The Spectro-Electro Array: A Novel Platform for the Measurement of Secondary Metabolites in Botanicals, Supplements, Foods and Beverages - Part : Theory and Concepts Paul A. Ullucci, Bruce Bailey, Ian N. Acworth, Christopher Crafts, Marc Plante Thermo Fisher Scientific, Chelmsford, MA, USA
verview Purpose: A gradient HPLC UV Diode Array (DAD) and multichannel electrochemical (EC) detection method was developed and evaluated for the deteration of phenols, phenolic acids and polyphenols in botanicals, supplements, foods, and beverages. Method: Gradient HPLC with DAD and multi-channel EC detection were used. Results: The method enables the separation and quantitation of different compounds commonly found in botanicals, supplements, foods, and beverages. Introduction Plants contain an amazingly diverse range of secondary metabolites, many of which are purported to offer health benefits. The challenge for the analytical chemist wanting to measure these compounds is two-fold: first, to develop an assay that can accurately discriate between compounds that often have similar physicochemical characteristics; second, to analyze such compounds in complicated matrices including botanicals, supplements, foods and beverages. Gradient HPLC with diode array detection (DAD) is often used for the deteration of natural products. However, this approach often suffers from a lack of specificity as compounds with similar structures may co-elute chromatographically and cannot be easily deconvoluted spectrally. Furthermore, this technique lacks sensitivity limiting its use for the study of natural products metabolism in animals and humans. Alternately, coulometric electrode array detection is selective and is able to distinguish between subtle changes in chemical structure with sub-picogram limits of detection. The Thermo Scientific Dionex CoulArray detector can be used to exae natural product absorption and metabolism. The combination of DAD and coulometric electrochemical array detection extends the range of compounds measured by either technology alone. The concept behind the spectro-electro array will be discussed and the power of this approach will be illustrated using a global method capable of measuring ~ 5 analytes simultaneously. Method Liquid Chromatography Pump: Thermo Scientific Dionex LPG-BM with SR- Solvent Rack Autosampler: Thermo Scientific Dionex WPS-TBSL UV Detector: Thermo Scientific Dionex DAD-RS Diode Array Detector Channel : nm Channel : nm Channel : 5 nm Channel : 75 nm EC Detector: Thermo Scientific Dionex CoulArray Detector with Thermal rganizer Module. EC Parameters: channel array from to + mv in + mv increments Mobile Phase A: mm monobasic sodium phosphate, % acetonitrile,.% tetrahydrofuran, ph.5 Mobile Phase B: mm monobasic sodium phosphate, 5% acetonitrile, % tetrahydrofuran, ph.5 Mobile Phase C: % methanol Gradient: - : %B/%C.; : 7%B/%C; 5 7% B/%C. Curve 7 Analytical Column: Thermo Scientific Acclaim, C, 5 mm, µm Flow Rate:.5 ml/ Injection: or µl Data Analysis and Processing Data were analyzed using Thermo Scientific Dionex Chromeleon Chromatography Data System ver.. (SR) and CoulArray software.. Standard Preparation Stock standards were prepared, depending upon solubility, in ethanol, methanol or methanol/water solutions at µg/ml or µg/ml. Working standards were prepared at.,.5 and. µg/ml in 5% methanol containing.% ascorbic acid. The Spectro-Electro Array: A Novel Platform for the Measurement of Secondary Metabolites in Botanicals, Supplements, Foods and Beverages - Part : Theory and Concepts
Results and Discussion Electrochemical detection is renowned for being both highly sensitive and selective, whereas UV detection is much less sensitive but more universal in nature. The platform described here (spectro-electro array) makes use of the benefits of both detection formats and is used to extend the range of compounds that can be measured by either detector alone. The unique resolution of the coulometric electrode array comes from the high sensitivity of the flow-through porous graphite working electrodes. When these electrodes are used in series in an array, compounds are resolved based on their voltammetric behavior. Compounds detected by the CoulArray detector are resolved based both on their chromatographic and voltammetric (redox) behavior. Furthermore, these parameters can be used to identify and authenticate a compound. Electrochemical detection is incredibly sensitive to small changes in chemical structure, UV detection much less so. Figure demonstrates that voltammetric data from the CoulArray detector and spectral data from the DAD are complementary. Spectral data is primarily dependent on the backbone of the chemical structure. For example, flavonones all have similar absorbance as shown in the top left of Figure. This can be an issue if these compounds co-elute. Electrochemcial detection, on the other hand, is extremely sensitive to the type, degree, and position of the substitution on the backbone of the chemical structure. Shown on the bottom left of the figure are the current-voltage curves (hydrodynamic voltammograms) for three comounds: catechin, with a catechol ring, is the most labile oxidixing at mv; hesperidin oxidizes at ~ mv; while naringin, containing a phenol ring, oxidizes at ~5 mv. Such structural/voltammetric relationships are used by the CoulArray detector to identify and authenticate analytes and to resolve co-eluting peaks. Spectral data are used when a phytochemical is not EC active. Figure. Comparison of the spectral and electrochemical properties of selected flavonoids. 5 Structures Typical Absorbance Max at -nm Response (AU) 5 Typical Broad Shoulder at nm H 5 Catechin Wavelength (nm) 5 Rh,Gl Rh = rhamnose Gl = glucose Response Ratios Characteristic Electrochemical Signature Naringin CH Rh,Gl E ½ Electrode Potential (mv) Catechin Hesperidin Naringin Hesperidin Rh = rhamnose Gl = glucose The wide variety of compounds that were measured in this study, along with their retention times and doant electrochemical channel are listed in Table. It should be noted that there are six compounds designated UV-UV. These compounds show poor response by EC detection but are readily detectable by UV. Figure shows their chromatographic separation and detection by UV at nm and 5 nm, while Figure shows their separation and response on the CoulArray detector. The CoulArray detector readily enables the resolution of co-eluting compounds, and response to gradient is imal. The Spectro-Electro Array: A Novel Platform for the Measurement of Secondary Metabolites in Botanicals, Supplements, Foods and Beverages - Part : Theory and Concepts
Table. Standard mixture - identification, retention time and array channel. Peak No. Compound Retention Time utes Doant EC Channel Gallic acid.5 -Hydroxybenzyl alcohol. p-aobenzoic acid.,-dihydroxybenzoic acid 5. 5 Gentisic acid 5.7 -Hydroxybenzyl alcohol 7.5 7 -Hydroxybenzoic acid. Chlorogenic acid. p-hydroxyphenyl acetic acid. Catechin. Vanillic acid.7 -Hydroxybenzaldehyde. 5 Syringic acid. Caffeic acid.5 5 Vanillin. Syringealdehyde. 7 Umbelliferone. p-coumaric acid. UV,-Dimethoxybenzoic acid.7 Salicylic acid 5. 5 Sinapic acid 5. Ferulic acid 5. Ellagic acid. UV Coumarin.7 Rutin. Ethyl vanillin 7. UV Methoxybenzaldehyde.7 5 -Hydroxycoumarin.5 5 Hesperidin. 7 Naringin. Rosemarinic acid. Fisetin. Myricetin. Trans-resveratrol. UV Cinnamic acid. Luteolin 5. Cis-resveratrol.5 Quercetin.5 UV5 Apigenin 7. 5 Kaempferol. Isorhamnetin. 7 Eugenol. Isoxanthohumol. UV Chrysin. Cavarcrol. Thymol. Carnosol.7 Xanthohumol 5.7 Carnosic acid.7 An example of the ability of the spectro-electro array to resolve co-eluting compounds is presented in Figure. The upper chromatogram shows the UV response at 5 nm and the co-elution of chlorogenic and -hydroxybenzoic acids. The lower chromatogram is from the CoulArray detector and shows that even though these two compounds co-elute, they can be resolved voltammetrically, with chlorogenic acid responding on channel (red tracing at mv) and -hydroxybenzoic acid responding on channel (green tracing at 7 mv). nly selected channels are shown for visual clarity. The Spectro-Electro Array: A Novel Platform for the Measurement of Secondary Metabolites in Botanicals, Supplements, Foods and Beverages - Part : Theory and Concepts
Doant EC Channel 5 5 5 o-eluting compounds V response at 5 nm he lower en though these two chlorogenic acid enzoic acid ed channels are FIGURE. UV detection of separation of Standard mixture at and 5 nm.. The limits of detection were typically by UV. The limits of quantification were 5-5 pg by UV. Response range w five by UV. Typical R values were ~. of calibration curves of selected compo retention time precision for all analytes with a range of..%. 5.. uv & uv5 5. 5 & 7. & uv 7 & 5. 5 7 uv uv nm uv 7 5nm.. 5. -.. FIGURE 5. Typical response curves f Response (Area) d array channel................. FIGURE. channel coulometric electrochemical array detection of separation of Standard mixture.. Conc dihydoxybenzoic a.5. 5.5.. 7 5 7 5 5 7 7.. FIGURE. The spectro-electro array.. FIGURE : Detection of chlorogenic and -hydroxybenzoic acid. Top: UV - shows co-elution. Bottom: EC array shows full resolution.. UV at 5 nm Chlorogenic acid/ -Hydroxybenzoic acid 7.5 5. Conclusion.5.. 7. 7. 7. 7. 7..... ECD -Hydroxybenzoic acid.. Catechin Chlorogenic acid... 7. 7.5..5. A multi-analyte method for the detera polyphenols was developed that combin channel electrochemical detection. The combination of the two detection te that can be detected by either technolog Analyte co-elutions are readily identifie This approach has application to both t The method demonstrates two importa detector: First, the CoulArray is the only elution chromatography. Second, the Co to permit simultaneous measurement of eliating manual gain adjustments or r All trademarks are the property of Thermo Fisher Sci This information is not intended to encourage use of intellectual property rights of others. 5 The Spectro-Electro Array: A Novel Platform for the Measurement of Secondary Metabolites in Botanicals, Supplements, Foods and Beverages - Part : Theory and Concepts
The limits of detection were typically -5 pg on-column by ECD and 5 pg by UV. The limits of quantification were pg (on-column) by ECD and 5-5 pg by UV. Response range was over seven orders of magnitude by ECD and five by UV. Typical R values were ~. or better for all compounds. Examples of calibration curves of selected compounds are given in Figure 5. Average intra-day retention time precision for all analytes averaged.55% RSD over a day period, with a range of..%. FIGURE 5. Typical response curves for representative analytes. Response (Area) 7 5 R² =. R² =.7 R² =...... Concentration (µg/ml) dihydoxybenzoic acid caffeic acid luteolin FIGURE. The spectro-electro array system used in this study. Conclusion A multi-analyte method for the deteration of phenols, phenolic acids, and polyphenols was developed that combined UV Diode Array detection with multichannel electrochemical detection. The combination of the two detection techniques extends the range of compounds that can be detected by either technology alone. Analyte co-elutions are readily identified and resolved. This approach has application to both targeted and metabolomic studies. The method demonstrates two important and unique features of the CoulArray detector: First, the CoulArray is the only EC detector fully compatible with gradient elution chromatography. Second, the CoulArray utilizes signal autoranging technology to permit simultaneous measurement of analytes with disparate concentrations thus eliating manual gain adjustments or re-analysis of samples. The Spectro-Electro Array: A Novel Platform for the Measurement of Secondary Metabolites in Botanicals, Supplements, Foods and Beverages - Part : Theory and Concepts
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