Chromatography-solid Phase Extraction-nuclear Magnetic Resonance Research Articles

Bioactive Bioflavonoids from Platonia insignis (Bacuri) Residues as Added Value Compounds

Platonia insignis fruit, popularly known as bacuri, is traditionally used in folk medicine for its anti-inflammatory and antioxidant properties. Therefore, this study determined the chemical composition and biological activities of the bacuri’s shell and seeds extracts, considered residues from its consumption and industrial uses. Four biflavonoids (GB-2a, GB-1a, morelloflavone, and volkensiflavone) were identified in the extracts by high-performance liquid chromatography-diode array detection (HPLC-DAD), liquid chromatography tandem mass spectrometry (LC-MS/MS), and liquid chromatography-solid phase extraction-nuclear magnetic resonance (LC-SPE-NMR) techniques. Morelloflavone was identified as the main compound in the shell ethyl acetate extract, being responsible for the high in vitro antioxidant (50% effective concentration (EC50) ranging from 8.0-10.5 µg mL−1 in different protocols), anti-glycant (80%), and moderate inhibition of nitric oxide (1.56 µg mL−1 for > 90% cell viability) activities. This extract showed promising in vivo anti-inflammatory activity evaluated through the paw edema protocol after its incorporation into a liquid-crystalline drug carrier system, reducing the edema by up to 31%. The results demonstrated the potential of the fruit for the development of drugs of natural origin and corroborated to add economic value to these discarded residues.

Isolation and identification of human metabolites from a novel anti-tumor candidate drug 5-chlorogenic acid injection by HPLC-HRMS/MSn and HPLC-SPE-NMR.

A novel anti-tumor candidate drug, 5-chlorogenic acid (5-CQA) injection, was used for the treatment of malignant glioma in clinical trial (phase I) in China. The isolation and identification of the metabolites of 5-CQA injection in humans were investigated in the present study. Urine and feces samples obtained after intramuscular administration of 5-CQA injection to healthy adults have been analyzed by high-performance liquid chromatography coupled with high-resolution mass and multiple-stage mass spectrometry (HPLC-HRMS/MSn). No metabolite was detected in human feces; however, in human urine, a total of six metabolites were identified including isomerized 5-CQA (P1 and P2), hydrolyzed 5-CQA (M1and M2), and methylated 5-CQA (M3 and M4). Among them, M3 and M4 were the main metabolites and target analytes for human mass balance study. Additionally, the structure of M3 and M4 was characterized by high-performance liquid chromatography-solid phase extraction-nuclear magnetic resonance (HPLC-SPE-NMR), and the results demonstrated that the methoxy group of M3 and M4 was exclusively attributed to C-3' and C-4', respectively. Due to the unavailability of commercial reference, the pure products of M3 and M4 were synthesized by 5-CQA methylation and followed by isolation and purification. Moreover, the potential activity of M3 and M4 on malignant glioma was predicted using a reverse molecular docking analysis on eight malignant glioma-related pathways. The results showed that M3 and M4 had various interactions against malignant glioma-related targets. Our study provides an insight into the metabolism of 5-CQA injection in humans and supports the clinical human mass balance study. Graphical abstract ᅟ.

Identification of some Bioactive Metabolites in a Fractionated Methanol Extract from Ipomoea aquatica (Aerial Parts) through TLC, HPLC, UPLC-ESI-QTOF-MS and LC-SPE-NMR Fingerprints Analyses.

The plant species Ipomoea aquatica contains various bioactive constituents, e.g. phenols and flavonoids, which have several medical uses. All previous studies were executed in Asia; however, no reports are available from Africa, and the secondary metabolites of this plant species from Africa are still unknown. The present study aims finding suitable conditions to identify the bioactive compounds from different fractions. Chromatographic fingerprint profiles of different fractions were developed using high-performance liquid chromatography (HPLC) and then these conditions were transferred to thin-layer chromatography (TLC). Subsequently, the chemical structure of some bioactive compounds was elucidated using ultra-performance liquid chromatography-quadrupole time of flight-tandem mass spectrometry (UPLC-QTOF-MS) and liquid chromatography-solid phase extraction-nuclear magnetic resonance (LC-SPE-NMR) spectroscopy. The HPLC fingerprints, developed on two coupled Chromolith RP-18e columns, using a gradient mobile phase (methanol/water/trifluoroacetic acid, 5:95:0.05, v/v/v), showed more peaks than the TLC profile. The TLC fingerprint allows the identification of the types of chemical constituents, e.g. flavonoids. Two flavonoids (nicotiflorin and ramnazin-3-O-rutinoside) and two phenolic compounds (dihydroxybenzoic acid pentoside and di-pentoside) were tentatively identified by QTOF-MS, while NMR confirmed the structure of rutin and nicotiflorin. The HPLC and TLC results showed that HPLC fingerprints give more and better separated peaks, but TLC helped in determining the class of the active compounds in some fractions. Bioactive constituents were identified as well using MS and NMR analyses. Two flavonoids and two phenolic compounds were tentatively identified in this species for the first time, to the best of our knowledge. Copyright © 2017 John Wiley & Sons, Ltd.

An enhanced targeted identification strategy for the selective identification of flavonoid O-glycosides from Carthamus tinctorius by integrating offline two-dimensional liquid chromatography/linear ion-trap-Orbitrap mass spectrometry, high-resolution diagnostic product ions/neutral loss filtering and liquid chromatography-solid phase extraction-nuclear magnetic resonance

Targeted identification of potentially bioactive molecules from herbal medicines is often stymied by the insufficient chromatographic separation, ubiquitous matrix interference, and pervasive isomerism. An enhanced targeted identification strategy is presented and validated by the selective identification of flavonoid O-glycosides (FOGs) from Carthamus tinctorius. It consists of four steps: (i) enhanced separation and detection by offline two-dimensional liquid chromatography/LTQ-Orbitrap MS (offline 2D-LC/LTQ-Orbitrap MS) using collision-induced dissociation (CID) and high-energy C-trap dissociation (HCD); (ii) improved identification of the major aglycones by acid hydrolysis and LC-SPE-NMR; (iii) simplified spectral elucidation by high-resolution diagnostic product ions/neutral loss filtering; and (iv) more convincing structural identification by matching an in-house library. An offline 2D-LC system configuring an Acchrom XAmide column and a BEH Shield RP-18 UPLC® column enabled much better separation of the easily co-eluting components. Combined use of CID and HCD could produce complementary fragmentation information. The intensity ratios of the aglycone ion species ([Y0-H]−/Y0− and [Y0-2H]−/Y0−) in the HCD-MS2 spectra were found diagnostic for discriminating the aglycone subtypes and characterizing the glycosylation patterns. Five aglycone structures (kaempferol, 6-hydroxykaempferol, 6-methoxykaempferol, carthamidin, and isocarthamidin) were identified based on the 1H-NMR data recorded by LC-SPE-NMR. Of the 107 characterized flavonoids, 80 FOGs were first reported from C. tinctorius. Unknown aglycones, pentose, and novel acyl substituents were discovered. A new compound thereof was isolated and fully identified, which could partially validate the MS-oriented identification. This integral strategy can improve the potency, efficiency, and accuracy in the detection of new compounds from medicinal herbs and other natural sources.

Preparative Separation and Structural Identification of Impurities of a New α2‑Adrenoceptor Agonist Using Stacking Injection, LC-MSn and LC-SPE-NMR

Identifying impurities in drug substances has become one of the most important issues in pharmaceutical analysis since it can have a significant impact on the efficacy of new pharmaceutical products. Due to the purity requirements, in this paper a new synthetic α2-adrenoceptor agonist, called LPSF-PT-31, was purified and its impurities were characterized by liquid chromatography-multistage mass spectrometry (LC-MSn) and liquid chromatography-solid phase extraction-nuclear magnetic resonance (LC-SPE-NMR). The purification step was conducted using a semi-preparative liquid chromatography and stacked injections as a new approach to drug purification. As a result, a total yield of 75% of the pure LPSF-PT-31 and 2.9 L day-1 in solvent reduction was obtained. The combination of semi-preparative stacking injection, LC-MSn, and LC-SPE-NMR, demonstrated to be efficient to purify active drugs and unambiguously identify its impurities. In addition, isolation and identification of drug impurities in the early stages of development can improve the synthetic pathway, preventing the formation of impurities or minimizing this formation to minimum levels.

Alkaloid analysis by high-performance liquid chromatography-solid phase extraction-nuclear magnetic resonance: New strategies going beyond the standard

The hyphenated technique HPLC-SPE-NMR is an important tool for rapid dereplication of complex mixtures of in particular small molecules and has been successfully employed in natural product research. However, positively charged alkaloids at low pH are often poorly trapped on the generally used SPE cartridge limiting the general application of the procedure. In this work, two new approaches for efficient SPE trapping of alkaloids and elution efficiencies were evaluated using 24 model alkaloids. Use of a 0.1M NaOH solution as the post-column dilution greatly enhanced trapping of alkaloids on the commonly used cartridge containing divinylbenzene polymer (GP resin). This procedure, however, was unsuitable for trapping phenolic alkaloids. Severe line broadening and immiscibility with water made chloroform-d1 unsuited as eluent. None of these problems occurred when methanol-d4 was used as eluent. Previously, mixed mode cation exchange sorbents have not been used in HPLC-SPE-NMR analysis of natural products. In contrast to GP resin this material showed good retention and elution characteristics for retention and elution of alkaloids. As well the use of methanol-d4 containing 1% aqueous NaOD (40%) as methanol-d4 containing 5% aqueous NH4OH (30%) as eluents were successful, even though elution of alkaloids with pKa of the corresponding acid above 10 proved difficult. Alkaloid extracts of Huperzia selago containing complex aliphatic alkaloids and Triclisia patens containing bisbenzylisoquinoline alkaloids were used for validation of the protocols for analysis of a diverse collection of alkaloids. Mixed mode cation exchange sorbent was efficient for trapping and elution of both types of alkaloids as evidenced by acquisition of 2D NMR data for all trapped compounds. In contrast, GP resin proved only viable for all the H. selago alkaloids whereas trapping and elution of bisbenzylisoquinoline alkaloids were dubious.

Characterisation of commercially available linear alkylbenzenesulfonates by LC-SPE-NMR/MS (liquid chromatography-solid phase extraction-nuclear magnetic resonance spectroscopy-mass spectroscopy)

Commercially available linear alkylbenzenesulfonates (LASs) are a mixture of various homologues and isomers, leading to 20 major species. In this work we investigated the commercial product by liquid chromatography-solid phase extraction-nuclear magnetic resonance spectroscopy-mass spectrometry (LC-SPE-NMR/MS). The commercial product was separated into 17 fractions by liquid chromatography (LC). After chromatographic separation, 5% of the flow was split to a mass spectrometer (MS) while 95% was send to post-column solid phase extraction cartridges for enrichment of the analytes (LC-SPE). After elution from the SPE-cartridges a NMR-spectrometer equipped with a cryo-probe was used for the characterisation of the different LASs species. For the first time 1H-1D and H-H-COSY spectra for 14 LASs species out of 20 major isomers are presented, whereas the 6 remaining species are detected as mixtures in 3 1H-1D and H-H-COSY spectra. These data were used to correlate the chromatographic retention of the LASs isomers to the substitution pattern of the alkyl chain.

Identification of major metal complexing compounds in Blepharis aspera

Verbascoside and isoverbascoside, present at 0.7% and 0.2% (w/w dryweight), were identified to be major compounds that could contribute to the metal complexation in Blepharis aspera collected in Botswana, Africa. The metallophyte B. aspera has high ability to cope with a high level of metal accumulation. The presence of metal complexing compounds and/or antioxidants can prevent oxidative reactions in lipids, proteins and DNA that take place due to the metal accumulation. On-line liquid chromatography–solid phase extraction–nuclear magnetic resonance (LC–SPE–NMR) was applied for the identification, while electrospray-mass spectrometry (ESI-MS) and UV-vis spectroscopy was used to assess whether these compounds can complex with metals. It was found that verbascoside and isoverbascoside may form complexes with nickel, iron (verbascoside only) and copper. Thus, the presence of verbascoside and isoverbascoside can explain the survival of B. aspera in mineral-rich areas.

Conventional sample enrichment strategies combined with high-performance liquid chromatography–solid phase extraction–nuclear magnetic resonance analysis allows analyte identification from a single minuscule Corydalis solida plant tuber

Identification of putative biomarker molecules within the genus Corydalis (Papaveraceae) was pursued by combining conventional off-line sample enrichment with high-performance liquid chromatography–solid phase extraction–nuclear magnetic resonance (HPLC–SPE–NMR) based structure elucidation. Off-line reversed phase solid phase extraction (SPE) was used to enrich the desired analytes from a methanolic extract (93 mg dry weight) of a miniscule single tuber (233 mg dry weight) of C. solida. An aliquot of the SPE fraction (2.1 mg) was subjected to separation in the HPLC–SPE–NMR hyphenation. Chromatographic peaks bearing the metabolites under investigation were trapped in the SPE device in a single experiment and transferred to a 600 MHz NMR spectrometer equipped with a 30 μl cryofit insert fed into a 3 mm cryoprobe. Recorded homo- and heteronuclear 1D and 2D NMR data allowed the identification of the three analytes under investigation as protopine, allocryptopine, and N-methyl-laudanidinium acetate. The latter is a rare alkaloid, which has been isolated only once before.