Identification of Anthocyanin Compounds in Butterfly Pea Flowers (Clitoria ternatea L.) by Ultra Performance Liquid Chromatography/Ultraviolet Coupled to Mass Spectrometry

Butterfly pea flower have great sensory attraction, but they have not yet been used widely in Vietnam. Extracts of butterfly pea flowers can be used conveniently as a natural blue colorant for food products. In this study, the identification of anthocyanin compounds in butterfly pea flowers was performed by UPLC coupled with a UV and Mass spectrometer instrument. Positive and negative ion electrospray MS/MS chromatograms and spectra of the anthocyanin compounds were determined. By analyzing the chromatograms and spectra for each ion, five anthocyanins were identified in the butterfly pea flower extract; these were delphinidin-3-(6″-p-coumaroyl)-rutinoside, cyanidin 3-(6″-p-coumaroyl)-rutinoside, delphinidin-3-(p-coumaroyl) glucose in both cis- and trans- isomers, cyanidin-3-(p-coumaroyl-glucoside) and delphinidin-3-pyranoside. Additionally, based on their intensity, it was determined that cyanidin-3-(p-coumaroyl-glucoside) was the most abundant anthocyanin, followed by cyanidin 3-(6″-p-coumaroyl)-rutinoside, delphinidin-3-(p-coumaroyl-glucoside), delphinidin-3-(6″-p-coumaroyl)-rutinoside and delphinidin-3-pyranoside. In this study, cyanidin derivatives were discovered in butterfly pea flower extract, where these compounds had not been detected in previous studies.


Introduction
Butterfly pea flowers, known Clitoria ternatea L., area plant species belonging to the Fabaceae family [1]. In Vietnam, butterfly pea flowers are usually used as food drinks and as a colorant. Delphinidin is the main anthocyanin responsible for the deep blue to purple color in this flower [2]. The different flower colors are mainly due to the chemical structure of the different anthocyanins or anthocyanidins synthesized in the flower [3]. In the discussion of copigmentation reactions and color stability of berry anthocyanins, it was observed that the color of anthocyanins changes from pink to blue as the number of hydroxyls increases and methoxyl groups replacing the hydroxyls reverse the trend [4]. There is increasing interest in the search and use of natural colorants, of which blue is rare and tends to be sensitive to processing and storage conditions [5]. Butterfly pea flower extract can be used as a natural blue colorant, is convenient to use, and has a longer shelflife than equivalent plant-based colorants [6]. Therefore, it is very important to identify the main coloring compounds in butterfly pea flowers. 2 of 13 Anthocyanins present in edible fruits, vegetables and flowers have protective effects against diseases, especially cardiovascular disease, certain types of cancer [7], and against several chronic diseases, such as hyperglycemia [8]. Anthocyanins also improve vision [9]. Because of their benefits, anthocyanins are becoming increasingly commercializedand used in foods [10]. Anthocyanins are an important class of water-soluble pigments belonging to the flavonoid family [11]. To date, more than 600 different anthocyanins have been characterized [12]. Structurally, anthocyanins are glycosylated polyhydroxy and polyethoxy derivatives of flavylium salts; they also possess an aglycone form called anthocyanidins. Cyanidin, delphinidin, pelargonidin, peonidin, malvidin, and petunidin are the most common anthocyanidins distributed in the plants [13]. Their different structures depend on the number and position of the hydroxyl and methoxyl groups on the flavilium ring. Normally, anthocyanidins are bound to sugars, giving stability and water solubility to the molecule [14]. Among the sugars, the most common are glucose, galactose, arabinose, rhamnose and xylose, which are usually present as 3-monoglycosides and 3,5-diglycosides. Other sugars such as rutinoside (6-O-α-Lramnosyl-D-glucoside), sophorosides (2-OD-glucosyl-D-glucosides) and sambubiosides (2-O-β-D-xylosyl-D-glucosides) [15,16] are also present.
Despite many studies regarding anthocyanins in berries [23,24], studies on anthocyanins in butterfly pea flowers are still limited. In this study, anthocyanin composition in butterfly pea flowers (grown in Can Tho, Vietnam) was determined by UPLC/UV/MS for the first time.
More recently, faster methods have been proposed for the quantification of polyphenols using more powerful chromatographic systems such as UHPLC in combination with a UV detector [25,26] or mass analyzer [27]. The aim of this study was to develop a UPLC method using a UV detector and an ESI-triple quadrupole (QQQ) mass analyzer for the simultaneous determination of the major anthocyanin compounds of butterfly pea flowers. This study will provide insight into the composition of anthocyanin in butterfly pea flowers so that the extract can be applied more effectively.

Results and Discussion
The identification and assignment of anthocyanin peaks was mainly performed based on the comparison between their retention times (RT) and mass spectrometry data with standards, references [28] as shown in Table 1. The chemical structures and molecular weights of six common anthocyanidins and the most common sugars and acylation groups are shown on the basis of preferences. The study of anthocyanin composition in butterfly pea flowers was started by using the basic anthocyanin structure as a reference, taking into account previous literature data on anthocyanin structure [29][30][31][32][33]. More recently, faster methods have been proposed for the quantification of polyphenols using more powerful chromatographic systems such as UHPLC in combination with a UV detector [25,26] or mass analyzer [27]. The aim of this study was to develop a UPLC method using a UV detector and an ESI-triple quadrupole (QQQ) mass analyzer for the simultaneous determination of the major anthocyanin compounds of butterfly pea flowers. This study will provide insight into the composition of anthocyanin in butterfly pea flowers so that the extract can be applied more effectively.

Results and Discussion
The identification and assignment of anthocyanin peaks was mainly performed based on the comparison between their retention times (RT) and mass spectrometry data with standards, references [28] as shown in Table 1. The chemical structures and molecular weights of six common anthocyanidins and the most common sugars and acylation groups are shown on the basis of preferences. The study of anthocyanin composition in butterfly pea flowers was started by using the basic anthocyanin structure as a reference, taking into account previous literature data on anthocyanin structure [29][30][31][32][33]. UPLC chromatograms ( Figure 1) for total ions in butterfly pea flowers were collected. It was observed that the acquisition times of anthocyanin peaks ranged from 5.5 to 7.0 min, approximately, in mass chromatogram straight after UV absorption. Unlike other groups of flavonoids or polyphenols, anthocyanins exist in the cationic form, so a positive ion mode has often been used to analyze them. Due to the positive charge and phenolic groups of anthocyanins, these compounds can readily donate protons to free radicals. However, it is difficult to distinguish anthocyanins from flavonol glycosides in active ionization mode using MS, for example between delphinidin glycoside and quercetin glycoside, or between cyanidin glycoside and kaempferol glycoside. These flavonoids may have the same molecular ions and mass fragmentation patterns as the corresponding anthocyanins. UPLC chromatograms ( Figure 1) for total ions in butterfly pea flowers were collected. It was observed that the acquisition times of anthocyanin peaks ranged from 5.5 to 7.0 min, approximately, in mass chromatogram straight after UV absorption. Unlike other groups of flavonoids or polyphenols, anthocyanins exist in the cationic form, so a positive ion mode has often been used to analyze them. Due to the positive charge and phenolic groups of anthocyanins, these compounds can readily donate protons to free radicals. However, it is difficult to distinguish anthocyanins from flavonol glycosides in active ionization mode using MS, for example between delphinidin glycoside and quercetin glycoside, or between cyanidin glycoside and kaempferol glycoside. These flavonoids may have the same molecular ions and mass fragmentation patterns as the corresponding anthocyanins.
Meanwhile, unlike the MS data collected in the positive ionization mode, the mass spectrometry data obtained with negative ionization provide a wide range of ions that are specific for anthocyanins [34,35]. Therefore, in this work, mass chromatograms of anthocyanins in both positive ion mode and negative ion mode were investigated.
The results in Figure 1a,b also show that in the negative ion mode, the total ion chromatogram for anthocyanins is simpler than in the positive mode. Additionally, the results also show that all ions exhibited the maximum UV absorption at 535 nm, which is consistent with the previous study by Wang et al. [36] on anthocyanins in the black tomato variety Indigo Rose. Therefore, the determination wavelength was 535 nm for all anthocyanins and the UV chromatogram for butterfly pea flowers extract was at 535 nm ( Figure 1c).
To ensure efficiency and accuracy, a combination of spectrophotometric methods, ultraviolet/visible (UV/Vis) and mass spectrometry (MS) methods were performed for anthocyanin identification. Therefore, after UV chromatographic analysis, mass spectrometry data for total ions in both positive and negative modes were also acquired ( Figure 2). To obtain the detailed structures of the detected anthocyanins, the MS/MS fragments of anthocyanins were further analyzed. The literature was also used concurrently to focus on and narrow down specific compounds. Meanwhile, unlike the MS data collected in the positive ionization mode, the mass spectrometry data obtained with negative ionization provide a wide range of ions that are specific for anthocyanins [34,35]. Therefore, in this work, mass chromatograms of anthocyanins in both positive ion mode and negative ion mode were investigated.
The results in Figure 1a,b also show that in the negative ion mode, the total ion chromatogram for anthocyanins is simpler than in the positive mode. Additionally, the results also show that all ions exhibited the maximum UV absorption at 535 nm, which is consistent with the previous study by Wang et al. [36] on anthocyanins in the black tomato variety Indigo Rose. Therefore, the determination wavelength was 535 nm for all anthocyanins and the UV chromatogram for butterfly pea flowers extract was at 535 nm ( Figure  1c).
To ensure efficiency and accuracy, a combination of spectrophotometric methods, ultraviolet/visible (UV/Vis) and mass spectrometry (MS) methods were performed for anthocyanin identification. Therefore, after UV chromatographic analysis, mass spectrometry data for total ions in both positive and negative modes were also acquired ( Figure 2). To obtain the detailed structures of the detected anthocyanins, the MS/MS fragments of anthocyanins were further analyzed. The literature was also used concurrently to focus on and narrow down specific compounds.   Table 2. Five anthocyanins were identified, including three delphinidin derivatives and two cyanidin derivatives, responsible for the blue color of butterfly pea flowers. The ion chromatograms of the MS/MS product in positive and negative mode and the spectrum of the first anthocyanin eluent component shown in Figure 3 are similar.   Showing a similar signal to the positive ion analysis, the negative ion electrospray tandem mass chromatogram and the spectrum of the second anthocyanin are shown in  Figure 3b.
Showing a similar signal to the positive ion analysis, the negative ion electrospray tandem mass chromatogram and the spectrum of the second anthocyanin are shown in Figure 4. Compound 2 exhibited a molecular ion [M + H] + with m/z 741.1 and produced a major fragment with m/z 286.9 that may correspond to cyanidin (m/z 287). In addition, the m/z transition from 594.9 to 286.9 implied the loss of rutinose (m/z 308). Thus, with a p-coumaroyl fragment (m/z 147), this compound could be tentatively identified as cyanidin 3-(6 -p-coumaroyl)-rutinoside [37].  Three peaks can be observed in the chromatogram in the positive ion mode, but only two in the negative ion mode ( Figure 5). That means that the third anthocyanin has two isomers. Peaks 1 and 2 have the same daughter fragment of delphinidin aglycone (m/z 302.9 and 302.8). In peak 1, the m/z transition from 464.4 to 302.9 indicates a loss of glucose. Meanwhile, peak 2 produces the main fragment, m/z 146.7, corresponding to p-coumaroyl. In addition, because the cis-p-coumaroyl derivative has a higher polarity, it elutes earlier  than its trans configuration [38]. Therefore, peak 1 was tentatively identifiedas delphinidin-3-(cis-p-coumaroyl-glucoside) and peak 2 was provisionally identified as delphinidin-3-(trans-p-coumaroyl-glucoside) [39]. The chromatograms and MS/MS negative and positive ion electrospray of the final anthocyanin are shown in Figure 6. Two peaks appeared in the mass chromatogram in positive ion mode, but only one peak appeared in negative ion mode. Compound 4 showed molecular ion [M + H] + with m/z 595.2 and produced two major fragments with m/z 287.0 (cyanidin) and m/z 146.9 (p-coumaroyl). Together with the m/z transition from 448.9 to 287.0, indicating glucose loss (m/z 162), peak 4 was identified as cyanidin-3-(pcoumaroyl)-glucose [39]. Three peaks can be observed in the chromatogram in the positive ion mode, but only two in the negative ion mode ( Figure 5). That means that the third anthocyanin has two isomers. Peaks 1 and 2 have the same daughter fragment of delphinidin aglycone (m/z 302.9 and 302.8). In peak 1, the m/z transition from 464.4 to 302.9 indicates a loss of glucose. Meanwhile, peak 2 produces the main fragment, m/z 146.7, corresponding to p-coumaroyl. In addition, because the cis-p-coumaroyl derivative has a higher polarity, it elutes earlier than its trans configuration [38]. Therefore, peak 1 was tentatively identifiedas delphinidin-3-(cis-p-coumaroyl-glucoside) and peak 2 was provisionally identified as delphinidin-3-(trans-p-coumaroyl-glucoside) [39].

Chemicals and Materials
HPLC methanol, acetonitrile, formic acid, and acetic acid were purchased from Merck KGaA, Darmstadt, Germany. All chemicals were analytical grade. Milli-Q water (Milli-Q IQ 7003/7005, Merck, NJ, USA) was used.
Butterfly pea flowers were grown at the College of Agriculture, Can Tho University, Vietnam. Butterfly pea flowers were freeze-dried in an Alpha 2-4 DL freeze dryer (Martin (1) Intensity Counts vs. Acquisition Time (min)

Chemicals and Materials
HPLC methanol, acetonitrile, formic acid, and acetic acid were purchased from Merck KGaA, Darmstadt, Germany. All chemicals were analytical grade. Milli-Q water (Milli-Q IQ 7003/7005, Merck, NJ, USA) was used.
Butterfly pea flowers were grown at the College of Agriculture, Can Tho University, Vietnam. Butterfly pea flowers were freeze-dried in an Alpha 2-4 DL freeze dryer (Martin Christ, Germany) at −80 • C and 0.001 mbar to 3−5% moisture content and finely ground before extraction.

Purification Procedure
The extraction solution (100 µL) prepared above was put into a centrifuge tube and 700 µL of ice ethanol was added. The tubes were vortexed for 15 s and kept at −80 • C for 60 min. The tubes were then centrifuged at 21,000× g for 30 min. The supernatant was filtered through a 17 mm (0.2 mm) PVDF syringe filter (VWR Scientific, Seattle, WA, USA) and dried at 40 • C under vacuum.
The residue was activated with 6 mL of methanol 100%. This mixture was passed through 200 mg C18 solid-phase extraction cartridge (Water, MA, USA) and washed with water (6 mL), then eluted with methanol (6 mL). The methanol fraction containing the parent anthocyanins was concentrated to dryness, and dissolved before HPLC analysis.

Conclusions
The mass spectrometry behaviors of anthocyanins in positive and negative ionic modes were studied and were demonstrated to be a valuable tool for identifying anthocyanins from butterfly pea flowers. A new strategy was developed based on observation to distinguish anthocyanin compounds in this flower. Data were generated from UPLC/MS using a developed method that is able to provide rapid and reliable anthocyanin determination with the use of a UV detector. The obtained results show the potential of butterfly pea flowers in anthocyanin extraction and further use as safe colorant in food processing.