Development of an Ultrasonication-Assisted Extraction Based HPLC With a Fluorescence Method for Sensitive Determination of Aflatoxins in Highly Acidic Hibiscus sabdariffa.

Xiaofei Liu,Meihua Yang,Qian Li,Lei Zhang,Shanshan Zhang,Chaonan Sun,Guangyao Ying,Xiaoyan Xing,Weijun Kong

doi:10.3389/fphar.2018.00284

Abstract

The high acidity and complex components of Hibiscus sabdariffa have provided major challenges for sensitive determination of trace aflatoxins. In this study, sample pretreatment of H. sabdariffa was systematically developed for sensitive high performance liquid chromatography-fluorescence detection (HPLC-FLD) after ultrasonication-assisted extraction, immunoaffinity column (IAC) clean-up and on-line post-column photochemical derivatization (PCD). Aflatoxins B1, B2, G1, G2 were extracted from samples by using methanol/water (70:30, v/v) with the addition of NaCl. The solutions were diluted 1:8 with 0.1 M phosphate buffer (pH 8.0) to negate the issues of high acidity and matrix interferences. The established method was validated with satisfactory linearity (R > 0.999), sensitivity (limits of detection (LODs) and limits of quantitation (LOQs) of 0.15–0.65 and 0.53–2.18 μg/kg, respectively), precision (RSD <11%), stability (RSD of 0.2–3.6%), and accuracy (recovery rates of 86.0–102.3%), which all met the stipulated analytical requirements. Analysis of 28 H. sabdariffa samples indicated that one sample incubated with Aspergillus flavus was positive with aflatoxin B1 (AFB1) at 3.11 μg/kg. The strategy developed in this study also has the potential to reliably extract and sensitively detect more mycotoxins in other complex acidic matrices, such as traditional Chinese medicines, foodstuffs, etc.

Highlights

Aflatoxins (AFs) are highly toxic secondary metabolites mainly produced by the toxigenic fungi Aspergillus flavus and Aspergillus parasiticus that display severe mutagenic, carcinogenic and teratogenic effects (Peraica, 1999; The Joint FAO/WHO Expert Committee on Food Additives, 2012)
The tolerance levels of aflatoxin B1 (AFB1) set by the European Commission are 5–20 μg/kg in various matrices (European Commission, 2010), while maximum levels (MLs) of AFB1 and AFs posed by Chinese pharmacopeia (Chinese Pharmacopoeia Commission, 2015) are 5 and 10 μg/kg, respectively
Because of their acute virulence and wide occurrence combined with ultra-low levels in various matrices, current analytical methods for analysis of AFs mainly include thin layer chromatography (TLC) (Stroka et al, 2009), enzymelinked immunosorbent assay (ELISA) (Li et al, 2009), and liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) (Xie et al, 2015; Pesek et al, 2017)

Summary

Introduction

Aflatoxins (AFs) are highly toxic secondary metabolites mainly produced by the toxigenic fungi Aspergillus flavus and Aspergillus parasiticus that display severe mutagenic, carcinogenic and teratogenic effects (Peraica, 1999; The Joint FAO/WHO Expert Committee on Food Additives, 2012). The tolerance levels of AFB1 set by the European Commission are 5–20 μg/kg in various matrices (European Commission, 2010), while MLs of AFB1 and AFs posed by Chinese pharmacopeia (Chinese Pharmacopoeia Commission, 2015) are 5 and 10 μg/kg, respectively Because of their acute virulence and wide occurrence combined with ultra-low levels in various matrices, current analytical methods for analysis of AFs mainly include thin layer chromatography (TLC) (Stroka et al, 2009), enzymelinked immunosorbent assay (ELISA) (Li et al, 2009), and liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) (Xie et al, 2015; Pesek et al, 2017). This alternative method provided rapid and sensitive detection of AFs in many different types of matrices (Wen et al, 2013; Kong et al, 2014; Kara et al, 2015; Golge et al, 2016; Campos et al, 2017)

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Conclusion