in environmental and biological matrices. It combines the high resolving power (RP) performance of the Orbitrap with the high performance selectivity of the quadrupole. Tetracyclines (TCs) are a family comprising some of the most widely used antibiotics in veterinary medicine. This study presents the quantitative performances of the Q-Exactive by illustrating a new approach to quantify TCs using liquid chromatography coupled to a HRMS in a complex matrix, i.e., swine manure. The Q-Exactive was used at high-resolution in both full scan (FS) and targeted ion fragmentation (tMS2) modes. These two modes were optimized and compared to determine the most reliable and efficient approach to quantify TCs with good accuracy. The proposed method was optimized to obtain the best selectivity and sensitivity, thus eliminating false positive and allowing the detection of trace levels of analyte. The TCs were extracted from the matrix by sonication using McIlvaine buffer followed by an off-line solid phase extraction method to concentrate and clean the extracts. Both FS and tMS2 modes presented good linearity (R2>0.991) and repeatability (RSD<15%). Mass accuracy was acceptable with values below 2ppm. The method detection limits (MLD) calculated from the calibration curves ranged from 2.0 to 12ngg−1 for FS mode and from 1.5 to 3.6ngg−1 for tMS2 mode. Accuracy and interday/intraday relative standard deviations were below 21% for both modes studied. TCs were quantified in real samples of swine manure with concentrations ranging from 29 to 75ngg−1. This study showed the possibility of using hybrid HRMS for trace detection and quantification of TCs in a complex matrix, thus avoiding false positive while achieving good selectivity and sensitivity.