Ion mobility mass spectrometry (IMMS) is a sensitive and fast analytical technique that can provide accurate quality information [1,2]. Compared to traditional mass spectrometry, IMMS offers one more dimension for identification and can be combined with one- or two-dimensional liquid phases to enhance analytical performance. In addition, the remarkable benefit of IMMS is that it can also acquire collision cross-section (CCS) values to boost the effectiveness of ion structure analysis [3,4]. Applications of IMMS are varied in the food, environmental and clinical medicine fields. Additionally, IMMS is also used for the analysis of natural ingredients because of its ability to precisely identify complex compounds and isomers in natural products. For example, Matz et al. employed IMMS to complete the separation and analysis of 7 kinds of opiates within 70 s, shortening the sample analysis time and significantly increasing sample throughput [5]. Furthermore, IMMS significantly improves peak capacity and signal-noise ratio, which Paglia et al. used for more accurate qualitative and quantitative analysis of lipids [6]. It is observed that IMMS is promising in the analysis of compounds and species identification, which is worth exploring in natural products.

Two-dimensional liquid chromatography (LC×LC) is based on two columns with independent separation mechanisms. Compared to one-dimensional chromatography, LC×LC has proven to be better with respect to separation efficiency, resolution, sensitivity and peak capacity [7]. Currently, LC×LC has been used widely in the analysis of environmental samples, biological substrates, food samples, and some other complex natural substances encountered in the life sciences. Due to the diversity and complexity of ingredients in herbs, LC×LC is quite prevalent in traditional medicine. For example, Yao et al. effectively accomplished baseline separation and accurate quantification of eight ginsenosides in proprietary Chinese medicines using multiple center-cut two-dimensional chromatography [8]. Moreover, 120 metabolites were found in total when Wong et al. used LC×LC to study the extract of Glycyrrhiza glabra L., which is a significant increase in the amount of metabolites found using one-dimensional chromatography [9]. Although these tested methods can effectively detect some substances, analytical techniques for natural medicines need further development. Therefore, the combination of LC×LC and IMMS with chemometrics was developed for the analysis of complex multicomponents, especially for the identification of species and quality control of natural products.

Honeysuckle is the dried flower bud or primrose flower of Lonicera japonica, a genus of the family Lonicerae, and is widely used in plant tea. There are numerous varieties of honeysuckle, which are known in China as Jinyinhua, Rendongteng and Shanyinhua [10]. Honeysuckle plays an important role in the treatment of various diseases, such as fever, sore throat, neurodegenerative diseases and diabetes [11], [12], [13]. The active compounds and their pharmacological efficacy are consistent. There are approximately 200 active ingredients, including organic acids, flavonoids, iridoids, saponins [14,15] and other substances. These compounds have antioxidant, antibacterial, anti-inflammatory and antiviral effects [16] and can all be found in honeysuckle. In the available literature, common detection techniques such as gas chromatography (GC), high-performance liquid chromatography (HPLC) [17], mass spectrometry (MS) [18], capillary electrophoresis (CE), HPLC‒MS, and GC‒MS [19] are widely used to analyze the active components in honeysuckle. In a previous study, using TOF-MS, Gao Wen [20,21] quickly analyzed the metabolite data for seven different honeysuckle species and successfully identified 51 batches of honeysuckle using chlorogenic acid as a control standard. Zhao et al. [17] used HPLC and inductively coupled plasma‒mass spectrometry (ICP‒MS) to thoroughly examine 13 substances of 40 batches of honeysuckle, which was first established to distinguish Lonicera japonica flos from four species of Lonicera flos. However, the analytical methods currently used focus on the analysis of one or two types and lack effective classification studies for numerous species. Because all of the abovementioned instruments have limited peak capacity and insufficient accuracy, technologies are required for more thorough detection. Therefore, it is meaningful to use LC×LC and IMMS for species identification of honeysuckle.

In this study, an RP-LC×RP-LC application was established, and after optimization of key parameters such as stationary phase and flow rate, the identification of the honeysuckle components was acquired by means of both LC×LC (two-dimensional liquid chromatography) and IMMS (ion mobility mass spectrometry) data. With the ability to provide CCS values, IMMS has proven to be a powerful tool to improve detection accuracy. Subsequently, in combination with statistical methods, the active ingredients could be accurately identified, and the species could also be analyzed categorically. Furthermore, a variety of honeysuckle flowers were used to evaluate the inhibitory activity of honeysuckle extracts against α-glucosidase.