HPLC alkamides

Bauer, R. and Remiger, P. 1989. TLC and HPLC analysis of alkamides in Echinacea drugs. Planta Med. 55, 367-371. 

The analysis of CAD and alkamides has been completed using a variety of analytical techniques that include high-performance liquid chromatography (HPLC), capillary electrophoresis, gas chromatography (GC), GC-mass spectrometry (GC-MS), liquid chromatography-mass spectrometry (LC-MS), and nuclear magnetic resonance (NMR). Sample preparation methodologies utilize hexane, methanol and ethanol as the primary extraction solvents. 

Two of the most common methods for the extraction of alkamides involve the use of hexane (Bauer et al., 1989 He et al., 1998) or acetonitrile (Perry et al., 1997). Fractionation of crude extract can be completed using column chromatography in which silica gel serves as the stationary phase and mixtures of hexane-ethyl acetate as the mobile phase. Perry et al. (1997) used a reverse-phase extraction column and acetonitrile-water solvent system. Further purification can be completed using reverse-phase HPLC. 

The evaluation of alkamides, as an indicator for standardization, in Echinacea preparations is commonly completed using HPLC. The method of Bauer (1999b) illustrates the most common method to evaluate alkamides using reverse-phase HPLC. In this method, the lipophilic components are separated by gradient elution using water (eluent A) and acetonitrile (eluent B) linearly from 40% to 80% eluent B at 1 ml/min. The separation was completed on a C18 reverse-phase column and detection was at 254 nm. Thin-layer chromatography using silica 60 plates with... 

Molgaard P, Ohnsen S, Christensen P, Cornett C. HPLC method validation for simultaneous analysis of cichoric acid and alkamides in Echinacea purpurea plants and products. J Agric Food Chem 2003 51 6922-6933. 

HPLC separation of an alcoholic extract from Echinacea angustifolia roots with alkamides. From [4]. 

By HPLC analysis (RP 18 solvent gradient 40-80% acetonitrile/water flov 1.0 ml/min) and photodiode array detection, the different types of alkamides can easily be identified by their retention times and UV-spectra [30]. HPLC analysis is therefore especially suitable for the identification of . angustifolia roots in phytopreparations (see Fig. 2). It is also the most reliable way for the discrimination of extracts from . angustifolia and . pallida roots (see Fig. 7) [31] and for the detection of adulterations with roots of Parthenium integrifolium (Fig. 10) [32]. Also TLC can be used (silica gel solvent -hexane-ethylacetate (2 1) detection anisalde-hyde/sulfuric acid), but with less significance [30]. 

HPLC can also be used for the quantitative determination of the alkamides using external standard calibration [30, 31]. Roots of . angustifolia were shown to contain 0.01-0.15% alkamides and therefore a higher amount than . purpurea roots [30]. Consequently, the alkamides are well suited for the standardization of corresponding phytopharmaceuticals. 

With the aid of HPLC analysis and photodiode array detection (DAD), the different types of alkamides can easily be identified by their different UV-spectra [30]. Hence, the roots of . purpurea and . angustifolia can clearly be discriminated by DAD-HPLC of these lipophilic constituents (see Figs. 2 and 9). Quantitative determination in phytopreparations is also possible by HPLC. The roots of E purpurea were found to contain 0.004-0.039% dodeca-2 ,4 ,8Z,10 /Z-tetraenoic acid isobutylamide [30]. HPLC analysis of the alkamides in extracts of Echinacea purpurea roots would therefore be especially suitable for standardization purposes. An RPLC procedure published recently for the quantitative analyses of alkamide levels in Echinacea purpurea extracts [80] is suspected of leading to elevated levels of alkamides and should therefore only be applied after careful recalibration. 

HPLC analysis with photodiode array detection is the best method for analysis of alkamides, because the different types can be identified via their UV-spectra. However, it is difficult to distinguish the aerial parts of the different Echinacea species, because they show no qualitative difference in the alkamide pattern (see Fig. 15) [30, 82]. HPLC is also useful for the determination of the contents of alkamides and was applied successfully for the analysis of fresh plant tinctures [99]. It could be shown that the yield in the aerial parts is 0.001-0.03% [30]. Small amounts of alkamides can also be found in the expressed sap of . purpurea [100]. It could be shown that the content varies considerably between the different products on the market and even in between the batches of one product (Fig. 16) [100]. One reason may be the seasonal variation of the alkamide content, which is low at the beginning of the vegetation period and becomes high only in the middle of August (data of 1996, see Fig. 17) [101]. Another reason may be the different yields of alkamides in the various parts of the plant. Alkamides are especially accumulated in the flower heads in particular in the tubulous flowers and achenes (Fig. 18) [82, 101, 102]. Therefore the date and mode of harvest play an important role and standardization is urgently needed. 

---