Solid-phase Extraction Nuclear Magnetic Resonance Research Articles

Biotransformation of pungent constituents from ginger (Zingiber officinale Roscoe) by Colletotrichum gloeosporioides yields oxidative ortho–ortho coupling products

This work investigated the biotransformation of ginger constituents (zingerone, [6]-shogaol, [6]-gingerol, and methyl-[6]-gingerol) by the pathogenic fungus Colletotrichum gloeosporioides. Experiments were carried out with and without deuterium-labelled compounds. The product metabolites were analyzed by liquid chromatography coupled to tandem mass spectrometry and liquid chromatography solid phase extraction-nuclear magnetic resonance. Substrates supplied to the fungus were incorporated into metabolic pathways mostly by oxidation reactions, including aromatic carbon–carbon coupling. Zingerone and [6]-gingerol biotransformation products included biphenol dimers. A biodegradation pathway for biphenol formation was proposed based on the presence of the intermediate 4-(2-hydroxyethyl)-2-methoxyphenol, commonly identified from [6]-gingerol and [6]-shogaol biodegradation. This intermediate likely originates from a Baeyer–Villiger reaction followed by hydrolysis. The C–C coupling of molecules could result in phenolic oxidative ortho–ortho coupling, suggesting that biphenol dimers are products of C. gloeosporioides laccase catalysis.

Solid-Phase Extraction Nuclear Magnetic Resonance (SPE-NMR): Prototype Design, Development, and Automation

Reliable measurements of oil-in-water (OiW) content is essential in the oil and gas industry. The current OiW analysis techniques deployed in the industry need frequent calibration and mostly depen...

Advances in the Biosynthesis of Pyranocoumarins: Isolation and 13C-Incorporation Analysis by High-Performance Liquid Chromatography-Ultraviolet-Solid-Phase Extraction-Nuclear Magnetic Resonance Data.

Citrus sinensis and Citrus limonia were obtained by germination from seeds, and isotopic-labeling experiments using d-[1-13C]glucose were performed with the seedlings. After 60 days, the seedlings were analyzed by high-performance liquid chromatography-ultraviolet-solid-phase extraction-nuclear magnetic resonance, data and the 13C enrichment patterns of xanthyletin and seselin indicated that the pyran ring was formed by the methylerythritol phosphate pathway and that the coumarin moiety was derived from the shikimate pathway in both compounds. This information regarding the biosynthetic pathway can be used to increase resistance against phytopathogens, because xanthyletin and seselin are reported to have antimicrobial activity on the growth of Xylella fastidiosa, which causes citrus variegated chlorosis in orange.

Quadruple high-resolution α-glucosidase/α-amylase/PTP1B/radical scavenging profiling combined with high-performance liquid chromatography–high-resolution mass spectrometry–solid-phase extraction–nuclear magnetic resonance spectroscopy for identification of antidiabetic constituents in crude root bark of Morus alba L.

In this paper, quadruple high-resolution α-glucosidase/α-amylase/PTP1B/radical scavenging profiling combined with HPLC-HRMS-SPE-NMR were used for studying the polypharmacological properties of crude root bark extract of Morus alba L. This species is used as an anti-diabetic principle in many traditional treatment systems around the world, and the crude ethyl acetate extract of M. alba root bark was found to inhibit α-glucosidase, α-amylase and protein-tyrosine phosphatase 1B (PTP1B) with IC50 values of 1.70 ± 0.72, 5.16 ± 0.69, and 5.07 ± 0.68 μg/mL as well as showing radical scavenging activity equaling a TEAC value of (3.82 ± 0.14) × 104 mM per gram extract. Subsequent investigation of the crude extract using quadruple high-resolution α-glucosidase/α-amylase/PTP1B/radical scavenging profiling provided a quadruple biochromatogram that allowed direct correlation of the HPLC peaks with one or more of the tested bioactivities. This was used to target subsequent HPLC-HRMS-SPE-NMR analysis towards peaks representing bioactive analytes, and led to identification of a new Diels-Alder adduct named Moracenin E as well as a series of Diels-Alder adducts and isoprenylated flavonoids as potent α-glucosidase and α-amylase inhibitors with IC50 values in the range of 0.60–27.15 μM and 1.22–69.38 μM, respectively. In addition, these compounds and two 2-arylbenzofurans were found to be potent PTP1B inhibitors with IC50 values ranging from 4.04 to 21.67 μM. The high-resolution radical scavenging profile also revealed that almost all of the compounds possess radical scavenging activity. In conclusion the quadruple high-resolution profiling method presented here allowed a detailed profiling of individual constituents in crude root bark extract of M. alba, and the method provides a general tool for detailed mapping of bioactive constituents in polypharmacological herbal remedies.

High-resolution PTP1B inhibition profiling combined with high-performance liquid chromatography–high-resolution mass spectrometry–solid-phase extraction–nuclear magnetic resonance spectroscopy: Proof-of-concept and antidiabetic constituents in crude extract of Eremophila lucida

Type 2 diabetes (T2D) constituted 90% of the global 387 million diabetes cases in 2014. The enzyme protein-tyrosine phosphatase 1B (PTP1B) has been recognized as a therapeutic target for treatment of T2D and its adverse complications. With the aim of accelerating the investigation of complex natural sources, such as crude plant extracts, for potential PTP1B inhibitors, we have developed a bio-analytical platform combining high-resolution PTP1B inhibition profiling and high-performance liquid chromatography–high-resolution mass spectrometry–solid-phase extraction–nuclear magnetic resonance spectroscopy, i.e., HR-bioassay/HPLC–HRMS–SPE–NMR. Human recombinant PTP1B enzyme was used for the microplate-based PTP1B inhibition assay, which was optimized for pH and substrate concentration to be compatible with rate measurements within the 10min incubation time. Subsequently, analytical-scale HPLC-based microfractionation followed by colorimetric microplate-based PTP1B bioassaying enabled construction of a high-resolution inhibition profile corresponding to the HPLC profile. The high-resolution PTP1B inhibition profiling was validated using an artificial mixture of known PTP1B inhibitors and non-inhibiting compounds as negative controls. Finally, a proof-of-concept study with a real sample was performed using crude ethyl acetate extract of the phytochemically hitherto unexplored plant Eremophila lucida. This led to the identification of the first viscidane type diterpene, i.e., 5-hydroxyviscida-3,14-dien-20-oic acid (9) as PTP1B inhibitor with an IC50 value of 42.0±5.9μM. In addition, a series of flavonoids, i.e., luteolin (1), dinatin (3a), tricin (3b), 3,6-dimethoxyapigenin (4), jaceidin (5), and cirsimaritin (6) as well as a cembrene diterpene, (3Z, 7E, 11Z)-15-hydroxycembra-3,7,11-trien-19-oic acid (8), were also identified for the first time from E. lucida.

Edible seaweed as future functional food: Identification of α-glucosidase inhibitors by combined use of high-resolution α-glucosidase inhibition profiling and HPLC–HRMS–SPE–NMR

Crude chloroform, ethanol and acetone extracts of nineteen seaweed species were screened for their antioxidant and α-glucosidase inhibitory activity. Samples showing more than 60% α-glucosidase inhibitory activity, at a concentration of 1mg/ml, were furthermore investigated using high-resolution α-glucosidase inhibition profiling combined with high-performance liquid chromatography–high-resolution mass spectrometry–solid-phase extraction–nuclear magnetic resonance spectroscopy (HR-bioassay/HPLC–HRMS–SPE–NMR). The results showed Ascophyllum nodosum and Fucus vesicolosus to be rich in antioxidants, equaling a Trolox equivalent antioxidant capacity of 135 and 108mMTroloxmg−1 extract, respectively. HR-bioassay/HPLC–HRMS–SPE–NMR showed the α-glucosidase inhibitory activity of A. nodosum, F. vesoculosus, Laminaria digitata, Laminaria japonica and Undaria pinnatifida to be caused by phlorotannins as well as fatty acids – with oleic acid, linoleic acid and eicosapentaenoic acid being the most potent with IC50 values of 0.069, 0.075 and 0.10mM, respectively, and showing a mixed-type inhibition mode.

Triple aldose reductase/α-glucosidase/radical scavenging high-resolution profiling combined with high-performance liquid chromatography–high-resolution mass spectrometry–solid-phase extraction–nuclear magnetic resonance spectroscopy for identification of antidiabetic constituents in crude extract of Radix Scutellariae

In this work, development of a new microplate-based high-resolution profiling assay using recombinant human aldose reductase is presented. Used together with high-resolution radical scavenging and high-resolution α-glucosidase assays, it provided the first report of a triple aldose reductase/α-glucosidase/radical scavenging high-resolution inhibition profile – allowing proof of concept with Radix Scutellariae crude extract as a polypharmacological herbal drug. The triple bioactivity high-resolution profiles were used to pinpoint bioactive compounds, and subsequent structure elucidation was performed with hyphenated high-performance liquid chromatography–high-resolution mass spectrometry–solid-phase extraction–nuclear magnetic resonance spectroscopy. The only α-glucosidase inhibitor was baicalein, whereas main aldose reductase inhibitors in the crude extract were baicalein and skullcapflavone II, and main radical scavengers were ganhuangemin, viscidulin III, baicalin, oroxylin A 7-O-glucuronide, wogonoside, baicalein, wogonin, and skullcapflavone II.

Fungal plasma membrane H+-ATPase inhibitory activity of o-hydroxybenzylated flavanones and chalcones from Uvaria chamae P. Beauv.

In our ongoing efforts of finding natural fungicides to fight food and feed spoilage during production and storage, the antifungal potential of Ghanaian Uvaria chamae P. Beauv. was investigated, with emphasis on plant metabolites targeting the fungal plasma membrane (PM) H+-ATPase. Ethyl acetate extract of U. chamae was subjected to high-resolution fungal PM H+-ATPase inhibition screening followed by structural elucidation by high-performance liquid chromatography–high-resolution mass spectrometry–solid-phase extraction–nuclear magnetic resonance spectroscopy (HPLC–HRMS–SPE–NMR). This led to identification of a series of uncommon o-hydroxybenzylated flavanones and chalcones, i.e., chamanetin (8), isochamanetin (9), isouvaretin (10), uvaretin (11), dichamanetin (12), and diuvaretin (15). Preparative-scale isolation of the active metabolites allowed determination of IC50 values for inhibition of the PM H+-ATPase, and growth inhibition of Saccharomyces cerevisiae and Candida albicans. These revealed a strong correlation between o-hydroxybenzyl substituents and PM H+-ATPase activity, with dichamanetin being the most potent compound, but showing moderate activity in the fungal growth inhibition assays.

Combined use of high-resolution α-glucosidase inhibition profiling and high-performance liquid chromatography-high-resolution mass spectrometry-solid-phase extraction-nuclear magnetic resonance spectroscopy for investigation of antidiabetic principles in crude plant extracts.

Type 2 diabetes is a metabolic disorder affecting millions of people worldwide, and new drug leads or functional foods containing selective α-glucosidase inhibitors are needed. Crude extract of 24 plants were assessed for α-glucosidase inhibitory activity. Methanol extracts of Cinnamomum zeylanicum bark, Rheum rhabarbarum peel, and Rheum palmatum root and ethyl acetate extracts of C. zeylanicum bark, Allium ascalonicum peel, and R. palmatum root showed IC50 values below 20 μg/mL. Subsequently, high-resolution α-glucosidase profiling was used in combination with high-performance liquid chromatography-high-resolution mass spectrometry-solid-phase extraction-nuclear magnetic resonance spectroscopy for identification of metabolites responsible for the α-glucosidase inhibitory activity. Quercetin (1) and its dimer (2), trimer (3), and tetramer (4) were identified as main α-glucosidase inhibitors in A. ascalonicum peel, whereas (E)-piceatannol 3'-O-β-D-glucopyranoside (5), (E)-rhapontigenin 3'-O-β-D-glucopyranoside (6), (E)-piceatannol (8), and emodin (12) were identified as main α-glucosidase inhibitors in R. palmatum root.

High-resolution α-amylase assay combined with high-performance liquid chromatography-solid-phase extraction-nuclear magnetic resonance spectroscopy for expedited identification of α-amylase inhibitors: proof of concept and α-amylase inhibitor in cinnamon.

Type 2 diabetes affects millions of people worldwide, and new improved drugs or functional foods containing selective α-amylase inhibitors are needed for improved management of blood glucose. In this article the development of a microplate-based high-resolution α-amylase inhibition assay with direct photometric measurement of α-amylase activity is described. The inhibition assay is based on porcine pancreatic α-amylase with 2-chloro-4-nitrophenyl-α-D-maltotriose as substrate, which this gives a stable, sensitive, and cheap inhibition assay as requested for high-resolution purposes. In combination with HPLC-HRMS-SPE-NMR, this provides an analytical platform that allows simultaneous chemical and biological profiling of α-amylase inhibitors in plant extracts. Proof-of-concept with an artificial mixture of six compounds-of which three are known α-amylase inhibitors-showed that the high-resolution α-amylase inhibition profiles allowed detection of sub-microgram amounts of the α-amylase inhibitors. Furthermore, the high-resolution α-amylase inhibition assay/HPLC-HRMS-SPE-NMR platform allowed identification of cinnamaldehyde as the α-amylase inhibitor in cinnamon (Cinnamomum verum Presl.).

Coupling HPLC-SPE-NMR with a microplate-based high-resolution antioxidant assay for efficient analysis of antioxidants in food – Validation and proof-of-concept study with caper buds

This work describes the coupling of a microplate-based antioxidant assay with a hyphenated system consisting of high-performance liquid chromatography–solid-phase extraction–nuclear magnetic resonance spectroscopy, i.e., HPLC-SPE-NMR/high-resolution antioxidant assay, for the analysis of complex food extracts. The applicability of the microplate-based antioxidant assay for high-resolution screening of common food phenolics as well as parameters related to their trapping efficiency, elution behavior, and recovery on/from SPE cartridges are described. It was found that the microplate-based high-resolution antioxidant assay is an attractive and easy implementable alternative to direct on-line screening methods. Furthermore, it was shown that Resin SH and Resin GP SPE material are superior to RP C18HD for trapping of phenolic compounds. Proof-of-concept study was performed with caper bud extract, revealing the most important antioxidants to be quercetin, kaempferol, rutin, kaempferol-3-O-β-rutinoside and N1,N5,N10-triphenylpropenoyl spermidine amides. Targeted isolation of the latter, and comprehensive NMR experiments showed them to be N1,N10-di-(E)-caffeoyl-N5-p-(E)-coumaroyl spermidine, N1-(E)-caffeoyl-N5,N10-di-p-(E)-coumaroyl spermidine, N10-(E)-caffeoyl-N1,N5-di-p-(E)-coumaroyl spermidine, and N1,N5,N10-tri-p-(E)-coumaroyl spermidine amides.

Targeting high-performance liquid chromatography–high-resolution mass spectrometry–solid-phase extraction–nuclear magnetic resonance analysis with high-resolution radical scavenging profiles—Bioactive secondary metabolites from the endophytic fungus Penicillium namyslowskii

The high-resolution radical scavenging profile of an extract of the endophytic fungus Penicillium namyslowskii was used to target analysis by high-performance liquid chromatography–high-resolution mass spectrometry–solid-phase extraction–nuclear magnetic resonance spectroscopy, i.e., HPLC–HRMS–SPE–NMR, for identification of anti-oxidative secondary metabolites. This revealed the two chromatographic peaks with the highest relative response in the radical scavenging profile to be griseophenone C and peniprequinolone. The HPLC–HRMS–SPE–NMR analysis was performed in the tube-transfer mode using a cryogenically cooled NMR probe designed for 1.7mm NMR tubes. To further explore the potential of the above HPLC–HRMS–SPE–NMR platform for analysis of endophytic extracts, six peaks displaying no radical scavenging activity were also analyzed. This allowed unambiguous identification of six metabolites, i.e., dechlorogriseofulvin, dechlorodehydrogriseofulvin, griseofulvin, dehydrogriseofulvin, mevastatin acid, and mevastatin. The high mass sensitivity of the 1.7mm cryogenically cooled NMR probe allowed for the first time acquisition of direct detected 13C NMR spectra of fungal metabolites, i.e., dechlorogriseofulvin and griseofulvin, directly from crude extract via HPLC–HRMS–SPE–NMR. Dechlorodehydrogriseofulvin was reported for the first time from nature.

HPLC–NMR Revisited: Using Time-Slice High-Performance Liquid Chromatography–Solid-Phase Extraction–Nuclear Magnetic Resonance with Database-Assisted Dereplication

Time-based trapping of chromatographically separated compounds onto solid-phase extraction (SPE) cartridges and subsequent elution to NMR tubes was done to emulate the function of HPLC-NMR for dereplication purposes. Sufficient mass sensitivity was obtained by use of a state-of-the-art HPLC-SPE-NMR system with a cryogenically cooled probe head, designed for 1.7 mm NMR tubes. The resulting (1)H NMR spectra (600 MHz) were evaluated against a database of previously acquired and prepared spectra. The in-house-developed matching algorithm, based on partitioning of the spectra and allowing for changes in the chemical shifts, is described. Two mixtures of natural products were used to test the approach: an extract of Carthamus oxyacantha (wild safflower), containing an array of spiro compounds, and an extract of the endophytic fungus Penicillum namyslowski, containing griseofulvin and analogues. The database matching of the resulting spectra positively identified expected compounds, while the number of false positives was few and easily recognized.

Comprehensive analysis of commercial willow bark extracts by new technology platform: Combined use of metabolomics, high-performance liquid chromatography–solid-phase extraction–nuclear magnetic resonance spectroscopy and high-resolution radical scavenging assay

Here, proof-of-concept of a new analytical platform used for the comprehensive analysis of a small set of commercial willow bark products is presented, and compared with a traditional standardization solely based on analysis of salicin and salicin derivatives. The platform combines principal component analysis (PCA) of two chemical fingerprints, i.e., HPLC and 1H NMR data, and a pharmacological fingerprint, i.e., high-resolution 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonate) radical cation (ABTS+) reduction profile, with targeted identification of constituents of interest by hyphenated HPLC–solid-phase extraction–tube transfer NMR, i.e., HPLC–SPE–ttNMR. Score plots from PCA of HPLC and 1H NMR fingerprints showed the same distinct grouping of preparations formulated as capsules of Salix alba bark and separation of S. alba cortex. Loading plots revealed this to be due to high amount of salicin in capsules and ampelopsin, taxifolin, 7-O-methyltaxifolin-3′-O-glucoside, and 7-O-methyltaxifolin in S. alba cortex, respectively. PCA of high-resolution radical scavenging profiles revealed clear separation of preparations along principal component 1 due to the major radical scavengers (+)-catechin and ampelopsin. The new analytical platform allowed identification of 16 compounds in commercial willow bark extracts, and identification of ampelopsin, taxifolin, 7-O-methyltaxifolin-3′-O-glucoside, and 7-O-methyltaxifolin in S. alba bark extract is reported for the first time. The detection of the novel compound, ethyl 1-hydroxy-6-oxocyclohex-2-enecarboxylate, is also described.

LC–SPE–NMR–MS: A Total Analysis System for Bioanalysis

Liquid chromatography (LC)-solid-phase extraction (SPE)-nuclear magnetic resonance (NMR)-mass spectrometry (MS) coupling is a key technology for fast and thorough structure elucidation of valuable mass-limited samples. Laborious serial isolation and purification procedures of metabolites, byproducts or impurities from complex biomatrices, natural product extracts or other mixtures of several components can be circumvented by the use of this integrated modular system. This combination of high-end analytical technology significantly accelerates the structure-elucidation process for valuable samples present in minute quantities in mixtures. The information depth is significantly increased by the concurrent availability of NMR and MS data of one chromatographic peak. Thus, this flexible technique is well on its way to becoming the gold standard in analytical chemistry of mixtures. LC-SPE-NMR-MS overcomes the limitations of directly coupled LC-NMR. Full flexibility regarding chromatographic conditions and NMR acquisition is gained by this modular technique. LC-SPE-NMR-MS allows for a rapid structure-elucidation process that would not be possible on the basis of MS or NMR data alone.

Evaluation of on-line solid-phase extraction parameters for hyphenated, high-performance liquid chromatography–solid-phase extraction–nuclear magnetic resonance applications

The hyphenated technique HPLC–SPE–NMR is proving to be a useful analytical tool for structure elucidation of mixture components, particularly for mass-limited samples where traditional isolation procedures are either time consuming or challenging. In this work, we investigated SPE trapping performance of 25 model natural products within a format corresponding to that of HPLC–SPE–NMR hyphenation. Six different silica-based bonded phases and two polymeric phases were evaluated. The trapping efficiency of polystyrene/divinylbenzene polymers was generally superior compared to silica bonded phases, which showed variable results and performed well only with hydrophobic analytes. Acetonitrile concentration in the loading solvent was critical for trapping on polymeric phase (Resin GP), as small changes of the organic solvent concentration (±3%) could alter the trapping efficiency significantly. Flow rate changes of the loading solvent within 0.8–5.0 mL/min did not affect trapping kinetics. Simulation of multiple trapping showed excellent performance of this approach for hydrophobic analytes, and moderate gain for more polar analytes that do not trap quantitatively in a single trapping step. Determination of 50% breakthrough levels by frontal chromatography analysis showed feasibility of accumulation of analyte amounts corresponding to about 0.5 μmol (10 mm × 2 mm i.d. Resin GP cartridge).

Comparison of analytical and semi-preparative columns for high-performance liquid chromatography–solid-phase extraction–nuclear magnetic resonance

The application of analytical and semi-preparative columns in reversed-phase liquid chromatography–solid-phase extraction–nuclear magnetic resonance (HPLC-SPE-NMR) was compared. The work was aiming at separating a higher sample amount in a single run and in this way to reduce the necessary NMR measurement time of separated compounds. Several parameters for compound separation and trapping procedures were optimised: flow rate of HPLC and make-up water pumps, choice of stationary phase cartridges and drying time. The separation and loadability of nine model compounds on analytical and semi-preparative columns was determined, as well as the focussing capacity of SH-type SPE cartridges. It was found that a semi-preparative column—or multiple peak trapping on analytical columns—gave better results than a standard 4.6 mm analytical column for non-polar compounds (e.g. flavonoid aglycones, sesquiterpene lactones, non-polar terpenes, log P > 2), but for polar compounds (log P < −2) did not offer any advantage over an analytical column, or was even disadvantageous. For intermediately polar compounds (−2 < log P < 2) this has to be investigated per compound.