Comprehensive Profiling of Phytohormones in Honey by Sequential Liquid–Liquid Extraction Coupled with Liquid Chromatography–Mass Spectrometry

Honey exhibits various nutritional and medicinal functions, which are highly related to the active components; thus, the exploration of new compounds in honey is of great importance. Because honey is a byproduct of flower nectar, which is rich in phytohormones, the existence of phytohormones in honey is anticipated. In this research, a method for comprehensive profiling of 49 phytohormones in honey was developed by sequential liquid-liquid extraction (LLE) coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS). Good linearities for 49 phytohormones were obtained with correlation coefficients (R) larger than 0.9913. The limits of detection (LODs) were in the range of 0.2-628.2 pg/mL. Satisfied reproducibility and reliability were achieved by evaluation of the intra- and interday precisions with relative standard deviations (RSDs) less than 15.8% and relative recoveries ranging from 80.4 to 123.7%. The method was further applied to analyze the phytohormones in 14 monofloral raw honey samples and 3 commercial honey samples. The existence of 34 phytohormones was confirmed, including 14 cytokinins (CKs), 8 gibberellins (GAs), 5 brassinosteroids (BRs), indole-3-acetic acid (IAA), indole-3-butyric acid (IBA), abscisic acid (ABA), salicylic acid (SA), jasmonic acid (JA), jasmonoyl-leucine (JA-Leu), and jasmonoyl-phenylalanine (JA-Phe). In addition, the content and species of phytohormones varies in different kinds of honey. The study is beneficial to fully illustrate the phytohormone profile of honey and contributive to elucidate the mechanism of its nutritional and medicinal functions.