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Journal of Chromatography A, 1128 (2006) 152–163

Application of high-performance liquid chromatography–nuclear magneticresonance coupling to the identification of limonoids from mahogany tree

(Switenia macrophylla, Meliaceae) by stopped-flow 1Dand 2D NMR spectroscopy

Alexandre B. Schefer a,∗, Ulrich Braumann a, Li-Hong Tseng a, Manfred Spraul a,Marisi G. Soares b, Joao B. Fernandes b, Maria F.G.F. da Silva b,

Paulo C. Vieira b, Antonio G. Ferreira b

a NMR Division, Bruker BioSpin GmbH, D-76287 Rheinstetten, Silberstreifen, Germanyb Chemistry Department, Federal University of Sao Carlos, Rod. Washington Luıs Km 235, C.P. 676, 13565-905 Sao Carlos-SP, Brazil

Received 14 July 2005; received in revised form 9 June 2006; accepted 19 June 2006Available online 14 August 2006

bstract

Separation and characterization of limonoids from Switenia macrophylla (Meliaceae) by HPLC–NMR technique has been described. Analysesere carried out using reversed-phase gradient HPLC elution coupled to NMR (600 MHz) spectrometer in stopped-flow mode. Separated peaks

ere collected into an interface unit prior to NMR measurements, which were performed with suppression of solvent signals by shaped pulses

equences. Structure elucidation of the limonoids was attained by data obtained from 1H NMR, TOCSY, gHSQC and gHMBC spectra withoutonventional isolation that is usually applied in natural products studies.

2006 Elsevier B.V. All rights reserved.

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eywords: HPLC–NMR; Loop storage; Swietenia macrophylla (Meliaceae); L

. Introduction

Direct coupling of high-performance liquid chromatogra-hy to nuclear magnetic resonance (HPLC–NMR) has turnednto an important analytical tool in many fields, which dealsith separation and identification of compounds in complexixtures [1–3]. Although HPLC–NMR techniques have mainly

een applied to pharmaceutical developments and clinical inves-igations with successful accomplishments [4,5], an increasingumber of papers has been published on their applications inatural products research, since they permit fast screening ofrude plant extract. Traditional off-line strategies have been

pplied to a variety of chromatographic techniques, includingPLC, to perform activity test and to aim the isolation of fewilligrams of pure compounds from crude extract followed by

∗ Corresponding author. Tel.: +49 721 5161 218; fax: +49 721 5161 297.E-mail address: alexandre.schefer@bruker-biospin.de (A.B. Schefer).

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pectroscopic measurements, such as mass spectrometry (MS)nd NMR, in order to determine the structure of a moleculef interest. This procedure is rather technically demanding andime consuming. Besides, in many circumstances, it may leado well-known compounds, which have no interesting spectro-copic or biological features. Thus, the ability to rapidly identifynown or undesirable compounds plays a crucial role in ordero prevent unnecessary investment of resources. Furthermore,roblems due to sensitivity of some compounds to the extrac-ion conditions are avoided as well. Therefore, HPLC–NMRepresents a potentially suitable technique in phytochemicalesearch for the detailed on-line analysis of natural products6–12].

High-resolution NMR spectroscopy is one of the most impor-ant methods of structure elucidation with an extensive range of

iochemical and chemical applications. The direct linking ofPLC with NMR spectroscopy has a remarkable success story

nd has transformed the technique from a research tool to thetage where routine analytical applications are possible.

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A related factor, which delayed the implementation of prac-ical HPLC–NMR spectroscopy, was the earlier need to useeuterated solvents for chromatography to overcome dynamicange problems. The solution to this difficulty for reversed-hase HPLC–NMR has come about because modern NMRpectrometer can perform solvent resonance suppression veryfficiently, thus avoiding the need for deuterated solvents. Withegard to the solvent suppression, the most critical problemsrise when continuous flow experiments are run under typicalC reversed-phase operating conditions when more than onerotonated solvent is used and when the resonances change fre-uencies during the analysis in gradient mode. These problemsave now been overcome thanks to the development of fast, reli-ble and powerful solvent suppression techniques such as Wateruppression Enhanced through T1 effects [13] (WET), whichroduces high-quality spectra in both on-flow and stopped-flowodes.The success of HPLC–NMR in recent years is mainly based

n advances in hardware, software and pulse programs relatedo NMR. These advances include the use of higher field mag-ets (500 MHz and greater) and digital signal processing whichave helped address the lack of sensitivity [14]. In addition, newrobe designs allow the use of gradient pulse sequences and pro-ide the efficient and specific suppression of the NMR signalsue to the HPLC solvents. However, the sensitivity remains onef the main limitations for the widespread use of this technique.n order to improve the signal-to-noise ratio (S/N) obtained inMR, several parameters can be adjusted: increase of the sam-le volume (generally HPLC–NMR cell volumes are between0 and 200 �L with 2–4 mm i.d., representing a good compro-ise with respect to satisfactory chromatographic performance);

ncrease of the magnetic field strength (ultra-high field NMRagnets up to 750–800 MHz have been used) [15]; increase oflling factor as well as the quality of the NMR coil; reductionf the receiver bandwidth; operation at low temperature; andmprovement of the pre-amplifier quality [16]. The maintenancef good magnetic field homogeneity, giving narrow NMR lines,s also a contribution to enhance the sensitivity, as well as these of new techniques for the acquisition of NMR data, such asigital filtering and oversampling.

In order to cope with these problems of sensitivity, and alsoo perform 2D correlation experiments, several modes of opera-ion are employed in HPLC–NMR. As continuous-flow mode isarticularly used for monitoring the chromatographic run, pro-iding NMR spectra that are stored as a set of scans as discretencrements, the stopped-flow mode is carried out by two dif-erent approaches, although in both cases, the flow is stoppedhen the top of a HPLC peak reaches the centre of the NMRow cell. Then multiple scans can be acquired for 1D and 2Dxperiments. In the direct stopped-flow mode, after the NMReasurement is finished, the flow is resumed until the next peak

f interest is detected. For HPLC peaks that still sit on the col-mn or in the transfer capillary, when a first stop occurs, this

eans they undergo diffusion and therefore the chromatographic

esolution for later eluting peaks is reduced. To bypass thisroblem, a loop collection unit has been introduced, that allowsransferring peaks into capillary storage loops, which retain the

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r. A 1128 (2006) 152–163 153

C-peakshape due to small inner diameter (e.g. 0.25 mm). Inhis case the flow is not stopped, but only valves are switchedo direct the HPLC peaks into the capillary storage loops. Afterhe separation is finished, the loop contents can be transferredequentially to the NMR using the HPLC pump.

The on-line combination of HPLC with 1H NMR data allows,n many cases, the structural elucidation of various knownompounds. These data, however, are not always sufficientor a full on-line identification of new constituents. If a com-lete structural identification has to be carried out, informationn the 13C signals is necessary and different 2D correlationxperiments have to be performed. These types of experi-ents can be run by HPLC–NMR in the stopped-flow mode,

rovided that the sample concentration in the NMR cell isigh enough. In this mode, 1H–1H correlations such as COSY,OCSY and NOESY can be measured, and information on 13Can be extracted from 1H–13C correlation experiments suchs gradient heteronuclear single quantum coherence (gHSQC)nd gradient heteronuclear multiple-bond correlation (gHMBC)17].

Despite the successful introduction of LC–NMR techniquesn the analysis of compounds in mixture, an alternative andomplementary innovation for improving the sensitivity of suchechniques is already available. This is possible due to the on-lineolid phase extraction (SPE) after chromatographic separationhat replaces loop collection of fractions. Such approach allows/N improvements of up to a factor of 4 because discrete analytere concentrated on individual SPE cartridges after the chro-atographic separation and prior to the on-line NMR analysis.nother important advantage of LC–SPE–NMR technique is the

act it that chromatographic separation can be performed withndeuterated solvents, as the SPE cartridges are dried with nitro-en before being transferred on-line to the NMR spectrometer,hich is done with a suitable fully deuterated solvent. Appli-

ation of this relatively new technology has been demonstratedostly on 400–600 MHz NMR instruments (refs.). The use of a

ryogenic flow probehead further increases the S/N to a factorf 3–4, making possible the study of analytes in the nanogramange.

In this report we describe the application of loop storagetopped-flow HPLC–NMR technique to the identification of 8imonoids from extracts obtained from the seeds of mahoganyree (Switenia macrophylla, Meliaceae).

This species belongs to the Swietenioideae subfamily, whichonsists of the genera Khaya and Entandrophragma in Africa,wietenia and Cedrela in Latin America and Toona in Asia andustralia, which are all attacked by the shoot borer Hypsipyla

Lepidoptera: Pyralidae). As a consequence, efforts to establisharge-scale homogeneous plantations of native Meliaceae havelmost invariably failed [18]. Recently, there have been someorks that show the remarkable resistance of the limonoids

gainst Hypsipila grandella [19–22]. Consequently, furthernvestigations on the chemical composition of extracts asso-

iated with S. macrophylla are of great interest. HPLC–NMRechniques seems to be a very suitable approach aiming thetructural elucidation of natural products constituents withoutheir individual isolation.

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54 A.B. Schefer et al. / J. Chro

. Experimental

.1. Instrumentation

The HPLC system consisted of a quaternary low-pressureradient-mixing LC22 solvent delivery pump from Bruker Sax-nia (Leipzig, Germany), a manual injector from Rheodyne,odel 7726i (Cotati, USA), equipped with a 1 mL sample loop

nd a diode array detector from J & M (Aalen, Germany).PLC–NMR measurements were carried out using the BPSU-6 interface coupled to a DRX 600 NMR spectrometer workingt 600.13 MHz 1H frequency equipped with a 1H/13C 4 mm

nverse LC probe head (120 �L) from Bruker Biospin GmbHRheinstetten, Germany) and controlled by the XWinNMR soft-are system from Bruker. The temporary sample storage unit

BPSU-36) was used for automated multiple NMR experiments

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ig. 1. (A and B) Chromatographic separations of the methanolic extract from driedbtained by pre-purification through a cellulose column. The chromatograms are reco

r. A 1128 (2006) 152–163

vernight. UV–vis detection was used to trigger the transfer ofPLC fractions to the BPSU (for temporary storage). All trans-

er connexions were made with polyether ether ketone (PEEK)apillary (0.25 mm i.d.).

.2. Chemical

CH3CN (NMR Chromasolv quality) was from Riedel-e-Haen (Seelze, Germany). Deuterium oxide (99.9%) wasbtained from Deutero GmbH (Kastellaun, Germany).

.3. Sample

Extract was obtained from 11.6 g macerated dried seeds ofahogany tree (Swietenia macrophylla) from Amazon forest

seeds of mahogany (Swietenia macrophylla). Samples A and B are fractionsrded at 230 nm; the identified peaks are denoted 1–8.

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Belem-PA, Brazil). The extraction was carried out with HPLCrade MeOH at room temperature in 24 h. The concentratedeOH extract was purified through a cellulose column yielding

wo major fractions in mixture (samples A and B), which wereltered and evaporated. 107 mg of each sample was re-dissolved

n 400 �L methanol–d4 and filtered through a 0.2 mm membranelter. Final solutions were used for HPLC–NMR analyses.

.4. Measurements. HPLC

One chromatographic HPLC–NMR run was performed forach sample at 35◦C using a 250× 4.6 mm Phenyl-Hexyl5 �m—particle size) Phenomenex column at a flow rate of.0 mL/min. The injection volume was 10 �L of the solution.he mobile phase consisted of 70% D2O and 30% CH3CNhanged to 90% CH3CN within 30 min and held for another0 min. The diode array detector was operated at 230 nm.

.5. NMR

Acquisition of all NMR experiments was performed in

topped-flow mode after transferring each peak volume col-ected in the BPSU-36 interface into the NMR flow cell. 2DMR spectra were acquired depending on the 1H NMR signal-

o-noise ration (S/N).

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ig. 2. Six hundred megahertz LC–1H NMR spectra of peaks 1 and 2 after loop stond residual HDO peak. Both spectra were acquired with 128 scans. Expansions excl

r. A 1128 (2006) 152–163 155

.6. 1H NMR

The spectra were obtained using the following parameters:ree induction decays (FIDs) were collected into 32 K data pointsith a spectral width of 12019.2 Hz. Ninety degrees pulses were

pplied with an acquisition time of 1.36 s and the relaxationime delay was set to 2.0 s. Depending on the concentrationf each peak analysed, different numbers of scans were accu-ulated, varying from 64 to 256 scans. A NOESYPRESAT

olvent suppression with 13C decoupling was performed on sig-als of CH3CN and 1HO2H (from D2O) using standard Brukerulse programs. Prior to Fourier transformation, an exponen-ial apodization function was applied corresponding to a lineroadening of 0.3 HZ. For calibration the residual signal of theH3CN was set to 2.0 ppm.

.7. TOCSY

2D TOCSY spectra were recorded in stopped-flow modesing shaped pulse of 2.4 s during relaxation time for off-

esonance pre-saturation of both the solvent residual signals,H3CN and 1HO2H (from D2O). Continuous wave 13C decou-ling was applied during spin-lock and acquisition time of22 ms. Spectra size was 2048 × 160 data points, zero-filled to

rage. The spectra were recorded with suppression of undeuterated acetonitrileude the suppressed solvent peaks.

156 A.B. Schefer et al. / J. Chromatogr. A 1128 (2006) 152–163

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ig. 3. Six hundred megahertz LC–1H NMR spectra of peaks 3 and 4 after loond residual HDO peak. Spectra were acquired with 64 and 256 scans, respecti

048 × 2048 data points over a spectral width of 8389.3 Hz inoth dimensions. The number of scans acquired depended onhe S/N ratio of each previously recorded 1H NMR spectrum,arying from 8 to 24 scans per increment. TOCSY mixing timeas 100 ms.

.8. WET-gHSQC

Gradient HSQC spectra were acquired using a 13C (f2) spec-ral width of 27173.9 Hz, a 1H (f1) spectral width of 8389.3 Hz,

relaxation delay of 2.0 s, 1024 data points with zero fillingo 2048, 128 time increments and the number of scans perncrement for each fraction varied from 48 to 128, with 13Cecoupling GARP pulse optimised for 1JCH = 125 Hz during thecquisition time of 90 ms. WET suppression was applied for theuppression of solvent resonances, CH3CN and 1HO2H (from2O).

.9. gHMBC

Gradient HMBC spectra were performed with a long-rangeH–13C coupling constant of 8 Hz. FIDs were collected into

048 data points with the spectral width of 7183.9 Hz (f2) andn acquisition time of 140 ms. The spectra were acquired using88 time increments of 128 scans each with a relaxation delayf 2 s.

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rage. The spectra were recorded with suppression of undeuterated acetonitrilexpansions exclude the suppressed solvent peaks.

. Results and discussion

Each sample obtained from the methanolic extract ofahogany seeds, S. macrophylla (Meliaceae), was dissolved ineOD-d3. 10 �L of the 267.5 mg/mL solutions was the volume

njected for chromatographic runs, which were carried out forhe stopped-flow HPLC–NMR experiments after loop collectionf fractions. That is a rather high concentration solution, whichas specially necessary to acquire 2D NMR spectra from most of

nalysed compounds. In spite of the large amount of the injectedample, it can be seen in the chromatograms (Fig. 1) that the col-mn was not overloaded and major constituents are relativelyell separated. The reasonable purity of the analysed fractions

an be observed on the acquired 1H NMR spectra (Figs. 2–5) Ashown in Fig. 1, the chromatographic runs might be improved sohat shorter analysis time would have been obtained. However,s the peaks were well separated, there was no need of chro-atographic optimisation, once most of peaks were collected

nto the BPSU-36 unit. By the results accomplished with theyphenated techniques, structures of eight limonoids (Fig. 6)ould be elucidated as follows.

From all the 13 peaks collected from sample A into the loops,

PLC–1H NMR data were acquired only from peaks indicatedith structures numbers 1–5, which were in a suitable concen-

ration to be detected by 1H NMR spectra by collecting up to56 transients in stopped-flow mode. Figs. 2–5 show expansions

A.B. Schefer et al. / J. Chromatogr. A 1128 (2006) 152–163 157

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ig. 4. Six hundred megahertz LC–1H NMR spectra of peaks 5 and 6 after loond residual HDO peak. Spectra were acquired with 128 and 344 scans, respect

f these spectra, from which it was possible to assess the puritynd concentration (through S/N ratio) of each fraction collectednto the loops. Prior to every NMR measurement of each peak,uning and gradient shimming were performed, leading to sym-

etric and narrow line shapes, making possible the observationf coupling constants smaller than 1 Hz.

In order to perform structure elucidation by HPLC–NMR,rucial information was provided by 2D NMR experiments.egarding the S/N determined for each 1H NMR spectrum,D experiments were obtained from compounds 1–3. 1H–1HOCSY was first acquired and provided information on the con-ectivity of the coupling hydrogens as well as the assignmentf peaks which were covered by solvent signals in 1H NMRpectra. Fig. 7 presents 1H–1H TOCSY spectrum of compoundand shows that despite the application of solvent suppression,

he spectrum quality allows assignment of most of hydrogen fortructure elucidation.

Injection of sample B in the HPLC–NMR system led to thedentification of six limonoids, from which three of them, 1–3,ad already been found in the study of sample A. Analyses ofeaks indicated with structures numbers allowed the identifica-

ion of three further limonoids (6–8). From peaks of 6 and 7,nly 1H NMR spectra were recorded, with 256 and 512 scans,espectively, due to their low concentration. Nevertheless, it wasossible to determine their structures, as well as for peak of 8.

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rage. The spectra were recorded with suppression of undeuterated acetonitrileExpansions exclude the suppressed solvent peaks.

13C data were extracted from gradient heteronuclear cor-elated experiments (gHSQC and gHMBC) and displayed inable 1. Differently from 1H homonuclear 1D and 2D spectraith solvent suppression, such heteronuclear correlated spectra

how loss of information only within the range of acetonitrileesidual peak. From gHSQC and gHMBC spectra of compound(Figs. 8 and 9), it was possible to assign the hydrogen H15�

t δ 3.93 (m), covered by the suppression of the HDO peak onhe 1H spectrum, as well as the 13C chemical shift of its corre-ponding carbon C15 (δ 33.6) on the gHSQC spectrum. Fromhe 2J heteronuclear coupling of the same hydrogen H15� on theHMBC spectrum, one could assign the carbonyl carbon C18t δ 173.9. Besides, gHMBC spectra allowed the determinationf the 13C–1H connectivities, which make structure elucidationasier to be accomplished.

After having recorded HPLC–1H NMR spectra, it was possi-le to conclude that the chromatographic peaks had high purity,ccording to resonances observed with limonoid 1H pattern fromiterature. 1H NMR spectrum of peak 1 indicated the presencef four tertiary methyl groups with singlets at δ 0.81, 0.87, 0.91nd 1.28 (Table 2), one methoxyl singlet (δ 3.72), three down-

eld shifted signals attributed to a �-substituted furan ring (δ.55, 7.51 and 6.48) and three signals characteristic of pro-ons attached to a carbon adjacent to an oxygen atom (δ 5.54 s,.49 s and 3.48 d, J = 9.5 Hz). The signal at δ 2.91, assigned

158 A.B. Schefer et al. / J. Chromatogr. A 1128 (2006) 152–163

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ig. 5. Six hundred megahertz LC–1H NMR spectra of peaks 7 and 8 after loond residual HDO peak. Spectra were acquired with 64 and 128 scans, respecti

o the hydrogen H2, showed a ddd multiplicity (J = 9.5, 5.9 and.6 Hz), correlating with a doublet at δ 3.48 (J = 9.5 Hz) and withdoublet of doublet at δ 3.13 (J = 2.6 and 14.7 Hz), which werebserved in the TOCSY spectrum, representing an ABB’X spinystem. Comparison with information from literature suggestshe swietenolide [23,24] limonoid for peak 1.

A drawback on the necessary application of solvent suppres-ion in LC–NMR is its effect on the observable spectrum range.lthough NOESY 1D and WET techniques with 13C decouplingrovides a good quality of solvent suppression, there is a lossf information under the irradiated signals if signals of interestas a chemical shift in the range of the irradiated region. Sincehe 13C satellites are typically much taller than the signals ofnterest and often have longer T1, the intensity of the t1 noiseecomes an issue. The t1 noise, and baseline distortions fromery large peaks, can actually mask signals of interest if therere coincident chemical shifts. The WET-gHSQC spectrum ofeak 1 showed a strong t1 noise over the 1.85–2.15 ppm range,hich prevented us from determining the chemical shifts of car-ons C9 and C11. Nevertheless, all the other hydrogen-bearingarbons could be assigned.

Peak 2 exhibited a very similar HPLC–1H NMR spectrums found for 1, indicating the structure of the unreported 3�,14-ihydroxymexicanolide [25]. The only two differences lie in thebsence of a hydroxyl group on carbon C6 and in the presence

(cc3

rage. The spectra were recorded with suppression of undeuterated acetonitrilexpansions exclude the suppressed solvent peaks.

f a hydroxyl group on carbon C14, instead of a double bondetween the C8–C14 carbons. Although C14 was not detected byhe stopped-flow gHMBC spectrum via long-range correlations,he hydrogen H8 (δ 2.37 m) was observed in a chemical shift,hich is characteristic of hydrogen vicinal to a C–OH group,

orroborating the assignment of the hydroxyl at the carbon C14.n addition, 1H–1H TOCSY showed a correlation between H8nd both methylenic hydrogens H30 (δ 3.17 and 2.47), confirm-ng the assignment proposed. A relevant and interesting point ishe absence of a signal at ∼4.5 ppm, characteristic of the hydro-en H6 when there is an O–R substitution attached to the carbon6, suggesting the existence of a (–CH2–) instead, which wasot observed in the 1H NMR spectrum due to the overlappingith the CH3CN residual peak at 2.0 ppm.The stopped-flow NMR data for peak 3 revealed the presence

f a tigloyl moiety attached to carbon C3. In the gHMBC spec-rum, the H34 and H35 methyl protons showed connectivitieso carbons C32 and C33. Besides, C35 methyl protons also cor-elated with the carbonyl C31. C5 carbon was readily identifiedδC 45.8) and its corresponding hydrogen showed connectivityo C3 (δC 79.6), C4 (δC 39.5), C7 (δC 177.1), C9 (δC 58.5), C10

δC 51.4), C19 (δC 16.6), C28 (δC 22.8) and C29 (δC 22.9). Theharacteristic olefinic hydrogen H30 (δH 5.21, d, J = 7.3), whichouples to H2 showed 3JCH correlation to C9 (δC 58.5) and 4JHH,JHH and 4JHH to H9 (δH 2.31, m), H2 (δH 3.40, d, J = 9.5 Hz)

A.B. Schefer et al. / J. Chromatogr. A 1128 (2006) 152–163 159

Fig. 6. Limonoids 1–8 identified by LC–NMR analysis of methanolic extract from dried seeds of mahogany tree (Swietenia macrophylla).

160 A.B. Schefer et al. / J. Chromatogr. A 1128 (2006) 152–163

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ig. 7. Six hundred megahertz LC–NMR 2D TOCSY spectrum of peak 1 after lnd residual HDO peak.

nd H3 (δH 4.55, d, J = 9.5 Hz), respectively (Table 2). Thus, thetructure of the limonoid 3 was characterized as swietenine [26].

Structure elucidation of compounds 4, 3-O-tigloylswi-enolide [24], and 5, febrifugina [27], was carried out bynalysing 1H NMR spectra only, since their low concentra-

ion prevented us from acquiring 2D spectra. Even though thenformation was limited to 1H chemical shift and 1H–1H cou-ling constant, comparison with data obtained from literaturend other identified limonoids allowed us to assign most of

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ig. 8. Six hundred megahertz LC–NMR 2D gHSQC spectrum of peak 1 after loop stnd residual HDO peak.

orage. The spectra were recorded with suppression of undeuterated acetonitrile

he hydrogens. Limonoids 4 and 5 are very similar, differingn only two aspects. Firstly, while 4 has a double bond between8–C14 carbons, 5 has a double bond between C8–C30 car-ons. This can be observed through the signals of H30 of bothtructures. The doublet at δH 5.25 (J = 7.0 Hz) characterizes the

lefinic hydrogen with a vicinal coupling constant in compound. As the structure of compound 4 has a methylenic carbon on30, its 1H NMR spectrum showed a doublet at δH 2.70 with aoupling constant of 15.3 Hz. Such value is characteristic of a

orage. The spectra were recorded with suppression of undeuterated acetonitrile

A.B. Schefer et al. / J. Chromatogr. A 1128 (2006) 152–163 161

Fig. 9. Six hundred megahertz LC–NMR 2D gHM

Table 113C data of limonoids 1–4 from extracts of Swietenia macrophylla (Meliaceae)

C Limonoid

1 (δC) 2 (δC) 3 (δC) 4 (δC)

1 n.o. n.o. 220.0 219.62 51.1 51.2 49.5 48.53 78.4 76.7 79.6 79.94 40.2 n.o. 39.5 39.85 44.8 75.6 45.8 44.96 73.7 n.o. 72.9 73.77 177.6 n.o. 177.1 172.48 n.o. 34.0 140.2 n.o.9 n.o. n.o. 58.5 54.410 54.9 n.o. 51.4 53.811 n.o. n.o. 21.6 19.112 29.6 29.6 35.0 30.013 38.4 n.o. 36.7 39.014 130.9 n.o. 45.1 135.315 33.5 33.6 29.9 34.016 173.9 n.o. 171.517 81.5 81.0 78.1 81.918 18.1 18.0 21.2 18.619 18.1 16.9 16.6 16.820 121.8 n.o. 122.2 121.521 143.2 141.7 141.7 142.322 110.6 110.4 110.3 110.723 143.8 142.2 144.3 144.228 23.6 24.3 22.8 23.329 23.9 20.4 22.9 23.130 34.6 34.1 122.9 34.4OCH3 53.4 52.5 53.3 53.731 – – 168.4 172.6

32 – – 128.2 20.821.3

33 – – 140.1 –34 – – 14.9 –35 – – 12.0 –

Assignments based on gHSQC and gHMBC acquired in stopped-flowHPLC–NMR experiments; n.o., not observed.

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eminal coupling and supports the assignment suggested. Thether hydrogen H30 was not observed, as the residue of theH3CN resonance must have overlapped its signal. The secondifference between 4 and 5 lies in the carbon C6. On compound, the characteristic 1H signal of H6 attached to a C–OR carbonas not observed, while the spectrum of compound 4 shows a

inglet at δH 4.56.The HPLC–NMR results obtained from sample B gave rise to

hree more different limonoids, in addition to 1–3. The structuref limonoid 8, 3,6-di-O,O-acetylswietenolide, was determinedsing data from 1D and 2D NMR spectra. The main differenceetween it and the other limonoids was the presence of twocetate groups attached to the carbons C6 and C3, instead ofither hydroxyl or tiglate groups which were the most commonubstitutions noticed.

A remarkable example of how powerful the use of couplingechniques can be the identification of limonoids 6 and 7, notnly because they are likely new compounds, but also because ofhe fact that these limonoids were assigned from 1H NMR datanly. This was accomplished by means of comparison of suchata with those obtained from literature [28–32] and the otherdentified limonoids in the present work. The 1H NMR spectraf 6 and 7 were recorded with 256 and 512 scans, respectively,evealing the infeasibility of acquiring 2D experiments.

In both the HPLC–1H NMR spectra recorded for 6 and 7,ignals of the tigloyl moiety could be assigned. For (6), signalsf hydrogenes H33, H34 and H35 were observed at δ 7.01 (q,= 7.0 Hz), 1.90 (d, J = 7.0 Hz) and 1.91 (s), respectively. For (7),

he same signals were observed at δ 6.93 (q, J = 7.0 Hz), 1.81 (d,= 7.0 Hz) and 1.82 (s). Broad singlets assigned to hydrogen H6ere observed at δ 4.48 (6), and δ 4.42 (7). The presence of aydroxyl attached to the carbon C2 was the only difference of

6) compared to (7). The singlet (δ 4.80) attributed to H3 sup-orts such assumption. Another fact observed in the 1H NMRpectrum of (6) is the absence of a signal to hydrogen H2, whichonfirms a substitution on carbon C2. This hydrogen is observed

162A

.B.Schefer

etal./J.Chrom

atogr.A1128

(2006)152–163

Table 2HPLC–1H NMR spectral data of limonoids 1–8

H Limonoid

1 δa J (Hz) 2 δa J (Hz) 3 δa J (Hz) 4 δa J (Hz) 5 δa J (Hz) 6 δa J (Hz) 7 δa J (Hz) 8 δa J (Hz)

2 2.91 ddd (2.6; 5.9; 9.5) 2.95 ddd (2.6; 5.9; 9.5) 3.40 tAB (9.5) 3.10 m 3.55 m – 3.41 dd (2.6; 9.1) 3.07 ddd (9.9; 5.6; 2.2)3 3.48 d (9.5) 3.68 d (9.5) 4.55 d (9.5) 4.65 d (9.7) 4.75 d (9.2) 4.80‘s 4.71 d (9.1) 4.87 d (9.9)5 3.17 s 3.70 s 3.47 s 3.35 s 3.45 dl (10.2) 3.22 sl 3.26 sl 3.36 sl6 4.49 s ND 4.58 s 4.56 s 2.10–2.35 m 4.48 s 4.42 s 5.46 sl8 – 2.37 m – – – – – –9 n.o. 3.15 m 2.31 m n.o. 2.10–2.35 m 1.80 m 1.80 m 2.16 m11� 1.73 m n.o. n.o. n.o. n.o. n.o. n.o. n.o.11� 1.84 m 1.79 m 1.78 m n.o. n.o. n.o. n.o. 1.8112� 1.05 m 1.03 m 1.47 dt (14.3; 4.4) n.o. n.o. 1.38 m 1.23 m 1.1812� 1.71 m 1.82 m 1.63 dt (3.3; 14.3) n.o. n.o. 1.58 dd (12.1; 7.0) 1.48 dd (12.5; 7.6) 1.7314 – – 2.32 m – 2.10–2.35 m n.o. n.o. –15� 3.43 dt (20.9; 2.6) 3.46 dt (2.6; 20.9) 2.87 dd (19.1; 6.6) 3.30 dt (21.0 e 3.2) 2.90 dd (18.7; 6.6) 3.12 dd (17.6; 12.5) 3.41 dd (12.5; 17.6) 3.51 dt (20.9; 2.9)15� 3.93 m 4.01 2.70 d (19.1) 3.49 d (21.0) 2.71 d (18.7) 2.86 dd (7.0; 17.6) 2.68 dd (17.6; 7.0) 3.70 d (20.9)17 5.54 s 5.64 s 5.53 s 5.50 s 5.60 s 5.13 s 5.15 s 5.57 s18 0.91 s 0.97 s 0.92 s 0.95 s 0.80 s 1.00 s 0.95 s 0.97 s19 1.28 s 1.08 s 1.36 s 1.34 s 1.10 s 1.33 s 1.17 s 1.10 s21 7.55 sl 7.60 s 7.65 sl 7.60 sl 7.75 sl 7.57 s 7.49 s 7.61 sl22 6.48 d (1.8) 6.53 d (1.1) 6.50 d (1.8) 6.51 sl 6.55 sl 6.46 sl 6.39 sl 6.52 d (1.8)23 7.51 tAB (1.8) 7.53 dd (1.1; 1.5) 7.55 tAB (1.8) 7.45 sl 7.55 sl 7.53 sl 7.46 sl 7.54 tAB (1.8)28 0.87 s 0.74 s 1.04 s 1.02 s 0.99 s 0.91 sl 0.83 sl 1.02 s29 0.81 s 0.66 s 0.87 s 0.84 s 0.77 s 0.84 sl 0.83 s 0.81 s

30 1.92 m 3.17 m 5.21 d (7.3) 2.70 d (15.3) 5.25 dl (7.0) 3.41 s 3.18 d (2.6) 2.123.13 dd (14.7; 2.6) 2.47 m – n.o. – – – 2.91 dd (15.8; 2.2)

OCH3 3.72 s 3.67 s 3.66 s 3.80 s 3.65 s 3.74 s 3.75 s 3.74 s32 – – – – – – – 2.11 s

– – – – – – – 2.15 s33 – – 6.85 dq (7.0; 1.5) 6.90 q (7.3) 6.80 q (7.3) 7.01 q (7.0) 6.93 q (7.0) –34 – – 1.67 dd (7.0; 1.1) 1.77 d (7.3) 1.68 d (7.3) 1.90 d (7.0) 1.81 d (7.0) –35 – 1.73 dd (1.5; 1.1) 1.83 sl 1.75 s 1.91 s 1.82 –

Assignments based on HPLC–1H NMR and 1H–1H TOCSY experiments; n.o., not observed, mainly due to overlap with ACN suppression residue.a Values in ppm (600 MHz). Solvent ACN–D2O.

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A.B. Schefer et al. / J. Chro

or (7) at d 3.41 (dd, J = 2.6 e 9.1 Hz), and couples with H30 (δ.18, d, J = 2.6 Hz) and H3 (δ 4.71 ppm, d, J = 9.1 Hz). Hydro-en H3, in the limonoid (7), shows a chemical shift, which is.90 ppm lower than to the limonoid (6), which is assumed toe caused by the electron withdrawing effect of the hydroxyl onarbon C2.

. Conclusions

It has been shown that hyphenated HPLC–NMR techniqueepresents an outstanding way of dealing with compounds inixture. In natural products research, coupling techniques allow

he identification of peaks without exhaustive isolation proce-ures. A very important practical advantage that one has bysing the chromatography/spectroscopy coupling approach ishat less laboratorial steps are required and therefore, the con-amination and degradation risks for certain peaks are drasticallyeduced. That is already an important issue especially in nat-ral products and bio-fluids metabolites investigations, onceost of unknown compounds of interest are present in rather

mall amounts. Although NMR spectroscopy has an intrinsicow sensitivity, the HPLC–NMR coupling allows the collectionf very important structure information, such as for stereo-hemical analysis. If in conjunction with MS spectroscopy, oneas an even more powerful analytical tool for rapid investi-ation of mixture, even when structure elucidation of minorompounds is wished. With the reported work, structure elu-idation of eight limonoids from extracts obtained form theeeds of mahogany tree (S. macrophylla, Meliacea) could beccomplished by acquiring 1D and 2D spectra from the chro-atographically separated peaks without isolation. Compounds

–7 were identified by comparing acquired 1H NMR spectraith data from literature and other structures in the mixture.hose limonoids were present in smaller amount, although

hey showed UV–vis absorption in the same order of the otherdentified ones in higher concentration. That makes LC–NMRoupling a powerful approach for natural products research,nce NMR can be considered a universal detector with richtructural information for most organic compounds. That is areat utility in vegetal extracts studies aiming new compoundsith biological activity of interest to be screened with more

fficiency.

cknowledgements

The authors thank Conselho Nacional de Desenvolvimentoientıfico e Tecnologico (CNPq)-Pronex, Fundacao de Amparo

` Pesquisa do Estado de Sao Paulo (FAPESP), Coordenacao

e Aperfeicoamento de Pessoal de Ensino Superior (CAPES)nd Financiadora de Estudos e Projetos (FINEP) for the finan-ial support. A.B.S. acknowledges FAPESP and CAPES for theesearch fellowships.

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r. A 1128 (2006) 152–163 163

eferences

[1] I.D. Wilson, J. Chromatogr. A 892 (2000) 315.[2] J.C. Lindon, J.K. Nicholson, I.D. Wilson, Prog. NMR Spectrosc. 29 (1996)

1.[3] K. Albert, J. Chromatogr. A 703 (1995) 123.[4] J.C. Lindon, J.K. Nicholson, I.D. Wilson, J. Chromatogr. B 748 (2000) 233.[5] M. Godejohann, L.-H. Tseng, U. Braumann, J. Fuchser, M. Spraul, J. Chro-

matogr. A 1058 (2004) 191.[6] M. Sandvoss, A. Weltring, A. Preiss, K. Levsen, G. Wuensch, J. Chro-

matogr. A 917 (2001) 75.[7] G. Bringmann, M. Wohlfarth, M. Heubes, J. Chromatogr. A 904 (2000)

243.[8] A. Lommen, M. Godejohann, D.P. Venema, P.C.H. Hollman, M. Spraul,

Anal. Chem. 72 (2000) 1793.[9] K. Holscher, B. Schneider, Phytochemistry 50 (1999) 155.10] J.-L. Wolfender, S. Rodrigues, K. Hostettmann, J. Chromatogr. A 794

(1998) 299.11] B. Ouattara, L. Angenot, P. Guissou, P. Fondu, J. Dubois, M. Frederich,

O. Jansen, J.-C. Heugen, J.-N. Wauters, M. Tits, Phytochemistry 65 (2004)1145.

12] V. Exarchou, M. Godejohann, T.A. Van Beck, I.P. Gerothanassis, J. Ver-voort, Anal. Chem. 75 (2003) 6288.

13] S.H. Smallcombe, S.L. Patt, P.A. Keifer, J. Magn. Reson. 117 (1995)296.

14] S.C. Bobzin, S. Yang, T.P. Kasten, J. Chromatogr. B 748 (2000)259.

15] U.G. Sidelmann, U. Braumann, M. Hofmann, M. Spraul, J.C. Lindon, J.K.Nicholson, S.H. Hansen, Anal. Chem. 69 (1997) 607.

16] M.E. Lacey, R. Subramanian, D.L. Olson, A.G. Webb, J.V. Sweedler,Chem. Rev. 99 (1999) 3133.

17] J.-L. Wolfender, K. Ndjoko, K. Hostettmann, Phytochem. Anal. 12 (2001)2.

18] J.R. De Paula, I.J.C. Vieira, M.F. Das, G.F. Da Silva, E. Rodrigues Fo,J.B. Fernandes, P.C. Vieira, A.L. Pinheiro, E.F. Vilela, Phytochemistry 44(1997) 1449.

19] B.S. Mootoo, R. Motilal, R. Pingal, A. Ramlal, A. Khan, W.F. Reynolds,S. Mclean, J. Nat. Prod. 62 (1999) 1514.

20] M. Nakatani, S.A.M. Abdelgaleil, H. Okamura, T. Iwagawa, A. Sato, M.Doe, Tetrahedron Lett. 41 (2000) 6473.

21] S. Abdelgaleil, H. Okamura, T. Iwagawa, A. Sato, I. Miyahara, M. Doe,M. Nakatani, Tetrahedron 57 (2001) 119.

22] M.G. Soares, L.G. Batista-Pereira, J.B. Fernandes, A.G. Correa, M.F. Das,G.F. Da Silva, P.C. Vieira, E. Rodrigues Fo, O.S. Ohashi, J. Chem. Ecol.29 (2003) 2143.

23] S. Kadota, L. Marpaung, T. Kikuchi, H. Ekimoto, Chem. Pharm. Bull. 38(1990) 639.

24] J. Bickii, N. Njifutie, J.A. Foyere, L.K. Basco, P. Ringwald, J. Ethnophar-macol. 69 (2000) 27.

25] T.R. Govindachari, G. Suresh, B. Banumathy, S. Masilamani, G.Gopalakrisshnan, G.N.K. Kumari, J. Chem. Ecol. 25 (1999) 923.

26] J.D. Connolly, R. Henderson, R. Mccrindle, K.H. Overton, TetrahedronLett. 37–38 (1964) 2593.

27] B. Banerji, S.K. Nigam, Fitoterapia 55 (1984) 3.28] D.A. Okorie, D.A.H. Taylor, J. Chem. Soc. C (1970) 211.29] A. Jimenez, C. Villarreal, R.A. Toscano, M. Cook, J.T. Arnason, R. Bye,

R. Mata, Phytochemistry 49 (1998) 1981.

30] D.A. Okorie, D.A.H. Taylor, Phytochemistry 10 (1971) 469.31] M.S.J. Simmonds, W.M. Blaney, S.V. Lwy, G. Savona, M. Bruno, B.

Rodriguez, Phytochemistry 28 (1989) 1069.32] B.S. Mootoo, R. Ramsewak, A. Khan, W.F. Tinto, W.F. Reynolds, S.

Mclean, M. Yu, J. Nat. Prod. 59 (1996) 544.