Flavonoids are a group of natural phenolic secondary metabolites that include flavones, flavonols, isoflavones and other subclasses [1,2]. Flavonoids are known for their excellent antioxidant capacity [3,4] and also possess a diverse range of potential physiological activities including anti-inflammatory [5], anti-cancer [6] and alleviating effects in chronic diseases such as diabetes mellitus [7], cardiovascular [8] and neurodegenerative diseases [9]. In the breeding industry, flavonoids are also recognized as functional feed additives to improve the production performance of livestock and aquatic animals [10,11]. However, excessive consumption of flavonoid-rich diets can lead to an accumulation of flavonoids in organisms, which can cause some adverse effects such as the inhibition of thyroid function [12]. Against this background, the quantitative analysis of flavonoids is an essential link in the quality assessment and a key step in the formulation of processing standards. The development of analytical methods for flavonoids is therefore of great importance and continuing interest.

To date, many analytical methods have been reported for flavonoids, of which chromatography is the most widely used one [13]. Due to their poor volatility, flavonoids are not suitable for direct analysis by gas chromatography (GC) and often require derivatization such as silylation or methylation to increase their volatility and thermal stability [14]. However, derivatization is often time-consuming and laborious, and may produce multiple derivatives for the polyhydroxyl groups of flavonoids, making subsequent analysis extremely difficult [15,16]. When analyzed by liquid chromatography (LC), flavonoids are well suited to conventional ultraviolet (UV) or diode array detection (DAD) as they all contain aromatic rings, but the sensitivity is often unsatisfactory [17,18]. Fluorescence detection can improve the sensitivity by 2–3 orders of magnitude compared to UV detection, while the number of flavonoids that exhibit intrinsic fluorescence is limited, and tedious derivatization is thus required [19,20]. Because of the polyphenolic hydroxyl groups in flavonoids, electrochemical detection is also used with high sensitivity, the detection electrodes are however susceptible to contamination and their surface is easily saturated, both of which are detrimental to practical analysis [21,22]. Mass spectrometry (MS), especially tandem mass spectrometry (MS/MS) as the LC detector, has dominated the analysis of flavonoids due to its high sensitivity and powerful qualitative capability, but the high analytical cost and strong reliance on well-experienced specialists have hindered its large-scale application [23], [24], [25], [26], [27]. In addition, chemical sensors or biosensors have been reported for the analysis of flavonoids based on colorimetry [28], fluorescence [29] and surface plasmon resonance [30] or enzyme [31]. However, sensing methods are often used to quantify only one flavonoid or the total content of flavonoids, and such sensors are also complex to construct and have short lifetimes.

Capillary electrophoresis (CE) is a micro- and nano-scale liquid-phase separation technique characterized by its greenness, high efficiency, high speed, and multiple modes [32]. However, the UV detection commonly used in CE suffers from poor sensitivity for flavonoid analysis [33], [34], [35]. While MS offers greater sensitivity, the separation ability of CE is often limited by the MS requirements for running buffer [36,37]. Benefiting from monochromaticity, high brightness and directionality, the laser is easy to focus into micro- and nano-channel and achieves very high fluorescence detection sensitivity, making laser-induced fluorescence detection (LIF) particularly suitable for CE analysis [38,39]. In the previous study, we found that tetraborate complexation could significantly sensitize the weak intrinsic fluorescence of salvianolic acids [40]. Since flavonoids are also polyhydroxy substances with weak intrinsic fluorescence, can the sensitizing effect of tetraborate be extended to flavonoids? Therefore, a range of flavonoids including flavones, flavonols and isoflavones were examined and the fluorescence results confirmed this hypothesis. On this basis, an integrated strategy of derivatization and separation was proposed for the universal analysis of flavonoids using the 405 nm CE-LIF system built in our laboratory. Combined with solid phase extraction (SPE) for sample clean-up, the developed CE-LIF method was applied to quantify some flavonoids in Medicago sativa (alfalfa) plants and granulated alfalfa. Also, this method was used to analyze the soaking fluid of single seed for alfalfa and Melilotus officinalis (sweet clover), two forage grass seeds with very similar apparent morphology. The resulting electropherograms of the seeds could be used for the non-destructive distinction of two types of seeds by the principal component analysis (PCA). Finally, this method was utilized to continuously monitor some metabolites in the soaking fluid at the level of single seed.