Npr REVIEW NMR Methods for Unravelling the Spectra of Complex Mixtures

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Cite this: Nat. Prod. Rep., 2011, 28,78 www.rsc.org/npr REVIEW NMR methods for unravelling the spectra of complex mixtures

Ramon Novoa-Carballal,a Eduardo Fernandez-Megia,a Carlos Jimenezb and Ricardo Riguera*a

Received 30th July 2010 DOI: 10.1039/c005320c

Covering: up to May 2010

The main methods for the simpliﬁcation of the NMR of complex mixtures by selective attenuation/ suppression of the signals of certain components are presented. The application of relaxation, diffusion and PSR ﬁlters and other techniques (J-resolution, TOCSY, etc.), to biological samples, pharmaceuticals, foods, living organisms and natural products are illustrated with examples.

1 Introduction 3.3.3 NMR of pharmaceuticals 2 Relaxation ﬁlters 3.3.4 NMR of foods 2.1 Basic concepts 3.3.5 NMR of humic substances

2.1.1 T1, T2 and T1r filters 3.3.6 NMR of biological fluids and tissues 2.1.2 T2 and T1r versus T1 filters 3.3.7 NMR of other complex mixtures 2.1.3 T2 versus T1r filters 4 Other methods 2.1.4 Limitations and practical considerations of relaxa- 4.1 Selective saturation and magnetization transfer tion editing 4.2 TOCSY 2.2 Methodology and state of the art 4.3 J-resolved spectroscopy (JRES) 2.2.1 Pulse sequences: Inversion recovery and spin-echo 4.4 ‘Virtual’ relaxation-edited spectroscopy (RESY) Downloaded on 30/04/2013 15:21:41. 2.2.2 The use of combined techniques 4.5 Paramagnetic spin relaxation (PSR) filtering 2.2.3 Advanced editing of spectra (TOSY and TOPSY) 5 Conclusions

Published on 11 October 2010 http://pubs.rsc.org | doi:10.1039/C005320C 2.3 Application of relaxation filters to the NMR analysis 6 Acknowledgements of mixtures 7 References 2.3.1 NMR of biological fluids 2.3.2 NMR of cells and tissues 2.3.3 NMR of whole specimens 1 Introduction 2.3.4 NMR of food 2.3.5 Relaxation vs. diffusion editing For chemists, it is unfortunate that Nature only very rarely 3 Diffusion methods presents its results in the form of pure compounds. Therefore, 3.1 Basic concepts separation and purification of components from complex 3.2 Methodology and the state of the art mixtures has historically occupied a central role in the effort of 3.2.1 Data acquisition researchers, particularly in the field of natural products 3.2.2 Data-processing approaches research. 3.2.3 The use of combined techniques Although very efficient separation methods have been devel- 3.2.4 Enhancement of diffusion coefficient differences oped that provide the pure components ready for structure 3.3 Applications of diffusion NMR spectroscopy to determination, most of the working time is devoted to the NMR analysis of complex mixtures separation/purification steps and not to the structural determi- 3.3.1 NMR of natural product extracts nation. A step further in the optimization of this research has 3.3.2 NMR of natural product mixtures been the implementation of hyphenated techniques such as HPLC–NMR, where the products of the HPLC column enter the NMR spectrometer directly so that no isolation or manip- aDepartment of Organic Chemistry and Centre for Research in Biological ulation of the sample is necessary to obtain the spectra. Still, the Chemistry and Molecular Materials, University of Santiago de investigation of complex mixtures relies on the classical para- Compostela, Jenaro de la Fuente s/n, 15782 Santiago de Compostela, Spain. E-mail: [email protected] digm that has presided over the work of natural products bDepartment of Fundamental Chemistry, Faculty of Sciences, University of chemists for years: first, to separate and purify the components, A Coruna,~ A Coruna,~ Spain and next, to submit the pure compounds to spectroscopic

78 | Nat. Prod. Rep., 2011, 28, 78–98 This journal is ª The Royal Society of Chemistry 2011 View Article Online

analysis. This protocol requires the separation of many ‘useless’ its constituents. The mixture is directly submitted to NMR components in order to isolate a single or just a few compounds and the acquisition is implemented in a way that allows the of interest, and stresses the increasing importance of der- data from the components to be obtained with no previous eplication. separation. Nowadays. and thanks to the developments of NMR, it is The most important procedures are based on differences in the not always necessary to separate the components of translational diffusion and NMR relaxation times of the a mixture in order to obtain spectroscopic information from components in the mixture. These differences allow one to obtain

Ramon Novoa-Carballal was BS (1984) and PhD (1988) in born in 1979, and received his Organic Chemistry at the BS (2002), MS (2004) and University of Santiago de Com- PhD (2009) in chemistry at the postela (USC), and did post- University of Santiago de Com- doctoral studies at the postela (USC) (supervisors R. University of California at Riguera and E. Fernandez- Santa Cruz (USA) with Dr Phil Megia). During his BS, he Crews (1988–1990) and at the joined the Friedrich Schiller Scripps Research Institute, La University Jena (Germany) for Jolla (USA) with Dr Alfonso two semesters (2000–2001). As Tramontano (1990–1991). He part of his PhD, he had two was assistant professor of short stays at the Imperial Chemistry at USC until 1992 Ramon Novoa-Carballal College London (supervisors A. Carlos Jimenez and then moved to University of Miller and M. Thanou) and the A Coruna,~ where he reached his Stockholm University (super- current position as Full visor G. Widmalm). In 2010 he obtained a Barrie de la Maza Professor of Organic Chemistry in 2009. His research focuses on Fellowship to undertake a postdoc at the University of Bayreuth. the chemistry of bioactive natural products (isolation, structural His research is focused on synthetic modiﬁcation and character- elucidation, and synthesis), mainly from marine organisms, and the ization (especially by NMR spectroscopy) of polysaccharides and applications of NMR techniques to structural problems, mainly in

Downloaded on 30/04/2013 15:21:41. dendrimers of pharmaceutical interest. the determination of the relative conﬁguration of organic molecules. He has authored some 80 scientiﬁc publications and mono- graphs. Published on 11 October 2010 http://pubs.rsc.org | doi:10.1039/C005320C

Eduardo Fernandez-Megia was Ricardo Riguera received his born in 1967 in Vigo, Spain, and PhD in Chemistry from the is currently a Professor Titular University of Santiago de Com- in the Organic Chemistry postela (USC) in 1973. He Department and Centre for carried out postdoctoral studies Research in Biological Chem- at University College London istry and Molecular Materials with Prof. P. Garrat (1974). He at the University of Santiago de was appointed Lecturer in 1978, Compostela (USC). After and in 1990 he became full completing a Ph.D. in 1995 at Professor of Chemistry at USC. USC (supervisor Prof. F. Javier His research is represented by Sardina), he undertook a post- around 200 papers and patents doctoral stay with Prof. Steven covering several topics: bioac- Eduardo Fernandez-Megia V. Ley at the University of Ricardo Riguera tive natural products, medicinal Cambridge (1997–1999). Then, chemistry and NMR methods he returned to the USC as for determination of absolute a Marie Curie Fellow and Prof. Asociado. In 2003, Eduardo conﬁguration. He is now interested on polymeric nanostructures for became a Ramon y Cajal Fellow, followed by a Prof. Contratado biomedical applications and on stimuli responsive dynamic poly- Doctor in 2008. His research has focused on the interface between mers. He has authored three textbooks for students and served the organic and polymer chemistry with emphasis on the preparation of University as Chairman of the Department, Dean of the Faculty well-deﬁned polymeric nanostructures for biomedical applications and Vice-Chancellor. and the development of NMR and SPR tools for their characterization.

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either edited spectra (i.e. spectra where only the components with widely used as a source of information on the dynamics of defined relaxation times or diffusion coefficients are observed), compounds,9 as well as on the study of interactions.10 or to obtain bidimensional spectra (one dimension being the In the case of complex mixtures, the dependence of the chemical shift and the other the diffusion coefficient or relaxation relaxation times on the dynamics and flexibility of each molecule time). Both options have been described for relaxation and opens the possibility to selectively attenuate, or even remove, the diffusion, but bidimensional spectra are only commonly used for signals of the components with shorter spin–lattice (also called

diffusion (diffusion-ordered spectroscopy, DOSY). longitudinal) relaxation times, T1 and T1r (longitudinal relaxa- Relaxation ﬁlters allow the attenuation/suppression of the tion under spin-lock pulse conditions), or spin–spin (transversal)

signals from compounds with high molecular weight, and diffusion relaxation times, T2. ﬁlters attenuate the signals from low molecular weight compounds. An illustrative example of relaxation edited spectra is shown in Therefore they can be used to simplify the spectra of a mixture by Fig. 1, where the signals from proteins and lipoproteins (with shorter

selectively acting on these two types of components. Other T1, T1r and T2) are almost completely removed from the spectra. procedures, such as the addition of paramagnetic spin relaxation

agents (PSR filters), are based on completely different mecha- 2.1.1 T1, T2 and T1r filters. A comprehensive description of nisms, and expand the usefulness of diffusion and relaxation filters the theoretical principles of NMR relaxation is out the scope of to other components independently of their molecular weight. the present review. The interested reader is referred to excellent These approaches have been demonstrated to work very books and reviews in the field.9 Although this section is devoted successfully with biological fluids (urine, serum, cells, tissues), to the applications in relaxation-edited NMR, some comments

with whole specimens and cells, pharmaceutical preparations, on the different methods that derive from the use of T1, T2 or T1r and in food analysis, but surprisingly, only a very few examples ﬁlters and their differences should be mentioned. of application to natural products research have been published.

In this review, we present the main characteristics and prac- 2.1.2 T2 and T1r versus T1 ﬁlters. The overall rotational tical aspects of methods for unravelling the NMR of mixtures, correlation time has a much greater importance in determining T2 illustrated with examples. We hope to provide practitioners of and T1r than T1, which is more sensitive to local motions such as natural products research with information useful to evaluate the segmental reorientations, rotations etc. Additionally, T2 and T1r use of these procedures when investigating the mixtures they values show a continuous decrease with decreasing mobility, while

usually come across. T1 diminishes until a minimum and increases with a further Accordingly, this review contains three main sections, the ﬁrst decrease of the mobility. As a consequence, low and high molecular

two being devoted to the use of relaxation and diffusion weight compounds differ much more in their T2 and T1r values parameters. A third section describes the application of other than in T1. This fact determines the different use of both types of NMR methods, such as J-resolved spectroscopy, TOCSY and filters depending on the nature of the molecules in the sample. the use of PSR agents. A good example is human plasma (Fig. 1) where proteins with Downloaded on 30/04/2013 15:21:41. T2, T1r and T1 values lower than those of peptides and glucose 2 Relaxation filters can be distinguished from them using these filters. Thus, the T2 and T values of the proteins are one order of magnitude lower Published on 11 October 2010 http://pubs.rsc.org | doi:10.1039/C005320C 1r Relaxation filters are pulse sequences that allow the selective than those of the peptides and the glucose, while T1 values are attenuation of signals of high molecular weight and/or rigid only 3–5 times lower. Therefore, the attenuation of the high compounds, eventually leading to their suppression (Fig. 1). molecular weight compounds is more efficient using T2 and T1r For the analysis of natural mixtures, this methodology was filters than with T1. This explains the much more extensive use of first proposed in the late 1970s with studies on adrenal medulla,2 the former filters in spectral editing.† 3 and metabolites in red blood cells. Later on, the group of Although the differences in T1 are lower, they are sufficient to Nicholson introduced several improvements converting this be exploited for the purpose of spectral editing (see Fig. 1). One NMR technique into a routine method in metabolism studies.4–8 typical example is water peak suppression, a topic that is out of For this reason, most of the relaxation filter applications are the scope of this review.11 related to metabolic profiling of cells, tissues, and biological fluids in human or animals. Moreover, some other interesting 2.1.3 T2 versus T1r filters. Although similar efficiency is applications have been recently described in other fields obtained using T2 or T1r filters, the T1r filter has found little or no (metabolism, taxonomy, food chemistry), illustrating the high application in the analysis of complex mixtures. This is probably potential of these techniques. because T2 editing allows long relaxation periods to be completed with total signal stability, while the use of T1r filters may lead to undesirable sample heating – and loss of stability – due to the spin- 2.1 Basic concepts lock. However, T1r filtering is commonly combined with NOE In an NMR experiment, a molecule is placed under a magnetic and STD (saturation transfer difference) experiments for the field leading to the splitting of the nuclear spin levels (Zeeman elimination of protein signals in protein–ligand interaction effect). The application of a radiofrequency pulse situates the studies, and is commonly used in solid-state NMR.12 system in an excited state that immediately begins its return to equilibrium through a complex process known as relaxation. † It must also be considered that due to the nature of the T1 filtering NMR relaxation is directly bound to the dynamics and flexibility experiment (inversion recovery experiment) some of the signals in the of the excited molecule; therefore, NMR relaxation has been experiment may appear inverted in the spectra.

80 | Nat. Prod. Rep., 2011, 28, 78–98 This journal is ª The Royal Society of Chemistry 2011 View Article Online

1 1 Fig. 1 H NMR spectra of a human blood plasma sample, (A) H with water presaturation; (B) T1-edited NMR spectrum, plotted inverted, relaxation filter 265 ms; (C) T1r-edited NMR spectrum; spin-locking time 120 ms; (D) T2-edited NMR spectroscopy; relax filter 160 ms. Reprinted from ref. 1 with permission. 2.1.4 Limitations and practical considerations of relaxation 3) Finally, when small molecules in the mixture are bound to editing. There are some considerations that must be taken into macromolecules, the ‘complex’ behaves as a single entity and account before using relaxation filters: responds to the filter as a whole. Indeed, the small molecule will be 1) The use of relaxation filters is restricted to situations where eliminated from the spectrum together with the big one, even when the relaxation times of the components are considerably a small filter – selected for the macromolecule – has being used.15 different. For example, if we have three NMR signals A, B and C in identical ratio 1/1/1 with T2 values of 10, 30 and 100 ms 2.2 Methodology and state of the art respectively, and apply a filter of 200 ms, the ratio will change to Downloaded on 30/04/2013 15:21:41. 0.1/0.3/1 (95%, 86% and 61% of attenuation respectively). The The use of relaxation-edited spectra is theoretically not restricted to 1 new signal intensities are calculated by the formula If ¼ H NMR spectroscopy, and can be applied to other nuclei. Nevertheless, the use of 13C NMR has been limited by its lower Published on 11 October 2010 http://pubs.rsc.org | doi:10.1039/C005320C Io exp(tf/T2), where If is the signal intensity after applying the T2 filter, Io is the signal intensity before filtering and tf is the T2 sensitivity. In the only example we found in the literature, Blackwell filter duration. This implies that A will be practically eliminated et al. successfully applied a T2 relaxation filter for the suppression of from the spectrum, while B will be only partially attenuated. For humic acid signals in the 13C NMR spectrum from a mineral soil.16 this reason, if precise quantitative information is required, we have to introduce a correction on the signal intensities based on 2.2.1 Pulse sequences: Inversion recovery and spin-echo. The the different attenuation of the signals coming from protons with pulse sequences commonly used for T1, T2 or T1r filters are those different relaxation times.‡13 used for the determination of relaxation times – namely, inver- In those cases where the signals to be suppressed are the most sion-recovery for the longitudinal relaxation time T1, spin-echo intense (such as in blood plasma, Fig. 1) the loss of signal for T2 measurements, and a simple spin-lock pulse for the experienced by those signals whose suppression is not desired is determination of T1r. In the case of biological fluids where compensated by the increased receiver gain. the sample is dissolved in water with a small proportion of D2O, 2) Additionally, the relaxation times are not only dependent on the problem of dynamic range forces these pulse sequences to be the overall rotational correlation time but also on the local combined with solvent suppression (usually by presaturation).4–8 motions. Different parts of a molecule may have very different Convenient pulse sequences are implemented in the NMR local mobility (i.e. flexibility); therefore different relaxation times libraries from Bruker and Varian, but they can also be taken and different sensitivity to the filter result: some parts of the from NMR books. Furthermore, adequate protocols have been molecule may remain present in the spectra after filtering while recently described.4,5 However, it is interesting to highlight some others are suppressed. Such is the case with the N-acetyl signals practical differences between the different spin-echo pulse of N-acetylglucosamine and N-acetylneuraminic acid residues in sequences and some combinations of relaxation filters with other mobile carbohydrate side-chains of glycoproteins.14 NMR methodologies.

Spin-echo pulse sequences ‡ Attention should be paid to the experimental conditions (temperature, sample viscosity, etc.) when relaxation times taken from the literature are The Hahn spin-echo pulse sequence, applied in the early works used for quantitative analysis. describing relaxation editing,7,17 results in phase-modulation of

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signals. Therefore it has been replaced by the Carr–Purcell– Relaxation-filtered bidimensional spectroscopy Meiboom–Gill (CPMG) spin-echo pulse sequence, which mini- Edited 1H NMR spectra give very interesting information on mizes the J-modulation and the loss of signal intensity caused by the composition of natural mixtures, but 2D NMR is often diffusion through field gradients.x,18 required for unambiguous assignments. In 2009, a modified CPMG pulse sequence (CPMG-z) was The first examples of relaxation-filtered bidimensional spec-

described. This experiment makes use of an additional gradient troscopy (T2-filtered COSY) were described for the identification filter on the z-axis to completely eliminate phase distortions and assignment of 1H NMR spectra of small molecules in intact caused by the J-modulation,19 Nevertheless, the benefit of erythrocytes using conventional spin echo.28 Few years later, moving from CPMG to CPMG-z on a general basis has still not Holmes et al. described the combination of CPMG and COSY, been demonstrated. demonstrating its applicability in the NMR spectra of cancer 29 cells and mouse abdominal fat. An example of T2-filtered edited 2.2.2 The use of combined techniques. Relaxation editing is total correlation spectroscopy (TOCSY) for the study of pig a powerful tool for spectral simplification, but its applicability is blood plasma has also been described.30 restricted to situations where the relaxation times of the More recently, Nicholson et al. have described sequences to components are considerably different. Combinations of enable an easier implementation of phase-sensitive 2D NMR and

different relaxation filters and combinations of relaxation filters to extend the concept to T1/T1r/T2-edited COSY and TOCSY. with diffusion filters and 2D NMR (forming part of the same Those experiments applied to a blood plasma sample,1 and pulse sequence), that extend the capabilities of relaxation editing, clearly favoured the detection of small metabolite signals. For are described next. example, after the relaxation editing, the signals of amino acids (valine, leucine, isoleucine) in the 0.7–2.0 ppm region (dashed Combination of inversion-recovery and spin-echo box in Fig. 2B–D) emerged much more clearly behind the intense Rabenstein et al. described the inversion-recovery spin-echo lipid peaks. (IRSE) sequence as a combination of the inversion-recovery and Two interesting experiments that used relaxation-filtered the spin-echo pulse sequences. With this pulse sequence, over- HSQC were recently described by Luy et al. for the measurement lapping resonances can be sorted first according to differences in of the residual dipolar couplings of strychnine in the presence of 31 their T1 values and then to differences in their T2 values. This a polymeric alignment media. In one of then, a spin-lock period 17 sequence has been used in NMR studies of red blood cells. It is introduced, where the magnetization is relaxed by T1r.In also permits the measurement of T1 from small molecules that a second approach, a z-filter period is added, where the relaxa- overlap with proteins.20 Later on, Lindon et al. described an tion is mainly determined by 1H–1H NOE. In both experiments, improved pulse sequence that eliminates the phase distortions suppression of the polymeric alignment media is accomplished, caused by spin–spin coupling during the spin-echo period and thus facilitating the measurement of 1H–13C dipolar couplings. simultaneously suppresses the water signal.21 Downloaded on 30/04/2013 15:21:41. 2.2.3 Advanced editing of spectra (TOSY and TOPSY). Relaxation combined with diffusion TOSY Lindon et al. described a new pulse sequence that allows The 2D-NMR experiment termed raTe of relaxation Ordered Published on 11 October 2010 http://pubs.rsc.org | doi:10.1039/C005320C

DIffusion and T2 RElaxation editing in a single NMR experi- SpectroscopY (TOSY) is a bidimensional method in which one ment (DIRE).21 The experiment allows the attenuation of low dimension is the chemical shift and the other the relaxation time.

and high molecular weight molecules to the desired extent (by TOSY can be T1/T1r/T2, displaying the distribution of rates of modulating the duration the relaxation and diffusion ﬁlters), and relaxation in the indirect dimension and the traditional spectrum in has been applied to biological ﬂuids. the direct dimension. In contrast to what happens in diffusion Other authors described different pulse sequences with the NMR where DOSY is very common, TOSY has been reported only same purpose but in other contexts: simultaneous elimination of in a few cases as application to pharmaceutical formulations.32 polymer and non-bound low molecular weight compounds in combinatorial chemistry,22 elimination of protein signals when TOPSY

determining the interaction with a low molecular weight Another interesting technique, T1 or T2 Ordered Projection compound by diffusion NMR,23,24 determination of the diffusion SpectroscopY (TOPSY) was described in 2009.33 The spectra are 25 coefﬁcients in mixture of humic substances, or removing colour-coded according to the T1 and T2 proton relaxation times polymer signals in a recent procedure for diffusion chromato- respectively, so that changes of relaxation time across multiple graphic NMR.26 Among these different possibilities, the one compounds can be followed. In this way, TOPSY allowed the described by Shapiro combines the more suitable pulse sequences differences in the relaxation times caused by the presence of the

for both the T2 relaxation and diffusion filters, minimizing phase borate used as food preservative to be easily visualized (Fig. 3). distortions. The group of Larive et al. has also described the combination of T as well as T filtering with diffusion.25,27 1 1r 2.3 Application of relaxation filters to the NMR analysis of mixtures As has been mentioned before, the great majority of the appli- x The length of the spin-echo delay in the CPMG pulse sequence should cations reported for relaxation filters are related to metabolic be very short (conventionally less than 1 ms) to avoid phase distortions. profiling of cells, tissues and biological fluids in human or Therefore, in order to obtain the required T2 relaxation filter, the number of loops of the cycle should be increased. animals.

82 | Nat. Prod. Rep., 2011, 28, 78–98 This journal is ª The Royal Society of Chemistry 2011 View Article Online

1 1 Fig. 2 Relaxation-edited H– H COSY NMR spectra of human blood plasma. (A) Normal COSY spectrum; (B) T1-edited spectrum; 265 ms filter; (C) T1r-edited spectrum, 120 ms filter; (D) T2-edited spectrum; 160 ms filter. Reprinted from ref. 1 with permission.

1H NMR spectroscopy is one of the preferred analytical such as J-resolved spectroscopy (see subsection 4.3), 1D and 2D techniques for metabonomic research because it requires little or TOCSY (see subsection 4.4) and 2D NMR experiments, no sample preparation and it is cost-effective, unbiased, rapid, including the differential analysis of 2D spectra, successfully robust, reproducible, quantitative, non-selective and non- applied to natural mixtures from bacteria, fungi, and other destructive. Although this technique is not as sensitive as others, sources.34 useful data can be generated from very small samples. In addi- Thanks to all these procedures, a large number of constituents tion, NMR avoids the drawbacks of traditional bioanalysis can be identified and catalogued because NMR provides detailed methods requiring extensive sample preparation based on structural information. Moreover, 1H NMR spectroscopy allows physical methods such as chromatography that may cause both perturbations of the concentrations of endogenous metabolites biological and physicochemical changes to the sample.4–8 to be detected, and therefore it has been employed to investigate Downloaded on 30/04/2013 15:21:41. However, these NMR studies are very challenging due to the changes in biochemical composition prior to and after treatment very strong signal overlap, considerable range of concentrations, with a drug or a toxin, or to monitor the metabolic responses to molecular weights, and molecular mobility (hence signal line stress, exercise, and certain diets, and even to relate metabolic Published on 11 October 2010 http://pubs.rsc.org | doi:10.1039/C005320C width) of the individual organic components of the biological phenotypes with dietary preferences.35–37 There is a growing samples. To overcome those difficulties, pseudo-separation optimism about the potential of 1H NMR as a diagnostic and based on relaxation times and molecular diffusion coefficients prognostic tool for cancer, heart disease, diabetes or inborn brings about considerable spectral simplification and serves also errors of metabolism and as an aid for personalized medicine.38 to simplify the signal assignment process. In addition, these In the following subsections, some more information is filters are commonly used together with other NMR methods given about these new uses of NMR on biological fluids

Fig. 3 600 MHz TOPSY spectra from urine. In A, the colour scale represents T1 values projected onto the 1D spectrum. In D, the colour scale represents T2 values projected onto the 1D spectrum. Reprinted from ref. 33 with permission.

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(subsection 2.3.1), and on cells and tissues (subsection 2.3.2). 2.3.2 NMR of cells and tissues. In addition to extracts and Some less common but very interesting examples of the NMR biological fluids, relaxation editing has been applied to cells and of living organisms (subsection 2.3.3) and of food (subsection tissues too. Initially, this methodology was used in the NMR 2.3.4) are also discussed. Finally, the combined use of relax- study of the adrenal medulla2 and red blood cells,3 and then ation and diffusion filters for the selective suppression of applied to leukemia cells and rat liver hepatocytes.50 In those certain types of components in mixtures is described in early studies, the experiments were carried out using the Hahn subsection 2.3.5. spin-echo pulse sequence and the cells where isolated and resuspended in the NMR tube to diminish the sample viscosity. 2.3.1 NMR of biological fluids. The idea of using relaxation The strong line broadening induced by such viscosity hampers editing as a way to diminish the heavy overlap of metabolite the application of this technique by conventional liquid state resonances in biological fluids was first introduced by Sadler in NMR spectroscopy. the early 1980s in his work on the NMR of plasma.39 With the emergence of high resolution magic angle spinning

Since then, T2 filtering has become a routine method for the (HR-MAS) NMR spectroscopy it was possible to solve those elimination or attenuation of macromolecule resonances (mainly difficulties improving the spectral quality of viscous cell and proteins and lipoproteins) in body fluids, and therefore tissue samples. HR-MAS NMR spectroscopy reduces the line enhancing the detection of small metabolites such as peptides, broadening associated with restricted molecular motion, chem- choline, taurine, acetate, ascorbate and many others. The Hahn ical shift anisotropy, dipolar couplings and field inhomogeneity spin-echo experiments (J modulation, see subsection 2.2.1) used by high-rate spinning of the sample at the magic angle.51 This in the early papers were gradually replaced with the more results in well-resolved NMR spectra with narrow linewidths convenient CPMG. Examples of the relaxation-edited NMR that are comparable to in vivo magnetic NMR spectra, albeit with spectra of urine, blood plasma, bile, and amniotic, cerebrospinal generally much higher resolution and sensitivity. HR-MAS and seminal fluids are known,37,40–42 but cell extracts have been NMR of tissues has been shown to be valuable in the non- reported too.43 destructive assessment of metabolite profiles in skin, brain, bone, A representative example of the use of relaxation filtering for cartilage, eye, gut, liver, kidney, muscle, and vascular tissues. the detection of small metabolites in blood plasma is shown in Additional information about the metabolites accessible from Fig. 1. Detailed information on the classification and listing of the 1H NMR of tissues and cells can be found in quite recent metabolites accessible from the 1H-NMR of biofluids has been comprehensive reviews.5,52 4–8 reviewed elsewhere. In analogy to what is common in the liquid state, the use of T2 Relaxation editing is not always necessary when carrying out filters using CPMG on viscous samples (HR-MAS-CPMG), the 1H NMR of biofluids – its necessity depends on the fluid allows the pseudo-separation of the low molecular weight studied and the information required. For example, blood metabolites from the macromolecules.5,52,53 Although the CPMG plasma and even serum contain a large quantity of macromole- pulse sequence can be adapted to the MAS probe,13,54 the group Downloaded on 30/04/2013 15:21:41. cules that should be removed in order to obtain high-quality of Nicholson et al. remarks that this is not always necessary.5,52 It metabolic information about the small molecules. In contrast, is also interesting to note that the filters to remove macromole- the NMR of urine, which contains smaller quantities of macro- cules in these viscous samples should be longer (100–300 ms) Published on 11 October 2010 http://pubs.rsc.org | doi:10.1039/C005320C molecules, is obtained by most authors without using relaxation than those used in the liquid state. A representative example of

editing. Nevertheless, Slupsky et al. have demonstrated that T2 the NMR of a liver biopsy is shown in Fig. 4. filtering of urine samples is very convenient to eliminate baseline artefacts due to protein and lipids, when precise quantitative 2.3.3 NMR of whole specimens. In addition to the well- information is needed.44 established use of HRMAS-CPMG as pseudo-separation tech- It is also interesting to highlight that by modulating the length nique, in human and other mammalians, cells and Tissues, other of the relaxation filter, the suppression of different types of less common examples can be found for whole biological speci- macromolecules can be accomplished. For example, most mens. It is the aim of this subsection to describe these examples proteins of blood plasma (albumin, immunoglobulin, etc.) can be and to show the unexploited potential of this technique to other 1 eliminated from the H NMR spectra with a T2 filter of around areas related to natural products. 100 ms,45 while the resonances from more mobile lipoproteins Thus, CPMG filters in HR-MAS have been exploited in the (specially the prominent peaks near 0.9 and 1.3 ppm due to study of unicellular organisms such as Mycobacterium smegma-

lipidic CH3 and CH2 groups; see also Fig. 1) are only partially tis. In that work, the authors propose that NMR may become attenuated even with longer filters.46 a unique tool to study conformation, dynamics, and interactions In practice, it is common to use filters of around 40–120 ms, among biologically important macromolecules in their native because the lipoprotein resonances contain very interesting state.56 information as diagnostic and prognostic tools.38 The analysis of the yeast Pichia anomala described by Schnurer€ Most of the studies that used relaxation editing in biological et al. was another example of a HR-MAS-CPMG.57 This allowed fluids have been performed in humans and other mammals the analysis of many soluble compounds, such as trehalose, (rodent, canine, pig). Nevertheless, the applicability of this arabitol, and glycerol, inside intact cells without the need for an technique goes well beyond that, and cases illustrating the extraction step. The variation of the levels of these metabolites potential of CPMG are also found in other fields exemplified by under conditions of limited oxygen demonstrated that oxygen algae extract,47 fish blood plasma,48 and hemolymphs from the availability affected the respiration and not the fermentation of Red abalone shellfish (Haliotis rufescens).49 the yeast. Analysis of four different marine microalgae by this

84 | Nat. Prod. Rep., 2011, 28, 78–98 This journal is ª The Royal Society of Chemistry 2011 View Article Online

An interesting HR-MAS-CPMG study on a living whole- specimen was conducted on the freshwater cladoceran Daphnia magna in order to monitor the changes in metabolism occurring during animal life. Survival of ten 24 h old daphnids after HR- MAS analysis at different speeds for 14 days was observed. The surviving daphnids displayed a normal life cycle, developing eggs and embryos as usual.61

2.3.4 NMR of food. Although the use of relaxation ﬁlters in food chemistry has been reported, to date it has not been fully 62 generalized. Water attenuation by T2 relaxation–Carr–Purcell– Meiboom–Gill pulse sequence (WATR-CPMG) has been demonstrated to be very effective in aqueous solution.63 This is

based on the reduction of the proton T2 relaxation time of water by proton-exchange reagents. In this example, it allowed Fig. 4 1H HR-MAS NMR spectra of a human liver biopsy sample a precise quantitative determination of the glucose content in (rotation rate 4 kHz): (a) standard 1H spectrum; (b) spin-echo (CPMG) spectrum. Assignments: L1–7 lipid resonances. Reprinted from ref. 55 different commercial juices. Yeung et al. used samples of orange with permission. and guava as a model to demonstrate the feasibility of the WATR-CPMG method to simultaneously eliminate the signals from water and macromolecules.64 tandem methodology demonstrated its usefulness in taxonomic Marsaioli et al. described the use of 1H NMR to monitor the classification studies.58 This application was also used by Ferreira profiles of fermentation and roasting processes of cupuassu et al., who demonstrated that 1H HR-MAS NMR and FT-IR (Theobroma grandiflorum), a Brazilian product similar to choc- ,65 provided a fast and economic method for lichen chemotaxonomy olate.{ The authors used a CPMG T2 filter to eliminate in a complete analysis of eleven different specimens from several residual protein signals that caused deleterious effects on acid families, genera and species.59 The use of CPMG was necessary and alcohol quantifications in normal 1H NMR spectra. The to obtain high-quality spectra from the low molecular weight quantification of lactic acid, acetic acid and 2,3-butanediol by carbohydrates used for the classification (Fig. 5). This method is NMR is 5- to 10-fold faster than by HPLC, with the advantage of much faster than the traditional colorimetric analysis requiring providing the identification of several chemical species in a single laborious and time-consuming sample pre-treatment. experiment. Application to the red alga Gracilariopsis lemaneiformisa More recently, Stocchero et al. showed the usefulness of NMR allowed to discriminate between small molecules – in solution in combined with chemometrics for the evaluation of the ageing of Downloaded on 30/04/2013 15:21:41. the plant cells – and larger rigid components such as cell wall balsamic vinegar of Modena.66 1H NMR acquired by the use of 60 material and membrane constituents. In this study, HR-MAS a T2 filter gave high quality spectra showing of organic acids (like NMR was shown to be a suitable tool for in situ studies when formic, tartaric, malic, succinic, and acetic), and carbohydrates Published on 11 October 2010 http://pubs.rsc.org | doi:10.1039/C005320C short experimental times are required. In fact, cultivation of G. (glucose and fructose) useful for the evaluation of the ageing lemaneiformisa could be continued after 1 h of spinning at 5 kHz, process. showing that the organisms survive in good condition after the experiment. However, after overnight experiments, some meta- 2.3.5 Relaxation vs. diffusion editing. As already mentioned, bolic changes were observed. relaxation filters allow the attenuation of the signals from compounds with high molecular weight (quickly relaxing) while diffusion-based filters (see section 3 for basic concepts and methodology) enable the attenuation of low molecular weight compounds (quickly diffusing). Thus, these two approaches are complementary and can be used in a combined way, making it possible to selectively observe the low or the high molecular weight components of a mixture, as shown in Fig. 6. This approach was demonstrated by Lindon et al. for blood plasma,21 by Fesik et al. for the screening of protein ligands,67 and is nowadays used for the selective observation of metabolites and proteins in biological fluids and tissues.1,35,37,41 It should also be mentioned that the suppression of high molecular weight components from the NMR spectra may also be carried out by the subtraction of two different spectra: one diffusion spectra in which only large molecules are detected, and

1 Fig. 5 Lichen H NMR spectra: (A) without magic angle spinning; (B) { Related to cacao, cupuassu is popular in Brazil, but little known HR-MAS with CPMG pulse; (C) HR-MAS without a CPMG pulse. elsewhere. In the production of ‘cupulate’ from cupuassu, the seeds are Reprinted from ref. 59 with permission. ﬁrst separated and then fermented.

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3.1 Basic concepts

Self-diffusion is the random translational motion of ensembles of particles (molecules or ions) as a consequence of their thermal energy. Diffusion behaviour is related to the properties of both individual molecules (size, shape, molecular weight, charge, etc.) as well as those related to the surrounding environment, such as concentration, solvent, temperature and aggregation state. In this way, each component of a mixture can be pseudo-separated, in accordance with its own diffusion coefficient. Quantitatively, this process is measured as the self-diffusion coefficient, D (m2 s1), which generally decreases with increasing molecular weight (MW). Therefore, for compounds to produce clearly different responses in NMR diffusion experiments, they should have sufficiently diverse molecular weights.

3.2 Methodology and the state of the art The quality of the final spectrum and its concomitant information content relies on both the quality of the acquired raw data and the appropriate choice of data processing methods. Fig. 6 800 MHz 1H NMR spectra of human amniotic fluid: (a) standard 1 H spectrum, (b) T2-edited spectrum, showing low molecular weight metabolite resonances, (c) diffusion-edited spectrum showing protein and 3.2.1 Data acquisition. The pulsed gradient spin-echo lipoprotein resonances. Reprinted from ref. 40 with permission. (PGSE) has become the method of choice for measuring diffusion in solution. A series of pulsed field gradient stimulated echo NMR spectra is recorded with increasing gradient pulse strength, resulting in signals decaying at rates determined by their diffu- another in which large and small molecules are detected. This sion properties. The PGSE NMR method was first devised by option suffers from longer experimental time requirements but Stejskal and Tanner71 in 1965, and was derived in turn from the has been claimed to be more useful than CPMG filtering for nuclear spin echo concept of Hahn, Carr and Purcell.18 68 automated spectral processing routines. Although, it was in the late 1960s and early 1970s that PGSE In another recent report, this diffusion difference protocol was NMR was proposed as a technique to measure diffusion coeffi- Downloaded on 30/04/2013 15:21:41. used in the detection of substoichiometric concentrations of cients in solution and to define domain size in emulsions, its use small molecules in protein samples. For example, it was possible for mixture analysis was not suggested until ten years later, and it to detect 30 mM imidazole in the presence of four different was not until the early 1990s that the method gained widespread Published on 11 October 2010 http://pubs.rsc.org | doi:10.1039/C005320C 69 proteins. use due to the development of NMR instruments. Extensive description of the theory of diffusion NMR experiments can be found in many of the published reviews.72,73 Several spectrometer 3 Diffusion methods parameters such as the baseline correction, overall stability and temperature must be controlled to obtain high-resolution Diffusion NMR spectroscopy is a collection of NMR experi- spectra. A very good detailed description of how to optimize the ments in which the resonances of a molecule are attenuated acquisition process of the PGSE NMR experiment and the according to its translational diffusion coefficient. This is different approaches to analyze the data was provided by Ant- possible thanks to the application of NMR field gradients that alek in 2002.72 By far the most used pulse sequence is the bipolar ‘label’ the spins along the direction of the applied gradient, and pulse pair longitudinal eddy-current delay (BPPLED).74 to the use of certain pulse sequences (based on a spin-echo).70 In Most diffusion NMR experiments are acquired for the 1H the case of a mixture, the spectra of the components can be nucleus, but the application of diffusion NMR to 13C NMR separated according to their apparent diffusion coefficients. spectra has also been described. Specific acquisition parameters Since the self-diffusion value is related to the size of molecules in must be used to account for the decoupling during the gradient solution, this technique relies on differences in the molecular size, pulses.75 The use of 13C implies an important improvement of and so it shows a lot of similarities to size-exclusion chroma- spectral resolution that can be useful in the case for complex tography. Indeed, molecules with different molecular masses but mixtures, as has been demonstrated for organic molecules and with the same chemical shifts can be well separated. For this 13C-enriched carbohydrates.75,76 reason, this technique is described as ‘in-tube chromatography’ or ‘NMR chromatography’, as it leads to virtual separation of 3.2.2 Data-processing approaches. As stated in the introduc- the species in a mixture. Although this technique has become one tion, diffusion NMR can be processed using DOSY (Diffusion- of the most powerful tools to characterize complex mixtures by Ordered SpectroscopY), either as a plain diffusion-edited NMR without first having to separate the components, it is spectrum (or 1D DOSY) or as a bidimensional spectrum (2D clearly still undervalued by most chemists. DOSY), the latter being the most frequently employed (Fig. 7).

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2D DOSY spectra Diffusion-edited spectra and diffusion difference spectra This representation was first proposed by Morris and John- In a simpler approach, 1D NMR can be carried out using son in 1992.77 Experimentally, it is achieved by acquiring a single field gradient. This method is analogous to relaxation a series of 1D NMR experiments with different amounts of editing but permits the selective observation of high molecular diffusion weighting. After transforming the acquisition dimen- weight components of complex mixtures (see subsection 2.3.5). sion, it is possible to extract the diffusion coefficients for each Another alternative is the acquisition of two 1D spectra with peak in the direct dimension by fitting the variation of the signal different diffusion weighting and the subtraction of both spectra. in the indirect dimension. From this information, it is then The application of this method to observe low molecular weight possible to generate a DOSY spectrum in which one axis metabolites in blood plasma has been described in subsection corresponds to the chemical shift and the other axis to the 2.3.5. The general applicability of this methodology has been apparent diffusion. 2D DOSY is an extremely useful data proposed by Dorner et al. and exemplified in the analysis of processing scheme because it enables one to quickly correlate carbohydrate mixtures.80 resonances with a specific component in solution, rather than The main advantage of diffusion-edited spectra and diffusion a specific spin system. difference spectra is the short experimental time required, while Actually, the term ‘2D DOSY’ describes a number of different the main drawback is that with these methods it is only processing schemes that result in such a 2D plot, but none of possible to differentiate between species of considerably these methods can be considered universal, a fact that has limited different size. the spread of this technique. The 2D DOSY processing techniques can be sorted in two 3.2.3 The use of combined techniques. The limits of high- classes: single-channel methods (SPLMOD, CONTIN are resolution DOSY are defined by the need to obtain resolved implemented in commercial software) and multivariate methods signals from individual species and by the signal-to-noise ratio of (DECRA, MCR, iRRT). Further information on the selection of the data obtained. The problem of resolution is a pressing one in the processing procedure can be found in several excellent complex mixtures, because of the relatively narrow range of reviews.9,72,73,78 proton chemical shifts. Various extensions to the basic proton The main advantages of 2D DOSY are: a) No a priori DOSY experiment have been developed. The most general knowledge of the system is necessary; b) there is no limit to the solution is to combine PFG methods with multidimensional number of resolved components, and c) it is possible to obtain NMR techniques where the spectral overlap is reduced by great precision in the diffusion dimension (differences as little as spreading NMR signals over the entire two-dimensional plane 1% have been reported). rather than along a single chemical axis. The addition of On the other hand, it has several limitations: a) the resonances a diffusion dimension to a 2D spectrum gives the so-called 3D- have to be completely resolved; b) if there are signals originating DOSY. The first experiment was a DOSY–NOESY reported by from more than one component, their measured diffusion coef- Gozansky and Gorenstein, where they were able to resolve Downloaded on 30/04/2013 15:21:41. ficient is the weighted average of those in all of components; c) overlapping resonances from a 14-mer DNA duplex and the method can only be applied with confidence for species within a nucleotide.81 Since then, numerous pulse sequences for 3D a diffusion window of 2 orders of magnitude;72 d) reference DOSY have been published: DOSY–COSY,82 DOSY–TOCSY,83 Published on 11 October 2010 http://pubs.rsc.org | doi:10.1039/C005320C deconvolution (small molecules such as TMS or TSP) must be DOSY–HMQC,84 2D-J-DOSY,85 COSY–iDOSY,86 constant- performed to obtain consistent line shape and high precision in time-HSQC–iDOSY,87 DQF–COSY–iDOSY,88 among others. the diffusion dimension.79 The most significant limitations of these 3D-DOSY experiments are the lengthy experimental time and large data storage requirements. Furthermore, in order to improve the ability of DOSY to distinguish between species, relaxation has been incorporated into diffusion experiments, as a further dimension.89 Additional combinations of relaxation editing and diffusion NMR have been described in subsection 2.2.2. Finally, the combination of diffusion-edited (1D DOSY) and TOCSY (named as DECODES) has been described for the detection of binding ligands to vancomycin in combinatorial chemistry.90

3.2.4 Enhancement of diffusion coefficient differences. Frequently, the mobility differences between compounds are not large enough for sufficient separation in the diffusion coefficient domain. Therefore, a series of strategies to solve this problem have been suggested. 1) Using chromatography stationary phases. Addition of chromatography stationary phases (silica gel and HPLC solid supports) slows down the diffusion of some components, and the spectral separation can be enhanced by one order of magnitude.91 Fig. 7 A 2D-DOSY plot of the glucose/sucrose solution with TSP at 21 A review covering the principles and applications of this C. Reprinted from ref. 72 with permission. approach to mixtures has been provided by Caldarelli et al.92

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2) Matrix-assisted DOSY (MAD) with surfactant micelles. 3.3 Applications of diffusion NMR spectroscopy to NMR Adding surfactant micelles to the mixtures has been shown to analysis of complex mixtures enhance resolution when the component molecules interact Although diffusion NMR measurements for analysis of mixtures differentially with the micelles in water solution. The approach are used in many different fields such as polymer mixtures, was demonstrated using perdeuterated surfactant micelles to supramolecular and combinatory chemistry,98 quick screening study mixtures of peptides with near-identical diffusion coeffi- methods for bioactivity, and mixture analysis in the solid state, cients.93 Using micellar SDS solutions, it was possible to resolve this review is focused on the analysis of complex mixtures in mixtures of isomers (such as dihydrobenzenes and mono- areas such as natural products, food chemistry, pharmaceuticals, methoxyphenols).94 biological fluids and other mixtures. In this section, we will 3) Using polymers. Morris et al. reported in 2001 the use of describe the most representative reports of applications of DOSY weakly acidic and basic polymers to detect association between analysis to unravel the NMR spectra of different types of the polymer and the components of a library on the basis of acid– mixtures characteristics of those areas. base interactions.95 In 2009, Heikkinen et al. reported one of the most attractive ways for the enhancement of diffusion coefficient 3.3.1 NMR of natural product extracts. A modern trend in differences by using the inexpensive, commercially available, and natural product chemistry aims at the direct spectroscopic very CDCl -soluble polymer polyvinylpyrrolidone (PVP), as the 3 analysis of crude mixtures without performing any preliminary ‘stationary phase’ due to its known ability to bind to a variety of chromatographic separation. In this sense, DOSY methods organic molecules. The interaction of the analytes (with similar have many applications to the direct analysis of commercial molecular weights) with PVP essentially follows normal-phase natural extracts such as quality control, batch-to-batch chromatography rules (Fig. 8).26 consistency, and stability studies. It has been applied to the 4) Using complexation agents. Another strategy to enhance the study of the traditional manufacturing procedures for the separation of mixtures by DOSY was developed by Sanstrom€ preparation of herbal medicines, to the identification of new et al. by using complexation agents such as b-cyclodextrin (b- natural products as well as to the study of new synergistic CD) and bovine serum albumin (BSA). In this method, b-CD effects. Furthermore, it is very powerful for the generation of and BSA were added to a mixture of catechins to mimic chro- multivariate fingerprints of complex mixtures. This has found matographic conditions and modify the average mobility, and application especially in mixtures where the identity of some of they were analyzed by DOSY using liquid and high-resolution the components is not known beforehand, such as adulteration magic-angle spinning (HR-MAS) NMR probes. The best spec- of herbal extracts. There are several reports on the application tral separation of the components was achieved with b-CD in the of DOSY to natural product extracts. A representative selec- liquid probe using a 15% CD CN–85% D O solution. This 3 2 tion of examples follows. methodology was also applied to the analysis of catechins from The comparison of hot- and cold-water extracts of cannabis, green tea extract.96

Downloaded on 30/04/2013 15:21:41. as well as of extracts made from ethanol–water mixtures (tinc- The separation of sugar mixtures using DOSY has been tures with different ethanol strength) were reported by Prieto enhanced on the basis of the complex formation abilities of some et al. using diffusion-edited DOSY 1H NMR along with 1H carbohydrates with cations. Thus, DOSY was used as a tool to Published on 11 October 2010 http://pubs.rsc.org | doi:10.1039/C005320C NMR experiments with suppression of the ethanol and water distinguish between the complexed and non-complexed forms in signals. The main aim of this study was to better understand how a mixture, due to the differences in their relative diffusion coef- these extracts differ in their overall composition and to distin- ﬁcients. This strategy was used for the separation of mixture of guish between tinctures from different cannabis cultivars. They sugars in the presence of lanthanide cations, because those sugars found that temperature of the water and polarity of the ethanol– having a particular relationship between their hydroxyl groups water mixtures used for the extraction were the two factors that are able to form complexes in water.97

Fig. 8 DOSY spectra (500 MHz) of a mixture of benzylalcohol, p-methylphenol and p-xylene before and after addition of 50 mg of polyvinylpyrrolidone (PVP). Reprinted from ref. 26 with permission.

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affected total amount of D9-THC in the extracts and its relative different ages, showing that there are compositional transitions quantity with respect to D9-THC-acid and other metabolites.99 depending on the age of the material. More specifically, DOSY The same authors applied both NMR experiments to the direct analysis was applied to distinguish the signals from low molec- analysis of commercial herbal tinctures derived from Echinacea ular weight compounds (i.e. amino acids) from those for large purpurea, Hypericum perforatum, Ginkgo biloba, and Valeriana molecular compounds (i.e. ginsenosides). It showed that the officinalis in order to obtain a metabolic fingerprint useful to methyl signals come from high molecular weight compounds and distinguish tinctures prepared from different plants. Batch-to- that the sugar signals are either from oligosaccharides or ginse- batch homogeneity, as well as degradation after the expiry date nosides. In terms of the composition of the Korean ‘Jeonra’ of a batch, was also investigated. In this case, single chemical ginseng and Chinese ginseng, these results suggest that the entities were difficult to identify due to the signal overlap from former contains more ginsenosides, whereas the latter may different compounds. The diffusion-edited DOSY 1H NMR (1D contain more oligosaccharides.102 DOSY) experiments and 1H NMR with suppression of the A new 2D DOSY experiment was described that is based on ethanol and water signals allowed them to identify some caffeic a Bipolar Pulse Pair Stimulated Echo (BPPSTE) including an acid conjugates from the aromatic protons, and the presence of Excitation Sculpting (ES) water signal suppression. It was carbohydrates/polysaccharides.100 applied to the analysis of black tea infusions, allowing the The first application of 2D DOSY and 3D DOSY–COSY assignment of their main constituents (caffeine, theogallin, the- experiments to a herbal mixture was published by Malet-Martino anine, and catechins, among others) (Fig. 9).103 and co-workers. The main objective of that work was to determine the synthetic drugs that some manufacturers have included 3.3.2 NMR of natural product mixtures. Despite the potential in formulations marketed as ‘herbal medicines’ or ‘dietary of DOSY as ‘NMR chromatography’, it seems that this tech- supplements’. Furthermore, they studied the composition of 17 nique is clearly still undervalued in the field of natural products different herbal drugs and dietary supplements for erectile research (for isolation monitoring, analysis of fractions and dysfunction and found eight formulations adulterated with extracts, etc.), and only a few reports have been published where synthetic PDE-5 inhibitors or analogues. Although the 2D DOSY analyses were used for screening new metabolites from DOSY 1H NMR analyses provide a lot of information about the natural resources. In this section, we will show some of those active pharmaceutical ingredients (API) in the extracts, the main examples together with others centered on the field of natural advantage of this 3D experiment was the ability to extract the polysaccharides. COSY spectrum of each component of the mixture from In order to find novel dereplication methods in natural prod- a selected line in the DOSY spectrum. This is indeed very useful ucts chemistry, Gerwick et al. developed two new diffusion-edi- for its structural determination. The results were confirmed by ted NMR experiments, improved DECODES and LC–MS–MS analysis and by comparison of 1D 1H NMR data of HETDECODES, that sort the constituents in a mixture by their standards.101 individual diffusion coefficients. These experiments allowed the Downloaded on 30/04/2013 15:21:41. The application of 2D DOSY analysis and other NMR partial NMR spectral assignment and cursory structure eluci- experiments allowed differentiation between Chinese and dation of many components of the mixtures.104 Korean ginseng root powder. In addition, the method was also In a research aiming to improve fermentation systems and to Published on 11 October 2010 http://pubs.rsc.org | doi:10.1039/C005320C able to identify Korean ginseng roots from a single region with speed up bioprocess development, DOSY experiments were used

1 Fig. 9 H DOSY-ES NMR spectrum of black tea infusion recorded at 298 K (10% D2O, pH 5.2): a) epigallocatechin gallate, b) epigallocatechin, c) epicatechin gallate, d) epicatechin, e) catechin. The blue and green colours represent weak and strong signal intensities. Reprinted from ref. 103 with permission.

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to identify both co-metabolites and impurities from different analytical ultracentrifugation and size exclusion chromatog- strains and culture conditions. This approach was validated in raphy (SEC).111 the production of lovastatin from Aspergillus terreus.105 Application of 2D DOSY analysis to the polysaccharidic Application of 2D DOSY to crude bromopyrrole fractions fraction from the mycelium of the basidiomycete mushroom from Okinawan marine sponges allowed the identification of two Lentinula edodes, grown on olive mill waste-water, showed the new bromopyrrole alkaloids, agesamides A and B, as well as presence of two polysaccharides of different sizes. They were several other known metabolites.106 identified as xylan and lentinan with the help of 1D and 2D 2D DOSY was also applied to the identification of the NMR techniques. Lentinan is known for its antitumor, anti- components of the ethyl acetate fraction of Bidens sulfurea, and bacterial, antiviral, anticoagulatory and wound-healing activi- allowed the identification for the first time in this plant of four ties.112 flavonoid glycosides derived from quercetin. Its 2D DOSY NMR A detailed study of the polysaccharides present in hot aqueous spectrum shows that the sample is composed of a mixture of extracts of Chlorella pyrenoidosa was performed by 2D DOSY substances presenting a high degree of structural similarity, NMR. These experiments measured the molecular weights of the spread over a logD between 9.8 and 10.0.107 arabinogalactans, extending the relationship between diffusion A very practical application of 1D DOSY was reported in the coefficients and molecular weights to different types of neutral characterization of three saponins isolated from the polar extract polysaccharides. DOSY experiments were also used to determine of Agave brittoniana leaves. Thus, an edited-spectrum DOSY was the molecular weight dispersions of various arabinogalactans.113 used to filter out the residual water signal and to obtain a clear Similarly, 2D DOSY analysis and diffusion-edited 1H NMR view of the signals near to water (anomeric protons of the spectra (1D DOSY) were applied to the study of high molecular saponin sugars). Other techniques for the suppression of solvent weight polysaccharides obtained from various mushroom signal such as presaturation of the water signal would affect the extracts and fractions. The main objectives were to monitor the anomeric resonances.108 fractionation procedure, and to check the purity of the isolated In a very recent report, a DOSY methodology was developed polysaccharides as well as their molecular size. It is worth noting to resolve mixtures of mono-, di-, and triacylglycerides (along that DOSY was also used to effectively filter out the signals of the with their methyl esters) derived from vegetable oils for biodiesel low molecular weight compounds.114 production.109 Also, 2D DOSY has been applied to the screening of different Diffusion-type experiments have also been proven to be useful batches of heparins and to obtain impurity profiles of the batches for the analyses of mixtures of polysaccharides. For example, (mainly comprising oversulfated chondroitin sulfate, OSCS). diffusion difference NMR spectroscopy was used in analysis of This method was also successfully applied to differentiate carbohydrate mixtures, including a mixture of two homoglycans between (a) unfractionated heparins, (b) the OSCS impurity (18 which differ only in chain length, a peptidoglycan for the char- kDa),12 and (c) the low molecular weight heparins (LMWHs, acterization of the linkage region between a bacterial cell wall around 4–8 kDa) obtained by partial depolymerization of Downloaded on 30/04/2013 15:21:41. polysaccharide and the surface-layer (S-layer) protein, and heparin. The 1H NMR DOSY traces of the N-acetyl region were finally, the monitoring of adenosine monophosphate (AMP) used to identify OSCS in the LMWHs.115 derivatization.80 In another interesting example, Mannina and Published on 11 October 2010 http://pubs.rsc.org | doi:10.1039/C005320C co-workers described in 2003 the use of 2D DOSY to estimate the 3.3.3 NMR of pharmaceuticals. The need for new and effi- average molecular weight of uncharged polysaccharides. DOSY cient analytical methods for quality control in the manufacture experiments on six well-characterized pullulan fractions of of drugs is essential. In this sense, DOSY is a powerful meth- different molecular weights were carried out on dilute aqueous odology for the complete characterisation of pharmaceutical solutions. The calibration curve was validated with a series of formulations because it allows the precise determination of compounds of known molecular weight, including oligo- and differences in drug composition. A major advantage of DOSY is polysaccharides as well as synthetic polymers and small mole- that this technique provides global information on the compo- cules. Furthermore, DOSY was shown to allow the spectral sition of a formulation and permits to consider the drug prepa- resolution of mixtures of two dextran homopolymers with ration as a whole. It allows detection of not only the active different molecular weights (1.27 and 668 kDa). The results pharmaceutical ingredients but also most excipients. Not only demonstrated that DOSY has, over other methodologies, the can it be used to distinguish between genuine and imitation advantage of allowing the measurement over a wider range of tablets, but it is also helpful in determining the relationship molecular weights.110 between different samples and so to assist in the investigation of An application of 2D DOSY to characterize the size of the sources of those drugs. A chemical fingerprint of different

different N-acetyl-chitooligosaccharides (GlcNAc)16 and their types of pharmaceutical samples can be obtained by DOSY, and complexes to protein receptors such as the small binding so it provides precise information on the samples analysed. protein hevein has been reported by Jimenez-Barbero et al. In Indeed, it is a recognised tool for screening pharmaceutical this study, DOSY has also been applied to two synthetic formulations and checking the quality of drugs.33,53

related oligosaccharides (GlcNH2-(GlcNAc)4 and GlcNH2- One of the ﬁrst examples of this type of 1D and 2D DOSY (GlcNAc)2-GlcNAcSO3Na), and their molecular weights could application was the analysis of the ‘signature’ of 5 commercial be estimated to within 10% error. The evaluation of the stoi- formulations of ciproﬂoxacin tablets purchased in different chiometry of the complexes formed between hevein and countries, including some with a non-regulated market. In

(GlcNAc)6 showed that 2D DOSY is a complementary method addition to ciproﬂoxacin, several excipients could be observed to other currently used physical biochemical methods, such as depending on the formulation studied. DOSY spectra clearly

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showed similarities and differences in the composition of the DOSY spectrum, allowing an easier structural determination. pharmaceutical formulations, thus giving a signature of the This study allowed the authors to distinguish imitations or manufacturer.116 counterfeit samples from the authentic formulation by detecting In the particular case of pharmaceutical formulations of the presence of sildenafil or adulterants and other excipients. The tadalafil, genuine Cialis and seven illegally manufactured precise global ‘signature’ of each drug indicated that the quality formulations of this drug were analysed by 2D DOSY NMR. of products manufactured in India and Syria was better than The samples displayed similarities and differences sufficient to those of the Chinese formulations, which were adulterated with produce a precise and global ‘signature’ of the manufacturer, vardenafil and homosildenafil.117 including not only the active pharmaceutical ingredient(s) but The integral characterization of counterfeit antimalarials also the excipients. The self-diffusion coefficient value was constitutes another demonstration of the power of 2D DOSY measured for each peak and an average self-diffusion coefficient 1H NMR to provide a highly comprehensive chemical finger- was determined for each formulation. The differences in the print of pharmaceutical formulations and complex mixtures. In values of the diffusion coefficients for the same component in the this study, 14 different artesunate-based antimalarial drugs, different formulations are due to the different viscosity of each representative of those that can be purchased from informal formulation. The data obtained in this study showed that the sources in southeast Asia, were investigated by a combination of quality of the Cialis ‘imitations’ manufactured in India and 2D DOSY and mass spectrometry techniques. The expected Syria was acceptable, whereas the Chinese formulation was active pharmaceutical ingredient (API), artemisinin, was detec- adulterated with other active pharmaceutical ingredients.118 ted in only five formulations along with common organic In another work, the specific ‘signature’ of several pharma- excipients such as sucrose, lactose, stearate, dextrin, and starch. ceutical formulations (solid and liquid) of fluoxetine and flu- The remaining formulations were shown to contain various voxamine was obtained with 2D DOSY NMR. This study ‘wrong’ APIs.120 allowed the characterisation of the active ingredients and excipients present in the formulations studied. The DOSY 3.3.4 NMR of foods. In food chemistry analyses, the case of spectra of brand and generic formulations of fluvoxamine were liquid natural foods is a challenging one because it perfectly identical, whereas some differences could be seen between the exemplifies many of the problems that make chemical analyses brand-named and the generic formulation of fluoxetine. Fluox- difficult due to the complexity of the matrix: overlap, low reso- etine and fluvoxamine have similar molecular weights, but by lution for larger components and low abundance of many others. adding b-cyclodextrin as a complexing agent, 2D DOSY sepa- In this context, DOSY spectra of liquid food samples have ration was obtained.119 provided useful information about the chemical composition of The first application of a 3D DOSY–COSY experiment to tomato pulp and juice, mango, apple juice, grape juice, beer, and a pharmaceutical formulation was the analysis of 17 formula- port wine. tions of sildenafil. One of them was the genuine Viagra, while A comprehensive assignment of the proton spectra of both Downloaded on 30/04/2013 15:21:41. the others were illegally manufactured formulations of this drug tomato pulp and juice was obtained by combination of 2D from India, Syria and China (Fig. 10). In this 3D DOSY–COSY DOSY and other 1D and 2D NMR experiments (J-resolved, 1H NMR experiment, the COSY spectrum of each component of COSY, TOCSY, HSQC, HMBC and HR-MAS). The compar- Published on 11 October 2010 http://pubs.rsc.org | doi:10.1039/C005320C the mixture was successfully extracted from a selected line in the ison between the juice and pulp spectra shows that essentially all

1 Fig. 10 2D DOSY H NMR spectra in D2O of tablets from (A) genuine Viagra and (B) the Syrian counterfeit formulation 13. (S) sildenaﬁl; (*) hypromellose; (-) triacetine; (Y) lactose; (B) polyethylene glycol (PEG). TMPS (trimethylsilylpropane sulfonic acid) is the internal reference. Reprinted from ref. 117 with permission.

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the water-soluble substances present in the pulp are present in the juice. On the basis of the assignments made, the qualitative and quantitative characterization of tomato juice from two tomato cultivars (Red Setter and Ciliegino) was also determined. The results suggest that NMR could be a very useful tool for the characterization of tomatoes, especially when statistical analysis is used, since quantitative determination of many compounds could be obtained by a single experiment.121 Very few PGSE NMR studies have been applied to the self- diffusion of small solutes in natural polysaccharide polymers used in the food industry as thickeners. Since aroma diffusion in food matrices could have an impact on flavour release, this study is an interesting and economic challenge. In this context, a 2D DOSY analysis to investigate the diffusion of two aroma- producing compounds, ethyl butanoate and linalool, in different gelling states was performed by using i-carrageenan matrix as 122 food model. Fig. 11 2D DOSY spectrum of ripe mango juice. Reprinted from ref. A DOSY analysis was also applied to fruit juices (apple and 125 with permission. grape) and beer. This technique was shown to be particularly useful for aliphatic, sugar and aromatic compounds. In the case of apple juice, DOSY spectra confirmed the expected predomi- 3.3.5 NMR of humic substances. The potential of DOSY nance of sucrose, glucose and fructose, the presence of some NMR in the analysis of natural mixtures has been shown in the organic acids (e.g. quinic, malic, citric and tartaric acids) in the aggregation of natural organic matter. Soils and sediments often aliphatic region, and chlorogenic acid and epicathechin in the termed humic substances are very complex mixtures of organic aromatic region. The grape juice showed greater complexity than matter and can be divided into three main fractions: humic acid, apple juice. Its aliphatic region was dominated by signals due to fulvic acid, and humin. the presence of many amino acids superimposed with several Humic substances have been traditionally thought to be high aliphatic organic acids; the sugar region is dominated by glucose molecular weight, cross-linked macromolecules with mostly and fructose, while the aromatic region displays evidence of undetermined structures. The application of 1D and 2D DOSY tyrosin, phenylalanine, gallic acid, niacin, uridine and hydrox- to two extracts of humic substances demonstrated the presence of ycinamic acids. Finally, two beer types were analysed by DOSY, numerous aliphatic and aromatic compounds, as well as sugars which showed some small differences between them.123 and amino acids. These studies demonstrated that fulvic and Downloaded on 30/04/2013 15:21:41. The first 2D DOSY study on wine was performed on samples humic acids are in fact aggregates composed by relatively simple of port wine. This analysis aimed not only to identify the main molecules that take on colloidal properties in the presence of components but also to achieve improved interpretation of the metal ions.126,127 Published on 11 October 2010 http://pubs.rsc.org | doi:10.1039/C005320C composition differences between wines of different ages. This Later on, different research groups confirmed its aggregate study showed the evolution of the size of the polyphenols after nature, described the presence of lignin, peptides and sugars, and aging. Indeed, 2D DOSY showed that the most notable differ- determined the differences in the main components in humic acid ence introduced by age was the decrease in the concentration of and fulvic acid.128 In the particular case of the humic substances polyphenolic molecules and the simultaneous appearance of new isolated from vermicompost, 2D DOSY served to study its small compounds, reflecting the molecular breakdown occurring composition and the changes produced after transformation.129 with wine aging.124 The application of 2D DOSY along with 1D and 2D NMR 3.3.6 NMR of biological fluids and tissues. The importance of and hyphenated NMR (LC–NMR and LC–NMR–MS) to the NMR spectroscopy as an analytical tool for metabonomic characterization of mango juice allowed the identification of studies of biological fluids, cells and tissues have been mentioned. more than 50 compounds. More specifically, the diffusivity Section 2.3.5 describes the applications of diffusion-edited information provided by 2D DOSY allowed some previous spectra (1D DOSY) to biological fluids and tissues.1,35,37,41 This ambiguous assignments, such as some singlets in the aromatic section is devoted to 2D DOSY only. region and the two broad signals in the same region, previously 2D DOSY has been applied to the study of a percholic acid assigned to polyphenols and now attributed to glutamine, to be extract from gerbil brain, allowing the identification of 15 clarified (Fig. 11). Furthermore, this work incorporated a study metabolites.130 of compositional changes taking place as a function of ripening. A new approach to aid complex biomixture analysis is statis- They found that as ripening proceeds, many compositional tical diffusion-ordered spectroscopy (S-DOSY). This technique changes occur, and by signal integration, absolute concentra- combines DOSY with statistical total correlation spectroscopy tions were obtained for organic acids, amino acids, and sugars. (STOCSY), and its usefulness was demonstrated in the charac- DOSY also proved to be of use in providing average relative terization of urinary biomarkers and in studies on plasma.131 molecular mass estimates for the pectic fraction of both unripe The new 2D DOSY experiment based on a bipolar pulse pair and ripe juices, reflecting the expected decrease in size as ripening stimulated echo (BPPSTE) sequence including an excitation (and hence pectin hydrolysis) proceeds.125 sculpting (ES) water signal suppression sequence, was applied to

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the analysis of human plasma, allowing the molecular weight- systems such as synthetic organic intermediates;136 the identifi- based separation of the compounds. The large difference between cation of heroin in street doses for forensic studies;137 the iden- the molecular weights of metabolites and macromolecules tification and quantification of minor components (amino acids) (proteins, lipids) leads to an excellent separation on the 2D in honey,138 in metabonomic studies of urine,139 and in the DOSY-ES spectrum. In this way, low molecular weight meta- analysis of balsamic vinegar of Modena (Fig. 12).66 bolites (amino acids, lactate, creatine, etc.) were localized at the In a different application, the association constant of an arti- top of the spectrum, while proteins or lipids were localized ficial receptor–carbohydrate complexation was determined by towards the bottom.103 TOCSY titration to overcome the severe overlapping in the 1H NMR spectra.140 3.3.6 NMR of other complex mixtures. Other studies using Murthy et al. have used 2D TOCSY experiments to analyze DOSY analysis have been carried out on petroleum distillates76 mixtures of inositol phosphates. Since all protons of the inositol and in the study of DNA secondary structural elements.132 ring are part of a connected spin system, it was possible to extract the individual 1H subspectra of each component without prior 141 4 Other methods separation. 2D TOCSY has also been employed to show three diastereomeric lanthionine-bridged peptides in a reaction 4.1 Selective saturation and magnetization transfer mixture.142 Bruschweiler et al. have optimized the processing and data analysis of 2D-TOCSY for the analysis of mixtures, and Rabenstein et al. described the use of presaturation to partially applied it to the metabolic profiling of an insect venom (from 1 eliminate the hemoglobin envelope from H NMR spectra of Anisomorpha buprestoides).143 intact erythrocytes, but retaining the signal of most low molec- 133 ular weight metabolites. The method is similar to the Advantages and drawbacks. The main advantage of TOCSY, suppression of water by selective presaturation, but in this case, compared to diffusion and relaxation editing, is that no differ- the saturation is transferred to protons of the less mobile parts of ence in molecular weight or mobility is required to obtain the a large protein (due to spin diffusion by cross-relaxation), and pseudo-separation. The unique requirement is the existence of therefore the suppression attains not only the region of the pre- a one spin multiplet clearly isolated from the rest of the spectra. saturation but also the whole protein spectrum. This method not On the other hand, the main drawback is that the spectra of only suppresses the hemoglobin envelope, but also any other a spin system and not of the whole molecule, is obtained. This resonances near the presaturation, and so does not allow such means that it is rather a method for selection than for suppres- good pesudo-separation as relaxation editing. sion. For example, in a mixture of proteins with low molecular weight metabolites, it is not possible to obtain the spectra of the 4.2 TOCSY overall mixture of metabolites (without the proteins), but only the spectra of each individual component separately. Downloaded on 30/04/2013 15:21:41. The TOtal Correlation SpectroscopY (TOCSY) experiment (1D and 2D versions) is conventionally used for the identification of resonances generated by a one-spin system in a molecule. In the 4.3 J-resolved spectroscopy (JRES) Published on 11 October 2010 http://pubs.rsc.org | doi:10.1039/C005320C 1D version, a single resonance is selectively irradiated, and the The 2D 1H J-resolved NMR (JRES NMR) experiment was 1 magnetization propagates through the H coupling network, introduced 1976 by Ernst et al.144 JRES consists of an array of resulting in the selective and clean visualization of the whole spin spin-echo pulse elements, in which an incremented delay period is network. used to define a second frequency dimension. After appropriate et al. Subramanian showed the high potential of 1D-TOCSY data processing,4,145,146 the chemical shift and J-coupling infor- experiments for the analysis of mixtures by separating the spectra mation are resolved on two different dimensions while the of two isomers (cyclic lactones).134 Lindon et al. first applied 1D resonance intensities are edited on the basis of the proton T2 TOCSY for the analysis of a complex mixture, allowing the relaxation times. From the 2D JRES experiment it is possible to detection and assignment of pyroglutamate and uracil in seminal extract a projection that is quite similar to an ordinary 1D 135 fluid. This method has been also applied to several other spectrum (p-JRES), where each proton appears as a single peak, irrespective of the multiplicity of the original signal and hence effectively yielding a 1H broadband-decoupled spectrum. This simplifies the 1D NMR spectra, facilitating the separation of the signals in crowded areas, although information on J-couplings is lost. JRES experiment has been applied to the analysis of biological fluids (blood plasma and urine) since the late 1980s, allowing (for example) the assignment of the 3–4 ppm spectral region in blood plasma for the first time.147 Since then, JRES has been applied in metabolomics studies on biological fluids, such as cerebro- 148 135 Fig. 12 (A) Selective TOCSY experiment on balsamic vinegar of spinal and seminal fluid, as well as to tissues (in this case 55,149 Modena obtained by using a selective Gaussian-shaped pulse centred at JRES to HR-MAS), for the elimination of polymer reso- 150 4.44 ppm. (B) T2-filtered spectrum of the same sample. Reprinted from nances in combinatorial chemistry, and for spectral simplifi- ref. 66 with permission. cation in complex reaction mixtures.151

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Although the J-resolved spectrum is always obtained in the 2D mode, the data analysis is more frequently carried out in the 1D p-JRES spectrum.4,5 The advantage lies in the combined action of relaxation ﬁltering and absence of couplings. This is exem- pliﬁed in Fig. 13, where proteins and lipoproteins are eliminated or strongly attenuated respectively in the JRES experiment. For their part, the groups of Verpoorte and Viant developed the application of JRES to extracts from plants152 and aquatic species.153 Examples can also be found in invertebrates (e.g. the hemolymph of tobacco hornworms).154

Advantages and drawbacks. The main advantages of p-JRES spectra versus CPMG editing are the increased resolution and attenuation of macromolecules with respect to that obtained

using T2 editing (Fig. 13). On the other hand, the main disad- vantages are the longer data acquisition times required (although Fig. 14 1H NMR from human blood plasma: black, 1H; blue and red, these are still shorter than those of other 2D NMR methods) and CPMG with 102.4 and 608 ms T2 filtering; green and magenta, D-RESY the phase-twisted line shapes which complicates the quantifica- and G-RESY, respectively. Reprinted from ref. 156 with permission. tion.146 Overall, JRES and CPMG editing should be taken as complementary techniques because the information contained in JRES projections is intrinsically different from that obtained in Advantages and drawbacks. The main advantages of this the CPMG spectra.145 approach vs. CPMG are the better signal-to-noise ratio and that it can be used to extract information hidden (or simply not 4.4 ‘Virtual’ relaxation-edited spectroscopy (RESY) previously analyzed) information in archived 1H NMR data sets. It should be stressed that these processing approaches are not A completely different approach for spectral simplification is a substitute for T editing. Indeed, if very closely spaced narrow based on the use of processing procedures that resemble a CPMG 2 signals coming from protons with long T times are present, they filter. These methods try to attenuate the signals from protons 2 could be observed in the CPMG but not in the D-RESY spec- with short transverse relaxation times and to enhance the signals trum, especially at lower fields. originating from small molecules. This methodology was first described by Lindon et al., who applied a Gaussian shaping to the free induction decay (G-RESY).155 In 2008 the same author

Downloaded on 30/04/2013 15:21:41. expanded this technique using a different mathematical func- 4.5 Paramagnetic spin relaxation (PSR) ﬁltering tion,156 named as D-RESY (Derivative-based RElaxation-edited SpectroscopY). This ‘virtual’ relaxation-edited spectrum was Although it is known that nuclei relax faster in the presence of Published on 11 October 2010 http://pubs.rsc.org | doi:10.1039/C005320C shown to be very useful in the identiﬁcation of biomarkers for paramagnetic spin relaxing metal ions,157 the application of this type 2 diabetes in human blood plasma, formerly accomplished concept for unravelling the spectra of complex mixtures was not by CPMG spectra. Fig. 14 shows the D-RESY and G-RESY disclosed until 2007 by the group of Fernandez-Megia and spectra together with the CPMG. Riguera.158

Fig. 13 Comparison of unedited, diffusion-edited, T2-edited and p-JRES spectra of plasma. Reprinted from ref. 4 with permission.

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The principle of PSR ﬁlters rests on: i) the inverse pro-

portionality between T2 and the spectral line width, and ii) the different ability of the various functional groups within a mixture to complex PSR metal ions through fast chemical exchange.157 Accordingly, these authors have described that the addition of increasing concentrations of a PSR metal ion to a mixture sharply reduces the relaxation times (and broadens the line width) of those constituents with the highest ion-complexing ability (acidic and polyanionic species). Eventually, at higher ion concentrations these signals could became embedded in the baseline,159 while leaving unaffected those of other less sensitive components (e.g., amides, ammonium and hydroxyl groups, and ethers). For this application, Gd3+ was selected as PSR ion based on its

high electronic relaxation time (ss) and the presence of a large 1 1 Fig. 16 H PSR filtering. H NMR spectra (D2O) of (a) santiagoside, (b) 3+ number of unpaired electrons. When Gd (0.4 mM) was added methyl-a-D-glucopyranoside, (c) a mixture of santiagoside and methyl-a- to a mixture of lactose and hyaluronic acid (HA), the resonances D-glucopyranoside in the absence of Gd3+, and (d) a mixture of santia- corresponding to HA were completely removed, and only those goside and methyl-a-D-glucopyranoside in the presence of Gd3+ of lactose were seen in the spectra, without loss of resolution or (0.5 mM). signal-to-noise ratio (Fig. 15). The same approach has been applied to many other mixtures where the signals corresponding diffusion coefficients that are greatly affected by molecular to the most strongly Gd3+-complexing species were selectively weight. This has allowed the implementation of combined PSR suppressed in 1H, 13C, and even 2D NMR experiments (COSY filters with CPMG and diffusion to produce multiple or and HMQC).158 sequential suppressions with unprecedented results. However, The same group has recently shown (unpublished results) that ways to extend this methodology to other less sensitive a PSR filter effectively suppresses the resonances of the sulfate substrates, and efforts to quantify the sensitivity of different containing triterpene and steroidal glycosides santiagoside and compounds to PSR ions, are needed in order to predict selective lefevreioside in the presence of neutral sugars.160 As an example, suppressions in mixtures. Fig. 16 shows the suppression of the signals of a marine triterpene asterosaponine in the presence of methyl-a-D-glucopyr- 161 anoside. 5 Conclusions Interestingly, PSR filters have been used in combination with Downloaded on 30/04/2013 15:21:41. CPMG (a PSR-CPMG filter) in cases where species with similar This review describes the most important ways to selectively 1 3+ H T2 values and sensitivities to Gd were not practical by simplify the NMR spectra of complex mixtures on the basis of applying the PSR filter alone. the relaxation, diffusion and other characteristics of the Published on 11 October 2010 http://pubs.rsc.org | doi:10.1039/C005320C components. These methods are complementary, so judicious Advantages and drawbacks. The main advantage of the PSR selection and adjustment of the acquisition parameters can allow filter is its high insensitivity to molecular weight, in contrast to the suppression of different classes of components in a mixture.

CPMG and diffusion filters that depend on differences of T2 and A convenient approach is to use a diffusion filter to attenuate/ suppress the signals from the small molecules leaving untouched those of the larger ones, and/or to use a relaxation filter to attenuate/suppress the signals due to the large molecules, enabling visualization of just the smaller ones. When a strong signal overlap exists and further simplification is needed, J- resolved spectroscopy (simultaneous relaxation filtering and effective 1H broadband-decoupling) and TOCSY (selective and clean visualization of a spin network) can be used. Finally, a completely different method relies on the addition of paramagnetic agents (PSR filtering), that allows the attenuation/ suppression of the signals coming from compounds interacting with the PSR agent, irrespective of their molecular weight. A summary is provided in Table 1. The above methods have been applied with success mainly to biological samples, pharmaceutical preparations, foods and beverages. In addition, a few examples of applications to living organisms and natural products have been also reported. In our

1 1 Fig. 15 H PSR ﬁltering. H NMR spectra (D2O, 500 MHz) of (a) view, it is only a matter of time that extracts will be investigated lactose, (b) HA, (c) a mixture of lactose and HA in the absence of Gd3+, using these ‘pseudo-separation’ techniques, instead of devoting and (d) a mixture of lactose and HA in the presence of Gd3+ (0.4 mM). such a huge effort to isolation and dereplication.

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Table 1 Principal methods for spectral simpliﬁcation of complex mixtures

Method Application Pseudo-separation basis Key parameters

Relaxation filtering Attenuation/suppression of Relaxation times Length and number of loops macromolecules (CPMG) Diffusion filtering Attenuation/suppression of low Translational motion Diffusion time, pulse length molecular weight molecules TOCSY Visualization of a spin network J coupling Mixing time J-Resolved spectroscopy Effective broadband decoupling J coupling Increment time, spectral processing and relaxation filtering PSR filtering Suppression of molecules Paramagnetic relaxation PSR agent concentration, interacting with the PSR agent enhancement complexing ability

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