Hyphenated NMR Methods in Natural Products Research, Part 2: HPLC-SPE-NMR and Other New Trends in NMR Hyphenation

Table of Contents

Abstract

This review describes the principles and performance of a novel and highly promising hyphenated technique, HPLC-SPE-NMR, which is based on post-column analyte trapping by solid-phase extraction. The analytes are subsequently eluted from the SPE cartridges using deuterated solvents. This indirect HPLC-NMR hyphenation offers numerous advantages compared to direct HPLC-NMR methods. Multiple trapping leads to a dramatic increase of analyte amounts available for NMR, enabling acquisition of high-quality 2D NMR data within a short time. Other new developments, including combination of solenoidal coil capillary flow-probes with microflow HPLC, are also discussed. Fast extract dereplication using these techniques enables focusing of isolation efforts on truly novel and promising natural products, based on precise structural data obtained with crude extracts or fractions.

Key words

Nuclear magnetic resonance - liquid chromatography - LC-NMR - HPLC-SPE-NMR - hyphenated techniques - microcoils - cryoprobes

Introduction

In the first part of this review [1], the use of direct NMR hyphenation in natural products research has been discussed. The direct HPLC-NMR methods were defined as methods in which the NMR data acquisition is performed on the HPLC column eluate [1]. This implies either a direct connection between the NMR spectrometer and the HPLC apparatus, or use of storage capillaries in which the HPLC eluate is temporarily stored prior to acquisition of NMR data.

Many useful results related to natural products have been obtained using direct HPLC-NMR methods [1] and the technique has been reviewed at various stages of its development [2], [3], [4], [5], [6], [7], [8], [9], [10], [11], [12], [13], [14], [15], [16], [17], [18]. Pharmaceutical, biomedical and other applications of NMR hyphenation have also been extensively reviewed [19], [20], [21], [22], [23], [24], [25], [26], [27], [28], [29], [30]. However, the direct combination of state-of-the-art separation technology with the most powerful and versatile tool for structure elucidation of organic molecules in solution suffers from various practical problems. In short, these practical limitations are due to somewhat contradictory requirements of HPLC and NMR with respect to solvents and time-scale of the experiment, and thus compromises are necessary in order to hyphenate these two techniques. For example, ¹H-NMR spectra are preferably acquired in deuterated solvents, but use of deuterated solvents for HPLC is expensive and the selection is limited. Some useful HPLC eluents, such as buffers with high ionic strengths, are directly undesirable in direct HPLC-NMR hyphenation. Gradient elution of HPLC columns, required in most separations, implies constantly changing magnetic susceptibility and field homogeneity in the NMR flow-probe, not to mention variations of analyte chemical shifts with solvent composition.

On the other hand, the limited sensitivity of NMR detection means that HPLC-NMR experiments are usually acquired in the stopped-flow mode [1]. This can result in difficulties caused by diffusion and can limit the possibility of obtaining 2D NMR data to one peak per chromatogram. The latter difficulty is circumvented by use of capillary loops for temporary storage of chromatographic peaks [1]; however, only a few studies relying on the loop storage technique have been reported so far [1]. To sum up, the outcome of a direct HPLC-NMR experiment is largely determined by chromatography, both in terms of the solvent used and because analyte from one injection only is available for NMR.

The recently introduced automated HPLC-SPE-NMR technique makes use of an entirely different approach to HPLC-NMR. Here, chromatographic peaks eluted from a reversed-phase HPLC column are passed one by one through small solid-phase extraction columns (SPE cartridges) in order to remove the analyte from the HPLC mobile phase. The SPE cartridges are subsequently dried with nitrogen gas, and the analytes desorbed with a deuterated solvent for NMR spectroscopy. The whole process is carried out automatically under software control (Fig. [1]).

Fig. 1 Schematic description of an HPLC-SPE-NMR system. After separation of the mixture components on a reversed-phase HPLC column with non-deuterated eluents, the chromatogram is recorded using a photodiode array detector, a mass spectrometer, or both. The HPLC column eluate is then diluted with water and the analytes present in the chromatographic peaks are trapped on individual solid-phase extraction (SPE) cartridges. The trapping is governed by signals from the PDA or the MS detector. The SPE cartridges are then dried with a stream of nitrogen gas, and the analytes eluted with a deuterated solvent into the NMR flow-probe.

Early Combinations of Post-Column SPE with HPLC-NMR

Solid-phase extraction is a standard method of sample preparation and analyte concentration prior to HPLC analysis [31]. Such analyte pre-concentration by SPE can also be combined on-line with HPLC-NMR analysis; for example, de Koning et al. used an SPE-HPLC-NMR experiment for the analysis of hop bitter acids [32].

The HPLC-SPE-NMR technique, where SPE is used after the HPLC separation, has its roots in the early phase-system switching (PSS) technique developed to solve mobile-phase and flow-rate incompatibilities in HPLC-MS. Kokkonen and colleagues were apparently the first to use post-column addition of water to the HPLC eluate in order to diminish the eluting power of the mobile phase and withhold the analyte on a trapping column prior to desorption to the MS interface [33]. Silica-based C₈ material was used in this HPLC-SPE-MS experiment both in the analytical and in the trapping columns [33]. With respect to NMR applications, Wilcox and Phelan reported already in 1986 a procedure consisting of a reversed-phase HPLC separation, collection of HPLC fractions using an automatic fraction collector, dilution of the fractions with water, solid-phase extraction of the diluted fractions using C₁₈ SPE cartridges, and elution of the analyte from the cartridges for ¹H-NMR at 250 MHz [34]. This off-line sequence of HPLC, SPE and NMR operations can be regarded as a prototype of modern HPLC-SPE-NMR experiments.

Griffiths and Horton [35] applied the concept of analyte concentration on a trapping column after water dilution of analytical column eluate [33] in connection with on-line NMR analysis. The analytes were desorbed from the trapping column with a back-flush of CD₃CN, but without prior drying of the trapping column [35]. Griffiths and Horton demonstrated that column trapping of chromatographic peaks leads to increased solute concentrations resulting in increased signal-to-noise ratios in the NMR spectra, showed the advantage of the use of deuterated solvents for analyte back-flush, and pointed out the similarity of their concept to the already introduced loop storage system [35]. The system of Griffiths and Horton can be regarded as an immediate forerunner of the state-of-the-art HPLC-SPE-NMR installations (Fig. [1]). Later, Nyberg et al. used SPE-NMR hyphenation, transferring analytes from SPE cartridges with a system of switching valves to the NMR flow-probe with CD₃OD, whereas the separation of sample constituents (Capsicum chinense fruit extract or Quillaja saponaria saponin fraction) was performed off-line [36], [37].

Advantages of HPLC-SPE-NMR Hyphenation

The signal-to-noise ratio in NMR spectra increases linearly with analyte amount but only with the square root of acquisition time. This emphasizes the importance of maximizing analyte amounts in the sensitive region of the NMR flow-cell. The sensitivity of detection in direct HPLC-NMR experiments will also depend on the ratio between the active volume of the NMR flow-cell and the peak elution volume (flow rate × peak width). Thus, in the majority of stopped-flow HPLC-NMR experiments performed with conventional columns with internal diameter of 4.6 mm or larger [1], only a small fraction of the analyte eluted from the column contributes to the NMR signal. For example, it can be calculated that for a Gaussian chromatographic peak with a standard deviation of 0.2 min (and hence the base-width of 0.8 min), eluted at a flow rate of 1 mL/min, the maximal percentage of the analyte present in the sensitive region of an NMR flow-probe is 23.6 %, 11.9 % or 6.0 % for flow-probes with sensitive volumes of 120, 60 or 30 μL, respectively. If the peak-width is reduced four-fold (peak standard deviation 0.05 min and the base-width 0.2 min), the percentage of the analyte present at the center of the peak in the above-mentioned sensitive volumes increases to 77.0 %, 45.2 % and 23.6 %, respectively. This emphasizes the importance of sharp peaks in direct HPLC-NMR experiments.

On the other hand, if the analyte can be trapped on an SPE cartridge quantitatively and eluted with about 30 μL of a solvent, most of the analyte will enter the NMR flow-cell and contribute to the NMR signal if a 30-μL flow-cell is used. In such a case, there will be an additional advantage of intrinsically higher mass sensitivity of the small-volume flow-cell. The only diminishing factor left will be the ratio between the sensitive volume and the total volume of the NMR cell (equal to about œ). This focusing of the analyte band is the basis of the HPLC-SPE-NMR technique. Additional benefits come from the use of pure deuterated solvents for cartridge elution, which will render solvent suppression easier or even unnecessary. Last but not least, multiple peak trapping may give a very substantial increase of the analyte amount available for NMR.

Standard HPLC-SPE-NMR installations (Fig. [1]) use 2 × 10 mm SPE trapping cartridges with a dead volume of about 30 μL that focus analytes eluted from standard HPLC columns into an NMR probe with a sensitive volume of 30 μL. Polystyrene- and/or divinylbenzene-based resins or C₁₈-bonded silica is normally used as the SPE material, and CD₃CN, CD₃OD or CDCl₃ as the eluting solvent. The prerequisites for successful operation of the HPLC-SPE-NMR system are 1) that the analyte is essentially quantitatively retained on the SPE cartridge, and 2) that it is quantitatively stripped from the cartridge as a sharp band, essentially with the solvent front. In order to increase affinity of compounds eluted from reversed-phase HPLC columns to the SPE material, dilution of the eluate with water is used prior to passage through the SPE cartridges, and the ratio of the post-column dilution is an adjustable parameter for optimization of HPLC-SPE-NMR experiments. Other post-column eluate modifications, such as pH adjustment, are in principle possible but have not yet been reported in conjunction with HPLC-SPE-NMR. Although a number of successful applications of the HPLC-SPE-NMR approach have been published (see below), quantitative studies of trapping and elution efficiency of various classes of natural products have yet to be reported.

Applications of HPLC-SPE-NMR

The first published study utilizing the automated HPLC-SPE-NMR technique described the identification of several flavonoids, rosmarinic acid and carvacrol in crude acetone extract of oregano using 1D ¹H-NMR spectra as well as TOCSY, HMQC and HMBC experiments [38]. The HPLC separation was performed on a 150 × 4.6 mm C₁₈ column, analytes were trapped on a divinylbenzene-type polymer, and the spectra recorded with a cryogenic probe-head [38]. Other published studies describe identification of radical-scavenging flavonoids from extracts of Rhaponticum carthamoides (Asteraceae) separated on a 100 × 7.8 mm C₁₈ column and employing polystyrene resin SPE cartridges [39], and structure determination of coumaric esters of iridoid glycosides from a purified fraction of Harpagophytum procumbens (Pedaliaceae), separated on a 250 × 2 mm C₁₈ column and trapped on C₁₈-silica or poly(divinylbenzene) [40]. The latter work demonstrated the complexity of trapping efficiency and analyte recovery as a function of SPE stationary phase and eluting solvent [40].

Subsequent reports described the identification of flavonoids and cardenolides from various parts of Kanahia laniflora (Apocynaceae) separated on a 150 × 4.6 mm C₁₈ column and trapped on C₁₈ SPE cartridges [41], and identification of 17 compounds, mainly isoflavonoids, present in a crude extract of Smirnowia iranica (Fabaceae) roots [42]. In the latter study, a linear accumulation of an isoflavan on C₁₈ SPE material in seven repeated trappings was demonstrated [42], illustrating the advantages of the multiple trapping approach; similar multiple trapping with a coumarin present in the root extract of Croton membranaceus (Euphorbiaceae) showed that poly(divinylbenzene) SPE material is more effective than C₁₈ material under the conditions used [43] (Fig. [2]). Moreover, inversion recovery experiments showed that the relaxation times determined in CD₃CN, a commonly used solvent for cartridge elution, are much longer than those in CDCl₃ [43]. Thus, not only SPE trapping and elution parameters, but also NMR acquisition parameters should be carefully optimized on a case-to-case basis for optimal performance of hyphenated NMR experiments. Constituents of a phenolic fraction of olive oil [44], lignans from Phyllanthus urinaria (Euphorbiaceae) [45], and oxygenated diterpenes from Rosmarinus officinalis (Labiatae) [46] were also analyzed using HPLC-SPE-NMR.

At present, the HPLC-SPE-NMR technique has been demonstrated to work well with many natural products classes including flavonoids, flavonoid glycosides and other glycosides, iridoids, various groups of terpenoids, simple phenolics, lignans and other phenylpropanoids, cardenolides, and alkaloids [38], [39], [40], [41], [42], [43], [44], [45], [46], [47]. Moreover, applications related to water and soil analysis [48] and drug metabolism [49], [50] have been reported. Xu and Alexander used HPLC-SPE-NMR in conjunction with a 150 × 10 mm C₁₈ column to concentrate trace constituents for NMR analysis; since SPE trapping necessitates post-column water addition to the column eluate, a high-capacity retention and mixing device was introduced in order to achieve a sufficient dilution ratio of the high eluent flow (5 mL/min) emerging from the column used [51].

Thus, even though the number of studies utilizing HPLC-SPE-NMR is still rather limited compared to those employing direct HPLC-NMR methods [1], its advantages are clearly apparent. The combined benefits of analyte concentration and accumulation and use of deuterated solvents enable acquisition of high quality 2D NMR data from multiple peaks, including minor peaks present in very complex chromatograms, without compromising the chromatographic separation process. Access to less-sensitive 2D NMR experiments such as NOESY/ROESY and availability of ¹³C chemical shifts (from HSQC and HMBC experiments) allow rigorous structure elucidation of rather complex natural products directly from extracts and mixtures (Figs. [3] and [4]). The large analyte amounts available by multiple trapping allow the recording of NMR spectra with excellent signal-to-noise ratios without solvent peak suppression, increasing the robustness of the experiment and eliminating possible suppression artifacts (Fig. [5]). The fact that chemical shift data are obtained in well-defined solvents (as opposed to mixed HPLC solvents) opens the possibility for the use of databases for computer-aided spectra interpretation.

For these reasons, HPLC-SPE-NMR is envisaged to be a prime future tool for extract dereplication. Cryogenically cooled probes result in a further sensitivity gain and were applied in several of the above-mentioned studies [38], [49]. The HPLC-SPE-NMR studies reported thus far have been performed using 400 MHz [38], [39], [45], [46], 500 MHz [49], [51] or 600 MHz magnets [40], [41], [42], [43], [44], [48], [50], but an increase in routinely available field strengths is expected as the cost of high-field magnets drops. No systematic comparisons have yet been published, but 30-μL (active volume) flow-probes together with 2 × 10 mm SPE cartridges are believed to be preferable and were used in most of the aforementioned investigations [38], [40], [41], [42], [43], [44], [50].

Obviously, the success of HPLC-SPE-NMR experiment depends on trapping efficiency and very polar compounds are not expected to be trapped on reversed-phase materials. Also, very apolar compounds may be difficult to elute from the SPE cartridges with CD₃CN [50] and the use of other solvents such as CDCl₃ may be necessary. However, most classes of natural products appear to have suitable trapping and elution properties and even alkaloids eluted from HPLC columns with acid-containing eluents as charged species were trapped on polystyrene-type cartridges [47]. Moreover, an extension of the range of SPE materials used for HPLC-SPE-NMR experiments and further optimization of trapping parameters [52] as well as of flow-cell design can be envisaged.

Fig. 2 Multiple trapping in HPLC-SPE-NMR experiments. Top: HPLC chromatogram of a crude extract of Smirnowia iranica roots (150 × 4.6 mm C₁₈ column eluted with CH₃CN gradient in H₂O). Center: Linear improvement of ¹H-NMR signal-to-noise ratio during seven trappings of the minor peak 10 from the above chromatogram on a 2 × 10 mm C₁₈ SPE cartridge. Bottom: Illustration of preferred trapping of a coumarin on poly(divinylbenzene) SPE cartridges (filled circles) as compared to C₁₈ SPE cartridges (open circles). All spectra were recorded at 600 MHz and in CD₃CN, using a 30-μL flow-probe. Reproduced from [42] and [43] by permission of the American Chemical Society and John Wiley and Sons, respectively.

Fig. 3 1D ¹H-NMR spectrum (top) and HSQC spectrum (bottom) of a 5α-cardenolide present in Kanahia laniflora. The spectra were recorded in the HPLC-SPE-NMR mode following eight trappings on a 2 × 10 mm C₁₈ SPE cartridge (150 × 4.6 mm C₁₈ column eluted with CH₃CN gradient in H₂O, 600 MHz, 30-μL cell). Reproduced from [41] by permission of the American Chemical Society.

Fig. 4 HMBC spectrum recorded in the HPLC-SPE-NMR mode from an extract of Rosmarinus officinalis (Lamiaceae) following single trapping on a 2 × 10 mm polystyrene-type SPE cartridge (150 × 4.6 mm C₁₈ column eluted with CH₃CN gradient in H₂O, 400 MHz, 120-μL cell, cartridge eluted with CDCl₃). Reproduced from [46] by permission of Elsevier.

Fig. 5 600 MHz ¹H-NMR spectrum obtained in the HPLC-SPE-NMR mode of a diterpene present in the ethyl acetate extract of roots of Perovskia atriplicifolia (Lamiaceae). The crude extract (1.5 mg per injection) was separated on a 150 × 4.6 mm C₁₈ column with CH₃CN gradient in H₂O. The arrow points to the peak analyzed (six trappings on a 2 × 10 mm C₁₈ SPE cartridge, elution with CD₃CN, 30-μL cell NMR flow-cell, no solvent peak suppression). Unpublished work from the author’s laboratory.

Hyphenation of Other Techniques with HPLC-SPE-NMR

Standard HPLC-SPE-NMR installations include a photodiode array detector monitoring HPLC separation and triggering SPE trappings (Fig. [1]), and hence the technique is sometimes referred to as HPLC-UV-SPE-NMR or HPLC-DAD-SPE-NMR. Thus, UV spectra are available along with the NMR data.

Already in the case of direct HPLC-NMR hyphenations [1], most of reported studies are supported by mass spectrometry, either using a separate HPLC-MS installation, or by introducing a split in the effluent flow and acquiring MS data simultaneously with the NMR data (HPLC-NMR-MS). It is of course possible to combine MS on-line with HPLC-SPE-NMR [38], [40], [49] (Fig. [1]). This extended hyphenation is useful not only by making MS or MS/MS data available simultaneously with the NMR data, but also because trapping of non-UV-absorbing constituents can be triggered by signals provided by MS (total ion or single ion monitoring). Furthermore, it can be mentioned that evaporative light scattering detection (ELS), already used in connection with stopped-flow HPLC-NMR [53], could be used to trigger trapping in a putative HPLC-ELS-SPE-NMR experiment.

In principle, other spectroscopic techniques such as CD [16], [54], [55], [56], or for special applications IR [57], might be hyphenated with HPLC-SPE-NMR. Chirality determination of natural products is especially important and CD spectra recorded with SPE-eluted material would benefit from increased concentration, use of pure solvents and the possibility of calibration of analyte concentration from the NMR response. Hyphenation of bioassays with HPLC-SPE-NMR is another exciting possibility [46].

Other New Trends in NMR Hyphenation

In an attempt to increase the sensitivity of NMR data acquisition, attention has been directed towards miniaturization of NMR coil design [58]. Small probe-volumes facilitate enclosing of a larger proportion of the total sample volume within the sensitive region of the probe (improved observe factor) and achieving maximal filling of the coil detection volume with the sample (improved filling factor). Moreover, the sensitivity per mass unit of miniaturized Helmholtz (saddle) coils used for vertical NMR tubes as well as vertical flow-cells used for direct HPLC-NMR hyphenation and for HPLC-SPE-NMR increases with reduction of coil length and diameter [59]. For these reasons, the 30-μL (sensitive volume) flow-probes used for HPLC-SPE-NMR experiments have a higher mass sensitivity than the 120-μL probes normally used for on-flow and stopped-flow HPLC-NMR.

Vertical capillary probes (with Helmholtz coils) having sensitive volumes down to 2.5 μL have already been used for some time instead of normal NMR tubes [58], [60]. However, additional sensitivity gain can be achieved with probes consisting of a solenoid wrapped around a horizontal capillary [58], [59], [61], [62]. The sensitivity of such probes is inversely proportional to the coil diameter and very short radiofrequency pulses at low power are possible with these microcoils, which can be as small as 50 μm in diameter.

The superior performance of solenoidal microcoils for the identification of natural products has been demonstrated using direct analyte injection [63], [64], [65], [66]. However, the main potential of microcoils is in their combination with microflow liquid chromatography, resulting in capillary HPLC-NMR [67], [68], [69], [70], [71], [72], [73], [74]. Stopped-flow capillary HPLC-NMR was recently used for the identification of isoflavonoids in plant material [75]. Use of microcoil NMR detectors together with microbore or capillary HPLC separations, which operate at considerably higher analyte concentrations in elution bands, is a very promising new development within HPLC-NMR that enables high-throughput applications [76]. Further improvements are expected from use of micromachined coils [77] and multiple coil probes [69], [78], [79]. Miniaturization, SPE interfaces as well as the already mentioned cryogenic probe technology [80], [81], [82] are expected to continue to improve the sensitivity of HPLC-NMR experiments. Both the cryoprobes and the microcoil probes show a capability of making direct observation of ¹³C NMR spectra (Fig. [6]) feasible with analyte amounts that can be achieved in the HPLC-NMR mode, especially when combined with SPE. In addition, microcoils facilitate NMR detection in electro-driven capillary separations [83], [84], [85], [86], [87].

Since analyte quantification is an important aspect of NMR hyphenation, use of the ERETIC method (Electronic REference To access In vivo Concentrations), originally introduced for internal calibration of in vivo NMR spectra [88], is of interest. The method, which relies on the generation of a synthetic reference signal with defined frequency and amplitude, was subsequently adapted for solution studies [89], [90], [91], and can be used as a means of quantification of HPLC-NMR data.

Another new development is the use of ultra-fast (single scan) acquisition of 2D NMR data relying on spatial encoding of spin interactions [92], [93], [94]; although this approach suffers from serious sensitivity limitations, its applicability for macro-scale on-flow LC-NMR has been demonstrated [95].

Fig. 6 Direct ¹³C observation using solenoidal capillary probe (cell volume 5 μL, sensitive volume 1.5 μL, 330 μg of menthol, 150 MHz observation frequency); broad signals between 80 - 120 ppm are probe background. Reproduced from [71] by permission of the American Chemical Society.

Conclusions

The HPLC-SPE-NMR technique is a very powerful tool for on-line extract dereplication, enabling routine access to high-quality 2D NMR data without distortion of the HPLC separation of the remaining extract constituents. In contrast to direct HPLC-NMR experiments, the amount of analyte available for NMR studies is not limited to that provided by a single injection to the HPLC column. The broad applicability of the technique to natural products has already been demonstrated, and the technique is envisaged to be an increasingly important analytical platform for natural products research. HPLC-SPE-NMR and other modern, hyphenated NMR techniques are expected to provide an increasing number of rigorously determined natural product structures directly from extracts and isolates, including compounds present in minor amounts. These developments will enable high-throughput extract analysis for the selection of those of extract constituents that deserve isolation and purification on a preparative scale, and in perspective on-line combinations of separation, automatic structure elucidation and bioassays.

References

• 1 Jaroszewski J W. Hyphenated NMR methods in natural products research part 1: direct hyphenation.  Planta Med. 2005;  71 691-700
• 2 Hostettmann K, Wolfender J L, Rodriguez S. Rapid detection and subsequent isolation of bioactive constituents of crude plant extracts.  Planta Med. 1997;  63 2-10
• 3 Wolfender J L, Ndjoko K, Hostettmann K. LC/NMR in natural products chemistry.  Curr Org Chem. 1998;  2 575-96
• 4 Wolfender J L, Rodriguez S, Hostettmann K. Liquid chromatography coupled to mass spectrometry and nuclear magnetic resonance spectroscopy for the screening of plant constituents.  J Chromatogr A. 1998;  794 299-316
• 5 Hostettmann K, Terreaux C. Search for new lead compounds from higher plants.  Chimia. 2000;  54 652-7
• 6 Wolfender J L, Terreaux C, Hostettmann K. The importance of LC-MS and LC-NMR in the discovery of new lead compounds from plants.  Pharm Biol Suppl. 2000;  38 41-51
• 7 Bobzin S C, Yang S, Kasten T P. Application of liquid chromatography-nuclear magnetic resonance spectroscopy to the identification of natural products.  J Chromatogr B. 2000;  748 259-67
• 8 Bobzin S C, Yang S, Kasten T P. LC-NMR: a new tool to expedite the dereplication and identification of natural products.  J Ind Microbiol Biotechnol. 2000;  25 342-5
• 9 Vogler B, Spring O. Application of LC-NMR measurements in phytochemical analysis.  Recent Research Develop Phytochemistry. 2000;  4 207-22
• 10 Hostettmann K, Wolfender J L, Terreaux C. Modern screening techniques for plant extracts.  Pharm Biol Suppl. 2001;  39 18-32
• 11 Wolfender J L, Ndjoko K, Hostettmann K. The potential of LC-NMR in phytochemical analysis.  Phytochem Anal. 2001;  12 2-22
• 12 Sanvoss M. Application of LC-NMR and LC-NMR-MS hyphenation to natural products analysis.  In: On-line LC-NMR and related techniques. Albert K, editor Chichester; Wiley & Sons; 2002.: p. 111-28.
• 13 Hostettmann K, Marston A. Twenty years of research into medicinal plans: results and perspectives.  Phytochem Rev. 2002;  1 275-85
• 14 Wolfender J L, Ndjoko K, Hostettmann K. Liquid chromatography with ultraviolet absorbance-mass spectrometric detection and with nuclear magnetic resonance spectroscopy: a powerful combination for the on-line structural investigation of plant metabolites.  J Chromatogr A. 2003;  1000 437-55
• 15 Wolfender J L, Ndjoko K, Hostettmann K. Application of LC-NMR in the structure elucidation of polyphenols.  In: Methods in polyphenol analysis. Santos-Buelga C, Williamson G, editors Cambridge; The Royal Society of Chemistry; 2003.: p. 128-56.
• 16 Bringmann G, Lang G. Full absolute stereostructures of natural products directly from crude extracts: the HPLC-MS/MS-NMR-CD ‘triad’.  Prog Mol Subcell Biol. 2003;  37 89-116
• 17 Urban S, Separovic F. Developments in hyphenated spectroscopic methods in natural product profiling.  Front Drug Des Discov. 2005;  1 113-66
• 18 Exarchou V, Krucker M, van Beek T A, Vervoort J, Gerothanassis I P, Albert K. LC-NMR coupling technology: recent advancements and applications in natural products analysis.  Magn Reson Chem. 2005;  43 681-7
• 19 Albert K. On-line use of NMR detection in separation chemistry.  J Chromatogr A. 1995;  703 123-47
• 20 Korhammer S A, Bernreuther A. Hyphenation of high-performance liquid chromatography (HPLC) and other chromatographic techniques (SFC, GPC, GC, CE) with nuclear magnetic resonance (NMR): a review.  Fresenius J Anal Chem. 1996;  354 131-5
• 21 Lindon J C, Nicholson J K, Wilson I D. The development and application of coupled HPLC-NMR spectroscopy.  Adv Chromatogr. 1996;  36 315-82
• 22 Albert K. Liquid chromatography-nuclear magnetic resonance spectroscopy.  J Chromatogr A. 1999;  856 199-211
• 23 Albert K, Dachtler M, Glaser T, Händel H, Lacker T, Schlotterbeck G. et al . On-line coupling of separation techniques to NMR.  J High Resolut Chromatogr. 1999;  22 135-43
• 24 Lindon J C, Nicholson J K, Wilson I D. Directly coupled HPLC-NMR and HPLC-NMR-MS in pharmaceutical research and development.  J Chromatogr B. 2000;  748 233-58
• 25 Wilson I D. Multiple hyphenation of liquid chromatography with nuclear magnetic resonance spectroscopy, mass spectrometry and beyond.  J Chromatogr B. 2000;  892 315-27
• 26 Albert K. , editor .On-line LC-NMR and related techniques. Chichester; Wiley & Sons; 2002.
• 27 Corcoran O, Spraul M. LC-NMR-MS in drug discovery.  Drug Discov Today. 2003;  8 624-31
• 28 Elipe M VS. Advantages and disadvantages of nuclear magnetic resonance spectroscopy as a hyphenated technique.  Anal Chim Acta. 2003;  497 1-25
• 29 Cardoza L A, Almeida V K, Carr A, Larive C K, Graham D W. Separation coupled with NMR detection.  Trends Anal Chem. 2003;  22 766-75
• 30 Wann M H. Application of LC-NMR in pharmaceutical analysis.  In: Handbook of pharmaceutical analysis by HPLC. Ahuja S, Dong MV, editors Amsterdam; Elsevier; 2005. Vol. 6: p. 569-79.
• 31 Pyrzynska K, Pobozy E. On-line coupling of solid phase extraction sample processing with high-performance liquid chromatography.  Crit Rev Anal Chem. 2002;  32 227-43
• 32 De Koning J A, Hogenboom A C, Lacker T, Strohschein S, Albert K, Brinkman U AT. On-line trace enrichment in hyphenated liquid chromatography-nuclear magnetic resonance spectroscopy.  J Chromatogr A. 1998;  813 55-61
• 33 Kokkonen P S, Niessen W MA, Tjaden U R, van der Greef J. Phase-system switching in liquid chromatography/continuous-flow fast-atom-bombardment mass spectrometry.  Rapid Commun Mass Spectrom. 1991;  5 19-24
• 34 Wilcox C D, Phelan R M. The use of solid-phase extraction columns to effect simple offline LC/MS, LC/NMR, and LC/FTIR.  J Chromatogr Sci. 1986;  24 130-3
• 35 Griffiths L, Horton R. Optimization of LC-NMR. III. Increased signal-to-noise ratio through column trapping.  Magn Reson Chem. 1998;  36 104-9
• 36 Nyberg N T, Baumann H, Kenne L. Application of solid-phase extraction coupled to an NMR flow-probe in the analysis of HPLC fractions.  Magn Reson Chem. 2001;  39 236-40
• 37 Nyberg N T, Baumann H, Kenne L. Solid-phase extraction NMR studies of chromatographic fractions of saponins from Quillaja saponaria .  Anal Chem. 2003;  75 268-74
• 38 Exarchou V, Godejohann M, van Beek T A, Gerothanassis I P, Vervoort J. LC-UV-solid-phase extraction-NMR-MS combined with a cryogenic flow probe and its application to the identification of compounds present in Greek oregano.  Anal Chem. 2003;  75 6288-94
• 39 Miliauskas G, van Beek T A, de Waard P, Venskutonis R P, Sudhölter E JR. Identification of radical scavenging compounds in Rhaponticum carthamoides by means of LC-DAD-SPE-NMR.  J Nat Prod. 2005;  68 168-72
• 40 Seger C, Godejohann M, Tseng L H, Spraul M, Girtler A, Sturm S. et al . LC-DAD-MS/SPE-NMR hyphenation, a tool for the analysis of pharmaceutically used plant extracts: identification of isobaric glycoside regioisomers from Harpagophytum procumbens .  Anal Chem. 2005;  77 878-85
• 41 Clarkson C, Stærk D, Hansen S H, Jaroszewski J W. Hyphenation of solid-phase extraction and nuclear magnetic resonance: application of HPLC-DAD-SPE-NMR to identification of constituents of Kanahia laniflora .  Anal Chem. 2005;  77 3547-53
• 42 Lambert M, Stærk D, Hansen S H, Sairafianpour M, Jaroszewski J W. Rapid extract dereplication using HPLC-SPE-NMR: Analysis of isoflavonoids from Smirnowia iranica . J Nat Prod, in press
• 43 Lambert M, Stærk D, Hansen S H, Jaroszewski J W. HPLC-SPE-NMR hyphenation in natural products research: optimization of analysis of Croton membranaceus extract.  Magn Reson Chem. 2005;  43 771-7
• 44 Christophoridou S, Dais P, Tseng L H, Spraul M. Separation and identification of phenolic compounds in olive oil by coupling high-performance liquid chromatography with postcolumn solid-phase extraction to nuclear magnetic resonance spectroscopy (LC-SPE-NMR).  J Agric Food Chem. 2005;  53 4667-79
• 45 Wang C Y, Lee S S. Analysis and identification of lignans in Phyllanthus urinaria by HPLC-SPE-NMR.  Phytochem Anal. 2005;  16 120-6
• 46 Pukalskas A, van Beek T A, de Waard P. Development of a triple hyphenated HPLC-radical scavenging detection-DAD-SPE-NMR system for the rapid identification of antioxidants in complex plant extracts.  J Chromatogr A. 2005;  1074 81-8
• 47 Jaroszewski J W. HPLC-SPE-NMR: A productivity tool for determination of natural products in plant material.  In: Magnetic resonance in food science, the multivariate challenge. Engelsen SB, Belton PS, Jacobsen HJ, editors Cambridge; The Royal Society of Chemistry; 2005.: p. 39-46.
• 48 Simpson A J, Tseng L H, Simpson M J, Spaul M, Braumann U, Kingery W L. et al . The application of LC-NMR and LC-SPE-NMR to compositional studies of natural organic matter.  Analyst. 2004;  129 1216-22
• 49 Godejohann M, Tseng L H, Braumann U, Fuchser J, Spraul M. Characterization of a paracetamol metabolite using on-line LC-SPE-NMR-MS and a cryogenic NMR probe.  J Chromatogr A. 2004;  1058 191-6
• 50 Sandvoss M, Bardsley B, Beck T L, Lee-Smith E, North S E, Moore P J. et al . HPLC-SPE-NMR in pharmaceutical development: capabilities and applications.  Magn Reson Chem. 2005;  43 762-70
• 51 Xu F, Alexander A J. The design of an on-line semi-preparative LC-SPE-NMR system for trace analysis.  Magn Reson Chem. 2005;  43 776-82
• 52 Poole C F, Gunatilleka A D, Sethuraman R. Contributions of theory to method development in solid-phase extraction.  J Chromatogr A. 2000;  885 17-39
• 53 Petritis K, Gillaizeau I, Elfakir C, Dreux M, Petit A, Bongibault N. et al . Evaporative light scattering detection for on-line monitoring of stopped-flow liquid chromatography-nuclear magnetic resonance analysis of compounds with weak or no chromophore groups.  J Sep Sci. 2002;  25 593-600
• 54 Bringmann G, Messer K, Wohlfarth M, Kraus J, Dumbuya K, Rückert M. HPLC-CD on-line coupling in combination with HPLC-NMR and HPLC-MS/MS for the determination of the full absolute stereostructure of new metabolites in plant extracts.  Anal Chem. 1999;  71 2678-86
• 55 Mistry N, Roberts A D, Tranter G E, Francis P, Barylski I, Ismail I M. et al . Directly coupled chiral HPLC-NMR and HPLC-CD spectroscopy as complementary methods for structural and enantiomeric isomer identification: application to atracurium besylate.  Anal Chem. 1999;  71 2838-43
• 56 Bringmann G, Messer K, Saeb W, Peters E M, Peters K. The absolute configuration of (+)-isoshinanolone and in situ LC-CD analysis of its stereoisomers from crude extracts.  Phytochemistry. 2001;  56 387-91
• 57 Somsen G W, Gooijer C, Brinkman U AT. Liquid chromatography-Fourier transform infrared spectrometry.  J Chromatogr A. 1999;  856 213-42
• 58 Lacey M E, Subramanian R, Olson D L, Webb A G, Sweedler J V. High-resolution spectroscopy of sample volumes from 1 nL to 10 μL.  Chem Rev. 1999;  99 3133-52
• 59 Hoult D I, Richards R E. Signal-to noise ratio of nuclear magnetic resonance experiment.  J Magn Reson. 1976;  24 71-85
• 60 Schlotterbeck G, Ross A, Hochstrasser R, Senn H, Kühn T, Marek D. et al . High-resolution capillary tube NMR. A miniaturized 5-μL high-sensitivity TXI probe for mass-limited samples, off-line LC NMR, and HT NMR.  Anal Chem. 2002;  74 4464-71
• 61 Peck T L, Magin R L, Lauterbur P C. Design and analysis of microcoils for NMR spectroscopy.  J Magn Reson B. 1995;  108 114-24
• 62 Olson D L, Peck T L, Webb A G, Magin R L, Sweedler J V. High-resolution microcoil ¹H NMR for mass-limited, nanoliter-volume samples.  Science. 1995;  270 1967-70
• 63 Hu J F, Yoo H D, Williams C T, Garo E, Cremin P A, Zeng L. et al . Miniaturization of the structure elucidation of novel natural products - two trace antibacterial acylated caprylic alcohol glycosides from Arctostaphylos pumila .  Planta Med. 2005;  71 176-80
• 64 Hu J F, Garo E, Yoo H D, Cremin P A, Zeng L, Goering M G. et al . Application of capillary-scale NMR for the structure determination of phytochemicals.  Phytochem Anal. 2005;  16 127-33
• 65 Yoo H D, Cremin P A, Zeng L, Garo E, Williams C T, Lee C M. et al . Suaveolindole, a new mass-limited antibacterial indolosesquiterpene from Greenwayodendron suaveolens obtained via high-throughput natural products chemistry methods.  J Nat Prod. 2005;  68 22-4
• 66 Wolfender J L, Queiroz E F, Hostettmann K. Phytochemistry in the microgram domain - an LC-NMR perspective.  Magn Reson Chem. 2005;  43 697-709
• 67 Wu N, Webb A, Peck T L, Sweedler J V. On-line NMR detection of amino acids and peptides in microbore LC.  Anal Chem. 1995;  67 3101-7
• 68 Subramanian R, Kelley W P, Floyd P D, Tan Z J, Webb A G, Sweedler J V. A microcoil NMR probe for coupling microscale HPLC with on-line NMR spectroscopy.  Anal Chem. 1999;  71 5335-9
• 69 Wolters A M, Jayawickrama D A, Sweedler J V. Microscale NMR.  Curr Opin Chem Biol. 2002;  6 711-6
• 70 Jayawickrama D A, Sweedler J V. Hyphenation of capillary separations with nuclear magnetic resonance spectroscopy.  J Chromatogr A. 2003;  1000 819-40
• 71 Olson D L, Norcross J A, O’Neil-Johnson M, Molitor P F, Detlefsen D J, Wilson A G. et al . Microflow NMR: concepts and capabilities.  Anal Chem. 2004;  76 2966-74
• 72 Sandvoss M, Roberts A D, Ismail I M, North S E. Direct on-line hyphenation of capillary liquid chromatography to nuclear magnetic resonance spectroscopy: practical aspects and application to drug metabolite identification.  J Chromatogr A. 2004;  1028 259-66
• 73 Hentschel P, Krucker M, Grynbaum M D, Putzbach K, Bischoff R, Albert K. Determination of regulatory phosphorylation sites in nanogram amounts of a synthetic fragment of ZAP-70 using microprobe NMR and on-line coupled capillary HPLC-NMR.  Magn Reson Chem. 2005;  43 747-54
• 74 Lewis R J, Bernstein M A, Duncan S J, Sleigh C J. A comparison of capillary-scale LC-NMR with alternative techniques: spectroscopic and practical considerations.  Magn Reson Chem. 2005;  43 783-9
• 75 Xiao H B, Krucker M, Putzbach K, Albert K. Capillary liquid chromatography-microcoil ¹H nuclear magnetic resonance spectroscopy and liquid chromatography-ion trap mass spectrometry for on-line structure elucidation of isoflavones in Radix astragali .  J Chromatogr A. 2005;  1067 135-43
• 76 Bailey N JC, Marshall I R. Development of ultrahigh-throughput NMR spectroscopic analysis utilizing capillary flow NMR technology.  Anal Chem. 2005;  77 3947-53
• 77 Walton J H, de Ropp J S, Shutov M V, Goloshevsky A G, McCarthy M J, Smith R L. et al . A micromachined double-tuned NMR microprobe.  Anal Chem. 2003;  75 5030-6
• 78 Macnaughtan M A, Hou T, Xu J, Raftery D. High-throughput nuclear magnetic resonance analysis using a multiple coil flow probe.  Anal Chem. 2003;  75 5116-23
• 79 Wang H, Ciobanu L, Edison A S, Webb A G. An eight-coil high-frequency probehead design for high-throughput nuclear magnetic resonance spectroscopy.  J Magn Reson. 2004;  170 206-12
• 80 Styles P, Soffe N F, Scott C A, Cragg D A, Row F, White D J. et al . A high-resolution NMR probe in which the coil and preamplifier are cooled with liquid helium.  J Magn Reson. 1984;  60 397-404
• 81 Spraul M, Freund A S, Nast R E, Withers R S, Maas W E, Corcoran O. Advancing NMR sensitivity for LC-NMR-MS using a cryoflow probe: application to the analysis of acetaminophen metabolites in urine.  Anal Chem. 2003;  75 1546-51
• 82 Kovacs H, Moskau D, Spraul M. Cryogenically cooled probes - a leap in NMR technology.  Prog Nucl Magn Reson Spectrosc. 2005;  46 131-55
• 83 Kautz R A, Lacey M E, Wolters A M, Foret F, Webb A G, Karger B L. et al . Sample concentration and separation for nanoliter-volume NMR spectroscopy using capillary isotachophoresis.  J Am Chem Soc. 2001;  123 3159-60
• 84 Wolters A M, Jayawickrama D A, Larive C K, Sweedler J V. Capillary isotachophoresis/NMR: extension to trace impurity analysis and improved instrument coupling.  Anal Chem. 2002;  74 2306-13
• 85 Jayawickrama D A, Sweedler J V. Dual microcoil NMR probe coupled to cyclic CE for continuous separation and analyte isolation.  Anal Chem. 2004;  76 4894-900
• 86 Wolters A M, Jayawickrama D A, Sweedler J V. Comparative analysis of a neurotoxin from Calliostoma canaliculatum by on-line capillary isotachophoresis/¹H NMR and diffusion ¹H NMR.  J Nat Prod. 2005;  68 162-7
• 87 Webb A G. Nuclear magnetic resonance coupled microseparations.  Magn Reson Chem. 2005;  43 688-96
• 88 Barantin L, Le Pape A, Akoka S. A new method for absolute quantification of MRS metabolites.  Magn Reson Med. 1997;  38 179-82
• 89 Akoka S, Barantin L, Trierweiler M. Concentration measurement by proton NMR using the ERETIC method.  Anal Chem. 1999;  71 2554-7
• 90 Akoka S, Trierweiler M. Improvement of the ERETIC method by digital synthesis of the signal and addition of a broadband antenna inside the NMR probe.  Instr Sci Technol. 2002;  30 21-9
• 91 Silvestre V, Goupry S, Trierweiler M, Robins R, Akoka S. Determination of substrate and product concentrations in lactic acid bacterial fermentations by proton NMR using the ERETIC method.  Anal Chem. 2001;  73 1862-8
• 92 Frydman L, Lupulescu A, Scherf T. Principles and features of single-scan two dimensional NMR spectroscopy.  J Am Chem Soc. 2003;  125 9204-17
• 93 Pelupessy P. Adiabatic single scan two-dimensional NMR spectroscopy.  J Am Chem Soc. 2003;  125 12 345-50
• 94 Shrot Y, Shapira B, Frydman L. Ultrafast 2D NMR spectroscopy using a continuous spatial encoding of the spin interactions.  J Magn Reson. 2004;  171 163-70
• 95 Shapira B, Karton A, Aronzon D, Frydman L. Real-time 2D NMR identification of analytes undergoing continuous chromatographic separation.  J Am Chem Soc. 2004;  126 1262-5