Multiple hyphenation of liquid chromatography with nuclear magnetic resonance spectroscopy, mass spectrometry and beyond

The advent of sensitive and reliable HPLC-NMR and HPLC-MS systems has revolutionised the identification of compounds eluting from chromatographic systems. More recently systems have been described wherein both NMR and MS are used together to provide an immensely powerful means of characterising compounds in chromatographic eluents. Here the construction and application of combined HPLC-NMR-MS systems to the analysis of mixtures of pharmaceuticals, drug metabolites in biological fluids and natural products in plant extracts is reviewed. In addition preliminary work with alternative systems such as HPLC-UV-NMR-FTIR-MS is highlighted and the prospects for such complex systems considered.     

Multiple component isolation in preparative multidimensional gas chromatography with characterisation by mass spectrometry and nuclear magnetic resonance spectroscopy

The preparative scale isolation of multiple components from an essential oil matrix is described using multidimensional gas chromatography (prep-MDGC) which allows their further characterisation by mass spectrometry and nuclear magnetic resonance (NMR) spectroscopy. Menthol, linalyl acetate, carvone and geraniol were isolated individually, and were also collected in various combinations. It was demonstrated to be possible to collect multiple selected components from numerous repeat injections of the sample, to permit increased mass recovery from an external cryotrap collection device. Peak retention times remained reproducible (<0.3 s) over the repeated injections and switching events. This methodology may be utilised to confirm peak identity or to produce unique mixed-component reference standards, for instance to allow their identification in other samples using GC/MS, or identify them in comprehensive two-dimensional gas chromatography (GC × GC) analysis.     

Reexamination of reduced pteridine cofactors behaviour by proton nuclear magnetic resonance spectroscopy and mass spectrometry

In aqueous solutions, the autoxidation by air of 2-amino-4-hydroxy-6,7-dimethyl-5,6,7,8-tetra-hydropteridine, a hydroxylase cofactor, leads to the corresponding 7,8-dihydro derivate. Oxidation by hydrogen peroxide and Horseradish peroxidase does not give a stable quinonoid form as previously claimed but affords two metabolic products. Kinetics of the two pathways and structures of the different final compounds were determined by ¹H nmr and mass spectrometry.     

Characterization of degradation products of poly[(3,3,3-trifluoropropyl)methylsiloxane] by nuclear magnetic resonance spectroscopy, mass spectrometry and gas chromatography

Poly[(3,3,3-trifluoropropyl)methylsiloxane] (PTFPMS) was treated with the solvents acetone, ethyl acetate and methanol and its degradation products were analyzed with multiple techniques. ¹H, ²⁹Si and ¹⁹F nuclear magnetic resonance (NMR) spectroscopy were useful for the characterization of the intact polymers and for the determination of cyclosiloxanes. Cyclosiloxanes with a ring size of up to 23 were quantified by gas chromatography. The only degradation products found were TFPmethyl-cyclosiloxanes. 1,3,5,7-Tetrakis(TFPmethyl)-cyclotetrasiloxane was predominant, and (TFPmethyl)cyclotri-, penta- and hexasiloxane could be detected at lower concentrations. The identity of cyclic degradation products with a ring size of up to 6 was unambiguously confirmed by direct infusion mass spectrometry. The TFPmethyl-cyclosiloxanes were successfully ionized by electrospray ionization in the negative mode. None of the techniques applied gave hints to other degradation products such as short linear oligomers. Almost complete degradation of PTFPMS occurred in acetone and methanol, while degradation is distinctly reduced in ethyl acetate.     

Novel glutathione conjugates of phenyl isocyanate identified by ultra‐performance liquid chromatography/electrospray ionization mass spectrometry and nuclear magnetic resonance

Phenyl isocyanate is a highly reactive compound that is used as a reagent in organic synthesis and in the production of polyurethanes. The potential for extensive occupational exposure to this compound makes it important to elucidate its reactivity towards different nucleophiles and potential targets in the body. In vitro reactions between glutathione and phenyl isocyanate were studied. Three adducts of glutathione with phenyl isocyanate were identified using ultra‐performance liquid chromatography/electrospray ionization mass spectrometry and nuclear magnetic resonance (NMR). Mass spectrometric data for these adducts have not previously been reported. Nucleophilic attack on phenyl isocyanate occurred via either the cysteinyl thiol group or the glutamic acid α‐amino group of glutathione. In addition, a double adduct was formed by the reaction of both these moieties. NMR analysis confirmed the proposed structure of the double adduct, which has not previously been described. These results suggest that phenyl isocyanate may react with free cysteines, the α‐amino group and also with lysine residues whose side chain contains a primary amine. Copyright © 2014 John Wiley & Sons, Ltd.     

Characterization of the aromatic composition of some liquid foods by nuclear magnetic resonance spectrometry and liquid chromatography with nuclear magnetic resonance and mass spectrometric detection

This paper describes the first application of NMR spectroscopy and LC-NMR/MS to the direct analysis of the aromatic composition of beer, grape juice and a wine phenolic extract. NMR spectroscopy provides non-invasive information on the overall aromatic profile and enables the identification of some compounds. However, a more comprehensive assignment is hindered by the low peak intensity and strong signal overlap in the low-field spectral region, as well as by the inherent lack of scalar coupling information for many aromatic compounds present. LC-NMR/MS can overcome these problems and is shown to aid significantly in the identification of aromatic compounds composing all samples analyzed. Some examples are the identification of several cinnamic acids (e.g. p-coumaric, trans-coutaric and trans-caftaric) in grape juice, the identification of 2-phenylethanol, tyrosol and tryptophol in beer and the detection of phenolics such as catechin, epicatechin, trans-resveratrol, tyrosol and caffeic acid in the wine extract.     

Separation and characterization of alkyl phenol formaldehyde resins demulsifier by adsorption chromatography, gel permeation chromatography, infrared spectrometry and nuclear magnetic resonance spectroscopy

This paper deals with the separation and characterization of alkyl phenol formaldehyde resins demulsifier by infrared spectrometry and nuclear magnetic resonance spectroscopy after separation of the different surfactants and low molecular additives by adsorption chromatography. Firstly, the types of surfactants are identified by methylene blue chloride-chloroform test method and the elemental analysis such as Ca, K, Mg, Na, P, S and N. Then, the different surfactants and low molecular components are separated by adsorption chromatography after parts of low molecular components are dried in an oven, and the molecular weight distribution is measured by gel permeation chromatography also. Finally, the separated surfactants are determined by Fourier transform infrared (FTIR) spectrometry and nuclear magnetic resonance (NMR) spectroscopy. The distortionless enhancement by polarization transfer (DEPT), H, C correlated spectroscopy (H, C-COSY), H, H correlated spectroscopy (H, H-COSY) and heteronuclear multiple-bond correlation (HMBC) spectroscopy are applied to determine the molecular structures.     

Tumour-inhibiting platinum(II) complexes with aminoalcohol ligands: biologically important transformations studied by micellar electrokinetic chromatography, nuclear magnetic resonance spectroscopy and mass spectrometry

(SP-4-2)-Bis[(R)-(-)-2-aminobutanol-kappaN]dichloroplatinum(II) and (SP-4-2)-bis[(R)-(-)-2-aminobutanolato-kappa2N,O]platinum(II) are promising cytotoxic agents exhibiting a strongly pH-dependent rate of reaction with the DNA-modeling nucleotide guanosine 5'-monophosphate (GMP). This potential mode-of-action binding, directly correlating with cytotoxicity, is influenced by the intramolecular chelation of bifunctional aminoalcohol ligands which was examined by means of micellar electrokinetic chromatography (MEKC) and nuclear magnetic resonance (NMR). While NMR clearly proves the existence of equilibrium between the ring-opened and ring-closed species, no such transformation was observed under MEKC conditions. In a kinetic study performed by MEKC, the half-lives of GMP bound to the platinum complexes were determined and compared to the kinetic data acquired by capillary zone electrophoresis. An appreciable increase in binding in the presence of sodium dodecyl sulfate (SDS) micelles was explained in terms of activation of (SP-4-2)-bis[(R)-(-)-2-aminobutanol-kappaN]dichloroplatinum(II). This apparently takes place due to the shifting of the equilibrium towards the ring-opened species, induced by adduct formation between SDS and the platinum complex that was confirmed by electrospray ionization mass spectrometry.     

Mechanistic study of enantiomeric recognition with native gamma-cyclodextrin by capillary electrophoresis,reversed-phase liquid chromatography,nuclear magnetic resonance spectroscopy,electrospray mass spectrometry and circular dichroism techniques

The possible mechanisms for the chiral recognition of 2(S)-(3,5-bis-trifluoromethyl-phenyl)-2-[3(S)-(4-fluorophenyl)-4-(1H-[1,2,4]triazol-3-ylmethyl)-morpholin-2(R)-yloxy]-ethanol (compound A) and its enantiomer with native gamma-cyclodextrin (gamma-CD) were investigated using capillary electrophoresis (CE), reversed-phase liquid chromatography (RPLC), proton (1H), fluorine (19F) and carbon (13C) nuclear magnetic resonance spectroscopy (NMR), electrospray mass spectrometry (ESI-MS) and circular dichroism (CD). All experiments provided clear evidence of the formation of diastereomeric complexes between the enantiomers and gamma-CD. Proton, fluorine and carbon NMR spectra suggested that both aromatic rings, with mono-fluoro and bis-tri-fluoro functional groups, on the guest molecule were partially included into the cavity of the gamma-CD. ESI-MS spectra indicated that the diastereomeric complexes have a 1:1 stoichiometric ratio. The binding constants of the diastereomeric complexes obtained by CE, RPLC and CD were compared. The effects of the gamma-CD concentration, organic modifiers and temperature on the CE-chiral separation were also investigated.     

Chiral high-performance liquid chromatography of N-octyl bicycloheptene dicarboximide and confirmatory studies using liquid chromatography-tandem mass spectrometry and two-dimensional nuclear magnetic resonance spectroscopy

N-Octyl bicycloheptene dicarboximide (MGK 264) has exo and endo diastereomers. Each structure has a chiral center at the nitrogen side chain. Enantioselective separation of MGK 264 was achieved by normal-phase high-performance liquid chromatography (HPLC) using cellulose-based Chiralcel OD column with diode-array and optical rotation detectors. Peaks were isolated with the purpose of identifying their stereochemical structures. Molecular mass of the HPLC peaks and their structural information was determined by liquid chromatography-electrospray tandem mass spectrometry (LC-ES-MS-MS). A two-dimensional nuclear magnetic resonance (NMR) spectroscopic technique was used to establish the structural features. Correlation of the data obtained from chiral separation and NMR facilitated in unambiguous assignment of the HPLC peaks.     

Mass spectrometrically detected directly coupled high performance liquid chromatography/nuclear magnetic resonance spectroscopy/mass spectrometry for the identification of xenobiotic metabolites in maize plants

Reconstructed ion chromatograms have been used to identify relevant high performance liquid chromatography (HPLC) peaks in a directly coupled high performance liquid chromatography/nuclear magnetic resonance spectroscopy/mass spectrometry (HPLC/NMR/MS) experiment. This has been applied to a study of the metabolism of a model compound, 5-nitropyridone (2-hydroxy-5-nitropyridine), in maize plants grown hydroponically. By monitoring the on-flow reconstructed ion chromatogram corresponding to the 5-nitropyridone fragment at m/z 143, and additional molecular ions corresponding to metabolites identified as products from similar compounds, relevant peaks were identified rapidly for subsequent stopped-flow 1H NMR spectroscopic analysis. The combination of coupled HPLC/NMR/MS enabled the direct identification of three metabolites, namely the N-glucoside, N-malonylglucoside, and O-malonylglucoside. This work demonstrates the power of HPLC/NMR/MS for the structural elucidation of xenobiotic metabolites in complex biological matrices (such as plant material) with minimal sample preparation. In particular, using mass spectrometry for the initial identification of relevant HPLC peaks allows the analysis of complex samples without the necessity for other spectroscopic markers, such as 19F NMR signal for fluorinated compounds or UV spectroscopy for molecules with strong UV chromophores.     

Combined use of liquid chromatography with mass spectrometry and nuclear magnetic resonance for the identification of degradation compounds in an erythromycin formulation

A commercial erythromycin formulation containing erythromycin A (EA) as the major compound showed the presence of an unknown degradation compound that was co-eluted with erythromycin E (EE) in the European Pharmacopoeia (Ph. Eur.) liquid chromatographic (LC) method. The amount of the degradation compound increased with respect to time. To separate this unknown (UNK1), investigation was performed with different LC methods coupled to ultraviolet detection (LC-UV). With the present Ph. Eur. method, the degradation compound could not be well separated. However, with the most selective LC-UV method (XTerra method), two more degradation products (UNK2 and UNK3) were found in the formulation which could not be observed using other methods because of their poor separation. By combining the results obtained with LC-UV, LC/MS and LC/NMR, the degradation products were identified as pseudoerythromycin A hemiketal (PsEAHK), erythromycin A enol ether carboxylic acid and erythromycin C enol ether carboxylic acid. PsEAHK is known to be a base-catalysed degradation product of EA, whereas the other two degradation products were newly identified.     

On-line identification of the antifungal constituents of Erythrina vogelii by liquid chromatography with tandem mass spectrometry,ultraviolet absorbance detection and nuclear magnetic resonance spectrometry combined with liquid chromatographic micro-fractionation

In our continuing search for new antifungal agents of plant origin, the investigation of Erythrina vogelii Hook. f. (Leguminosae), a plant used in the traditional medicine of Ivory Coast to treat various infectious ailments, was undertaken. In order to rapidly identify the active principles, the crude extract was analysed by low-flow LC-1H nuclear magnetic resonance spectrometry (NMR) which gave a sensitive detection of all the main peaks. LC microfractionation was performed just after LC-NMR detection and all peaks collected were submitted to antifungal bioautography assays against Cladosporium cucumerinum. By this means, the antifungal activity could be efficiently linked to three of the LC peaks. In order to obtain complementary on-line structural information for all peaks of interest, high-resolution LC-MS-MS together with LC-UV with post-column addition of UV shifts reagents was undertaken on the crude extract. This chemical screening strategy with integrated antifungal bioassays has permitted the on-line identification of numerous constituents and has given useful information for an efficient peak-guided isolation procedure.     

Identification of anthocyanins in wines by liquid chromatography, liquid chromatography-mass spectrometry and nuclear magnetic resonance

Liquid chromatography (LC) was used for the fractionation of particular anthocyanins in glycoside form from methanol extracts of red grape skins and solid phase extracts of red wine. By the combination of nuclear magnetic resonance spectroscopy and LC-mass spectroscopy the identification of 13 anthocyanins in a particular LC fraction and hence the in particular peaks in chromatograms were obtained. Peaks areas in the chromatograms obtained under the semi-quantitaive conditions of the solid phase extracts of red wines Pinot Noir, Cabernet Sauvignon and Merlot from the Coastal wine-growing region in Slovenia, produced in 1999, were used as input data in chemometric analysis. The chemometric methods used were hierarchical clustering analysis and regularised discriminant analysis. The results of both methods give 100% correct classification of wines regarding the vine variety.     

Analysis of nucleotides binding to chromatography supports provided by nuclear magnetic resonance spectroscopy

The epitope mapping of nucleotides bound to three chromatography supports is accomplished using saturation transfer difference (STD)-NMR spectroscopy. This experiment involves subtracting a spectrum in which the support was selectively saturated from one recorded without support saturation. In the difference spectrum only the signals of the ligands that bind to the support and received saturation transfer remain. The nucleotide protons in closer contact with the support have more intense signals due to a more efficient transfer of saturation. We investigate the effects on the binding to the nucleotides by the introduction of a spacer arm between l-histidine and Sepharose. Our NMR experiments evidence a clear contribution of the spacer to the interaction with all the nucleotides, increasing the mobility of the amino acid and giving different STD responses. This enhanced mobility originates the reinforcement of the interactions with the sugar moiety and phosphate group of 5'-CMP and 5'-TMP or the base of 5'-GMP and 5'-UMP. Hence, with this study we show that by using STD NMR technique on chromatographic systems it is possible to provide a fast, robust and efficient way of screening the atoms involved in the binding to the supports.     

Determination of enantiomer separation factors by nuclear magnetic resonance spectroscopy and by chiral liquid chromatography

The equilibrium constants K+ and K- for formation of the diastereomeric complexes of the two enantiomers of O,O'-dibenzoyltartaric acid (DBTA) with the chiral selector N,N'-diallyltartardiamide bis-(4-tert.-butylbenzoate) (TBB) have been determined by 1H-NMR. The experiments were performed at different temperatures in CDCl3 or in cyclohexane-d12/2-propanol-d8 mixtures. The equilibrium constants from the 1H-NMR results have been compared with the retention factors (k') obtained from the chromatographic resolution of rac. DBTA on a Kromasil CHI-TBB column with the same solvents as mobile phases. A satisfactory correlation between the 1H-NMR data and the chromatographic data was found.     

High performance liquid chromatography coupled to nuclear magnetic resonance spectroscopy and mass spectrometry applied to plant products: Identification of ecdysteroids fromSilene otites

Summary HPLC coupled in parallel to nuclear magnetic resonance (NMR) and mass spectrometry (MS) has been used to obtain¹H NMR and mass spectra of a number of ecdysteroids present in an extract of the plantSilene otites. Reversed phase gradient chromatography was performed using a D₂0-acetonitrile-based solvent system. NMR and mass spectra were obtained for integristerone A, 20-hydroxyecdysone, 2-deoxy-20-hydroxyecdysone and 2-deoxyecdysone to provide structural confirmation using continuous and stopped flow HPLC-NMR. The combined HPLC-NMR-MS system described here provided a more comprehensive analysis of the ecdysteroids present in the extract than HPLC-NMR alone.     

Identification of derivatives of bisphenol A diglycidyl ether and novolac glycidyl ether in can coatings by liquid chromatography/ion trap mass spectrometry

A reversed-phase liquid chromatographic method combined with fluorescence and multiple mass spectrometric detection in series is presented for the separation and structure elucidation of bisphenol A diglycidyl ether (BADGE) and novolac glycidyl ether (NOGE) derivatives. Atmospheric pressure chemical ionization in the positive ion mode and collision induced fragmentation in the ion trap allowed identification of BADGE- and NOGE-related compounds originating from reactions of the glycidyl ethers with bisphenols, solvents, and chain stoppers. Two extracts from food-can coatings were investigated in detail. It was possible to elucidate the structures of many substances and consequently to draw conclusions about the production of the lacquers.