title: Strategy for comparative untargeted metabolomics reveals honey markers of different floral and geographic origins using ultrahigh-performance liquid chromatography-hybrid quadrupole-orbitrap mass spectrometry.
abstract: Honey discrimination based on floral and geographic origins is limited by the ability to determine reliable markers because developing hypothetical substances in advance considerably limits the throughput of metabolomics studies. Here, we present a novel approach to screen and elucidate honey markers based on comparative untargeted metabolomics using ultrahigh-performance liquid chromatography-hybrid quadrupole-orbitrap mass spectrometry (UHPLC-Q-Orbitrap). To reduce metabolite information losses during sample preparation, the honey samples were dissolved in water and centrifuged to remove insoluble particles prior to UHPLC-Q-Orbitrap analysis in positive and negative electrospray ionization modes. The data were pretreated using background subtraction, chromatographic peak extraction, normalization, transformation and scaling to remove interferences from unwanted biases and variance in the experimental data. The pretreated data were further processed using principal component analysis (PCA) and a three-stage approach (t-test, volcano plot and variable importance in projection (VIP) plot) to ensure marker authenticity. A correlation between the molecular and fragment ions with a mass accuracy of less than 1.0ppm was used to annotate and elucidate the marker structures, and the marker responses in real samples were used to confirm the effectiveness of the honey discrimination. Moreover, we evaluated the data quality using blank and quality control (QC) samples based on PCA clustering, retention times, normalized levels and peak areas. This strategy will help guide standardized, comparative untargeted metabolomics studies of honey and other agro-products from different floral and geographic origins.
authors.affiliation: Institute of Apicultural Research, Chinese Academy of Agricultural Sciences, Beijing 100093, PR China; Laboratory of Risk Assessment for Quality and Safety of Bee Products, Ministry of Agriculture, Beijing 100093, PR China; Institute of Quality Standard and Testing Technology for Agro-Products, Chinese Academy of Agricultural Sciences, Beijing 100081, PR China.
authors.forename: Yi
authors.initials: Y
authors.lastname: Li
authors.affiliation: Institute of Apicultural Research, Chinese Academy of Agricultural Sciences, Beijing 100093, PR China; Laboratory of Risk Assessment for Quality and Safety of Bee Products, Ministry of Agriculture, Beijing 100093, PR China.
authors.forename: Yue
authors.initials: Y
authors.lastname: Jin
authors.affiliation: Institute of Apicultural Research, Chinese Academy of Agricultural Sciences, Beijing 100093, PR China; Laboratory of Risk Assessment for Quality and Safety of Bee Products, Ministry of Agriculture, Beijing 100093, PR China; Key Laboratory of Bee Products for Quality and Safety Control, Ministry of Agriculture, Beijing 100093, PR China.
authors.forename: Shupeng
authors.initials: S
authors.lastname: Yang
authors.affiliation: Institute of Apicultural Research, Chinese Academy of Agricultural Sciences, Beijing 100093, PR China.
authors.forename: Wenwen
authors.initials: W
authors.lastname: Zhang
authors.affiliation: Institute of Apicultural Research, Chinese Academy of Agricultural Sciences, Beijing 100093, PR China; Laboratory of Risk Assessment for Quality and Safety of Bee Products, Ministry of Agriculture, Beijing 100093, PR China; Bee Product Quality Supervision and Testing Centre, Ministry of Agriculture, Beijing 100093, PR China.
authors.forename: Jinzhen
authors.initials: J
authors.lastname: Zhang
authors.affiliation: Institute of Apicultural Research, Chinese Academy of Agricultural Sciences, Beijing 100093, PR China; Laboratory of Risk Assessment for Quality and Safety of Bee Products, Ministry of Agriculture, Beijing 100093, PR China; Bee Product Quality Supervision and Testing Centre, Ministry of Agriculture, Beijing 100093, PR China.
authors.forename: Wen
authors.initials: W
authors.lastname: Zhao
authors.affiliation: Institute of Apicultural Research, Chinese Academy of Agricultural Sciences, Beijing 100093, PR China; Laboratory of Risk Assessment for Quality and Safety of Bee Products, Ministry of Agriculture, Beijing 100093, PR China.
authors.forename: Lanzhen
authors.initials: L
authors.lastname: Chen
authors.affiliation: Institute of Apicultural Research, Chinese Academy of Agricultural Sciences, Beijing 100093, PR China.
authors.forename: Yaqin
authors.initials: Y
authors.lastname: Wen
authors.affiliation: Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing 100700, PR China.
authors.forename: Yongxin
authors.initials: Y
authors.lastname: Zhang
authors.affiliation: Thermo Fisher Scientific Co., Ltd., Shanghai 201206, PR China.
authors.forename: Kaizhi
authors.initials: K
authors.lastname: Lu
authors.affiliation: Thermo Fisher Scientific Co., Ltd., Shanghai 201206, PR China.
authors.forename: Yaping
authors.initials: Y
authors.lastname: Zhang
authors.affiliation: Institute of Apicultural Research, Chinese Academy of Agricultural Sciences, Beijing 100093, PR China; Laboratory of Risk Assessment for Quality and Safety of Bee Products, Ministry of Agriculture, Beijing 100093, PR China; Key Laboratory of Bee Products for Quality and Safety Control, Ministry of Agriculture, Beijing 100093, PR China; Bee Product Quality Supervision and Testing Centre, Ministry of Agriculture, Beijing 100093, PR China. Electronic address: zhoujinhui@caas.cn.
authors.forename: Jinhui
authors.initials: J
authors.lastname: Zhou
authors.affiliation: Institute of Quality Standard and Testing Technology for Agro-Products, Chinese Academy of Agricultural Sciences, Beijing 100081, PR China. Electronic address: yangshumingcaas@sina.com.
authors.forename: Shuming
authors.initials: S
authors.lastname: Yang
mesh_terms: D015415:Biomarkers / Q000032:analysis; D002851:Chromatography, High Pressure Liquid; D005524:Food Technology / Q000379:methods*; D006722:Honey / Q000032:analysis*; D013058:Mass Spectrometry; D055432:Metabolomics; D025341:Principal Component Analysis
chemical_list: D015415:Biomarkers
keywords: Comparative untargeted metabolomics; Floral origin; Geographic origin; Honey; Markers