Rapid screening and identification of α-glucosidase inhibitors from mulberry leaves using enzyme-immobilized magnetic beads coupled with HPLC/MS and NMR

α-Glucosidase plays important roles in the digestion and absorption of carbohydrates in the small intestine. The inhibition of α-glucosidase is regarded as a potential way to treat diabetes. We established an approach to screening α-glucosidase inhibitors from medicinal plants using enzyme-coated magnetic bead. Using 1-(3-dimethyl-aminopropyl)-3-ethylcarbodiimide and N-hydroxysuccinimide as reaction reagents, α-glucosidase was immobilized on the magnetic beads by covalent linkage. The conjugation of α-glucosidase to the magnetic beads was characterized using scanning electron microscope and X-ray diffractometer. The proposed approach was applied in fishing potential α-glucosidase inhibitors from extract of Morus alba, a Chinese medicinal plant. The structures of potential active compounds were identified via liquid chromatography-mass spectrometry and nuclear magnetic resonance. The results demonstrated that two flavonoids (isoquercitrin and astragalin) could bind to α-glucosidase, which was confirmed via conventional α-glucosidase inhibitory assay. Our findings suggested that enzyme-coated magnetic beads may be suitable for discovering active compounds from medicinal plants.     

Activated persulfate by chelating agent Fe·/complex for in situ degradation of phenol: intermediate identification and optimization study

Research on Chemical Intermediates - Due to its high toxicity, phenol is classified as a priority. In this paper, the removal of phenol from aqueous solutions was investigated using EDTA and...     

Simultaneous identification and quantification of five flavonoids in the seeds of Rheum palmatum L. by using accelerated solvent extraction and HPLC–PDA–ESI/MSn

In this study, flavonoids were extracted using accelerated solvent extraction (ASE) with 80% aqueous methanol. A high-performance liquid chromatography equipped with photodiode array detection and coupled with an electrospray ionization tandem mass spectrometry (HPLC–PDA–ESI/MSⁿ) method has been developed for rapid identification and quantification of flavonoids in the plant extract of Rheum palmatum L. (RPL) seeds from Qinghai. Ten flavonoids from methanolic extract of RPL seeds were screened, of which epicatechin, myricetin, hyperoside, quercitrin and quercetin were identified and quantitatively analyzed for the first time. The absorbance was monitored at 280 nm for epicatechin and 360 nm for other four flavonoids. It was found that the calibration curves for all five analytes showed a good linearity (r² > 0.999) within the test range and the data of the limit of detection (LOD) and limit of quantification (LOQ) for all investigated compounds were less than 2.98 μg mL^?1 based on PDA. The relative standard deviation (R.S.D) values of the intra- and inter-day precisions were 0.98% and 1.65%, respectively. The range of mean recoveries of the five components was 91.3–93.9%, and the R.S.D was 1.3–2.9%. It was also found that the content of quercitrin is the highest in RPL seeds while the contents of epicatechin and myricetin are relatively lower. In addition, the validation procedure confirmed that this method was suitable for providing quality control evaluation of RPL seeds to ensure the therapeutic benefits.     

Determination of n-octanol–water partition coefficients by hollow-fiber membrane solvent microextraction coupled with HPLC

A novel direct method has been developed for determination of n-octanol–water partition coefficients by hollow-fiber membrane solvent microextraction (HFMSME) combined with high-performance liquid chromatography (HPLC). The compound of interest is dissolved in water with sonication and a hollow fiber containing octanol inside is placed in the sample solution to perform microextraction. After microextraction the concentrations in both the aqueous and n-octanol phases are analyzed by HPLC with UV detection. The method was evaluated with ten reference compounds and shown to be suitable for determination of the partition coefficients of organic compounds accurately, cheaply, simply, and quickly. Previously unknown n-octanol–water partition coefficients have been obtained for other compounds by use of the hollow-fiber membrane solvent-microextraction technique. Figure Schematic diagram of equipment used for hollow-fiber solvent microextraction. (1) hollow cone-shaped support; (2) aqueous sample; (3) porous hollow-fiber membrane tube (PHFMT); (4) n-octanol phase; (5) sealed end of PHFMT; (6) container (15 mL); (7) stir bar; (8) magnetic stirrer     

Favipiravir (SARS-CoV-2) degradation impurities: Identification and route of degradation mechanism in the finished solid dosage form using LC/LC–MS method

Favipiravir finished dosage was approved for emergency use in many countries to treat SARS-CoV-2 patients. A specific, accurate, linear, robust, simple, and stability-indicating HPLC method was developed and validated for the determination of degradation impurities present in favipiravir film-coated tablets. The separation of all impurities was achieved from the stationary phase (Inert sustain AQ-C18, 250 × 4.6 mm, 5-μm particle) and mobile phase. Mobile phase A contained KH₂PO₄ buffer (pH 2.5 ± 0.05) and acetonitrile in the ratio of 98:2 (v/v), and mobile phase B contained water and acetonitrile in the ratio of 50:50 (v/v). The chromatographic conditions were optimized as follows: flow rate, 0.7 mL/min; UV detection, 210 nm; injection volume, 20 μL; and column temperature, 33°C. The proposed method was validated per the current International Conference on Harmonization Q2 (R1) guidelines. The recovery study and linearity ranges were established from the limit of quantification to 150% optimal concentrations. The method validation results were found to be between 98.6 and 106.2% for recovery and r² = 0.9995–0.9999 for linearity of all identified impurities. The method precision results were achieved below 5% of relative standard deviation. Forced degradation studies were performed in chemical and physical stress conditions. The compound was sensitive to chemical stress conditions. During the study, the analyte degraded and converted to unknown degradation impurities, and its molecular mass was found using the LC–MS technique and established degradation pathways supported by reaction of mechanism. The developed method was found to be suitable for routine analysis of research and development and quality control.     

Separation and identification of trinucleotide–melphalan adducts from enzymatically digested DNA using HPLC–ESI–MS

Melphalan is a bifunctional alkylating agent that covalently binds to the nucleophilic sites present in DNA. In this study we investigated oligonucleotides prepared enzymatically from DNA modified with melphalan. Calf thymus DNA was incubated in-vitro with melphalan and the resulting modifications were enzymatically cleaved by means of benzonase and nuclease S1. Efficient sample preconcentration was achieved by solid-phase extraction, in which phenyl phase cartridges resulted in better recovery of the modified species than C₁₈. The applied enzymatic digestion time resulted in production of trinucleotide adducts which were efficiently separated and detected by use of reversed-phase HPLC coupled to an ion-trap mass spectrometer with electrospray ionization. It was assumed that melphalan could act as both a monofunctional and bifunctional alkylating agent. Mono-alkylated adducts were much more abundant, however, and the alkylation site was located on the nucleobases. On the other hand, we unequivocally identified cross-link formation in DNA, even though at low abundance and only a few adduct types were detected. Figure Different Alkylation reactions of Melphalan with DNA     

Determination of antifouling pesticides and their degradation products in marine sediments by means of ultrasonic extraction and HPLC–APCI–MS

A method has been developed for the simultaneous determination of antifouling pesticides and some of their degradation products, e.g. dichlofluanid, diuron, demethyldiuron, 1-(3,4-dichlorophenyl)urea, sea-nine, Irgarol 1051 and one of its metabolites (2-methylthio-4-tert-butylamino-s-triazine) in marine sediments. The determination of these compounds in sediment samples was performed by means of methanolic ultrasonic extraction then clean-up on an Isolute ENV+ solid phase extraction (SPE) cartridge. The resulting extract was then analyzed by reversed-phase high-performance liquid chromatography coupled with atmospheric-pressure chemical-ionization mass spectrometry in negative and positive ion modes (HPLC–APCI–MS). Recovery ranged from 54–109% for the antifouling agents and their degradation products. The determination limits for the different compounds varied between 0.2 and 1.6 μg kg^–1 dry sediment. The analytical procedure was successfully applied to the determination of these pesticides and their degradation products in marine sediment samples from different marinas of the Catalan coast. The compounds detected were: diuron, dichlofluanid, demethyldiuron, sea-nine, and Irgarol 1051. The highest concentrations were those of diuron and Irgarol 1051 – 136 and 88 μg kg^–1, respectively.     

LC–high resolution MS in environmental analysis: from target screening to the identification of unknowns

This article provides an overview of the state-of-the-art and future trends of the application of LC–high resolution mass spectrometry to the environmental analysis of polar micropollutants. Highly resolved and accurate hybrid tandem mass spectrometry such as quadrupole/time-of-flight and linear ion trap/orbitrap technology allows for a more reliable target analysis with reference standards, a screening for suspected analytes without reference standards, and a screening for unknowns. A reliable identification requires both high resolving power and high mass spectral accuracy to increase selectivity against the matrix background and for a correct molecular formula assignment to unknown compounds. For the identification and structure elucidation of unknown compounds within a reasonable time frame and with a reasonable soundness, advanced automated software solutions as well as improved prediction systems for theoretical fragmentation patterns, retention times, and ionization behavior are needed.     

Identification of volatile degradation products from Baltic amber by headspace solid-phase microextraction coupled with gas chromatography–mass spectrometry

The aim of this study was to test and develop techniques for the detection and identification of volatile compounds released as degradation products by Baltic amber. During a preliminary investigation, the off-gassing of acidic volatiles was detected through the corrosion of lead coupons. The corrosive compounds released by the material were then identified as formic acid and acetic acid by headspace solid-phase microextraction coupled with gas chromatography–mass spectrometry. During an advanced investigation, based on the use of artificial ageing to initiate degradation of model amber samples in different microclimates, the detected formic acid and acetic acid off-gassing appeared to be more intense in a dry environment with normal oxygen concentration. The release of formic and acetic acids by the amber was likely the result of radical reactions which should be investigated in further studies.     

Synthesis and characterization of polyaniline–zirconium dioxide and polyaniline–cerium dioxide composites with enhanced photocatalytic degradation of rhodamine B dye

For the first time, chloroform and 2-butanol were used as solvent systems for the preparation of ZrO₂–PANI and CeO₂–PANI composites. Solubility of the synthesized composites was studied in chloroform, N-methyl-2-pyrrolidinone (NMP), and in mixture of toluene?+?2-propanol (2:1). XRD and cyclic voltammetry data showed that the ZrO₂–PANI and CeO₂–PANI composites possess both crystalline and amorphous domains indicating some sort of conductivity. TGA results showed that ZrO₂–PANI composite have a better thermal stability than pure PANI; however, CeO₂–PANI composite has lower thermal stability than pure PANI. The conjugated unsaturated structure of PANI is responsible for the enhanced photocatalytic properties of ZrO₂–PANI and CeO₂–PANI. Photocatalytic results showed that, at photolysis time of 60 min, rhodamine B (RhB) dye was degraded up to 34 and 35% by ZrO₂–PANI and CeO₂–PANI, respectively. The degradation products of RhB were quantified by LC–MS and GC–MS, and accordingly, a detailed pathway was proposed.     

A sensitive and selective HPLC–MS3 method for therapeutic drug monitoring of meropenem and its validation by comparison with HPLC–MS2 methods

Meropenem, a representative β-lactam antibiotic, is widely used to treat complicated and serious infections. Therefore, it is of great significance to monitor the plasma drug concentration for individualized antimicrobial therapy. This study first describes the development and validation of high-performance liquid chromatography–tandem mass spectrometry cubed method for monitoring meropenem in human plasma. Protein precipitation with methanol and a chromatographic analysis time of 7 min make this method simple and of high throughput. Meropenem was extracted from human plasma with recoveries >94.1%. Calibration curves were linear (R² > 0.995) in the concentration range of 0.5–50 μg/mL. Overall accuracy and precision did not exceed 8.0% as well as no significant matrix effect was observed. The novelty of this method is that the triple-stage mass spectrometry technology improves the selectivity and sensitivity. A comparison of the presented method and traditional liquid chromatography–tandem mass spectrometry method was assessed in 44 patients treated with meropenem and Passing–Bablok regression coefficients and Bland–Altman plots showed that no significant difference between the two methods. So the triple-stage mass spectrometry method developed in this study is appropriate and practical for the monitor of meropenem in the daily clinical laboratory practice.     

Fingerprinting and source identification of an oil spill in China Bohai Sea by gas chromatography-flame ionization detection and gas chromatography–mass spectrometry coupled with multi-statistical analyses

This paper describes a case study in which advanced chemical fingerprinting and data interpretation techniques were used to characterize the chemical composition and determine the source of an unknown spilled oil reported on the beach of China Bohai Sea in 2005. The spilled oil was suspected to be released from nearby platforms. In response to this specific site investigation need, a tiered analytical approach using gas chromatography–mass spectrometry (GC–MS) and gas chromatography-flame ionization detection (GC-FID) was applied. A variety of diagnostic ratios of “source-specific marker” compounds, in particular isomers of biomarkers, were determined and compared. Several statistical data correlation analysis methods were applied, including clustering analysis and Student's t-test method. The comparison of the two methods was conducted. The comprehensive analysis results reveal the following: (1) The oil fingerprinting of three spilled oil samples (S1, S2 and S3) positively match each other; (2) The three spilled oil samples have suffered different weathering, dominated by evaporation with decrease of the low-molecular—mass n-alkanes at different degrees; (3) The oil fingerprinting profiles of the three spilled oil samples are positive match with that of the suspected source oil samples C41, C42, C43, C44 and C45; (4) There are significant differences in the oil fingerprinting profiles between the three spilled oil samples and the suspected source oil samples A1, B1, B2, B3, B4, C1, C2, C3, C5 and C6.     

A simplified method for the HPLC resolution of α-carotene and β-carotene (trans and cis) isomers

A new HPLC/DAD (Diode Array Detector) method is proposed for the identification of some carotene isomers. The operating conditions adopted permit the resolution of alpha-carotene, all-trans-beta-carotene, 9-cis-beta-carotene, 13-cis-beta-carotene and 15-cis-beta-carotene. Moreover, the chromatographic conditions reported are simplified in respect of those reported up to now. The method is applied to the determination of carotenoids in a dried Dunaliella salina extract, but it could be also applied to other organic matrices such as eggs.     

Chemometric processing of second-order liquid chromatographic data with UV–vis and fluorescence detection. A comparison of multivariate curve resolution and parallel factor analysis 2

Second-order liquid chromatographic data with multivariate spectral (UV–vis or fluorescence) detection usually show changes in elution time profiles from sample to sample, causing a loss of trilinearity in the data. In order to analyze them with an appropriate model, the latter should permit a given component to have different time profiles in different samples. Two popular models in this regard are multivariate curve resolution-alternating least-squares (MCR-ALS) and parallel factor analysis 2 (PARAFAC2). The conditions to be fulfilled for successful application of the latter model are discussed on the basis of simple chromatographic concepts. An exhaustive analysis of the multivariate calibration models is carried out, employing both simulated and experimental chromatographic data sets. The latter involve the quantitation of benzimidazolic and carbamate pesticides in fruit and juice samples using liquid chromatography with diode array detection, and of polycyclic aromatic hydrocarbons in water samples, in both cases in the presence of potential interferents using liquid chromatography with fluorescence spectral detection, thereby achieving the second-order advantage. The overall results seem to favor MCR-ALS over PARAFAC2, especially in the presence of potential interferents.     

Separation and identification of selenotrisulfides in epithelial cell homogenates by LC–ICP–MS and LC–ESI-MS after incubation with selenite

To elucidate how selenite is metabolised in the intestine after oral intake, it was incubated with homogenized epithelial cells from pigs. When the metabolites were analysed by LC–ICP–MS, two major selenium metabolites were separated in the supernatant from the homogenate. These metabolites were formed instantly but disappeared within 15 min. No other selenium-containing compounds appeared during this time. Hence, the secondary reaction products were either volatilised or precipitated. To verify the identity of the compounds, a larger amount of selenite was incubated with epithelial cells. The presence of Cys-Se-SG and GS-Se-SG was verified by LC–ESI-MS. Selenotrisulfides were synthesized by reaction of L-cysteine and L-glutathione with sodium selenite. The reaction mixture contained three main products: selenodicysteine (Cys-Se-Cys), selenocysteine glutathione (Cys-Se-SG), and selenodiglutathione (GS-Se-SG). The two transient selenium compounds in the epithelial cell incubation mixture co-eluted with the synthesized Cys-Se-SG and GS-Se-SG, respectively. The identities of these compounds were verified by LC–ESI-MS. Hence, these selenium metabolites have now been identified by ESI-MS after isolation from epithelial cells.     

A new ICP–TOFMS. Measurement and readout of mass spectra with 30 μs time resolution, applied to in-torch LA–ICP–MS

In-torch LA–ICP–MS was implemented into an in-house-built ICP–TOFMS system. The fast data acquisition capabilities of the new configuration allowed simultaneous multi-element measurement and readout of in-torch LA–ICP–MS signals with 30 μs time resolution. The measurements confirmed previously observed fine structures of in-torch generated signals and provided new insights in the dynamic processes in the plasma on a microsecond time scale. The new setup is described in detail and first figures of merit are given. Figure Time dependent multi element signal after laser ablation in the torch of an ICP-TOFMS instrument     

Investigation into mercury bound to biothiols: structural identification using ESI–ion-trap MS and introduction of a method for their HPLC separation with simultaneous detection by ICP-MS and ESI-MS

Mercury in plants or animal tissue is supposed to occur in the form of complexes formed with biologically relevant thiols (biothiols), rather than as free cation. We describe a technique for the separation and molecular identification of mercury and methylmercury complexes derived from their reactions with cysteine (Cys) and glutathione (GS): Hg(Cys)₂, Hg(GS)₂, MeHgCys, MeHgGS. Complexes were characterised by electrospray mass spectrometry (MS) equipped with an ion trap and the fragmentation pattern of MeHgCys was explained by using MP2 and B3LYP calculations, showing the importance of mercury–amine interactions in the gas phase. Chromatographic baseline separation was performed within 10 min with formic acid as the mobile phase on a reversed-phase column. Detection was done by online simultaneous coupling of ES-MS and inductively coupled plasma MS. When the mercury complexes were spiked in real samples (plant extracts), no perturbation of the separation and detection conditions was observed, suggesting that this method is capable of detecting mercury biothiol complexes in plants. Figure Separation and structural identification of Hg and MeHg biothiols A part of this work was presented as a poster at the European Winter Conference on Plasma Spectrochemistry, 2007, held in Taormina, Italy.     

Gelatin and starch as stabilizers of the coupled enzyme system of luminous bacteria NADH:FMN–oxidoreductase–luciferase

We have studied the effects of a gel-like environment on the characteristics of enzyme preparations based on the coupled enzyme system of luminous bacteria, NADH:FMN–oxidoreductase–luciferase, to design a stable immobilizing reagent for bioluminescent analysis. Natural polymers, gelatin and starch, were used to create a viscous, structured microenvironment. The stability of the coupled enzyme system to such physical and chemical environmental factors as temperature, pH, and ionic strength in gelatin and starch-containing media was examined. It was shown that both gelatin and starch have a stabilizing effect on the enzymes of luminous bacteria under specific conditions. In particular, the enzymes’ activity is increased twofold in the presence of 1 and 5 % of gelatin at 20 °C and 25 °C, respectively (temperatures lower than the gel point). Also, the acceptable pH range of the coupled enzyme system expands into the alkaline region and becomes 6.8–8.1. Stabilization at low ionic strength (0.01–0.06 mol L^?1) is observed. At the same time, microenvironments based on either gelatin or starch do not change the enzymes’ thermal inactivation rate constants in the temperature range from 25 to 43 °C. Finally, gelatin and starch are suitable for development of a reagent for immobilization of enzymes which would be stable and resistant to physical and chemical environmental conditions.     

A facile preparation of (±)-β-hydroxy nitriles and their enzymatic resolution with lipases

A simple and efficient method for the preparation of racemic 4-aryloxy-3-hydroxybutanenitriles is described. Lipase mediated kinetic resolution in organic media was then utilised to effect enantioseparation. Lipases from different sources were screened in the resolution reaction using a number of organic solvents. Enantiomeric excesses of up to 99% were obtained by employing lipase from Pseudomonas cepacia in di-iso-propyl ether medium.