Reversed-phase ultrahigh-performance liquid chromatography-mass spectrometry (RP-UHPLC/MS) method is optimized for the quantitation of a large number of lipid species in biological samples, primarily in human plasma and serum. The method uses a C18 bridged ethylene hybrid (BEH) column (150 × 2.1 mm; 1.7 μm) for the separation of lipids from 23 subclasses with a total run time of 25 min. Lipid species separation allows the resolution of isobaric and isomeric lipid forms. A triple quadrupole mass spectrometer is used for targeted lipidomic analysis using multiple reaction monitoring (MRM) in the positive ion mode. Data are evaluated by Skyline software, and the concentrations of analytes are determined using internal standards per each individual lipid class.
- Klíčová slova
- High-throughput lipidomics, Mass spectrometry, Plasma, Quantitation, Reversed-phase, Serum, Ultrahigh-performance liquid chromatography,
- MeSH
- chromatografie s reverzní fází * metody MeSH
- hmotnostní spektrometrie metody MeSH
- kapalinová chromatografie-hmotnostní spektrometrie MeSH
- lidé MeSH
- lipidomika * metody MeSH
- lipidy * analýza MeSH
- rychlé screeningové testy metody MeSH
- software MeSH
- tandemová hmotnostní spektrometrie metody MeSH
- vysokoúčinná kapalinová chromatografie metody MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- lipidy * MeSH
Ultrahigh-performance supercritical fluid chromatography-mass spectrometry (UHPSFC/MS) method is optimized for the high-throughput quantitation of lipids in human serum and plasma with an emphasis on robustness and accurate quantitation. Bridged ethylene hybrid (BEH) silica column (100 × 3 mm; 1.7 μm) is used for the separation of 17 nonpolar and polar lipid classes in 4.4 min using the positive ion electrospray ionization mode. The lipid class separation approach in UHPSFC/MS results in the coelution of all lipid species within one lipid class in one chromatographic peak, including two exogenous internal standards (IS) per lipid class, which provides the optimal conditions for robust quantitation. The method was validated according to European Medicines Agency and Food and Drug Administration recommendations. UHPSFC/MS combined with LipidQuant software allows a semiautomated process to determine lipid concentrations with a total run time of only 8 min including column equilibration, which enables the analysis of 160 samples per day.
- Klíčová slova
- High-throughput lipidomics, Mass spectrometry, Plasma, Quantitation, Serum, Ultrahigh-performance supercritical fluid chromatography, Validation,
- MeSH
- hmotnostní spektrometrie metody MeSH
- lidé MeSH
- lipidomika * metody MeSH
- lipidy * analýza krev MeSH
- superkritická fluidní chromatografie * metody MeSH
- vysokoúčinná kapalinová chromatografie metody MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- lipidy * MeSH
The structure, function, and molecular mechanisms of lichen survival in harsh habitats like Antarctica and Alpine localities, where environmental extremes change frequently, are highly interesting yet largely underexplored. We used high resolution microscopy, Raman spectroscopy, and chlorophyll a fluorescence to investigate the basic structure and function, i.e., intrathalline distribution and allocation of photobionts, as well as the heat dissipation process in three Antarctic lichens: Dermatocarpon polyphyllizum (DP), Umbilicaria antarctica (UA), and Leptogium puberulum (LP). Microscopic images of their transverse slices revealed visual insights into the heterogeneous distribution of photobionts within their structurally distinct thalli. Raman spectra showed shifts in the carotenoid Raman ν1(CC) band between lichens with algal (DP and UA) and cyanobacterial (LP) photobionts, and interestingly, they revealed biosynthesis of scytonemin, a UV-screening pigment, in cyanolichen LP. We found that increasing actinic irradiance has a nearly equal effect on the shape of chlorophyll fluorescence transients also during dark relaxation in lichens with algal photobionts, but it differed greatly for cyanolichen LP. The dark relaxation kinetics of non-photochemical quenching (NPQ) in experimental lichens differed significantly between lichens with algal photobionts DP and UA; however, this parameter could not be calculated in cyanolichen LP. The components of NPQ revealed that rapidly relaxing energy dependent quenching, ΦqE, is active and protects the thallus of DP predominantly; however, in UA state transition quenching, ΦqT, predominates. The diversity in NPQ across the three examined lichens revealed intriguing aspects of heat dissipation in their photobionts as a mechanism for survival under Antarctica conditions.
- Klíčová slova
- Carotenoids, Chlorophyll a fluorescence transient, High resolution microscopy, Optical signal, Raman spectra, Scytonemin,
- MeSH
- chlorofyl a * metabolismus MeSH
- chlorofyl * metabolismus analýza MeSH
- lišejníky * metabolismus chemie fyziologie MeSH
- Ramanova spektroskopie * MeSH
- sinice metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- Geografické názvy
- Antarktida MeSH
- Názvy látek
- chlorofyl a * MeSH
- chlorofyl * MeSH
In this work, we present the synthesis and application of fluorescent rhodamine B hydrazide for the derivatization of simple oligosaccharides and complex glycans using a hydrazone formation chemistry approach. The labeling conditions and the experimental setup of CE/LIF were optimized by analyzing oligosaccharide standards. The CE/LIF separations were performed in polybrene-coated capillaries eliminating the need for the purification step after derivatization. The addition of methanol to the background electrolyte significantly increased the LIF detection sensitivity reaching the limits of detection in the attomole range. The resolution of carbohydrate samples was improved by using long (98 cm) capillaries and polymer additives (polybrene). The developed method was applied for CE/LIF and CE-MS analysis of N-linked glycans released from bovine ribonuclease B and the therapeutic monoclonal antibody of trastuzumab.
- Klíčová slova
- Capillary electrophoresis, Fluorescence, Glycan, Labeling, Mass spectrometry, Rhodamine B hydrazide,
- MeSH
- elektroforéza kapilární * metody MeSH
- fluorescenční barviva * chemie MeSH
- fluorescenční spektrometrie metody MeSH
- hmotnostní spektrometrie metody MeSH
- lasery MeSH
- oligosacharidy * chemie analýza MeSH
- polysacharidy * analýza chemie MeSH
- rhodaminy * chemie MeSH
- skot MeSH
- trastuzumab chemie analýza MeSH
- zvířata MeSH
- Check Tag
- skot MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- fluorescenční barviva * MeSH
- oligosacharidy * MeSH
- polysacharidy * MeSH
- rhodamine B MeSH Prohlížeč
- rhodaminy * MeSH
- trastuzumab MeSH
BACKGROUND: This study introduces an advanced 8-well loop-mediated isothermal amplification (LAMP) system specifically designed for the automated colorimetric detection of SARS-CoV-2. Incorporating two distinct configurations having either three light-emitting diodes (LEDs) with varying emission wavelengths per well, paired with a photodiode detector, or utilizing white LED illumination with a red, green, and blue (RGB) sensor. The colorimetric LAMP aims to provide a more accessible and rapid diagnostic tool than traditional fluorescence methods due to the system's simplicity. RESULTS: We designed, assembled, and compared two colorimetric home-assembled LAMP systems, the first one based on three LEDs, each with a different color with a photodiode, and the second one having RGB and a white LED, with traditional fluorescence-based LAMP method performed on a commercial qPCR instrument. Results demonstrated that the colorimetric RT-LAMP assays achieved critical threshold time (CT), closely matching the CT value of fluorescence-based detection accomplished by the qPCR instrument. We performed the fundamental experiment employing an identical RNA copy number of 1,570copies·μL-1, getting the CT value of (16.70 ± 0.43) min (mean ± standard deviation from 23 measurements). Then, we also performed different RNA numbers of copies between the highest and lowest RNA contents of ≈ 157,000 copies·μL-1 and ≈ 1570 copies·μL-1, respectively, getting CT values from (13.30 ± 0.04) min to (13.75 ± 0.30) min and (17.04 ± 0.02) min to (17.26 ± 0.02) min, all (mean ± standard deviation from three measurements). The colorimetric systems demonstrated rapid response and precision across varied viral loads while keeping the system simple due to the colorimetric detection method. SIGNIFICANCE AND NOVELTY: The LAMP system's rapid and precise detection capabilities underscore its potential as an effective tool for point-of-need diagnostics. It is crucial for timely responses in ongoing and future pandemic scenarios. This system enhances testing accessibility and provides a robust platform for potential adaptation to other pathogenic threats, making it a valuable asset in global health diagnostics.
- Klíčová slova
- Biosensors and actuators, Colorimetric detection, Loop-mediated isothermal amplification (LAMP), Point-of-care diagnostics, Rapid testing technologies, SARS-CoV-2 diagnostics,
- MeSH
- COVID-19 * diagnóza virologie MeSH
- diagnostické techniky molekulární metody MeSH
- kolorimetrie * metody MeSH
- lidé MeSH
- RNA virová analýza genetika MeSH
- SARS-CoV-2 * genetika izolace a purifikace MeSH
- techniky amplifikace nukleových kyselin * metody MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- RNA virová MeSH
BACKGROUND: Fatty acid esters of hydroxy fatty acids (FAHFAs) are a recently discovered class of endogenous bioactive lipids with promising therapeutic potential for diabetes and inflammation. They represent complex mixtures of different isomers whose biological functions are the subject of investigation. Highly selective methods are required to characterize the composition of enantiomers in biological samples composed of many isobars and regioisomers. We aimed to develop a method for characterizing the enantiomeric composition of FAHFAs in biological samples using supercritical fluid chromatography-mass spectrometry (SFC-MS). RESULTS: The influence of key chromatographic parameters, such as column chemistry, mobile phase composition, and gradient, on the separation efficiency of 21 commercially available FAHFA regioisomers without stated absolute configuration and 4 FAHFA enantiomers was assessed. The optimized SFC-MS method utilizes a chiral column based on a tris-(3-chloro-5-methylphenylcarbamate) derivative of amylose (Lux i-Amylose-3) and acetonitrile-methanol mobile phase modifier, enabling fast enantioseparation of most FAHFA racemic pairs in 5 min. However, the SFC separation of FAHFA regioisomers was less effective, limiting its applicability to complex biological samples. To address this, we propose an offline two-dimensional separation approach with reversed-phase liquid chromatography for isolating FAHFA regioisomers, followed by chiral SFC-MS analysis of fractions. The suitability of the method was demonstrated by characterizing the enantiomeric composition of FAHFA in white adipose tissue and rice samples. The chiral analysis revealed the presence of both R- and S-FAHFA isomers in the samples, with one enantiomer being predominant. SIGNIFICANCE: The developed approach represents a proof of concept for the use of SFC-MS with LC prefractionation for the characterization of FAHFA enantiomeric composition in complex biological samples, providing a valuable tool for future research on the biological roles of bioactive lipids in health and disease.
- Klíčová slova
- Chiral separation, FAHFA, Isomers, Lipids, Offline two-dimensional chromatography, Supercritical fluid chromatography,
- MeSH
- chromatografie kapalinová metody MeSH
- estery * chemie analýza izolace a purifikace MeSH
- hmotnostní spektrometrie * metody MeSH
- hydroxykyseliny chemie analýza izolace a purifikace MeSH
- mastné kyseliny * chemie analýza izolace a purifikace MeSH
- myši MeSH
- stereoizomerie MeSH
- superkritická fluidní chromatografie * metody MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- estery * MeSH
- hydroxykyseliny MeSH
- mastné kyseliny * MeSH
A more recent insight into the structural differences of sustainable poly(lactic acid)-based materials was revealed through a solvolysis reaction under environmentally friendly conditions. The decomposition process clarified the heterogeneous structure of the investigation polymers. It was found that materials used for nanofiber production degraded 25 % more poorly compared to materials suitable for packaging materials. The resultant product, ethyl lactate, demonstrated high purity and yield (up to 900 mg·L-1, 98 %). The degree and effect of decomposition of the poly(lactic acid) were monitored by employing the gel permeation chromatic method, differential scanning calorimetry and thermal gravimetric analysis. X-ray diffraction was conducted to assess differences between the crystalline portions of polymers. The yield and purity of the product were verified by gas and liquid chromatography. The kinetic studies evaluated the rate of polymers degradation connected with chemical structure and temperature. A structural difference was observed in the studied polylactides, with approximately a 15 % deviation in crystallinity. This observed variation resulted from differences in arrangement and chain lengths, as well as the terminal functional groups, leading to non-uniform degradation of both polymers. This study offers a new insight on the degradation efficiency of polymers highlights the non-uniformity of their structure. Converting biodegradable polymer waste into a suitable and reusable product as part of an environmentally friendly approach will contribute to the sustainability of polymer materials.
- Klíčová slova
- Architecture, Crystallinity, Degradation, sustainable materials, Environmentally friendly, Poly(lactic acid), Solvolysis,
- MeSH
- diferenciální skenovací kalorimetrie MeSH
- difrakce rentgenového záření MeSH
- kinetika MeSH
- laktáty chemie MeSH
- polyestery * chemie MeSH
- teplota MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- ethyl lactate MeSH Prohlížeč
- laktáty MeSH
- poly(lactide) MeSH Prohlížeč
- polyestery * MeSH
BACKGROUND: Alkylphenols are water contaminants of strong endocrine disruptive potential. Sample preparation is generally imperative to improve sensitivity and minimize matrix effects. Dispersive solid phase extraction is a powerful alternative to cartridge-based sorbent extraction omitting backpressure problems and reducing procedural time. Herein, solvent-dissolvable sorbents offer the advantages of easy and cost-efficient production, efficiency, and full analyte recovery, while eluates can be directly submitted to instrumental determination. Despite the potential to reduce environmental impact and enhance reproducibility, there is a lack of automation attempts. RESULTS: A fully automated solvent-assisted dispersive solid phase extraction method was developed for selected alkylphenols based on the technique Lab-In-Syringe. The void of automatic bidirectional syringe pump was used as mixing and extraction vessel. The iron(III) thenoyltrifluoroacetonate complex was used as novel dissolvable sorbent. 40 μL complex solution was dispersed in the sample, leading to the precipitation of 0.4 mg sorbent. Extraction occurred within 40 s and was accelerated by in-syringe magnetic stirring. The sorbent was retained on a melamine foam packing in the syringe inlet, dissolved in a methanolic solution of ascorbic acid, and injected into online-coupled HPLC. Linear working ranges were achieved from 1 to 1000 μg/L with sub-ppb detection limits and accuracies ranging from 98.3 to 110 %. SIGNIFICANCE: In this work, we explored for the first time automated in-syringe automated dispersive SPE based on a dissolvable sorbent. Parallel operation of sample pretreatment and separation enabled throughputs of 4.5/h with typically <5 % RSD and preconcentrations of 16.4-21.2. AGREE greenness evaluation yielded a score of 0.59.
- Klíčová slova
- Alkylphenols, High performance liquid chromatography, Iron(III) thenoyltrifluoroacetonate complex, Lab-in-syringe automation, Solvent-assisted dispersive solid phase Extraction,
- MeSH
- automatizace MeSH
- chemické látky znečišťující vodu analýza izolace a purifikace MeSH
- chromatografie kapalinová metody MeSH
- fenoly * izolace a purifikace analýza chemie MeSH
- injekční stříkačky * MeSH
- limita detekce MeSH
- mikroextrakce na pevné fázi * metody MeSH
- vysokoúčinná kapalinová chromatografie MeSH
- železité sloučeniny chemie MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- chemické látky znečišťující vodu MeSH
- fenoly * MeSH
- železité sloučeniny MeSH
MicroRNAs (miRNAs) are small non-coding RNAs (18-22 nucleotides) that regulate gene expression and are associated with various diseases, including Laryngeal Cancer (LCa), which has a high mortality rate due to late diagnosis. Traditional methods for miRNA detection present several drawbacks (time-consuming steps, high cost and high false positive rate). Early-stage diagnosis and selective detection of miRNAs remain challenging. This study proposes a 3D flexible biosensor that combines nanofibers (NFs), gold nanoparticles (AuNPs), and an inverse molecular sentinel (iMS) for enzyme-free, SERS-based detection of miRNA-223-3p, evaluated as a potential LCa biomarker. The electrospun flexible nanofibers decorated with AuNPs enhance Raman signal. Selective detection of miRNA-223-3p is achieved by immobilizing an iMS-DNA probe labeled with a Raman reporter (Cyanine 3) on the AuNPs. The iMS distinctive stem-and-loop structure undergoes a conformational change upon interaction with the miRNA-223-3p, producing an "on to off" SERS signal. The proposed sensor demonstrated a linear detection range from 10 to 250 fM, with a limit of detection (LOD) of 19.50 ± 0.05 fM. The sensor selectivity was confirmed by analyzing the SERS signal behaviour in the presence of both Non-complementary miRNA and miRNA with three mismatched base pairs. This easily fabricable sensor requires no amplification and offers key advantages, including sensitivity, flexibility, and cost-effectiveness.
- Klíčová slova
- Flexible sensors, Laryngeal Cancer, Nanofiber, SERS, miRNA-223-3p,
- MeSH
- biosenzitivní techniky * metody MeSH
- časná detekce nádoru metody MeSH
- kovové nanočástice * chemie MeSH
- lidé MeSH
- limita detekce MeSH
- mikro RNA * analýza genetika MeSH
- nádory hrtanu * diagnóza genetika MeSH
- nanovlákna * chemie MeSH
- Ramanova spektroskopie * metody MeSH
- zlato * chemie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- mikro RNA * MeSH
- MIRN223 microRNA, human MeSH Prohlížeč
- zlato * MeSH
This work focuses on profiling N-linked glycans by capillary electrophoresis coupled to mass spectrometry using a novel fluorescent and mass spectrometry (MS) active derivatization tag. The label is based on 2-phenylpyridine bearing tertiary amine and hydrazide functionalities. It provides efficient labeling via hydrazone formation chemistry, promising fluorescence properties, and ionization efficiency in the positive ion MS mode. Electrophoretic analysis in a neutral-coated capillary allowed baseline separation of maltooligosaccharides with limits of detection in nanomolar concentrations. The developed labeling method was successfully applied to the analyses of N-linked glycans released from several glycoproteins such as bovine ribonuclease B, human immunoglobulin G, or chicken albumin.
- Klíčová slova
- Capillary electrophoresis, Glycans, Labeling, Mass spectrometry, Oligosaccharides, Phenylpyridine,
- MeSH
- elektroforéza kapilární * metody MeSH
- glykoproteiny analýza chemie MeSH
- hmotnostní spektrometrie * metody MeSH
- imunoglobulin G chemie analýza MeSH
- kationty chemie MeSH
- kur domácí MeSH
- lidé MeSH
- polysacharidy * analýza chemie MeSH
- pyridiny * chemie MeSH
- ribonukleasy MeSH
- skot MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- skot MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- glykoproteiny MeSH
- imunoglobulin G MeSH
- kationty MeSH
- polysacharidy * MeSH
- pyridiny * MeSH
- ribonuclease B MeSH Prohlížeč
- ribonukleasy MeSH
