**1. Introduction**

Honey is a natural sweet substance formed by mixing nectar or honeydew collected by bees and their own secretions [1]. It is a mixture of water and sugar and is rich in nutrients such as amino acids, minerals and essential trace elements and vitamins [2–4]. In our country, honey is a kind of medicine and food with a long history. "Shennong Materia Medica Classic" records that honey has many effects such as beneficial gas filling, pain relief and detoxification. Modern pharmacological studies have shown that honey has a variety of biological activities, such as anti-inflammatory, antioxidant, immune regulation and wound repair [5]. Due to its nutritional value and pharmacological activity, there is a great market demand. According to the data of the Food and Agriculture Organization of the United Nations, China is the world's largest producer of bee culture and bee products, ranking first in the world in both the total number of bee colonies and the output of bee products [6]. There are about 300,000 beekeepers, 9.2 million bee colonies and about 500,000 tons of honey on the market [7]. However, some toxic substances are frequently introduced into honey products during the gathering honey process [8]. Therefore, the exact composition and contaminants in any batch of honey depend on the crops around the hives [9]. As early as 2000 years ago, many Greek soldiers accidentally ate the "mad honey", resulting in a poisoning incident [10,11]. In recent years, in Yunnan, Guizhou and other places, incidents of accidentally eating toxic honey have also occurred [12–14]. It has been proven that honey poisoning is mainly caused by nectariferous plants, including

**Citation:** Ma, X.; Zuo, M.-T.; Qi, X.-J.; Wang, Z.-Y.; Liu, Z.-Y. Two-Dimensional Liquid Chromatography Method for the Determination of *Gelsemium* Alkaloids in Honey. *Foods* **2022**, *11*, 2891. https://doi.org/10.3390/ foods11182891

Academic Editor: Isabel Sierra Alonso

Received: 16 August 2022 Accepted: 9 September 2022 Published: 17 September 2022

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**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

*Gelsemium elegans, Common Threewingnut Root, Tripterygium hypoglaucum* and so on [15,16]. Therefore, it is very important to establish a method for the detection of toxic ingredients in honey.

*Gelsemium elegans* is a whole herb of the genus Mackeraceae, and it ranks first among the nine poisons in ancient China. Whole plants are highly toxic to nectariferous plants. According to their different distribution areas, the snout can be roughly divided into three categories. One is the *Gelsemium sempervirens,* mainly distributed in the south of the United States to Central America; another one is the *Asia Gelsemium*. Current research shows that a total of 121 indole alkaloids have been found in *Asia Gelsemium* [17]. According to the skeleton structure of the compounds, they are mainly divided into six categories of alkaloids, including gelsemine-type, koumine-type, gelsedine-type, humantenine-type, yohimbine-type and sarpagine-type alkaloids. According to a report, koumine has the highest content of indole alkaloids, which belongs to the koumine-type; this is followed by gelsemine, which belongs to the gelsemine-type. The most toxic indole alkaloid is humantenmine, which belongs to the most toxic alkaloid in *Gelsemium,* which belongs to the gelsedine-type [18–20]. In recent years, there have been many cases of poisoning caused by ingestion of *Gelsemium* honey. On the one hand, *Gelsemium* honey poisoning can lead to strong and rapid poisoning reactions, such as dyspnea and convulsions, and even death. On the other hand, there is no specific detoxification drug. Therefore, it is necessary to establish a rapid, sensitive method for the detection of the phytotoxin *Gelsemium* alkaloid in honey, Which it is very important for the prevention and diagnosis of *Gelsemium* poisoning.

At present, there are only methodological studies of gelsemine, koumine and humantenmine in biological and plant samples [21–23]. Detection methods for *Gelsemium* poisoning are also limited to biological samples, such as blood and urine [24,25]. However, there are few reports on alkaloid detection in honey. Recently, the solid-phase extraction method (SPE), QuEChERs method (Quick, Easy, Cheap, Effective, Rugged, Safe) and the liquid–liquid extraction (LLE) method have been used for the determination of toxic substance residues in honey. Honey is an extraordinarily complex matrix containing more than three hundred chemicals, and it is rich in proteins and lipids [26]. Each sample of honey has a specific set of twenty-five ingredients because it comes from a different plant [27]. Thus, honey substrates require complex and extensive pre-treatment to eliminate or reduce the matrix effects. The traditional LLE method has complicated steps and requires many expensive organic solvents. It takes 150–180 min to process a sample, and the extraction recovery rate of polar substances in honey is low [28]. Pau Calatayud et al. compared the effects of SPE, QuEChERs and LLE on the extraction recovery of 52 pesticides in honey, and the results showed that SPE was superior to QuEChERs in terms of precision and accuracy. In addition, the plasma effect of SPE was lower than that of QuEChERs [29]. Therefore, the SPE method has more advantages for the pretreatment of the honey matrix than other methods.

It has been reported that liquid chromatography–tandem mass spectrometry (LC–MS) was used to detect the alkaloids in honey, including gelsemine and koumine [30]. However, due to the cost of mass spectrometry and the high requirement for the laboratory, the general laboratory cannot meet the requirements. Recently, our team established a two-dimensional liquid chromatography (2D-LC) method for *Gelsemium* in biological samples [31]. On this basis, this study aims to establish a 2D-LC method for the simultaneous determination of three standards of *Gelsemium* alkaloids (gelsemine, koumine and humantenmine) in honey samples. This method provides a technical means for the detection of *Gelsemium* alkaloids in food species of animal origin and can be used for the rapid detection of nectariferous plant poisoning events and the traceability of alkaloids.
