Benefits and Pitfalls of HPLC Coupled to Diode-Array, Charged Aerosol, and Coulometric Detections: Effect of Detection on Screening of Bioactive Compounds in Apples.

Marcela Hollá,Aneta Bílková,Stanislava Košková,František Švec,Pavel Jakubec,Hana Sklenářová,Dalibor Šatínský,Hana Kočová Vlčková

doi:10.3390/molecules26113246

Abstract

The new screening method for rapid evaluation of major phenolic compounds in apples has been developed. Suitability of coupling HPLC/UHPLC separation with the diode-array detection and universal charged aerosol detection with respect to the presence of interfering substances was tested. Characteristics of both detection techniques were compared and method linearity, limits of detection and quantitation, and selectivity of them determined. Student t-test based on slopes of calibration plots was applied for the detailed comparison. The diode-array detection provided the best results regarding sensitivity and selectivity of the developed method in terms of evaluation of phenolics profiles. The response of the charged aerosol detector was negatively affected by co-eluting substances during rapid-screening analyses. Coulometric detection was used for advanced characterization of extracts in terms of antioxidant content and strength to obtain more complex information concerning sample composition. This detection also allowed evaluation of unidentified compounds with antioxidant activity. HPLC/UHPLC separation using a combination of diode-array and coulometric detectors thus represented the best approach enabling quick, yet complex characterization of bioactive compounds in apples.

Highlights

Interfering sample components are the common problem in analyses of complex matrices
Total polyphenol content was based on the sum of the detected main polyphenolic compounds
A single detection method such as diode-array detection (DAD) and charged aerosol detection (CAD) is unlikely to be sufficient for the evaluation of phenolic compounds

Summary

Introduction

Interfering sample components are the common problem in analyses of complex matrices. The presence of the sample matrix and its potential co-elution with analytes can affect detection response. This problem is well known in the field of mass spectrometry (MS) as matrix effects mainly while applying the electrospray ionization (ESI). The matrix alters the signal of the analyzed components causing overestimation or underestimation of their content and results in poor analytical accuracy, reproducibility, and linearity [1]. Use of isotope labelled internal standards is widely applied in MS. These standards are usually expensive and not always available [10,11]. Amount, composition, physicochemical properties, and concentration of target analytes should be considered

Methods

Results

Conclusion