Hyphenated HPLC-NMR and its applications in drug discovery

Hyphenated HPLC-NMR is a fast growing technology, allowing rapid and detailed structural characterization of unknown mixtures. The technical aspects of the technology are reviewed on the basis of system configuration, operation, solvent suppression, HPLC and NMR optimization, and detection. The combined use of HPLC-NMR and HPLC-MS is also described and discussed. Various applications of HPLC-NMR and integrated HPLC-NMR-MS in drug discovery, especially in the separation and structure elucidation of drug impurities, reaction mixtures, degradation products, in vitro and in vivo metabolites, and combinatorial library samples, are illustrated.     

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.     

HPLC-SPE-NMR in pharmaceutical development: capabilities and applications

High-performance liquid chromatography-solid phase extraction-NMR spectroscopy (HPLC-SPE-NMR) has recently become commercially available and has been evaluated with regard to its applicability in a pharmaceutical environment. The addition of an automated SPE unit to an HPLC-NMR system for peak trapping results in an improved NMR signal-to-noise ratio (S/N) and also has other practical advantages. The trapping efficiency is shown to depend on compound polarity and is highest for compounds eluting late on reversed-phase HPLC systems. Multiple peak trapping further increases the S/N, again with the best results for less polar compounds. For polar compounds, multiple peak trapping resulted in no S/N gain as the amount of material retained on the SPE cartridge was equivalent to that from a single injection. When compared with conventional HPLC-NMR, a S/N gain of up to five-fold could be achieved for some compounds in a single trapping step. A major advantage of the technique is the independence of the chromatographic step from the NMR step, resulting in greater versatility than conventional HPLC-NMR in the HPLC solvents and NMR solvents that can be used. Practical applications from both drug metabolite and drug impurity identification are presented.     

Implementation of real-time two-dimensional nuclear magnetic resonance spectroscopy for on-flow high-performance liquid chromatography

Directly coupled HPLC-NMR has become a powerful tool for separation and structural elucidation of unknown compounds. However, there are only a few reports on application of on-flow two-dimensional (2D) NMR in HPLC-NMR. Here we present an alternative method for recording real-time 2D-NMR spectrum (total correlation spectroscopy, TOCSY) on a commercial HPLC-NMR system. The method is based on well-established Hadamard matrix for 2D-NMR frequency encoding. In addition, a modified/improved solvent suppression approach is incorporated. This makes it possible to carry out the experiment with both polar and gradient eluents, the widely used chromatographic conditions. The method is example using a synthesized mixture of three amino acids (His, Phe and Try) and a human urine sample. The method demonstrated here may be utilized for high-throughput structural or unknown component identification and fast dynamic study in a variety of applications.     

Observation of exchangeable protons by high-performance liquid chromatography-nuclear magnetic resonance spectroscopy and high-performance liquid chromatography-electrospray ionization mass spectrometry: a useful tool for the hyphenated analysis of natural products

The first high-performance liquid chromatography-nuclear magnetic resonance (HPLC-NMR) investigation of exchangeable protons of low-molecular-mass natural products is reported. Model alkaloids or crude plant extracts were dissolved in 2H2O-1H2O-MeCN (deuterium oxide-water-acetonitrile) or 2H2O-MeCN and, after direct injection or chromatographic separation, examined in a 60-microl NMR flow probe. Exchangeable amino protons initially detected by HPLC-electrospray ionization mass spectrometry were subsequently identified and investigated by stop-flow 1H-NMR, two-dimensional (2D) total correlation spectroscopy (TOCSY), and 2D nuclear Overhauser effect spectroscopy (NOESY). These experiments extend the applicability of HPLC-NMR for the investigation and structure elucidation of natural products.     

Polar compounds isolated from the leaves of Albertisia delagoensis (Menispermaceae)

Aqueous infusions of the leaves of the shrub Albertisia delagoensis (Menispermaceae) are used in South Africa in traditional Zulu medicine to alleviate a variety of symptoms, including fever, and intestinal problems. We report the analysis of such an aqueous extract using the HPLC-NMR technique. A number of polar compounds were identified, including proto-quercitol, nicotinic acid, allantoic acid, 3,4-dihydroxy-benzoic acid, phthalic acid and the aporphine alkaloid derivative roemrefidine. Allantoic acid and roemrefidine have been fully characterised by (1)H- and (13)C-NMR and mass spectrometry. Earlier reports of antiplasmodial activity of roemrefidine and of A. delagoensis extracts are correlated with this study and with the antipyretic properties of neutral aqueous extracts.     

HPLC-NMR在化学品检测中的应用前景

简要介绍高效液相色谱-核磁共振(HPLC-NMR)联用技术。综述了目前HPLC-NMR联用技术在药物代谢研究,天然产物分析以及食品分析等方面的应用。展望了HPLC-NMR在化学品分类鉴定中的应用前景。     

Real-time separation of natural products by ultrafast 2D NMR coupled to on-line HPLC

Hyphenated HPLC-NMR is an extremely efficient analytical tool, which makes it possible to perform on-flow experiments where 1D NMR spectra are obtained in real time as the analytes are separated and eluted from the chromatographic column. However, it is incompatible with multidimensional NMR methods that form an indispensible tool for the study of complex mixtures. Recently, Frydman and co-workers have proposed an ultrafast 2D NMR approach, where a complete 2D NMR correlation can be recorded in a single scan, thus providing a solution to the irreversibility of hyphenated techniques. This paper presents the first implementation of on-line ultrafast HPLC-NMR. Ultrafast COSY spectra are acquired every 12 s in the course of a chromatographic run performed on a mixture of natural aromatic compounds. The results, obtained on a commercial HPLC-NMR setup, highlight the generality of the ultrafast HPLC-NMR methodology, thus opening the way to a number of applications in the numerous fields in which HPLC-NMR forms a routine analytical tool.     

HPLC–MS determination of the oxidation products of the reaction between α- and β-pinene and OH radicals

Biogenic non-methane hydrocarbons such as isoprene, alpha-pinene, and beta-pinene, are emitted by forests in very large quantities. To evaluate the role of alpha- and beta-pinene and their contribution to the global production of trace gases and especially aerosol precursors, a study of the oxidation mechanism of alpha- and beta-pinene with hydroxyl radicals must be conducted.The degradation products of both monoterpenes with hydroxyl radicals were identified and quantified in a fast-flow reactor. The products were collected on a liquid-nitrogen trap coated with a 2,4-DNPH solution to which two internal standards (benzaldehyde-2,4-DNPH and tolualdehyde-2,4-DNPH) had been added. The collection method was based on the in situ conversion of aldehyde and/or ketone compounds to their 2,4-dinitrophenylhydrazone derivatives. The derivatives were analyzed by HPLC-MS using APCI(-). TIC chromatograms and mass spectral data for the various oxidation products are presented.For alpha-pinene, pinonaldehyde is the most important degradation product, with smaller amounts of acetone, formaldehyde, campholenealdehyde, and acetaldehyde. For beta-pinene, nopinone and formaldehyde are the most abundant products, of almost equal importance, whereas acetone and acetaldehyde are minor compounds.     

艾蒿柱层析馏分成分分析

将甲苯浸泡获得的艾蒿提取物置于硅土层析柱上,再用乙酸乙酯淋洗获得一个柱层析馏分。用气相色谱对该馏分进行分离,用质谱法和红外光谱法对分离组分进行了结构鉴定。共鉴定出20个纽分,其中倍半萜3个,单萜6个,在这9个萜类组分中,有3个是首次报道。同样,用液相色谱-质谱联用仪(LC—DAD-MSD)对该馏分进行了分析鉴定,结果显示这些组分中可能存在大量的萜类物质。     

Identification and evaluation of radiolysis products of irradiated chloramphenicol by HPLC-MS and HPLC-DAD

Summary The radiolysis products of chloramphenicol under γ-radiation sterilization were investigated systematically. Eight main radiolysis products were identified and quantified by HPLC-MS and HPLC-DAD, including two compounds never before reported. The minor radiolysis products were quantified, which shows that they are at concentration levels<threshold for identification. Carbon-carbon rupture reaction and oxidation reaction are proposed as the main radiolysis reactions of chloramphenicol powder. The applicability of γ-sterilization for chloramphenicol products was evaluated quantitatively with qualitative and quantitative data that were compared to the threshold requirements of international regulations for identification. It was concluded that toxicities of the radiolysis products of chloramphenicol produced by γ-radiation sterilization can be neglected, the radiolysis products are safe for human health from the chemical view.     

Molecular characterization of organically-bound sulfur in crude oils. A feasibility study for the application of Raney Ni desulfurization as a new method to characterize crude oils

Five crude oils with varying sulfur contents (0.1 – 4.7%) were characterized on a molecular level for organically-bound sulfur. Aromatic fractions were analyzed by GC-(MS) and asphaltene and polar fractions were analyzed by flash pyrolysis-GC-(MS). The polar fractions were also desulfurized with Raney Ni and the hydrocarbons formed were analyzed by GC-MS. Major sulfur compounds in the aromatic fractions were identified as alkylbenzo- and alkyldiben-zothiophenes. The flash pyrolyzates of the asphaltene contained alkylthiophenes and alkylbenzothiophenes as major compounds, depending on the thermal maturity of the oil. Generally, the sulfur-rich crude oils contained relatively more sulfur compounds. The flash pyrolyzates of polar fractions contained a variety of sulfur compounds (alkylthiolanes, alkylthianes, terpenoid sulfides, alkylbenzothiophenes) with substantial differences between different crude oils. Raney Ni desulfurization of the polar fraction yielded hydrocarbons dominated by n-alkanes, but isoprenoid alkanes, n-alkylcyclohexanes, mid-chain methylalkanes, tricyclic terpanes, hopanes and steranes were also present. These hydrocarbons show a potential to fingerprint crude oils since their distribution patterns are more characteristic than those of the hydrocarbons present in the saturated hydrocarbon fraction.     

HRGC-MS and HPLC-MS identification of new ketosterols in an extract of wheat straw

An ethereal extract of wheat (Triticum aestivum) straw with a positive biological assay for allelopathic activity, has been analyzed by HRGC. By means of HRGC-MS the extract composition was assessed as a complex mixture of organic acids and sterolic compounds. The sterolic fraction was subsequently separated and further studied by HRGC-MS and HPLC-MS. Among the thirty seven components identified, some described for the first time in wheat straw, two new ketostero's were characterized by means of their MS spectra. The potential of the particle-beam (PB) interface in HPLC-MS studies of unknown mixtures is exemplified and compared with that of HRGC-MS.     

毛细管电泳结合高效液相色谱-质谱用于天然产物活性成分的筛选

发展了毛细管电泳(CE)和高效液相色谱-质谱(HPLC-MS)相结合的用于天然产物中活性成分筛选和鉴定的方法。该方法中,用HPLC半制备柱对天然产物粗提物进行分离纯化,再用CE对HPLC纯化后的组分进行活性测试。根据HPLC-MS/MS提供的二级质谱数据,即可确定活性成分的化学结构。以乙酰胆碱酯酶为实验模型,对我们发展的筛选方法进行了验证。从黄连粗提物中确定了药根碱、巴马汀等7种活性成分,并通过CE测定了它们的半抑制率(IC₅₀)值。与传统的天然产物分离纯化和活性筛选方法相比,该方法具有简单、微量、快速、准确的优点。本文建立的方法为天然产物粗提物中活性成分的筛选提供了新技术。     

Identification of the oxidation products of the reaction between alpha-pinene and hydroxyl radicals by gas and high-performance liquid chromatography with mass spectrometric detection

In this paper an identification method is described for determining the degradation products of the reaction of alpha-pinene with hydroxyl radicals. The study is carried out in a fast-flow reactor equipped with a specially designed microwave cavity (type Surfatron) allowing to operate at pressures up to 100 Torr (1 Torr=133.322 Pa). The semivolatile products are collected on a liquid nitrogen trap (LN2 trap) and the batch samples are subsequently analysed by GC-MS and HPLC-MS. Some samples were also collected directly on a LpDNPH-cartridge, followed by HPLC-MS analysis. When experiments were carried out at 50 Torr both GC-MS and HPLC-MS measurements showed that campholenealdehyde and pinonaldehyde were identified as condensable oxidation products for the alpha-pinene/OH reaction, with pinonaldehyde being the main product. Furthermore, the LN2 trap collection method based on the in situ conversion of aldehydes and ketones to their 2,4-dinitrophenylhydrazone derivatives allowed the determination of formaldehyde, acetaldehyde and acetone. Although formaldehyde and acetone are present in small amounts in blank samples it could be established that formaldehyde and acetone are also formed in the alpha-pinene/OH reaction.     

Identification and Quantification of Polar Nitroaromatic Compounds in Explosive-Contaminated Waters by means of HPLC-ESI-MS-MS and HPLC-UV

In the area of former ammunition plants, contaminations caused by explosives and their degradation products are of great environmental relevance because of the immediate vicinity of ground- and drinking water reservoirs. Beside the determination of selected explosive-related compounds by means of HPLC with UV detection, a LC-MS-MS coupling utilizing electrospray ionisation was developed, which is particularly suited for the determination of highly polar compounds. Therewith, 12 different explosive-related compounds were identified and quantified on the basis of specific precursor-/ product-ion traces using the high selectivity and sensitivity of multiple reaction monitoring mode (MRM) of a triple quadrupole mass spectrometer. The LC-MS-MS-technique was applied to water samples from the area of a former ammunition plant. In addition to dinitrophenols and aminodinitrotoluenes which could be determined also by HPLC-UV, some highly polar compounds were found in the contaminated water, for example 3,5-dinitrobenzoic acid and 2-amino-4,6-dinitrobenzoic acid. The highest concentrations (14–40 μg L⁻¹) were found for two isomers of dinitrotoluenesulfonic acids. In order to assure the quantitative determination of polar substances in real samples with different composition, external calibration and standard addition method were compared. The presented LC-MS-MS-method is suited for screening and quantification of polar and hydrophilic explosive-related compounds from different classes of substances in a single analytical protocol.     

Analytical characterisation of glutardialdehyde cross-linking products in gelatine-gum arabic complex coacervates

Encapsulates having shells of cross-linked mixtures of proteins and polysaccharides are widely used in the food and pharmaceutical industry for controlled release of actives and flavour compounds. In order to be able to predict the behaviour and the release characteristics of the microcapsules, a better understanding of the nature and extent of the cross-linking reaction is needed. Several analytical techniques were applied for the characterisation of glutardialdehyde (GDA) cross-linked encapsulates made of gelatine and gum arabic. To allow the use of sensitive, high-resolution methods such as chromatography and mass spectrometry, the sample first had to be hydrolysed. In this way, a mixture of amino acids, small peptides and the cross-link moieties was obtained. High-resolution liquid chromatography coupled to high-resolution mass spectrometry (HPLC-MS) was applied to detect possible cross-link markers through a comparison of HPLC-MS mass-chromatograms obtained for cross-linked and non-cross-linked coacervates. HPLC-MS/MS was used to identify the species responsible for the differences. Cross-linking occurred between GDA molecules and lysine and hydroxylysine epsilon-amino groups, and up to eight cross-link products of different nature could be identified. They included pyridinium ions and Schiff bases, and also unreacted GDA condensation products. Next, based on the insight gained in the possible chemical structures present in the cross-link markers, methods for selective labelling of these functionalities were employed to allow easier detection of related reaction products. Both liquid chromatography (LC) and gas chromatography (GC) were used in these experiments. Unfortunately, these approaches failed to detect new cross-link markers, most likely as a result of the low levels at which these are present.     

Identification of highly polar nitroaromatic compounds in leachate and ground water samples from a TNT-contaminated waste site by LC-MS,LC-NMR,and off-line NMR and MS investigations

Leachate and ground water samples from a trinitrotoluene-contaminated waste disposal site near a former ammunitions plant in Stadtallendorf, Germany, were analyzed by liquid chromatography (LC)-mass spectrometry (MS) and LC-NMR hyphenated techniques to comprehensively characterize the range of highly polar nitroaromatic compounds. Wherever unknown components could not be identified by comparison with a multistandard, the spectroscopic data obtained on-line were used to make initial structure proposals, which were later confirmed by comparison with authentic reference materials. In those cases where reference materials were not commercially available, unknown compounds were isolated by HPLC cuts and their structures were elucidated by off-line NMR and MS investigations. A variety of previously unknown compounds, including nitrophenols, nitrobenzyl alcohols, methylnitrobenzoic acids, and hydroxynitrobenzoic acids, could be identified. The NMR and MS data are presented here. The main polar compounds were additionally quantified.     

Investigations by HPLC-electrospray mass spectrometry and NMR spectroscopy into the isomerisation of salinomycin

HPLC-MS studies have indicated that certain polyether ionophore veterinary drugs are prone to degradation when stored as water-methanol solutions at ambient temperature. Salinomycin and narasin were particularly susceptible, disappearing completely within weeks to produce more polar species, which were identified as isomers of the original compounds. Lasalocid appeared to be stable under such conditions. Structural elucidation of the principal ultimate salinomycin isomerisation product was achieved by 2D NMR spectroscopy. This indicated that the isomerisation process consists of the opening of the spiro rings in the salinomycin structure with the concomitant formation of a furan moiety. The MS data indicated that the isomers retain the ability to complex alkali metal ions and may therefore retain their pharmacological activity. These discoveries may have implications both for the development of legislation covering acceptable levels of polyether ionophore residues in foodstuffs and also for analytical protocols designed to detect them.     

Effort to improve the quantitative determination of oxidation and hydrolysis compound classes in edible vegetable oils.

This paper proposes an analytical method to evaluate the classes of products of polymerization, oxidation and hydrolysis as well as the polar compounds present in refined edible oils in a more reliable fashion. The polar compounds of a marketed refined peanut oil were analyzed by preparative gel permeation chromatography and the classes of substances corresponding to single chromatogram peaks were collected by means of a fraction collector, purified and used as standards for high-performance size-exclusion chromatographic analysis. The linearity of detector response, the precision and accuracy of the method for each class of compounds and for polar compounds were assessed. Another aim was to verify whether this method may be applied to other refined peanut oils and to edible vegetable oils in general, even of different botanical origin, using the standards that had already been prepared for that particular peanut oil. The results obtained showed that this was possible and the analytical method developed can be extended to the most common edible vegetable oils.