**1. Introduction**

The evergreen woody shrub genus *Rhododendron* is one of the largest genera in the family Ericaceae, and more than 1000 species are currently recognized; of these, 567 species representing 6 subgenera are known from China [1,2]. *Rhododendrons* are not only of high ornamental value but also good medicinal plants. Flowers of *Rhododendron* provide a large number of bioactive natural chemical products, including diterpenoids [3], flavonoids [4], and phenols [5], which are known to be effective for the treatment of rheumatism [6] and to have anti-inflammatory [7], anti-cancer [8], and antioxidant [9] properties. Volatile compounds from flowers also provide some ecological functions [10], including in the role of pollinators [11] and as defenders against nectar-thieving ants [12]. Aside from their ecological functions, flower volatiles have some aesthetic and emotional benefits for humans [13]. On the other hand, different volatile compounds may influence the odor, both in an individual and in a synergistic or antagonistic way, which in turn could be related to one or more chemical compounds or compound classes [14]. In order to investigate the aroma characteristics of *Rhododendron* flowers, it is important to research specific volatile constituents as thoroughly as possible. *Rhododendron irroratum*, *R. delavayi*, *R. annae*, and *R. agastum* are ecologically and horticulturally important alpine flowers and are also the pioneer and constructive species in Baili *Rhododendron* National Forest Reserve in the Guizhou province of China [15]. *R. delavayi* belongs to the subsection Arborea, while *R. irroratum*,

*R. annae*, and *R. agastum* belong to the subsection Irrorata. *R. irroratum* is one of the large-flowered *Rhododendron* species [16]. These species were chosen for the investigation of volatile odor constituents in different *Rhododendron* flowers.

Gas chromatography–mass spectrometry (GC-MS) has long been the primary technique used to detect the aroma components of various plants [17,18]. However, GC-MS can only identify a limited number of separable compounds due to its insufficient peak capacity, limited resolved power, and low sensitivity [19]. The combination of gas chromatography with high-resolution quadrupole time-of-flight mass spectrometry (QTOFMS) has been demonstrated for analysis in different fields, including flavor research [20] and volatile profiling [21], and has proved to be a powerful analytical tool. However, the limited chromatographic separation power inevitably causes co-elution problems for complex samples. Compared with traditional one-dimensional gas chromatography (1DGC), comprehensive two-dimensional gas chromatography (GC×GC), which has appeared as a new analytical technique based on the application of two GC columns with different stationary phases, provides substantially enhanced resolving power and peak capacity. GC×GC leads to linear distributions of homologous series in 2D chromatograms, thus greatly reducing the coelution problem [22]. GC×GC can thus be a more suitable tool for analysis of the complex chemical systems of plant aroma, where the number of volatile aroma compounds is large and some of them are present at trace levels [23]. Recently, GC×GC technology has been successfully applied for the assessment of various plants such as teas [24], berries [25], and tobacco [26]. To date, few reports have studied the volatile chemical components in *Rhododendron* flowers by 1DGC. With the 1DGC technique, 9,12,15-octadecatrienoic acid,[Z,Z,Z]-, phytol, and *n*-hexadecanoic acid were found to be the major compounds in flowers of *R. mucronatum* and *R. simii* [27]; while *R. ponticum* comprises mostly α-pinene, β-pinene, and linalool [28], in flowers of *R. schlippenbachii*, only 39 hydrophilic compounds could be detected by 1DGC [29]. A previous study reported the volatile compounds in the leaves, stems, and roots of six *Rhododendron* species [15]. However, the volatile components in flowers of the four *Rhododendron* species in the present study have never been investigated by the GC×GC approach before. Therefore, it is necessary to study the flowers' volatile compounds in order to explore odor characterizations.

In this study, GC×GC-QTOFMS was used in combination with headspace solid-phase microextraction (HS-SPME) to conduct in-depth analysis of the volatile aroma constituents in different *Rhododendron* flowers. The advantages of GC×GC–QTOFMS were exploited for high-throughput, untargeted chromatographic profiling of complex samples. The volatile compounds and their corresponding contents in various representative *Rhododendron* samples were examined. The obtained results provide useful information for establishing a volatile aroma chemical database from *Rhododendron* flowers.

#### **2. Results and Discussion**
