Liquid Chromatography Nuclear Magnetic Resonance Spectroscopy Research Articles

Analytical Methods for Standardization of Ayurvedic Asavas and Aristas; A Review

Plants serve as a rich source of bioactive molecules, which are used to treat various diseases in Ayurveda. Medicinal plant materials are formulated into valuable Ayurvedic medicines by application of modern scientific techniques, where standardization plays a pivotal role for authentication. Standardization confirms the identity, quality and purity of drugs. World Health Organization has set up suitable specific standardization parameters to evaluate the crude drugs and their finished products. These include various evaluation techniques such as pharmacognostical, physico-chemical, phytochemical, analytical, biological and biotechnological. Nowadays, application of several modern analytical techniques have become inevitable for evaluating the polyherbal Ayurvedic formulations to ensure quality, safety and efficacy. Various spectroscopic and chromatographic methods applicable for this purpose are ultra-violet spectroscopy, Fourier-transform infrared spectroscopy, nuclear magnetic resonance spectroscopy, thin-layer chromatography, high performance thin layer chromatography, high performance liquid chromatopgraphy, gas chromatography, mass spectroscopy and hyphenated techniques such as gas chromatography-mass spectroscopy, liquid chromatography-mass spectroscopy, liquid chromatography-nuclear magnetic resonance spectroscopy. Out of several formulations available in Ayurveda, asavas and aristas are considered as unique dosage forms due to their indefinite shelf life. Standardization of different asava-arista formulations using various analytical techniques are vividly discussed in this review.

Authentic compound-free strategy for simultaneous determination of primary coumarins in Peucedani Radix using offline high performance liquid chromatography–nuclear magnetic resonance spectroscopy–tandem mass spectrometry

Herein, a strategy is proposed for the simultaneous determination of primary coumarins in Peucedani Radix (Chinese name: Qianhu). The methodology consists of three consecutive steps: 1) Semi-preparative LC in combination with a home-made automated fraction collection module to fragment the universal metabolome standard into ten fractions (Frs. I–X); 2) LC–accurate MS/MS and quantitative 1H NMR spectroscopy conducted in parallel to acquire the qualitative and quantitative data of each fraction; 3) Robust identification and quantification of components by use of LC coupled to multiple reaction monitoring. In this final step, the most significant fractions (Frs. III–X) were pooled to serve as the pseudo-mixed standard solution. Meticulous online parameter optimization was performed to obtain the optimal parameters, including ion transitions and collision energies. Concerns were particularly paid onto pursuing the parameters being capable of monitoring regio-specific isomers, notably praeruptorin E vs. 3′-isovaleryl-4′-angeloylkhellactone. The quantitative performance of the method was validated according to diverse assays. Eleven primary coumarins (1–11) were unambiguously identified and absolutely quantified, even though no external reference compound was used. Above all, the integrated strategy not only provides a feasible pipeline for the quality assessment of Peucedani Radix, but more importantly, shows the potential for authentic compound-free quantitative evaluation of traditional Chinese medicines.

Sensitivity enhancement by chromatographic peak concentration with ultra-high performance liquid chromatography–nuclear magnetic resonance spectroscopy for minor impurity analysis

High performance liquid chromatography can be coupled with nuclear magnetic resonance (NMR) spectroscopy to give a powerful analytical method known as liquid chromatography-nuclear magnetic resonance (LC-NMR) spectroscopy, which can be used to determine the chemical structures of the components of complex mixtures. However, intrinsic limitations in the sensitivity of NMR spectroscopy have restricted the scope of this procedure, and resolving these limitations remains a critical problem for analysis. In this study, we coupled ultra-high performance liquid chromatography (UHPLC) with NMR to give a simple and versatile analytical method with higher sensitivity than conventional LC-NMR. UHPLC separation enabled the concentration of individual peaks to give a volume similar to that of the NMR flow cell, thereby maximizing the sensitivity to the theoretical upper limit. The UHPLC concentration of compound peaks present at typical impurity levels (5.0–13.1 nmol) in a mixture led to at most three-fold increase in the signal-to-noise ratio compared with LC-NMR. Furthermore, we demonstrated the use of UHPLC-NMR for obtaining structural information of a minor impurity in a reaction mixture in actual laboratory-scale development of a synthetic process. Using UHPLC-NMR, the experimental run times for chromatography and NMR were greatly reduced compared with LC-NMR. UHPLC-NMR successfully overcomes the difficulties associated with analyses of minor components in a complex mixture by LC-NMR, which are problematic even when an ultra-high field magnet and cryogenic probe are used.

Absorption, distribution, metabolism and excretion of novel phosphodiesterase type 4 inhibitor ASP3258 in rats.

The potent and selective phosphodiesterase 4 inhibitor ASP3258 is a novel therapeutic agent for asthma and chronic obstructive pulmonary disease (COPD). After a single oral administration to rats, ASP3258 is rapidly absorbed with a bioavailability of 106%. In situ absorption data indicated that ASP3258 is mainly absorbed in the small intestine. Tissue distribution data after oral administration of (14)C-ASP3258 showed rapid and extensive distribution to various tissues. Excluding the gastrointestinal tract, the tissues with the highest concentrations were liver, heart and plasma. Liquid chromatography-nuclear magnetic resonance spectroscopy data revealed that O-glucuronidation of the carboxylic acid moiety of ASP3258 (formation of an acyl glucuronide) plays a key role in metabolism. No indication was found that the acyl glucuronide reacted with proteins in plasma or tissues. When (14)C-ASP3258 was orally administered to intact rats, urinary and fecal excretion accounted for 1.3% and 100.6% of the administered radioactivity, respectively. After a single oral administration of (14)C-ASP3258 to bile-cannulated rats, urinary and biliary excretion accounted for 0.7% and 93.8% of the administered radioactivity, respectively. These findings suggest that fecal excretion via bile plays an important role in the elimination of ASP3258-derived radioactivity. In vitro metabolic profiles were relatively similar among the species examined, suggesting that our findings in rats may help us to understand pharmacokinetics, efficacy and safety profiles in humans and other species.

Comparative Analyses of Stilbenoids in Canes of Major Vitis vinifera L. Cultivars

Grapevine canes are rich in resveratrol and its complex derivatives. These compounds have many biological activities and are needed mainly for health purposes. Canes, which are often wasted, can be used to produce these high-value compounds at low cost. We studied sixteen Vitis vinifera L. cultivars among the most widely cultivated ones worldwide. Polyphenols were extracted from their canes and identified by liquid chromatography-nuclear magnetic resonance spectroscopy. We accurately determined the content of E-ε-viniferin, E-resveratrol, E-piceatannol, and vitisin B and, for the first time, that of hopeaphenol and miyabenol C. The canes did not contain these major stilbene compounds in similar proportions, and their abundance and order of abundance varied according to the cultivar. For instance, Pinot noir has very high levels of E-resveratrol and E-ε-viniferin; Gewurztraminer has very high levels of vitisin B, and Carignan and Riesling have very high levels of hopeaphenol. These findings suggest that the right cultivar should be used to obtain the highest yield of a polyphenol of interest.

Chemical dereplication of wine stilbenoids using high performance liquid chromatography–nuclear magnetic resonance spectroscopy

Wine is a major dietary source of numerous potentially health promoting stilbenoids that have been the subject of many qualitative and quantitative studies. However, our initial HPLC–MS analyses of crude wine samples demonstrated the presence of compounds with molecular weights matching characteristic stilbenoid dimers, trimers, and tetramers that were unaccounted for in the literature. Due to the likelihood that these are known compounds, a chemical dereplication method is highly desirable. We developed such a method using LC-DAD–MS monitored fractionation steps, using adsorption and centrifugal partition chromatography (CPC), to obtain fractions rich in stilbenoids for analysis in stopped-flow LC–NMR. 1H NMR spectra and MS data were cross-referenced with our laboratory database and the literature for identification. This method yielded highly useful structural information, allowing the characterization of previously unidentified stilbenoids in wine, ampelopsin C, isohopeaphenol, quadrangularin A, and E-ω-viniferin. These results demonstrate the usefulness of stop-flow LC–NMR in conjunction with LC–MS guided fractionation for the dereplication of compounds of interest in general, and specifically for expanding the current knowledge of wine chemistry.

Antifungal Azole Metabolites: Significance in Pharmaceutical and Biomedical Analysis

Antifungal Azole Metabolites: Significance in Pharmaceutical and Biomedical Analysis Individualised therapy and factors determining such variability among patients are confusing to both physicians and their patients because of the observed therapeutic, metabolic and toxic response. The same is true about antifungal azoles. They are under the influence and become targets of metabolic drug-drug interactions where more than one active form of the drug may be involved. The clinical relevance of these interactions may vary upon the azole involved and upon the intention of drug administration. The pharmacodynamics and pharmacokinetics of azole drugs as indicated by the reviewed data make the need for characterization of all their metabolites even more evident. The health care systems also emphasize the identification and quantitation of the metabolites for a comprehensive understanding of the biological safety of individual metabolites, thus, revealing the need and scope of bioanalytical research in metabolite and toxicity profiling of drugs. Availability of protocols for qualitative and quantitative characterization of all metabolites will have many applications for therapeutic drug monitoring, bioequivalence, toxicological and all related studies. Identification of metabolites may be done by a variety of chromatographic and spectroscopic techniques, either alone or in combination with other techniques. Conventional liquid chromatography has been exploited widely in the field of metabolite profiling. The arrival of hyphenated techniques has revolutionized metabolite profiling, by not only separating but also generating data for the structural identification of metabolites as well. Among all techniques, the most exploited are Liquid Chromatography-Mass Spectroscopy, Nuclear Magnetic Resonance spectroscopy, Liquid Chromatography-Nuclear Magnetic Resonance spectroscopy, Liquid Chromatography-Nuclear Magnetic Resonance spectroscopy-Mass Spectroscopy and Extraction-Nuclear Magnetic Resonance spectroscopy. This compilation provides a tool for the metabolic, bioanalytical and biomedical understanding of antifungal azole metabolites.

Identification of degradation products in loxoprofen sodium adhesive tapes by liquid chromatography–mass spectrometry and dynamic pressurized liquid extraction–solid-phase extraction coupled to liquid chromatography–nuclear magnetic resonance spectroscopy

Rapid and unambiguous identification of three degradation products (DP-1, DP-2 and DP-3) found in heat-stressed loxoprofen sodium adhesive tapes (Loxonin tapes) was achieved by LC–MS and dynamic pressurized liquid extraction (PLE)–solid-phase extraction (SPE) coupled to LC–NMR without complicated isolation or purification processes. The molecular formulae of the degradation products were determined by accurate mass measurements and product ion analyses and on-line hydrogen/deuterium (H/D) exchange experiments provided information about changes in the degradation of loxoprofen. To compensate for the low sensitivity of NMR, on-line dynamic PLE–SPE was employed and higher concentrations of degradation products trapped on the SPE column were afforded in a shorter time than they would be in such time-consuming sample preparations as pre-concentration after extraction. The loop-storage procedure was used in the LC–NMR analysis to allow the acquisition of the 1H spectra of the three degradation products in one chromatographic run without affecting the peak separation and to avoid the carry-over of previously eluted DP-1 of high concentration by washing the NMR detection cell prior to the measurement of the DP-2 spectrum. Based on the resulting 1H NMR spectra in combination with the MS results, DP-1 was successfully identified as an oxidation product having an oxodicarboxylic acid structure formed by the cleavage of the cyclopentanone ring of loxoprofen, DP-2 as a cyclopentanone ring-hydroxylated loxoprofen and DP-3 as a loxoprofen l-menthol ester.

Application of liquid chromatography–nuclear magnetic resonance spectroscopy for the identification of ethyldimethylpyrazine, a food flavouring agent

The application of liquid chromatography–nuclear magnetic resonance spectroscopy (LC–NMR) for the direct identification of ethyldimethylpyrazine, a food flavouring agent, has been studied. The commercial product is a mixture of two regio-isomers, 2-ethyl-3,5-dimethylpyrazine (1) and 2-ethyl-3,6-dimethylpyrazine (2); however, the exact composition of the mixture is unknown. Structural characterization by LC–MS and GC–MS was not possible because both regio-isomers yield the same molecular related ion and ion fragmentation. To rapidly identify the two regio-isomers, the product was analyzed by LC–NMR with on-flow and fraction loop modes. From the results, the structure elucidations of the two regio-isomers could be carried out without the need to isolate the isomers by the usual procedures.

Determination of number of significant components and key variables using genetic algorithms in liquid chromatography-nuclear magnetic resonance spectroscopy and liquid chromatography-diode array detection

A new method is proposed for determination of the number of significant components in hyphenated chromatographic data. The method is based on the application of genetic algorithms (GA). The method is applied to five datasets from on-flow liquid chromatography with nuclear magnetic resonance spectroscopy (LC-NMR) and on three datasets from liquid chromatography with diode array detector (LC-DAD). The effect of different factors such as the application of technique on time or spectral direction, noise level, baseline, chromatographic resolution and relative peak height has been studied. The method is compared with other chemometric approaches for rank analysis and variable selection. The results produced by GA are promising on both types of data.

The application of LC–NMR and LC–MS for the separation and rapid structure elucidation of an unknown impurity in 5-aminosalicylic acid

This work has demonstrated the usefulness of combining liquid chromatography–nuclear magnetic resonance spectroscopy (LC–NMR) and liquid chromatography–mass spectrometry (LC–MS) methodologies for a rapid identification of an unknown impurity ( N1) in the drug 5-aminosalycilic acid. Complementary information obtained from the two methods has revealed plenty of structural information and led to the fast on-line structure determination of N1 prior to its isolation and purification. The analysis of LC–NMR and LC–MS spectra revealed that N1 and 5-aminosalycilic acid are structurally closely related compounds. The structure of N1 was later confirmed by high-resolution NMR spectroscopy of the isolated compound and the atom assignment was made. The approach described here has potential for 5-aminosalycilic acid impurity profiling and monitoring the production process.

Metabolome analysis: the potential of in vivo labeling with stable isotopes for metabolite profiling

Metabolome analysis technologies are still in early development because, unlike genome, transcriptome and proteome analyses, metabolome analysis has to deal with a highly diverse range of biomolecules. Combinations of different analytical platforms are therefore required for comprehensive metabolomic studies. Each of these platforms covers only part of the metabolome. To establish multiparallel technologies, thorough standardization of each measured metabolite is required. Standardization is best achieved by addition of a specific stable isotope-labeled compound, a mass isotopomer, for each metabolite. This suggestion, at first glance, seems unrealistic because of cost and time constraints. A possible solution to this problem is discussed in this article. Saturation in vivo labeling with stable isotopes enables the biosynthesis of differentially mass-labeled metabolite mixtures, which can be exploited for highly standardized metabolite profiling by mass isotopomer ratios.

Combined use of liquid chromatography–nuclear magnetic resonance spectroscopy and liquid chromatography–mass spectrometry for the characterization of an acarbose degradation product

Directly coupled LC–MS and LC–NMR were applied to identify and structurally characterize an acarbose degradation product A in acidic media. A comparative analysis of the stop-flow LC–NMR ( 1 H and TOCSY) and LC–MS data provided evidence that A is structurally related to acarbose, differing from the parent compound in a number of subunits present in the molecule. Spectral analysis revealed that A was the α-glucosidase inhibitor amylostatin XG. Complementary information obtained from the two methods led to the structural elucidation of A which was later corroborated by high-resolution NMR spectroscopy of the isolated molecule.

Identification of Gemfibrozil Metabolites, Produced as Positional Isomers in Human Liver Microsomes, by On-line Analyses Using Liquid Chromatography/Mass Spectrometry and Liquid Chromatography/Nuclear Magnetic Resonance Spectroscopy

Liquid chromatography/mass spectrometry (LC/MS) and liquid chromatography/nuclear magnetic resonance spectroscopy (LC/NMR) were applied to the identification of the positional isomers of gemfibrozil metabolites produced in vitro by human liver microsomes. Gemfibrozil was metabolized to two major oxidative metabolites (M1 and M2) by human liver microsomes in the presence of an nicotinamide adenine dinucleotide phosphate, reduced form (NADPH)-generating system. M1 and M2 were characterized as metabolites formed by the oxidation of the 2′,5′-dimethylphenoxy moiety, which has potentially 5 different positions for hydroxylation, based on the results of negative-ion tandem mass spectrometry in combination with those of accurate mass measurements by quadrupole time-of-flight mass spectrometry. Separation and identification of M1 and M2 were conducted by LC/NMR analyses at 500 MHz. An on-flow 1H NMR analysis demonstrated that M1 was produced by hydroxylation at either the 2′-methyl group or the 5′-methyl group and M2 by hydroxylation at either the 3′-C or the 4′-C position of the aromatic ring. A stop-flow nuclear Overhauser effect spectroscopy (NOESY) analysis demonstrated that M1 and M2 were metabolites hydroxylated at the 5′-methyl group and the 4′-C position of the aromatic ring, respectively.

Identification of major metabolites in Aloe littoralis by high-performance liquid chromatography-nuclear magnetic resonance spectroscopy.

Examination of the leaf exudate of the South African species Aloe littoralis by reversed-phase HPLC revealed the presence of two major metabolites. The identification of the two compounds without isolation was attempted by HPLC-NMR based on separation using a C18 column eluting with a deuterium oxide:acetonitrile solvent gradient and an inverse HPLC-NMR probe. For each compound, one-dimensional proton spectra, and two-dimensional homonuclear COSY and TOCSY, and heteronuclear HSQC and HMBC, spectra were collected. On the basis of the data obtained, the metabolites were characterised as 10-hydroxyaloin B and deacetyllittoraloin.

The use of hyphenated techniques in comparative phytochemical studies of legumes

The coupling of instruments performing chromatographic separations to those providing structural data has had an enormous impact in analytical chemistry. These ‘hyphenated techniques’ are enabling compounds to be detected in plant extracts more effectively than ever before. At the same time, the rapid development of DNA sequencing technology and cladistic data analysis have provided taxonomists with the means to produce testable systematic hypotheses. These parallel developments in analytical chemistry and systematics have transformed the often criticised discipline of chemotaxonomy into modern integrated studies in comparative phytochemistry that aim to test cladistic hypotheses or gain insights into the biochemical evolution of plants. In this paper the key developments in the main hyphenated techniques, gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-mass spectrometry (LC-MS), are considered together with some of the challenges facing the further development of capillary electrophoresis-mass spectrometry (CE-MS) and liquid chromatography-nuclear magnetic resonance spectroscopy (LC-NMR). The application of GC-MS and LC-MS in comparative phytochemical studies in legumes is reviewed both from selected research in the literature and from the authors’ own experiences, with an emphasis on nitrogen-containing and phenolic compounds. The use of GC-MS has provided an extensive data set on the occurrence of quinolizidine alkaloids in legumes and this character is now assuming taxonomic significance at a high level. GC-MS also provides the means to separate the numerous isomeric forms of polyhydroxyalkaloids and hydroxypipecolic acids as their volatile trimethylsilyl derivatives and surveys of these compounds are supporting systematic work at lower taxonomic levels. LC-MS is enabling the metabolic profiles of intact flavonoid glycosides to be obtained from small fragments of material while recent methods to analyse non-protein amino acids by LC-MS without derivatisation hold much promise in surveys of these important taxonomic characters. Tandem mass spectrometry (MS/MS) provides a rapid means of sequencing peptides and so, as we enter the era of proteomics, LC-MS/MS is likely to play a central role in the analysis of legume proteins.

Quantitative and structural determination of pseudoephedrine sulfate and its related compounds in pharmaceutical preparations using high-performance liquid chromatography

A simple isocratic reversed-phase high performance liquid chromatograghic method for the separation of pseudoephedrine and its related compounds in pharmaceutical formulations is described. The separation is achieved in less than 35 min on a C-18 column (4.6 mm I.D.×25 cm length, 5-μm particle size) using a mobile phase consisting of a mixture of ammonium acetate and methanol. The results described in this report demonstrate that the method is sensitive and selective. Structural elucidation of two new pseudoephedrine degradation products is described. On the basis of structural analysis by liquid chromatography-mass spectrometry (LC-MS) and liquid chromatography-nuclear magnetic resonance spectroscopy (LC-NMR), the two newly elucidated degradation products were identified to be 2-(carboxyamino)propiophenone (molecular ion of m/ z=194) and 2-formyl-2-(methylamino)-acetophenone (molecular ion of m/ z=178).

On-line liquid-chromatography–nuclear magnetic resonance spectroscopy–mass spectrometry coupling for the separation and characterization of secoisolariciresinol diglucoside isomers in flaxseed

Two secoisolariciresinol diglucoside (SDG) diastereomers were extracted from flaxseed and liberated through alkaline hydrolysis. Anion-exchange and reversed-phase chromatography were successfully employed to purify the hydrolyzed flaxseed extract. On-line LC–NMR–MS analyses revealed the structure of the isolated and purified SDG diastereomers, [2 R,2′ R]-2,3-bis[(4-hydroxy-3-methoxyphenyl)-methyl]-1,4-butanediyl-bis-β-glucopyranoside the predominant flaxseed lignan and [2 R,2′ S]-2,3-bis[(4-hydroxy-3-methoxyphenyl)methyl]-1,4-butanediyl-bis-β-glucopyranoside, a previously incompletely characterized minor flaxseed lignan. Circular dichroism (CD) analyses confirmed the presence of two distinguished optically active compounds present in the flaxseed extract.

Characterization by liquid chromatography-nuclear magnetic resonance spectroscopy and liquid chromatography-mass spectrometry of two coupled oxidative-conjugative metabolic pathways for 7-ethoxycoumarin in human liver microsomes treated with alamethicin.

The microsomal metabolism of 7-ethoxycoumarin (7-EC) was investigated using liquid chromatography (LC)-NMR and liquid chromatography-mass spectrometry (LC-MS) to characterize the coupling of oxidative-conjugative metabolism events. Within microsomes, cytochromes P450 (P450s) and UDP-glucuronosyltransferases (UGTs) are spatially disparate, each having surface and luminal localization, respectively. To optimize cofactor and substrate transit to UGT without compromising P450 activity, the pore-forming peptide alamethicin was used for microsomal perforation. Aqueous extracts of microsomal incubations containing NADPH and UDP-glucuronic acid were injected for LC-NMR and LC-MS analysis. The analytical complementarity of LC-NMR and LC-MS permitted the identification of four metabolites (M1 to M4). The metabolites M1 and M2 are novel microsomal metabolites for 7-EC, consistent with 3-hydroxylation and subsequent glucuronidation, respectively. Metabolites M3 and M4 were 7-hydroxycoumarin (7-HC) and 7-HC glucuronide, respectively. Viewed collectively, these results illustrate the utility of alamethicin in the examination of coupled oxidative-conjugative metabolism and the synergy of LC-NMR and LC-MS in metabolite identification.

Use of on-line liquid chromatography–nuclear magnetic resonance spectroscopy for the rapid investigation of flavonoids from Sorocea bomplandii

This paper reports the identification of di- and triglycosylated flavonoids from Sorocea bomplandii (Moraceae) by liquid chromatography coupled on-line to nuclear magnetic resonance (LC–NMR). These glycosylated flavonoids may be used as a taxonomic marker in future work.