Evaluation of Ultrafast 2D NMR for Quantitative Analysis - Analytical

Anal. Chem. 2009, 81, 479–484

Evaluation of Ultrafast 2D NMR for Quantitative Analysis Patrick Giraudeau,* Ge ´ rald S. Remaud, and Serge Akoka Universite´ de Nantes, CNRS, CEISAM UMR 6230, B. P. 92208, 2 Rue de la Houssinie`re, F-44322 Nantes Cedex 03, France Recent ultrafast methods make it possible to obtain twodimensional (2D) nuclear magnetic resonance (NMR) spectra in a fraction of a second. This paper presents the first evaluation of ultrafast 2D NMR for quantitative analysis. On the basis of optimized conditions presented in recent studies, two homonuclear ultrafast techniques, J-resolved spectroscopy and TOCSY, are evaluated on model mixtures in terms of repeatability, long time stability, and linearity. The results are compared to conventional 1D 1H NMR spectroscopy. Repeatabilities better than 1% for ultrafast J-resolved spectra and better than 7% for TOCSY spectra are obtained. The long-term stability is better than 4% for J-resolved spectroscopy and between 2% and 11% for TOCSY. Moreover, both methods are characterized by excellent linearities. This new analytical method opens important perspectives for fast, precise, and accurate quantitative analysis of complex mixtures and for the quantitative study of short time scale phenomena. One-dimensional (1D) 1H nuclear magnetic resonance (NMR) is a powerful tool for quantitative analysis in a wide range of domains, from drug analysis1 to natural products analysis2 or in vivo spectroscopy.3 However, precise quantitative analysis of complex mixtures is often made difficult by the presence of large overlap between peaks. Twodimensional (2D) spectroscopy4,5 offers an interesting alternative, as it brings a resolution enhancement that is essential for quantitative studies of complex spectra. Recent papers have highlighted the potential of 2D NMR for quantitative analysis;6-9 however, despite its high potentialities, quantitative 2D NMR is restricted by two major limitations. First, it is hampered by long acquisition durations, from several minutes to several hours, due to the necessary t1 incrementation. Moreover, 2D peak volumes are influenced by a number of factors such as J-couplings or transverse relaxation times (T2), which * Corresponding author. Dr. Patrick Giraudeau, Chimie et Interdisciplinarite´: Synthe`se, Analyse, Mode´lisation (CEISAM), UMR 6230, Faculte´ des Sciences, BP 92208, 2 Rue de la Houssinie`re, F-44322 Nantes Cedex 03, France. Phone: +33(0)2 51 12 57 02. Fax: +33(0)2 51 12 57 12. E-mail: patrick.giraudeau@ univ-nantes.fr. (1) Holzgrabe, U. J. Pharma. Biomed. Anal. 2005, 38, 806–812. (2) Pauli, G. F.; Jaki, B. U.; Lankin, D. C. J. Nat. Prod. 2005, 68, 133–149. (3) Podo, F.; Henriksen, O.; Bove´e, W. M. M. J.; Leacj, M. O.; Leibfritz, D.; De Certaines, J. D. Magn. Reson. Imaging 1998, 16, 1085–1092. (4) Aue, W. P.; Bartholdi, E.; Ernst, R. R. J. Chem. Phys. 1976, 64, 2229–2246. (5) Jeener, J. Lecture presented at Ampere International Summer School II, Basko Polje, Yugoslavia, 1971. 10.1021/ac8021168 CCC: $40.75  2009 American Chemical Society Published on Web 11/21/2008

leads to very inaccurate results. This drawback can be bypassed by performing a calibration using various mixtures with different concentration ratios.6 Unfortunately, this procedure is very time-consuming due to the experiment duration. Consequently, conventional 2D NMR appears unsuitable for quantitative studies in a reasonable time. Moreover, the long experiment duration makes it inappropriate for studies of short time scale phenomena. Fortunately, a new method was recently proposed by Pr. Frydman and co-workers,10,11 allowing the acquisition of 2D NMR spectra within a single scan, thus reducing the experiment duration to a fraction of a second. In this so-called “ultrafast 2D NMR” approach, the usual indirect-domain temporal encoding of conventional 2D NMR is replaced by a spatial encoding. The latter is imparted in a single scan by applying alternating magnetic field gradients synchronized with selective, frequency-swept rf pulses. When an appropriate mixing period is employed, any type of homo- or heteronuclear correlation can be observed after an adapted postprocessing.10 The principles of ultrafast techniques, as well as their experimental implementation, have been presented in recent papers,10-17 and consequently they will not be detailed here. Ultrafast 2D experimental parameters are specific to this methodology and they strongly influence the resolution and the sensitivity of 2D spectra. Fortunately, several recent studies18-21 determined the optimal conditions (temperature and solvent conditions, pulse sequence parameters, etc.) in order to perform (6) Giraudeau, P.; Guignard, N.; Hillion, H.; Baguet, E.; Akoka, S. J. Pharm. Biomed. Anal. 2007, 43, 1243–1248. (7) Koskela, H.; Kilpela¨inen, I.; Heikkinen, S. J. Magn. Reson. 2005, 174, 237– 244. (8) Lewis, I. A.; Schommer, S. C.; Hodis, B.; Robb, K. A.; Tonelli, M.; Westler, W.; Sussman, M.; Markley, J. L. Anal. Chem. 2007, 79, 9385–9390. (9) Massou, S.; Nicolas, C.; Letisse, F.; Portais, J.-C. Metab. Eng. 2007, 9, 252–257. (10) Frydman, L.; Lupulescu, A.; Scherf, T. J. Am. Chem. Soc. 2003, 125, 9204– 9217. (11) Frydman, L.; Scherf, T.; Lupulescu, A. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 15858–15862. (12) Frydman, L. Bull. Isr. Chem. Soc. 2005, 19, 33–44. (13) Frydman, L. C. R. Chimie 2006, 9, 336–345. (14) Giraudeau, P.; Akoka, S. J. Magn. Reson. 2007, 186, 352–357. (15) Pelupessy, P. J. Am. Chem. Soc. 2003, 125, 12345–12350. (16) Shrot, Y.; Frydman, L. J. Chem. Phys. 2008, 128, 052209. (17) Shrot, Y.; Shapira, B.; Frydman, L. J. Magn. Reson. 2004, 171, 163–170. (18) Giraudeau, P.; Akoka, S. J. Magn. Reson. 2008, 190, 339–345. (19) Giraudeau, P.; Akoka, S. J. Magn. Reson. 2008, 192, 151–158. (20) Giraudeau, P.; Akoka, S. J. Magn. Reson. 2008, in press, DOI: 10.1016/ j.jmr.2008.1008.1001. (21) Shrot, Y.; Frydman, L. J. Chem. Phys. 2008, 128, 164513.

Analytical Chemistry, Vol. 81, No. 1, January 1, 2009

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2D ultrafast experiments with optimum resolution, line shape, and sensitivity. Thanks to its single scan nature and to these recent optimizations, ultrafast 2D NMR appears very promising for quantitative analysis. It should find applications in various areas where conventional 2D NMR fails because of the high experimental durations involved. However, to our knowledge, ultrafast 2D NMR has never been evaluated for quantitative analysis. The aim of this study is to present the first quantitative evaluation of homonuclear ultrafast 2D techniques, by evaluating their repeatability, their long-time stability, and their linearity on a simple model mixture. Two widely used 2D NMR methods, J-resolved14,22 spectroscopy and total correlation spectroscopy (TOCSY)15,23 are evaluated under optimized conditions defined in previous studies,18-20 and the results obtained are correlated to 1D 1H NMR spectroscopy. The choice of these two pulse sequences was dictated by their potential usefulness: conventional J-resolved spectroscopy has been widely studied over the past few years for its quantitative properties,6,24,25 whereas TOCSY is one of the most widespread homonuclear techniques for analyzing complex biological samples9,26,27 with large 1H overlaps. EXPERIMENTAL SECTION Sample Preparation. Ethanol-methanol mixtures in DMSOd6 were prepared to evaluate ultrafast NMR techniques for quantitative analysis. Methanol concentration was kept constant at 100 mmol L-1 to set the methanol signal as an internal reference. Six mixtures of 1 mL were prepared with the following ethanol-methanol concentration ratios determined by quantitative 1D 1H NMR: 0.23; 0.49; 0.72; 0.99; 1.43; 2.19. After homogenization, each sample was filtered and analyzed in a 5 mm tube. NMR Spectroscopy Experiments. NMR spectra were recorded at 298 K on a Bruker Avance 500 DRX spectrometer, at a frequency of 500.13 MHz with a triple resonance TBI probe including z-axis gradient. Successive experiments were separated by a 120 s delay. 1D 1H NMR spectra were recorded using 90° pulses with a single transient. Free induction decays (FIDs) were recorded in 20 K channels and zero-filled to 32 K, with a spectral width of 1965 Hz and an acquisition time of 5 s. Ultrafast 2D parameters were set to obtain optimum excitation and detection conditions, as described in previous studies.18-20 All experiments were performed using the encoding scheme proposed by Pelupessy,15 starting with a 90° hard pulse followed by two Wurst-100 180° encoding pulses28 with a sweep range of 11 kHz, applied during Ge ) ±0.011 T m-1 alternating excitation gradients. Wurst pulse durations were set to 30 ms for J-resolved spectroscopy and to 15 ms for (22) Aue, W. P.; Karhan, J.; Ernst, R. R. J. Chem. Phys. 1976, 64, 4226–4227. (23) Braunschweiler, L.; Ernst, R. R. J. Magn. Reson. 1983, 53, 521–528. (24) Mutzenhardt, P.; Guenneau, F.; Canet, D. J. Magn. Reson. 1999, 141, 312– 321. (25) Nuzillard, J.-M. J. Magn. Reson., Ser. A 1996, 118, 132–135. (26) Massou, S.; Nicolas, C.; Letisse, F.; Portais, J.-C. Phytochemistry 2007, 68, 2330–2340. (27) Morvan, D.; Demidem, A.; Papon, J.; Madelmont, J.-C. Magn. Reson. Med. 2003, 49, 241–248. (28) Kupce, E.; Freeman, R. J. Magn. Reson., Ser. A 1995, 115, 273–276.

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TOCSY, leading to the best compromise between sensitivity and resolution and to a 1965 Hz spectral width. The detection block for J-resolved spectroscopy14 was formed of 128 gradients (Ga ) 0.088 T m-1) applied during 5.8 ms each and separated by 180° hard pulses. For TOCSY spectra, a 38 ms MLEV-17 mixing period29 was added after excitation, followed by 256 alternating detection gradients (Ga ) ±0.517 T m-1) applied during 256 µs. Data Processing. All spectra were acquired and analyzed using the Bruker program Topspin 1.3. For 1D spectra, an exponential apodization of 0.3 Hz was applied to the FID prior to Fourier transform (FT). A polynomial baseline correction (n ) 3) was applied to the spectrum after phasing. The specific processing for ultrafast spectra was performed using our home-written routine in Topspin, including zero-filling and appropriate apodization functions (π/6 shifted square sinebell function for J-resolved spectra and π/4 shifted sine-bell function for TOCSY spectra). It is important to note that for J-resolved spectra, the common 45°-tilt operation was not performed, as it was shown recently that the ultrafast pulse sequence employed here directly leads to a nontilted J-spectrum19 (i.e., to 1 H decoupled 1H spectra), contrary to the conventional Jresolved technique. The integration of 1D signals was performed using the integration routine in Topspin. The integration of ultrafast 2D volumes was performed with the elliptic integration tool included in MestRe-C 4.7. Statistical Analysis. Repeatability was evaluated by calculating the standard deviation (SD) and coefficient of variation (CV) on five successive experiments. For long time stability evaluation, five series of measurements were performed every week during 5 consecutive weeks. Between each series of measurements, the probe was changed and the spectrometer was used for other experiments. Linearity was evaluated by plotting ultrafast 2D volume ratios as a function of 1D area ratios and by calculating the linear regression parameters (slope and y-intercept) and the coefficient of determination r2. RESULTS AND DISCUSSION The ultrafast pulse sequences employed are presented in Figure 1. The resulting ultrafast 2D J-resolved and TOCSY spectra are presented in Figure 2 for sample no. 6, together with the signal attributions. The J-resolved spectrum, obtained in 800 ms, and the TOCSY spectrum, obtained in 100 ms, both present the expected 2D patterns. In spite of the exceptionally short acquisition durations, the resolution in both dimensions is high enough to separate the signals originating from both compounds. We evaluated both ultrafast 2D methods for precise and accurate quantitative analysis. For 1D and 2D J-resolved spectroscopy, the ethanol CH3 and CH2 peak areas (or volumes) were measured relatively to the methanol CH3 signal. For TOCSY spectra, the volume of ethanol correlation peaks between CH3 or CH2 and OH was measured relatively to the correlation signal between methanol CH3 and OH. Repeatability was evaluated for each sample and each method by repeating five experiments successively. The results (29) Levitt, M. H.; Freeman, R.; Frenkiel, T. J. Magn. Reson. 1982, 47, 328– 330.

Figure 1. Pulse sequences for the acquisition of J-resolved (A) and TOCSY (B) ultrafast 2D spectra, using the phase-modulated encoding scheme of duration Te proposed by Pelupessy, followed by an EPI-based detection block. For J-resolved spectroscopy (A), a 180° hard pulse refocuses chemical shift effects in the course of the acquisition process. The 180° pulse phase is alternated (y, y, -y, -y) to avoid undesirable stimulated echoes. For the TOCSY pulse sequence (B), a MLEV-17 mixing period is added between excitation and detection. The Gc gradient prior to acquisition is adjusted to set the middle of the chemical shift range in the middle of the detection period Ta.

Figure 2. Ultrafast 500 MHz J-resolved (A) and TOCSY (B) spectra acquired in 800 (A) and 100 ms (B) on a methanol (100 mmol L-1) and ethanol (200 mmol L-1) mixture in DMSO-d6 at 298 K, with the pulse sequences indicated in Figure 1. Bold characters indicate signal attributions.

are presented in Table 1. The values of CV and SD obtained for J-resolved spectroscopy are of the same order of magnitude as the values obtained for conventional 1D NMR, highlighting the excellent repeatability (better than 1%) of ultrafast J-resolved spectroscopy. Lower CV and SD values are obtained for CH3 than for CH2, due a higher signal-to-noise ratio (S/N ) 372 ± 60 for CH3 and S/N ) 101 ± 9 for CH2 for sample no. 4). For the same reason, the more concentrated samples are characterized by lower CV and SD values. For TOCSY experiments, SD and CV are higher than for J-resolved or 1D spectroscopy, with an average repeatability around 3% for CH3 and 5% for CH2. This is due to a lower S/N ratio (S/N ) 13 ± 3 for the CH2-OH correlation peak for sample no. 4). Table 1 also shows that mean peak volume ratios measured on ultrafast 2D spectra are far different from relative peak areas measured on 1D spectra. Actually, 2D peak volumes depend on a variety of factors (J-couplings, T2, etc.), like in conventional 2D NMR. As a consequence, ultrafast 2D NMR is not intrinsically accurate. Taking all the above factors into account in order to correct peak volumes would be very tedious; however, the short experiment duration now makes it possible to circumvent this drawback by obtaining a calibration plot in a very short

time. Accurate results may be obtained after calibration if a good linearity is obtained. In the same way, we evaluated the long time stability of ultrafast methods by performing five series of experiments every week during 5 weeks, as indicated in the Experimental Section. The results are presented in Table 2. The same evolutions as for short time repeatability are observed. Lower CV values are generally obtained for CH3 than for CH2, and TOCSY spectra are characterized by higher CV than Jresolved spectra. The values of CV are between 0.4% and 3.6% for J-resolved spectroscopy and between 2.0% and 11.0% for TOCSY, which highlights the better long time stability of ultrafast J-resolved spectroscopy. Finally, one can observe the unexpectedly high CV obtained for 1D CH2 signals. It is probably due to a slight overlap between CH2 and H2O peaks. The linearity of ultrafast methods was also evaluated by plotting calibration curves of ultrafast volume ratios as a function of area ratios determined by conventional 1D NMR. The calibration curve parameters are shown in Table 3. The very high coefficients of determination show the excellent linearity of both ultrafast J-resolved and TOCSY experiments. This result shows their high potentialities for quantitative Analytical Chemistry, Vol. 81, No. 1, January 1, 2009

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Table 1. Repeatability of 1H and Ultrafast NMR Ethanol Signalsa method CH3

1D 1H NMR ultrafast J-resolved ultrafast TOCSYb

CH2


sample

1

2

3

4

5

6

mean SD CV % mean SD CV % mean SD CV %

0.229 0.000 0.09 0.551 0.002 0.28 0.182 0.009 4.94

0.493 0.000 0.05 0.798 0.003 0.43 0.292 0.008 2.77

0.721 0.001 0.08 1.000 0.002 0.22 0.395 0.009 2.32

0.988 0.001 0.05 1.290 0.001 0.08 0.540 0.023 4.19

1.434 0.001 0.04 1.704 0.002 0.11 0.748 0.024 3.16

2.186 0.001 0.03 2.540 0.003 0.12 1.059 0.011 1.06


0.153 0.001 0.60 0.265 0.002 0.81 0.100 0.004 4.21

0.331 0.001 0.36 0.349 0.001 0.16 0.159 0.010 6.41

0.481 0.001 0.24 0.425 0.003 0.62 0.207 0.013 6.27

0.658 0.001 0.13 0.527 0.002 0.40 0.266 0.015 5.62

0.958 0.001 0.15 0.694 0.002 0.23 0.355 0.018 4.96

1.462 0.001 0.09 0.980 0.001 0.13 0.500 0.013 2.56

a Methanol was used as an internal reference. The methanol CH3 peak was used as a reference for 1H and J-resolved spectra, whereas the reference for TOCSY spectra was the correlation peak between CH3 and OH. b For TOCSY spectra, correlation peaks between CH3 or CH2 and OH were considered.

Table 2. Long Term Stability of 1H and Ultrafast NMR Ethanol Signalsa method CH3


CH2


sample

1

2

3

4

5

6


0.229 0.000 0.05 0.548 0.016 2.95 0.200 0.015 7.56

0.493 0.000 0.08 0.786 0.016 2.04 0.307 0.023 7.63

0.721 0.000 0.05 0.996 0.016 1.64 0.390 0.009 2.24

0.989 0.001 0.12 1.282 0.017 1.29 0.529 0.010 1.97

1.434 0.002 0.12 1.690 0.015 0.91 0.743 0.016 2.10

2.186 0.003 0.14 2.505 0.030 1.20 1.082 0.031 2.87


0.154 0.002 1.30 0.274 0.010 3.60 0.116 0.013 10.98

0.330 0.003 1.02 0.352 0.008 2.16 0.168 0.013 7.78

0.481 0.002 0.42 0.431 0.006 1.42 0.205 0.013 6.18

0.662 0.004 0.63 0.528 0.003 0.64 0.255 0.009 3.56

0.959 0.004 0.39 0.699 0.005 0.77 0.362 0.011 3.10

1.460 0.005 0.36 0.982 0.004 0.40 0.520 0.020 3.87

a Methanol was used as an internal reference. The methanol CH3 peak was used as a reference for 1H and J-resolved spectra, whereas the reference for TOCSY spectra was the correlation peak between CH3 and OH. b For TOCSY spectra, correlation peaks between CH3 or CH2 and OH were considered.

analysis, as a calibration plot can now be obtained in a very short time compared to conventional 2D NMR. The very good linearity proves that ultrafast experiments can lead to accurate measurements after preliminary calibration. Moreover, the small standard deviations on slope and y-intercept indicate the long-time stability of the calibration plots, which can still be used after 5 weeks for the titration of an unknown sample. For quantitative purposes, it is also interesting to estimate the sensitivity of ultrafast methods. Following the ICH guidance,30 a signal-to-noise ratio of 3 is generally acceptable for estimating the limit of detection (LOD), and a value of 10 is typical for the limit of quantification (LOQ). According to the signal-to-noise ratios measured on the weakest signal (CH2) and indicated above, the values obtained for ultrafast (30) ICH-Q2A. http://www.fda.gov/cder/guidance/index.htm, 1995.

482


J-resolved spectroscopy are approximately LOD ) 3.0 mmol L-1 and LOQ ) 9.9 mmol L-1. The LOQ is below the lower concentration employed for the calibration curve. For TOCSY spectra, the same calculation leads to LOD ) 22.8 mmol L-1 and LOQ ) 76.2 mmol L-1. This result shows the lower sensitivity of the ultrafast TOCSY method, due to the low intensity of correlation peaks compared to diagonal peaks. Moreover, three of the samples used for the calibration procedure are under the LOQ; however, we were still able to obtain a repeatability better than 7% for these mixtures, as indicated in Table 1. Actually, we calculated LOQ using signal-to-noise considerations, but the ICH guidance also suggests that LOQ may be evaluated by “visual evaluation”, i.e., by “the minimum level at which the analyte can be quantified with acceptable accuracy and precision”.30 With the

Table 3. Calibration Curvea Parameters Obtained by Plotting the Ethanol/Methanol Signal Ratios Measured by Ultrafast 2D NMR as a Function of Signal Ratios Measured by Quantitative 1H NMR method CH3

J-resolved (r ) 0.999) 2

TOCSYb(r2 ) 0.997)

CH2

J-resolved (r2 ) 0.999) TOCSYb(r2 ) 0.995)

slope

y-intercept

mean SD CV % mean SD CV %

1.000 0.013 1.3 0.457 0.010 2.2

0.293 0.016 5.5 0.081 0.009 11.1

mean SD CV % mean SD CV %

0.548 0.005 0.9 0.312 0.008 2.6

0.175 0.009 5.1 0.061 0.007 11.5

a Each calibration curve was obtained using six different mixtures with ethanol/methanol concentration ratios ranging from 0.22 to 2.19. Every point in the curve is the average of five successive experiments. Uncertainties correspond to standard deviations of the slope and y-intercept values, measured on five series of experiments during 5 weeks. b For TOCSY spectra, correlation peaks between CH3 or CH2 and OH were considered.

use of this definition and considering a 7% repeatability as acceptable, the actual LOQ for the ultrafast TOCSY method employed here may be decreased to 23.0 mmol L-1, corresponding to the lower concentration measured in this study. It is also important to underline that LOQ and LOD are very dependent on the experimental conditions and should be significantly different for experiments performed on other spectrometers. The results presented above show that both ultrafast techniques are very promising for quantitative analysis. Both methods present excellent linearities; moreover, the repeatability and long time stability obtained for ultrafast J-resolved spectroscopy highlight the very good precision level for this method. Ultrafast TOCSY spectra are characterized by a lower sensitivity and precision; however, the latter remains under an acceptable level (7% for repeatability and 11% for long time stability), which makes ultrafast TOCSY an interesting tool for fast quantitative analysis of complex mixtures. In particular, TOCSY spectra offer a better signal discrimination than J-resolved spectroscopy; consequently, TOCSY will probably be more adapted to quantitative studies of very complex mixtures with important 1H overlap. On the contrary, Jresolved spectroscopy appears more convenient for very precise quantitative analysis of simple mixtures. The precision and linearity values measured in this study may originate from various hardware instabilities, which could explain why higher SD and CV values are obtained for the ultrafast methods than for quantitative 1D analysis. In particular, the repeatability may be affected by variations of the gradient amplitudes during the course of the long oscillating gradient train. Instabilities of the radiofrequency transmitter during the generation of the frequency swept chirp pulses may also alter precision. Moreoever, for long time repetitions where the probehead and magnet are removed from the spectrometer between experiments, variations may be caused by different

shimming levels, even though shimming was carefully adjusted before each experiment. Finally, it is also interesting to notice that the repeatability and linearity values are of the same order of magnitude than the values obtained in previous studies for optimized conventional 2D experiments.6,8 However, the experiment duration involved in these studies could not be decreased under a few minutes, whereas the duration of the ultrafast experiments remains under 1 s. CONCLUSION This paper shows that ultrafast J-resolved and TOCSY methods are reliable for very fast and precise quantitative analysis. Their relatively low sensitivity is probably the main limitation at the time of the writing; however, many experimental improvements can be expected to face this drawback. First, working at higher fields will contribute to decrease the LOD and LOQ values indicated above. The increasing use of cryoprobes31 is also expected to improve the sensitivity of ultrafast quantitative analysis. Finally, future investigations will consider the use of ultrafast 2D experiments together with dynamic nuclear polarization (DNP)32 in order to decrease the detection limit by several orders of magnitude. In this study, we have chosen to evaluate two of the most widespread homonuclear 2D NMR methods (J-resolved spectroscopy and TOCSY). However, in some cases (like in vivo localized spectroscopy), it is nearly impossible to implement the TOCSY mixing period due to technical constraints and the COSY experiment is preferred. Consequently, it could be useful in later works to evaluate ultrafast 2D COSY for quantitative analysis. Actually, for an ultrafast COSY spectrum, the spectral width would be the same as for the TOCSY experiment, and moreover, the global intensity would be spread over a smaller number of peaks. Moreover, the COSY experiment does not include any spin-lock period, which could alter precision in TOCSY experiments. Consequently, one could expect the precision of ultrafast COSY experiments to be at least as good as the one measured for TOCSY. As reported in recent studies, the resolution of ultrafast spectra is still lower than the one obtained with conventional 2D NMR, which could limit the efficiency of ultrafast methods for the analysis of mixtures presenting significant 1H overlap. However, resolution improvements are expected by performing several interleaved ultrafast acquisitions, as suggested by Frydman and co-workers.10 This method, even if it is not purely “single-scan”, should lead to a resolution equivalent to the one observed in conventional 2D acquisitions but in a shorter duration. This procedure is currently under investigation. After evaluating the high potentialities of ultrafast quantitative NMR, it seems useful to define a new abbreviation to describe this ensemble of techniques. We propose to call it “ufo-qNMR” (ultrafast optimized quantitative NMR), in reference to the concept of qNMR proposed by Pauli.33 Applications of ufo-qNMR are expected in many fields where it could (31) Kovacs, H. Prog. Nucl. Magn. Reson. Spectrosc. 2005, 46, 131–155. (32) Frydman, L.; Blazina, D. Nat. Phys. 2007, 3, 415–419. (33) Pauli, G. F. Phytochem. Anal. 2001, 12, 28–42.


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progressively replace conventional 2D NMR, leading to considerable time-saving. Ultrafast quantitative methods should be particularly useful for applications requiring very short experimental durations, such as kinetic studies of short time scale phenomena (chemical transformation,34 exchange process35) or as a detector after chromatographic separation.36 Further developments of ufo-qNMR will consider the evaluation of (34) Gal, M.; Mishkovsky, M.; Frydman, L. J. Am. Chem. Soc. 2006, 128, 951– 956. (35) Shapira, B.; Frydman, L. J. Magn. Reson. 2003, 165, 320–324. (36) Shapira, B.; Karton, A.; Aronzon, D.; Frydman, L. J. Am. Chem. Soc. 2004, 126, 1262–1265.

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heteronuclear techniques, which offer a better signal discrimination due to the larger chemical shift range of heteronuclei. ACKNOWLEDGMENT The authors would like to thank Michel Giraudeau for linguistic assistance. Received for review October 7, 2008. Accepted October 31, 2008. AC8021168

Evaluation of Ultrafast 2D NMR for Quantitative Analysis - Analytical

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