Food Research International 138 (2020) 109754 Available online 2 October 2020 0963-9969/© 2020 Elsevier Ltd. All rights reserved. Review Validated liquid chromatography separation methods for identifcation and quantifcation of anthocyanins in fruit and vegetables: A systematic review Mamatha Chandra Singh a, b, * , Celine Kelso b, c , William E. Price c , Yasmine Probst a, b a School of Medicine, Faculty of Science, Medicine and Health, University of Wollongong, Wollongong, NSW 2522, Australia b Illawarra Health and Medical Research Institute, University of Wollongong, Wollongong, NSW 2522, Australia c School of Chemistry and Molecular Bioscience, Faculty of Science, Medicine and Health, University of Wollongong, Wollongong, NSW 2522, Australia A R T I C L E INFO Keywords: Mass analyzer Quantifcation Food composition Cyanidin Anthocyanin Extraction Chemical compounds studied in this article: Acetonitrile (CID: 6342) Formic acid (CID: 284) Ethanol (CID: 702) Tartaric acid (CID: 875) Phosphoric acid (CID: 1004) Methanol (CID: 887) Ammonium formate (CID: 2723923) Dichloromethane (CID: 6344) Trifuoroacetic acid (CID: 6422) Acetic acid (CID: 176) Anthocyanin (CID: 145858) ABSTRACT Food composition data are challenged by data availability and quality. Anthocyanins are the bright colored pigments found in fruits and vegetables with growing evidence for health benefts. For the estimation of anthocyanin content in the foods, it is imperative to fnd an ideal analytical method. To quantify anthocyanin components, liquid chromatography-based methods are commonly used. This review addresses the variability of liquid chromatography (LC) mass spectrometry (MS) methods for the identifcation and quantifcation of an- thocyanins. Published studies for all years until February 2020 reporting LC methods for anthocyanins in fruits and vegetables were screened from 7660 studies. Only 29 studies met the eligibility criteria of method type and of these, only 13 studies reported a validated LC method. A wide range of validation parameters were identifed including specifcity, calibration, stability, and limits of detection. Differences in the sampling amounts for extraction were observed in all of the included studies. The quantifcation of multiple anthocyanin types without their corresponding analytical standards was observed in eight studies. The included 13 studies used reverse phase liquid chromatography separation with C 18 type or similar stationary phases and acidifed aqueous or acidifed aqueous: organic (usually methanol or acetonitrile) binary gradient mobile phases. Although all of the studies used mass spectrometry for identifcation, ultraviolet absorbance quantifcation was often used in conjunction with a photo-diode array (DAD/PDA) detector using reference standards where available. Extraction and preparation of samples remains the key concern for analysis as the oxidative stability of anthocyanins are a major impediment for accurate quantifcation of the components in foods. This review provides a summary of validated LC methods to assist analysts and nutritionists in the quantifcation of anthocyanin food components as the nutrient profles of foods are challenged by the variability of the analytical methods. 1. Introduction Nutrition research is a vast and rapidly evolving area. “Food composition is the science behind the differentiation of the complex food matrix” (Probst & Cunningham, 2015) and food composition data allows researchers to study and measure population intakes, their bioavail- ability, and their potential biological effects. Researchers use food composition data to understand how nutrients affect the human body. However, it is also important to understand how analytical methods developed to study the quantitative results of a nutrient/bioactive component can infer a relationship between food intakes and health outcomes. Nutrition analysts must be cautioned to ensure that the values collected for the development of food composition data must be valid and meaningful. In this context, this systematic literature review (SLR) is focused on anthocyanins, a major sub-class of phytochemicals found in foods. Anthocyanins are bioactive plant chemicals present in fruits and vegetables and have been shown to have strong antioxidant effects (Eker et al., 2020). They are highly reactive towards reactive oxygen species and their structural complexity with electron defcient form is thought to be the base for their various health benefts (Martín, Kuskoski, Navas, & Asuero, 2017). Further, this sub-class of more than 600 natural com- pounds (Zhao, Duan, & Wang, 2010), in the plant kingdom consists of sugar molecules generally conjugated to the anthocyanidin structure * Corresponding author at: School of Medicine, Faculty of Science, Medicine and Health, University of Wollongong, NSW 2522 Australia. E-mail addresses: mcs931@uowmail.edu.au (M. Chandra Singh), celine@uow.edu.au (C. Kelso), wprice@uow.edu.au (W.E. Price), yasmine@uow.edu.au (Y. Probst). Contents lists available at ScienceDirect Food Research International journal homepage: www.elsevier.com/locate/foodres https://doi.org/10.1016/j.foodres.2020.109754 Received 15 April 2020; Received in revised form 4 September 2020; Accepted 28 September 2020