1.4 Analytical Methods
1.4.6 High-Performance Liquid Chromatography–Nuclear Magnetic Resonance
provide sufficient online information for the identification of small molecules such as flavo- noids. However, in many cases, more data are required for an in-depth structural investiga- tion and this can be supplied by the addition of an LC–NMR analytical capability (Figure 1.13). For practical purposes, LC–UV–MS and LC–UV–NMR are generally run as separate
1 2 4
1 2 3 4
9.8⫻10−1
1.5310−1
200 400 600 800 1000 scans = 25 min
UV 350 nm
RIC 100
20
1 2 3 4
1 Isoorientin, R=OH 3 Isovitexin, R=H
2 Orientin, R=OH 4 Vitexin, R=H O
R
OH
O OH HO
Glc
O
R
OH
O OH HO
Glc
200 400
FIGURE 1.10 TSP LC–MS analysis of a methanol extract ofDissotis rotundifolia. Column Novapak C18
(3003.9 mm, 4mm); CH3CN–H2O (14:86) (containing 0.05% TFA); flow rate 0.8 ml/min; detection 350 nm; RIC reconstructed ion current.
operations. The coupling of HPLC with NMR spectroscopy, introduced around 1978, is one of the most powerful methods for the combined separation and structural elucidation of unknown compounds in mixtures.99,100
At first, LC–NMR was little used because of its lack of sensitivity. However, recent progress in pulse field gradients and solvent suppression, improvement in probe technology, and the construction of high-field magnets have given a new impulse to the technique. While HPLC–NMR coupling is relatively straightforward (the samples flow in a nonrotating 60 to 180ml glass tube connected at both ends with HPLC tubing) compared to LC–MS, the main problem of LC–NMR is the difficulty of observing analyte resonances in the presence of the much larger resonances of the mobile phase. This problem is magnified under typical reversed-phase HPLC operating conditions, where more than one protonated solvent is used and where the resonances change frequencies during analysis in the gradient mode.
Furthermore, the continuous flow of sample in the detector coil complicates solvent suppres- sion. These problems have now been overcome by the development of fast, reliable, and powerful solvent suppression techniques, such as WET (water suppression enhanced through T1effects),101which produce high-quality spectra in both on-flow and stopped-flow modes.
These techniques consist of a combination of pulsed-field gradients, shaped radiofrequency pulses, shifted laminar pulses, and selective13C decoupling, and are much faster than classical presaturation techniques previously used in the field. Thus, for typical reversed-phase HPLC analyses, nondeuterated solvents, such as methanol and acetonitrile, can be used, while water is replaced by D2O.
The information provided by LC–NMR consists mainly of1H NMR spectra or 1H–1H correlation experiments. Access to13C NMR is possible but is restricted only to a very limited number of cases where the concentration of the LC peak of interest is high and13C NMR data can be deduced indirectly from inverse detection experiments. Due to the low natural
TSP-MS of isoorientin
TSP-MS of orientin
MS-MS of [M+H–120]+
MS–MS of [M+H–120]+
100 200 300 400 500 m/z
100 200 300 400 500 m/z 100 200 300 400 500 m/z
100 200 300 400 500 m/z
20 40 60 80 100
20 40 60 80 100
287
287 329
329 359
359 395
395 431
431
137
137 149
177
300
300 311
311 329
329 Rel. intensity 449
20 40 60 80 100
Rel. intensity
20 40 60 80 100
Rel. intensity
Rel. intensity
[M+H]+
449 [M+H]+
5⫻
5⫻
(m/z 329)
(m/z 329)
for isoorientin
for orientin
FIGURE 1.11 Online mass spectra of isoorientin (1) and orientin (2) in the TSP mode. The respective TSP MS–MS (CID) analyses of the parent [MþH120]þ(m/z329) ions are also shown.
Separation and Quantification of Flavonoids 25
[M] [M]
Wessely–Moser rearrangement
Vitexin, R=H
Orientin, R=OH Isovitexin, R=H
Isoorientin, R=OH
[M–120] [M–120]
RDA RDA
m/z 149
m/z 177 X
O R
OH
O OH
HO O
R OH
O OH HO
O R
OH
O OH
HO O
R OH
O OH HO HO O
HO OH
CH2OH
HOCH2 O HO
OH OH
+OH
+OH
A
HO O
C
O O
+H2C A C8H5O3
FIGURE 1.12 Specific fragmentations of theC-glycosylflavones isoorientin and isovitexin.
HPLC column INJECTION
– Extract – Fraction – Mixture
LC–MS Interface HPLC
Postcolumn pump
addition of the LC–MS additives
Splitting dependent on the LC–MS interfaces used Waste
Waste LC probe
UV MS
NMR MS
NMR
Spectra
1
Computer 2
control of pump, UV and NMR for stop-flow experiments
HPLC UV pump
FIGURE 1.13 Schematic representation of the instrumentation used for LC–UV–MS (1) and LC–UV–
NMR (2) analyses.
abundance of the13C isotope (1.1%), the sensitivity for direct measurement in the LC–NMR mode is insufficient.
LC–NMR can be operated in two different modes: on-flow and stopped-flow. In the on- flow mode, LC–NMR spectra are acquired continuously during the separation. The data are processed as a two-dimensional (2D) NMR experiment. The main drawback is the inherent low sensitivity. The detection limit with a 60ml cell in a 500 MHz instrument for a compound with a molecular weight around 400 amu is 20mg. Thus, on-flow LC–NMR runs are mainly restricted to the direct measurement of the main constituents of a crude extract and this is often under overloaded HPLC conditions. Typically, 1 to 5 mg of crude plant extract will have to be injected on-column.102In the stopped-flow mode, the flow of solvent after HPLC separation is stopped for a certain length of time when the required peak reaches the NMR flow cell. This makes it possible to acquire a large number of transients for a given LC peak and improves the detection limit. In this mode, various 2D correlation experiments (COSY, NOESY, HSQC, HMBC) are possible.
The combination of HPLC with online UV, MS, and NMR detection has proved to be a very valuable tool for the analysis of natural products in extracts or mixtures.102,103The field of flavonoids is no exception. The LC–NMR information obtained comes from the1H NMR spectra of selected peaks in the HPLC chromatogram. From LC–MS, A- or B-ring substitution can be deduced from the fragmentation pattern but the exact location of the substituent cannot be determined. However, for a flavonoid like apigenin, where only one hydroxyl group is located on the B-ring,1H NMR will give the substitution position because each of the three possibilities of localization of the hydroxyl group will give a unique splitting pattern. Much information can be derived about the nature and linkage positions of sugars. However, since D2O is present in the eluent, exchangeable signals are not observed in the NMR spectrum.
An example of the LC–NMR stop-flow procedure is provided in the analysis of polyphe- nolics from the Chilean plantGentiana ottonis(Gentianaceae).104Preliminary LC–UV analysis of a methanol extract of the roots showed the presence of several xanthones (2,4,6–8; Figure 1.14), an iridoid (1), and two compounds (3,5) with UV spectra typical of flavonoids. TSP LC–
MS provided the molecular weights of the latter two compounds and gave fragments charac- teristic forC-glycosides (losses of 90 and 120 amu). According to their UV spectra,3 and5 (MW 448 and 446) were, respectively, tri- and tetra-oxygenated flavoneC-glycosides. In order to obtain further information for characterization of the polyphenols in the extract, LC–NMR was performed under the same conditions used for LC–UV–MS. However, water was replaced by D2O and the amount injected was increased to 0.4 mg, which did not cause a noticeable loss in resolution. LC–1H NMR spectra were recorded for all the main peaks in the stop-flow mode and the number of transients for a good signal-to-noise ratio varied between 128 and 2048. For flavoneC-glycoside5(MW 446), the LC–1H NMR spectrum (Figure 1.15) gave signals for six aromatic protons (d6.8 to 8.1), one methoxyl group (d4.0), and theC-glycoside moiety (d3.5 to 5.0). A pair of symmetric ortho-coupled protons (2H, d 7.06, J¼8.3, H-3’,5’and 2H, d 8.00, J¼8.3, H-2’,6’) was characteristic for a B-ring substituted at C-4’. The singlet atd6.8 was attributed to H-3. A singlet atd6.9 was due to a proton either at position C-6 or C-8 on the A-ring. Thus, LC–UV–MS and stop-flow LC–NMR were not sufficient to fully ascertain the structure of 5. In order to ascertain the position of C-glycosylation, an LC–MS–MS experiment was performed by choosing [MþH120]þas parent ion. This gave fragments at m/z191 and 163, characteristic for 6-C-glycosylated flavones (described earlier). The fragment at m/z 121 indicated a monohydroxylated B-ring, confirming the methoxyl group to be on the A-ring. This combination of data allowed identification of5as 5,4’-dihydroxy-7-methoxy- 6-C-glucosylflavone (swertisin).104
If full metabolite profiling of a plant extract has to be performed, LC–NMR can be run in the on-flow mode. In order to obtain adequate NMR spectra of all constituents, the amount
Separation and Quantification of Flavonoids 27
of sample injected has to be increased — this produces overloading when compared with normal analytical HPLC conditions but gives the possibility of testing for biological activity (in conjunction with a microfractionation procedure). This was the approach adopted for the investigation of new antifungal constituents from Erythrina vogelii(Leguminosae), a medi- cinal plant of the Ivory Coast.105In order to rapidly identify the active principles from the antifungal dichloromethane extract of the roots, preliminary analysis by LC–UV and Q-TOF LC–MS was performed. Approximately 12 major peaks were observed in the HPLC chro- matogram and from UV, MS, and MS–MS online data, these were shown to be prenylated isoflavones and isoflavanones. In order to obtain more information, on-flow LC–1H NMR was performed by injecting 10 mg of crude extract onto an 8 mm C18 radial compression column connected to the NMR instrument. At a low flow rate (0.1 ml/min), acquisition of ten LC–NMR spectra was possible. Of these ten peaks, five were found by simultaneous HPLC microfractionation to be associated with the antifungal activity of the extract. Interpretation of all online data, with emphasis on LC–NMR, allowed the identification of eight flavonoids, including a known isoflavone with antifungal activity and two putative new isoflavanones, also with antifungal activity. This dereplication procedure allowed the targeted isolation of the new antifungal compounds.105
Applications of LC–NMR for the online identification of flavonoids are still few and far between, one reason probably being the high cost of the apparatus. However, several other
0 10 20 30 40
0 500 1000 1500 2000
mAU 1
2 3 4 5
6
7 8
UV trace (254 nm)
400 300
200 nm
400 300
200 nm 200 300 400 nm 200 300 400 nm 200 300 400 nm
400 300
200 nm 200 300 400 nm 200 300 400 nm
1 2
5 6 8
4 3
7 [M+H]+= 375
Loss: 162
[M+H]+= 423
Loss: 18,90,120,162 [M+H]+= 449 Loss: 18,90,120
[M+H]+= 423 Loss: 162
[M+H]+= 447
Loss: 18,90,120 [M+H]+= 437
Loss: 162 [M+H]+= 261 [M+H]+= 275
min
FIGURE 1.14 Online LC–UV of a methanol extract ofGentiana ottonis, with protonated molecular ions obtained for the main constituents by TSP LC–MS. Column Novapak C18 (150 3.9 mm, 4 mm);
gradient CH3CN–H2O (containing 0.05% TFA) 5:95!65:35 in 50 min; flow rate 0.8 ml/min. (From Wolfender, J.-L., Ndjoko, K., and Hostettmann, K., inMethods in Polyphenol Analysis, Santos-Buelga, C. and Williamson, G., Eds., Royal Society of Chemistry, Cambridge, 2003. With permission.)
8 7 6 5 4 3 2 d ppm H-2',6' H-3',5'
H-3 H-8
OCH3-7
HOD MeCN
Suppr.
Suppr.
H-1'
H-2'-H-6'
50 30
10 20 40
10
8
6
2 4
327 [M+H–120]+
447 [M+H]+
8E6
50 30
10 20 40
10
8
6
2 4
121 191 163
327 [M+H–120]+
3E4 m/z 327
[M+H-120]+
a) LC/TSP-MS spectrum of 5
b) LC–TSP MS–MS of [M–120]+
A C
B
A B
m/z 121 m/z 191
m/z 163
Determination of the substituents on B-ring Fragments exclusively observed for 6-C glycosylflavones - CO
Swertisin (5)
Pathway I Pathway II
3'
3 8
2'
6'
20 30 40 n 5'
UV spectrum of 5
Stop-flow LC–1H-NMR of 5 O
OH
OH O O
HO HO
OH OH CH3O
O
OH
OH O CH3O
H2O+
C CH3O
O H2C+
O
O
C
OH
+O
O
O HO CH3O
Glc
OH
FIGURE 1.15 Stop-flow LC–1H NMR spectrum of swertisin (5) from the methanol extract of the roots ofGentiana ottonis, together with the TSP LC–MS (a) and TSP LC–MS–MS (b) spectra. The LC–MS–
MS analysis was performed using the fragment [MþH120]þ(spectrum a) as parent ion. Charac- teristic daughter ions atm/z121, 163, and 191 were observed, indicating the substitution on the A- and B-ring of theC-glycosylflavone. (From Wolfender, J.-L., Ndjoko, K., and Hostettmann, K., inMethods in Polyphenol Analysis, Santos-Buelga, C. and Williamson, G., Eds., Royal Society of Chemistry, Cambridge, 2003. With permission.)
Separation and Quantification of Flavonoids 29
examples do exist, in addition to those mentioned above. The technique has been successfully applied to the analysis ofHypericum perforatum(Guttiferae). Online identification of querce- tin, several of its glycosides, and the biflavonoid I5,II8-biapigenin in an extract was possible.106