The Spectro-Electro Array: A Novel Platform for the Measurement of Secondary Metabolites in Botanicals, Supplements, and Beverages

Jun 01, 2012
Volume 30, Issue 6, pg 492–503

This installment describes combining diode-array detection (DAD) and coulometric electrochemical-array detection to overcome sensitivity and specificity limitations often encountered when analyzing natural products.

Plants contain an amazingly diverse range of secondary metabolites that may offer important health benefits or risks (1,2). The challenge facing analytical chemists seeking to identify and measure these compounds is twofold: first, to accurately discriminate between compounds sharing similar physicochemical characteristics; and second, to tease this information out of complicated matrices including botanicals, supplements, foods, and beverages.

Figure 1: Comparison of the spectral and electrochemical properties of selected flavonoids. Compounds with similar structures have similar absorbance spectra and changes in functional groups have limited effect on this response (inset). Electrochemical detection, however, is extremely sensitive to the type, degree, and position of substitution as shown by the different current voltage curves (hydrodynamic voltammograms) of catechin (oxidizing at approximately +100 mV), hesperidin (oxidizing at approximately +400 mV), and naringin (oxidizing at approximately +700 mV).
Gradient high performance liquid chromatography (HPLC) with diode-array detection (DAD) is often used to measure the major components present in botanicals. HPLC–DAD is a robust, relatively simple technique that quantifies known analytes that can be resolved in simple matrices, but it suffers from a lack of specificity whenever compounds with similar structures that cannot be deconvoluted spectrally are coeluted (Figure 1). Furthermore, this technique is generally not sensitive enough for the study of natural product metabolism in animals and humans. Electrochemical detection (ECD), on the other hand, is highly sensitive and selective and can distinguish between subtle changes in chemical structure (Figure 1).

Although ECD is selective, many chemicals are electrochemically active, including compounds containing an aromatic backbone with alcohol, amine, or thiol groups attached directly to an aromatic ring, quinones, and conjugated polyenes (with three or more conjugated double bonds) (3). Phytochemical examples include some alkaloids (such as lysergic acid and morphine), carotenoids (such as carotenes and lycopene), fat soluble antioxidants (such as tocopherol and phylloquinine), phenolic acids, and various polyphenols such as the flavonoid group (for example, catechins, daidzein, and genistein).

Figure 2: In this application both chlorogenic acid and 4-hydroxybenzoic acid are coeluted and cannot be resolved spectrophotometrically (UV at 254 nm). However, the electrochemical profiles of these two compounds differ substantially, so they can be resolved voltammetrically, with chlorogenic acid responding exclusively at an upstream (low potential) electrode shown in red and 4-hydroxybenozoic acid responding exclusively at a downstream (high potential) electrode shown in green.
The structure–activity relationships of polyphenolics found in foods and beverages and their antioxidant properties have been extensively studied and are correlated to the ease of electrochemical oxidation. For example, the structural characteristics of polyphenolic phytochemicals that influence their oxidation potential include the type of substitution (such as hydroxy and methoxy), degree of substitution (such as phenol, catechol, and gallo), and the position of substitution relative to other groups (that is, ortho, para, and meta). Thus, the oxidation potential for different substitutions can be ranked from the most labile (lowest oxidation potential) to the most stable (highest oxidation potential): trihydroxy > dihydroxy > methoxy > monohydroxy. Such differences in electrochemical behavior are presented in Figure 1, using catechin, hesperidin, and naringin as examples. Because the compounds require different applied potentials to produce an electrochemical response, this attribute can provide voltammetric resolution when using an array of coulometric electrochemical sensors. Such electrochemical arrays are typically used with the first electrode set to a low potential and each electrode in the series set at increasing, but fixed potentials. Easily oxidized compounds can be selectively detected at upstream, low-potential sensors, and compounds that are more difficult to oxidize respond at downstream sensors set at higher potentials. Coulometric electrochemical-array detection uses a series of as many as 16 highly efficient sensors to voltammetrically resolve those compounds that are coeluted chromatographically (Figure 2) and can identify these analytes based on their voltammetric profile (in an analogous fashion to spectral information obtained from DAD) (3,4). This is a practical and robust form of ECD.

This approach has been successfully applied to the targeted measurement of numerous phytochemicals in a number of matrices such as botanicals (5,6), juice and alcoholic beverages (7–13), food (14–16), and tobacco (11,17). The sensitivity and selectivity of this technique are crucial when studying mammalian phytochemical metabolism (18–28).

Gradient HPLC with coulometric array detection is capable of resolving and measuring several hundred analytes simultaneously (3,29–32). Changes in the pattern of metabolites, when evaluated using chemometric modeling software, can be used to study product adulteration, contamination, composition, and stability, and, in the case of wine and fruit juice, the effect of growing region and differences between varietals (33).

Although electrochemical detection offers analysts many advantages, it is limited to the measurement of electrochemically active compounds only. The combination of DAD and coulometric electrochemical-array detection extends the range of compounds measureable by either technique alone. This platform, called the spectro-electro array, places the diode-array detector before the coulometric array, thereby enabling spectral characterization of analytes before their electrochemical detection. As discussed below, this technique was evaluated for its ability to resolve and quantify targeted phytochemicals in crude extracts of a variety of botanical supplements (for example, ginseng, black cohosh, and gingko), beverages (black and green tea), culinary herbs (oregano, rosemary, sage, and thyme) and spices (clove and nutmeg), as well as its ability to differentiate between samples based on their metabolite patterns.

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