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FIGURE 7.10 Typical chromatograms obtained by GC-MS of E. angustifolia (top) E. pallida (middle), and E. purpurea (bottom) roots extracted by maceration with dichloromethane:pentane (1:1, v/v). (From Lienert, D. et al., 1998, Phytochem. Anal., 9: 88-98. With permission.)

HPLC coupled to diode array detection and followed by ESI- or TSP-MS has been successfully applied to discriminate between the three Echinacea species. The first paper was published by He et al. (1998), who succeeded in detecting nine alkamides in the roots of E. purpurea, and several other alkamides not previously described. The HPLC analysis was performed on a Waters Symmetry C18 column (150 x 2.1 mm, 5 mm) using water (eluent A) and acetonitrile (eluent B) as mobile phases by the following elution profile: 0 to 30 minutes, 45% to 80% B; 30 to 32 minutes, 80% to 100% B; 32 to 35 minutes, 100% to 45% B. The flow rate was 0.2 ml/minute and the temperature was set at 45°C. UV spectra were taken in the range 200 to 500 nm; the MS spectra were acquired in the positive ion mode by an electrospray ionization interface, and the mass ranges were 200 to 700 m/z. As shown in Figure 7.11, nine alkamide peaks (Table 7.1) were well separated, while the isomeric pair 8/9 was not resolved. Most peaks yielded protonated molecular ions, sodiated molecular ions, and sodiated molecular dimer ions, exemplified as peaks (Figure 7.12). Purified alkamides 8/9 were employed as external standards to determine the content of these tetraenes in E. purpurea root (0.037%) and in E. pallida, E. purpurea, and E. angustifolia achene samples (0.08%, 0.75%, and 1.06%, respectively).

Sloley et al. (2001) applied the HPLC-UV-MS approach for the rapid characterization of alcoholic extracts from roots and leaves of E. pallida, E. purpurea, and E. angustifolia. Chromatographic separations were accomplished by gradient elution on a Zorbax 300 SB-C8 (25 cm x 4.1 mm) column, employing two eluents: A, 0.1% trifluoroacetic acid in 5% acetonitrile; and B, 0.1% trifluoroacetic acid in 70% acetonitrile. The gradient profile was 0 to 20 minutes, from 100% A to 85% A plus 15% B; 20 to 40 minutes, to 100% B; 40 to 45 minutes, 100% B; and 45 to 50 minutes, from 100% B to 100% A. The flow rate was 1 ml/minute. The peaks eluting from the column after UV detection (254 and/or 205 nm) were monitored by an electrospray mass spectrometer. The peaks devoid of strong mass signals (some alkamides) were individually collected and reevaluated by direct injection into the mass spectrometer. According to this study, the major UV-absorbing compounds (254 nm) in E. angustifolia roots were alkamides 6 to 9 followed by echinacoside and cynarin. Conversely, E. pallida roots had echinacoside and 6-O-caffeoylechinacoside as major UV-absorbing constituents. The chromatogram of E. purpurea roots showed mainly cichoric acid.

Leaf extracts from the three Echinacea species also provided distinct HPLC UV and electro-spray mass spectra profiles. Thus, E. purpurea and E. angustifolia were differentiated from E. pallida for the presence of alkamides 8/9. Cichoric acid was detected in both E. pallida and E. purpurea but not in E. angustifolia. Echinacoside was found in extracts from leaves of E. pallida but not in E. purpurea and E. angustifolia leaf extracts. In addition, rutin was detected in leaf extracts of all three species.

Fuzzati et al. (2001) recently developed an improved HPLC procedure that allows separation of echinacoside and 14 different alkamides from E. angustifolia root alcoholic extracts. Interestingly, this method permits satisfactory resolution of the critical isomeric pair 8/9 (Figure 7.6). The eluents are water (A) and 0.01% trifluoroacetic acid in acetonitrile. The elution is on a Zorbax SB C18 (250 x 4.6 mm, 5 mm) by a gradient mode. The peaks after UV detection (200 to 500 nm) are fed directly into a mass spectrometer equipped with a TSP-2 interface, and monitored between 200 to 900 m/z in positive ion mode. Similar to ESI-MS, the TSP mass spectra revealed only the protonated molecules ([M + H]+) without fragmentation. This method is currently applied to detect the presence of E. pallida roots as adulterant of E. angustifolia roots. Indeed, falsification with E. pallida roots is easily recognized from the presence of the ketoalkyne pentadeca-8Z,13Z-dien-11-yn-2-one (24), which is specific to this species (Figure 7.13). Finally, echinacoside and alkamide 8 were used to obtain calibration curves for the quantitation of echinacoside and total alkamides (calculated as alkamide 8) in different batches of E. angustifolia roots.

MS Chromatogram

Abundance TIC: HALKMS6.D

MS Chromatogram

Abundance TIC: HALKMS6.D

LC Chromatogram

Abundance LC Chromatogram: HALKMS6.D

LC Chromatogram

Abundance LC Chromatogram: HALKMS6.D

FIGURE 7.11 HPLC and HPLC-ESI-TIC chromatograms of E. purpurea roots. (From He et al., 1998, J. Chromatogr. A, 815: 205-211. With permission.)

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