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


Liquid Chromatography

A Thermo Scientific Dionex LPG-3400BM pump with an SR-3000 solvent rack was used. The autosampler used was a Thermo Scientific Dionex WPS-3000TBSL. The UV detector used was a Thermo Scientific Dionex DAD-3000RS diode-array detector, with the following settings: channel 1: 218 nm; channel 2: 240 nm; channel 3: 254 nm; and channel 4: 275 nm. A Thermo Scientific Dionex CoulArray detector with Thermal Organizer was used as the electrochemical detector, consisting of a 16-channel array at potentials ranging from 0 to +900 mV in 60-mV increments. Mobile-phase A consisted of 20 mM monobasic sodium phosphate, 3% acetonitrile, and 0.2% tetrahydrofuran at pH 3.35. Mobile-phase B consisted of 20 mM monobasic sodium phosphate, 50% acetonitrile, and 10% tetrahydrofuran at pH 3.45. Mobile-phase C consisted of 90% methanol. The gradient was as follows: 0–2 min at 2%B/3%C, then 2–30 min ramp to 97%B/3%C, hold until 45 min at 97%B/3%C. A 150 mm × 3 mm, 3-µm Thermo Scientific Acclaim 120 C18 HPLC column was used with a flow rate of 0.65 mL/min and an injection volume of 10 or 20 µL.

Data Analysis

Data were analyzed using Thermo Scientific Dionex Chromeleon chromatography data system 6.8 (SR9) and CoulArray software 3.1. Electrochemical-array data were transferred to Pirouette chemometrics software (Infometrix, Inc.) for chemometric analysis using a CoulArray version 2.0 software utility (Pattern Recognition Setup Wizard).

Standard Preparation

Figure 3: Measurement of standard mixture by UV detection at 218 and 254 nm (upper figure) and 16-channel coulometric electrochemical-array detection (lower figure).
Stock standards, depending on solubility, were prepared in ethanol, methanol, or methanol and water solutions at 1000 µg/mL or 100 µg/mL. Working standards were prepared at 0.2, 0.5, and 1.0 µg/mL in 10% methanol containing 0.2% ascorbic acid and 0.02% EDTA.

Sample Preparation

Table I: Analyte identity (UV1–UV6 are analytes that have strong UV but weak EC response)
Supplements and culinary herbs were prepared for analysis by extracting 100 mg of the material with 20 mL of methanol. The samples were sonicated for 30 min and subsequently centrifuged to obtain a clear solution. The solution was diluted 5× with a preservative solution (10% methanol containing 0.2% ascorbic acid and 0.02% EDTA) for injection into the HPLC system. Wine samples were diluted 1:50 (v/v) with the preservative solution.

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