Hard modeling methods for the curve resolution of data from liquid chromatography with a diode array detector and on-flow liquid chromatography with nuclear magnetic resonance spectroscopy

Hard modeling methods have been performed on data from high-performance liquid chromatography with a diode array detector (LC-DAD) and on-flow liquid chromatography with 1H nuclear magnetic spectroscopy (LC-NMR). Four methods have been used to optimize parameters to model concentration profiles, three of which belong to classical optimization methods (the simplex method of Nelder-Mead, sequential quadratic programming approach, and Levenberg-Marquardt method), and the fourth is the application of genetic algorithms using real-value encoding. Only classical methods worked well for LC-DAD data, while all of the methods produced good results when LC-NMR data were divided into small spectral windows of peak clusters and parameters were optimized over each window.     

Identification of impurities in acarbose by using an integrated liquid chromatography-nuclear magnetic resonance and liquid chromatography-mass spectrometry approach

The usefulness of applying an integrated LC-NMR and LC-MS approach to acarbose bulk drug impurity profiling is demonstrated. LC-MS and LC-NMR methodologies were employed for the online separation and structural elucidation of a final drug product. Combining data provided by the stop-flow LC-NMR and LC-MS experiments made it possible to identify the main components present in the acarbose sample. Spectral analysis revealed that A and B were known impurities while C was an unknown compound. LC-MS and LC-NMR analyses revealed that C was a pentasaccharide differing from the acarbose in number and nature of sugar subunits in the molecule. It was subsequently isolated and its structure was confirmed by the offline 1- and 2-D NMR experiments, and atom assignment was made.     

An improvement of window factor analysis for resolution of noisy HPLC-DAD data

Window factor analysis (WFA) is a powerful tool in analyzing evolutionary process. However, it was found that window factor analysis is much sensitive to the noise involved in original data matrix. An error analysis was done with the fact that the concentration profiles resolved by the conventional window factor analysis are easily distorted by the noise reserved by the abstract factor analysis (AFA), and a modified algorithm for window factor analysis was proposed. Both simulated and experimental HPLC-DAD data were investigated by the conventional and the improved methods. Results show that the improved method can yield less noise-distorted concentration profiles than the conventional method, and the ability for resolution of noisy data sets can be greatly enhanced.     

液相色谱–核磁共振联用技术研究进展

介绍了液相色谱–核磁共振(LC–NMR)联用技术发展状况。讨论了LC–NMR技术应用中面临的问题和解决方法,评述了LC–NMR和LC–SPE–NMR两种工作模式。介绍了LC–NMR在天然产物分析、生物代谢、异构体的鉴定和多聚物分析领域的应用情况,对其发展动态进行了展望。     

The rapid identification of isoflavonoids from Belamcanda chinensis by LC-NMR and LC-MS

The use of hyphenated LC-NMR and LC-MS techniques for the purpose of directly identifying the major constituents of Belamcanda chinensis was investigated. Reversed-phase isocratic chromatography was performed using an acetonitrile-water solvent system on a C18 column. The NMR spectrum yielded five main peaks, whose analysis revealed them to be 5, 6, 7, 3'-tetrahydroxy-4'-methoxyisoflavone (1), tectorigenin (2), iristectorigenin A (3), irigenin (4), and irisflorentine (5). The identification of these constituents was confirmed by performing LC-ESI-MS experiment. This study shows that hyphenated LC-NMR and LC-MS can be used for the rapid (70 min) identification of the isoflavonoids.     

High-performance liquid chromatography with nuclear magnetic resonance detection applied to organosilicon polymers. Part 2. Comparison with other methods

LC-NMR utilizing (1)H and (29)Si NMR spectroscopy is ideally suited for the analysis of silicones. It is shown that reversed phase gradient LC-NMR surpasses standard gel permeation chromatography (GPC) and diffusion ordered spectroscopy (DOSY) in the analysis of model hydride terminated polydimethylsiloxane. (1)H and (29)Si NMR in the stopped-flow arrangement leads to full identification of the components. Concentration gradient introduces a dependence of the (29)Si shifts on solvent composition, this dependence can be substantially reduced by a proposed method of referencing. It is shown that the ADEQUATE version of powerful but insensitive 2D INADEQUATE experiment can be used for complete line assignment.     

LC-NMR coupling technology: recent advancements and applications in natural products analysis

An overview of recent advances in nuclear magnetic resonance (NMR) coupled with separation technologies and their application in natural product analysis is given and discussed. The different modes of LC-NMR operation are described, as well as how technical improvements assist in establishing LC-NMR as an important tool in the analysis of plant-derived compounds. On-flow, stopped-flow and loop-storage procedures are mentioned, together with the new LC-SPE-NMR configuration. The implementation of mass spectrometry in LC-NMR is also useful on account of the molecular weight and fragmentation information that it provides, especially when new plant species are studied. Cryogenic technology and capillary LC-NMR are the other important recent developments. Since the plant kingdom is endless in producing potential drug candidates, development and optimization of LC-NMR techniques convert the study of natural products to a less-time-consuming task, speeding up identification.     

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 (1H 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 alpha-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.     

Real-time 2D NMR identification of analytes undergoing continuous chromatographic separation

We have recently proposed and demonstrated an approach that enables the acquisition of multidimensional nuclear magnetic resonance (NMR) spectra within a single scan. A promising application opened up by this new accelerated form of data acquisition concerns the possibility of monitoring in real time the chemical nature of analytes subject to a continuous flow. The present paper illustrates such potential, with the real-time acquisition of a series of 2D 1H NMR spectra arising from a mixture of compounds subject to a continuous liquid chromatography (LC) separation. This real-time 2D NMR identification of chemicals eluted minutes apart under usual LC-NMR conditions differs from the way in which LC-2D NMR has hitherto been carried out, which relies on stopped-flow modes of operations whereby fractions are first collected and then subject to individual, aliquot-by-aliquot analyses. The real-time LC-2D NMR experiment hereby introduced can be implemented in a straightforward manner using modern commercial LC-NMR hardware, thus opening up immediate possibilities in high-throughput characterizations of complex molecules.     

(29)Si NMR in LC-NMR analysis of silicones

It is demonstrated using a practical example that indirect detection of (29)Si NMR signals is sufficiently sensitive in LC-NMR stop-flow arrangement to analyze mixtures of siloxane polymers. New cryogenic probes with better signal-to-noise ratio will turn this version of LC-NMR into a routine method for analysis of siloxane polymers.     

Phytochemistry in the microgram domain - a LC-NMR perspective

Plants represent an extraordinary reservoir of novel molecules and there is currently a resurgence of interest in the vegetable kingdom as a possible source of new lead compounds for introduction into therapeutical screening programs. In order to discover potential new bioactive natural products, the dereplication of crude plant extracts performed prior to isolation work is of crucial importance for avoiding the isolation of a known constituent. In this respect, chemical screening strategies have been developed using hyphenated techniques (LC/UV-DAD, LC-MS and LC-NMR). In our laboratory, these techniques have been fully integrated into the isolation process and are used for the chemical screening of crude plant extracts in complement with on-line or at-line bioassays. LC-UV-MS is used as a first dereplication step in combination with UV and MS databases, while LC-NMR is performed in a second step for de novo on-line structure determination. This approach enables the partial or the complete on-line identification of natural products in complex matrices such as crude plant extracts. These methods also give a unique possibility to study unstable compounds, which rapidly degrade or which are not separable at a preparative level.In the multi-hyphenated approach used (hypernation), LC-NMR plays a key role since it provides the most detailed structural information. The relatively low sensitivity of this technique, however, requires that strategies for high loading of plant extracts are developed and compromises for solvent selection have to be made. For more demanding experiments, at-line strategies based on the microfractionation of the LC-peak of interest and recording of spectra in fully deuterated solvents in microflow probes represent a promising alternative.     

High-performance liquid chromatography with nuclear magnetic resonance detection--A method for quantification of alpha- and gamma-linolenic acids in their mixtures with free fatty acids

While many naturally occurring mixtures of free fatty acids are conveniently analyzed by hyphenated technique of LC-NMR, a complete separation of alpha- and gamma-linolenic acids for their quantitative determination appears impossible at least by the methods of reversed phase HPLC. However, they can be differentiated and quantified from 1H NMR spectra measured in the course of isocratic acetonitrile-chloroform (90:10, with C8 and C18 columns in series) LC-NMR analysis without the need for any derivatization.     

Resolution of the essential constituents of Ramulus cinnamomi by an evolving chemometric approach

The analysis of complex mixtures, such as those of traditional Chinese medicine (TCM), is difficult by conventional methods of analysis. Chemometric methods provide a new way to solve such problems. Subwindow factor analysis (SFA) paired with the evolving window orthogonal projection (EWOP) method, has been used as a new evolving approach to the resolution of volatile components of Ramulus cinnamomi (RC). Compared with conventional chromatographic analysis, the chromatographic separation conditions necessary are greatly mitigated in our approach, yet the accuracy of qualitative and quantitative results is improved, because the measured data matrix has been resolved into chromatograms and mass spectra of the chemical components. Our method is, moreover, friendly to use and easy to program. Experimental results show the efficiency and convenience of the proposed approach. Forty-seven of the seventy-eight separated constituents in an essential oil, accounting for 89.55% of the sample, were identified by mass spectroscopy (MS).     

On the use of transition matrix methods with extended ensembles

Different extended ensemble schemes for non-Boltzmann sampling (NBS) of a selected reaction coordinate lambda were formulated so that they employ (i) "variable" sampling window schemes (that include the "successive umbrella sampling" method) to comprehensibly explore the lambda domain and (ii) transition matrix methods to iteratively obtain the underlying free-energy eta landscape (or "importance" weights) associated with lambda. The connection between "acceptance ratio" and transition matrix methods was first established to form the basis of the approach for estimating eta(lambda). The validity and performance of the different NBS schemes were then assessed using as lambda coordinate the configurational energy of the Lennard-Jones fluid. For the cases studied, it was found that the convergence rate in the estimation of eta is little affected by the use of data from high-order transitions, while it is noticeably improved by the use of a broader window of sampling in the variable window methods. Finally, it is shown how an "elastic" window of sampling can be used to effectively enact (nonuniform) preferential sampling over the lambda domain, and how to stitch the weights from separate one-dimensional NBS runs to produce a eta surface over a two-dimensional domain.     

Building-Up a Comprehensive Database of Flavonoids Based on Nuclear Magnetic Resonance Data

The improvements in separation and analysis of complex mixtures by LC-NMR during the last decade have shifted its emphasis from data acquisition to data analysis. For correct data analysis, not only high quality datasets are necessary, but adequate software and adequate databases for semi (or fully)-automated assignments of complex molecules are needed. Only by using NMR, when necessary in combination with MS, the identification of molecules, as present for example in natural products, can be achieved. Here we report on the ongoing efforts required for the construction of an NMR database of flavonoids, implemented for automated assignments of flavonoids. The procedure is demonstrated for a series of flavonoids.     

二维重叠色谱峰解析方法的比较

利用渐进因子分析方法(EFA)和固定尺寸移动窗口因子分析方法(FSMWEFA)与局部正交投影方法(LOPA)相结合,用于模拟的HPLC—DAD二维数据解析。通过比较谱峰部分重叠、光谱完全重叠、色谱拖尾3种情况的结果显示,在谱峰部分重叠和色谱拖尾两种情况下两种方法都可以较好的解析重叠峰。但是在光谱完全重叠时,前一种方法不能得出正确的结论,而后一种方法仍能得到满意的结果。     

Combination of matrix solid-phase dispersion extraction and direct on-line liquid chromatography-nuclear magnetic resonance spectroscopy-tandem mass spectrometry as a new efficient approach for the rapid screening of natural products: application to the total asterosaponin fraction of the starfish Asterias rubens

A combination of matrix solid-phase dispersion extraction (MSPD) and LC-NMR-MS hyphenation is proposed as a rapid screening method of natural products for unknown compounds. In this report, this new analytical approach is applied for the first time. MSPD represents a significant simplification compared to classical extraction procedures and is thus an excellent complement to the fast and powerful LC-NMR-MS: MSPD yields extracts suitable for LC-NMR-MS in one simple preparation step, while LC-NMR-MS yields a wealth of information in one single chromatographic run. The suitability of this technique to characterise glycosidic compounds in the molecular mass range of 1200 to 1400 a.m.u. is demonstrated. The information on the number of exchangeable protons provided by an additional back-exchange experiment proved to be particularly valuable for structural elucidation. The possibility of semi-quantitative LC-NMR measurements through methyl signals H(3)-18 and 19 of the steroidal skeleton is demonstrated and is ensuingly used to provide relative quantitative data of the steroid oligosaccharide fraction.     

Mobile phase compensation to improve NMR spectral properties during solvent gradients

A solvent compensation method based on flow injection analysis is used to obtain high quality nuclear magnetic resonance (NMR) spectra during solvent gradients. Using a binary solvent system containing D2O and CD3OD, NMR line broadening and chemical shift changes are observed with a 10% methanol per min solvent composition gradient. However, by creating a second equal but reverse gradient and combining the two solvent gradients before the NMR detector, the composition of solvent reaching the NMR flow cell is kept constant. We demonstrate a system using flow injection analysis of combining solvent gradients and show constant NMR spectral performance as a function of time as the combined flow has a constant solvent composition irrespective of the initial solvent gradient. Using this approach, methods can be developed to measure high quality NMR spectra during on-flow gradient LC-NMR experiments. The ultimate ability of this approach depends on the ability to compensate for the disturbance of the solvent gradient and reverse gradient by a pair of LC columns (the analytical and reverse gradient columns).     

交互移动窗口因子分析法(AMWFA)用于二维数据重叠色谱峰的比较分析

采用交互移动窗口因子分析法(AMWFA), 通过挖掘两个体系中的选择性信息, 获得了不同样本间的共有组分数, 还同时得到了各物质对应的光谱或质谱信息. 详细阐述了本法的原理和计算方法, 并用一个模拟的GC-MS 数据对方法进行了验证.