Simultaneous determination of various platycosides in Four Platycodon grandiflorum cultivars by UPLC-QTOF/MS

Abstract

In Platycodi Radix (root of Platycodon grandiflorum), there are a number of platycosides that consist of a pentacyclic triterpenoid aglycone and two sugar moieties. Due to the pharmacological activities of platycosides, it is critical to assess their contents in PR, and develop an effective method to profile various platycosides is required. In this study, an analytical method based on ultra performance liquid chromatography coupled with quadrupole time-of-flight/mass spectrometry (UPLC-QTOF/MS) with an in-house library was developed and applied to profile various platycosides from four different Platycodi Radix cultivars. As a result, platycosides, including six isomeric pairs, were successfully analyzed in the PRs. In the principal component analysis, several platycosides were represented as main variables to differentiate the four Platycodi Radix cultivars. Their different levels of platycosides were also represented by relative quantification. Finally, this study indicated the proposed method based on the UPLC-QTOF/MS can be an effective tool for identifying the detail characterization of various platycosides in the Platycodi Radix.

Introduction

Platycodi Radix, the root of Platycodon grandiflorum A. De Candolle (Campanulaceae), has been widely used for both cuisine and traditional herbal medicine. The benefits of Platycodi Radix for health and biological activities have been reviewed previously [1]. Platycodi Radix saponins, called platycosides, are the primary constituents of Platycodi Radix, having pharmacological activities such as anti-oxidant, anti-obesity, anti-inflammatory, and anti-cancer effects [2,3,4,5,6,7,8,9]. Thus, it is critical to determine platycosides for the quality control and clinical use of Platycodi Radix. Furthermore, well-constructed analytical platforms are necessary for the effective analysis of platycosides.

Platycosides consist of a pentacyclic triterpenoid aglycone and two sugar moieties; one is a glucose unit attached at C-3 of a triterpene and the other is a series of three sugars (arabinose, rhamnose, and xylose in sequence) attached at C-28 through an ester linkage with the arabinose. In order to profile platycosides, analytical methods have been previously established by high-performance liquid chromatography (HPLC) coupled to a UV detector [10, 11] or by an evaporative light scattering detector (ELSD) [12, 13]. However, HPLC–UV, or ELSD, shows its limitations in the low sensitivity and the insufficient separation of compounds. Furthermore, these traditional methods need all reference standards for the identification and quantification of each analyte. Thus, LC coupled to the electrospray ionization (ESI)/mass spectrometry (MS) has been used as an alternative tool to analyze the platycosides in Platycodi Radix [14,15,16,17]. The mass measurement using MS has provided the selective and sensitive detection of platycosides from the crude extracts of Platycodon grandiflorum. Moreover, the tandem MS (MS/MS) has been applied for the structural analysis of various platycosides [18,19,20].

Platycosides have various structural isomers, according to types of C-3 and C-28 glycosyl groups, attached to triterpenoid aglycone [21]. Although MS/MS has been used to identify the linkage positions and compositions of oligosaccharides, the fragment ions of reducing sugar ring-cleavage have limitations in determining the isomers [18, 20]. Jeong et al. has recently applied the enzymatic hydrolysis and MSⁿ analysis to elucidate the structure of isomers, and nineteen platycosides were successfully identified [22]. However, the need to develop an effective method to comprehensively profile various platycosides still remains.

In this study, we used the ultra-performance LC (UPLC) coupled with the quadrupole time-of-flight (QTOF)/MS to analyze various platycosides. The UPLC system is useful to conduct a quick and effective separation of compounds in a complex mixture. Furthermore, the QTOF/MS is a sensitive and high-resolution detector that effectively provides the exact mass measurement of compounds [23, 24]. In our previous study, an optimal UPLC-QTOF/MS has been applied to profile various metabolites including platycosides [25]. The UPLC system, with its small particle size column, enables a fast and effective separation of various compounds. Furthermore, the QTOF/MS provides a sensitive and highly accurate mass measurement. Finally, we applied the platform based on UPLC-QTOF/MS and an in-house library to perform the comprehensive profiling of high- and low-abundance platycosides, including several isomers.

Materials and methods

Reagents and standard compounds

HPLC grade water (H₂O), acetonitrile (MeCN), and methanol (MeOH) were purchased from Merck (Darmstadt, Germany). Formic acid was products of Sigma-Aldrich (St. Louis, MO, United States) and reference standards of seven compounds were isolated from P. grandiflorum roots by a series of column chromatography and medium pressure liquid chromatography (MPLC) in our laboratory. Structure of compounds were elucidated based on the mass spectroscopic (MS) and nuclear magnetic resonance (NMR) data with the literature database: platycodin D3 [27], platycoside E [26], platycodin D [28], polygalacin D [27], platyconic acid A [29], and platycodin D2 [27]. The quality of the compounds was > 98.0% (determined by normalization of the peak areas detected by HPLC with DAD).

Platycodon grandiflorum samples

Platycodon grandiflorum roots (cultivars: Fuji Blue, Astra Pink, Astra Blue, Jangbaek) were cultivated and preserved for 2 years in the experimental field of the National Institute of Horticultural and Herbal Science (NIHHS-150128), Rural Development Administration (RDA), Chungbuk, Province, Korea, in 2014 (Fig. 1). Voucher specimen (NIHHS1601) was deposited at the herbarium of the Department of Herbal Crop Research, NIHHS, RDA, Chungbuk, Province, Korea. Three P. grandiflorum cultivars, including Fuji Blue, Astra Pink, Astra Blue, which were obtained from Swallowtail Garden Seeds (Santa Rosa, CA, USA), and Jangbaek were collected plants grown in a field in NIHHS, Korea.

Fig. 1

Morphological characteristics of floral leaf and root in Platycodon grandiflorum cultivars (a Fuji blue, b Astra pink, c Astra blue, d Jangbaek)

Sample preparation

Platycodon grandiflorum roots were dried at 35–40 °C in a forced-air, convection-drying oven for 2 days after they were washed and weighed. The main roots were used in extraction after removing the fine roots and rhizome. The main roots were pulverized using a cutting mixer (Hanil, Seoul, Korea) and were thoroughly blended, after which the samples were further homogenized using a ball mill (Retsch MM400, Haan, Germany) for the UPLC analyses. An accurately weighed portion (50 mg) of each fine powder was suspended in 40 mL of 70% (v/v) ethanol (EtOH), and ultrasonically extracted for 60 min at 50 °C. The extract was filtered and evaporated in a vacuum, and the residue was dissolved in 70% MeOH. The methanol solution was filtered through a 0.22 µm membrane, then analyzed directly by UPLC system.

UPLC conditions

UPLC was performed with a Waters UPLC system (ACQUITY H-Class, Waters Corp., Milford, MA, USA). Separation was achieved on an ACQUITY BEH C₁₈ column (130 Å, 2.1 mm × 100 mm, 1.7 µm). The column temperature was maintained at 40 °C and the mobile phase was 5% MeCN containing 0.1% aqueous formic acid (solvent B) and 95% MeCN containing 0.1% aqueous formic acid (solvent B). The gradient elution was as follows: 0–3 min, B 10–20%; 3–11 min, B 20–23%; 11–20 min, B 23–95%; 20–21 min, B 95–100%; 21–25 min, B 100%. The flow rate was 0.45 mL/min and the injection volume was 2 μL for each run.

QTOF/MS conditions

MS was performed by using a QTOF/MS (Xevo G2-S, Waters Corp., Milford, MA, USA) that operated in the negative ion mode. The mass detection conditions were optimized in our previous work [25]. The accurate mass measurement and MS/MS analysis were performed at different collision energies ranging from 4 to 45 eV with MS^E acquisition mode. The operating parameters were set as follows: capillary, 3.0 kV; cone voltage, 40 V; source temperature, 120 °C; cone gas flow, 30 L/h; desolvation temperature, 300 °C; and desolvation gas flow, 600 L/h. The Q-TOF results were collected between m/z 100–2000 with contains the internal reference. All analytes were acquired using an independent reference mass using the LockSpray interference to ensure accuracy and reproducibility; Leucine-enkephalin was used as the lock mass 554.262 m/z (ESI−). The accurate mass was calculated using software UNIFI (Ver. 1.8, Waters Corp., Milford, USA) incorporated in the instrument.

Data processing and multivariate analysis

After metabolite profiling of P. grandiflorum roots by UPLC-QTOF/MS, each set of MS^E data was processed using the UNIFI software. Data within UNIFI were passed through the apex peak detection and alignment processing algorithms. The intensity of each ion was normalized with respect to the total ion count to generate a data matrix having retention time, m/z value, and the normalized peak area. Charged cultivars, salt adducts, and fragments were all automatically aligned and grouped. The three-dimensional data including peak number (RT-m/z pair), sample name, and normalized peak areas were exported to the EZinfo software 3.0.3 (Umetrics, Umeå, Sweden) for multivariate statistical analyses. Multivariate statistical analyses were performed on the Pareto-scaled and mean-centered Mass spectra of all samples. Principal component analyses (PCA) of the mass spectra were conducted for a spectral pattern comparison of cultivars.

Results and discussion

Profiling of various platycosides in Platycodi Radix by UPLC-QTOF/MS

To develop an effective method to profile various platycosides from Platycodi Radix, we used the analysis conditions of UPLC-QTOF/MS method constructed in our previous study [25]. By using this method, seven standard samples including platycodin D3, platycoside E, platycodin D, polygalacin D, platyconic acid A, and platycodin D2 were efficiently separated in 15 min. In the negative mode of ESI, these standards were mainly detected as [M+HCOO]⁻ ions and [M−H]⁻ ions. The analytical performance of this method has also been proven by validation study. In this study, we applied the method to analyze the various platycosides from four Platycodi Radix cultivars [i.e. Fuji Blue (FB), Astra Pink (AP), Astra Blue (AB), and Jangbaek (JB)]. First, 70% (v/v) EtOH was used to extract the platycosides from four Platycodi Radix samples, FB, AP, AB, and JB, individually; next, each Platycodi Radix extract was analyzed by UPLC-QTOF/MS; and then processed the data using the UNIFI software. The combination of UPLC separation, Q/TOF–MS detection and UNIFI software has been frequently applied in the characterization of chemical constituents of traditional herbs [30]. The total ion chromatogram (TIC) showed the separation and detection of various metabolites, including platycosides from four Platycodi Radix samples (Additional file 1: Figure S1). The extracted ion chromatogram (EIC) and retention time (RT) of various platycosides were listed in the Additional file 1: Figure S2. The m/z of ions from the platycosides profile data were automatically matched to the in-house library compounds [25].

In the complex mixture of the Platycodi Radix extract, it is challenging to identify the various platycosides. The precursor ion m/z of the compounds obtained by the exact mass measurement based on QTOF/MS is critical for the identification of platycosides. Furthermore, the alternative high- and low-energy scans conducted by MS^E mode represents the product ions that assign the structure of the compounds. Thus, we analyzed several standard samples to find the patterns in platycosides of adduction and fragmentation. For example, in the low-energy scan data, platycodin D was detected as both [M+HCOO]⁻ and [M−H]⁻ ions. Furthermore, a specific product ion obtained by the cleavage of a sugar moiety was also found in the high-energy scan data (Additional file 1: Figure S3). To identify the platycosides, it is required to estimate the preceding and product ions in the MS^E data. However, manually determining each platycoside would be time-consuming.

For a fast and exact identification of the various platycosides, we attempted to construct an in-house library using the UNIFI software. First, the information of the compound’s name and molecular formulas was added into the preliminary library for platycosides. The profiling data of four Platycodi Radix samples was then automatically processed by the preliminary library. As a result, twenty platycosides that do not have isomers were determined based on their exact mass values and product ion data. Otherwise, the information was insufficient to identify six pairs of isomers (C₄₂H₆₈O₁₇, C₅₈H₉₄O₂₉, C₅₉H₉₄O₂₈, C₆₃H₁₀₂O₃₂, C₆₃H₁₀₂O₃₃, and C₆₅H₁₀₄O₃₄). Although the MS^E acquisition mode was used to assess the product ion data of six isomeric pairs, it was insufficient to differentiate the isomers by only the glycosidic bond cleavage (Additional file 1: Figure S4).

Determination of platycoside isomers

To determine seven pairs of platycoside isomers, we focused on examining the elution order of molecules. UPLC-QTOF/MS was used to analyze two standards, platycodin D2 and platycodin D3, which are two isomers with the same formula (C₆₃H₁₀₂O₃₃). As a result, platycodin D2 (RT: 9.23) had a longer retention time (RT) than platycodin D3 (RT: 5.53) (Fig. 2). According to the position of glucose, platycodin D2 belongs to Glc-(1 → 3)-Glc platycosides, and platycodin D3 belongs to Glc-(1 → 6)-Glc platycosides. Finally, this indicated that Glc-(1 → 3)-Glc platycosides are eluted later than Glc-(1 → 6)-Glc platycosides. This rule is then applicable to differentiate Glc-(1 → 3)-Glc and Glc-(1 → 6)-Glc platycoside isomers. To identify the isomeric pairs, we manually estimated the processed data representing the observed m/z, RT, molecular formula, and adducts. For example, two peaks having different RTs were shown in the BPI chromatogram of m/z 843.43 [M−H]⁻. They might be platycoside K and platycoside L having the same formula (C₄₂H₆₈O₁₇). According to the chemical structure, platycoside K belongs to Glc-(1 → 3)-Glc platycosides, and platycoside L belongs to Glc-(1 → 6)-Glc platycosides. Thus, the two peaks were determined as platycoside K (RT: 4.35) and platycoside L (RT: 2.59). Platycoside A and deapioplatycodin D3 are also two isomers with the same formula (C₅₈H₉₄O₂₉), and they have Glc-(1 → 3)-Glc and Glc-(1 → 6)-Glc, respectively. Hence, platycoside A must be eluted later than deapioplatycodin D3, and the two peaks in the BPI chromatogram of m/z 1299.58 [M–H]⁻ were determined as platycoside A (RT: 8.3) and deapioplatycodin D3 (RT: 5.18). Polygalacin D2 and platycoside G3, which are two isomers with the same formula (C₆₃H₁₀₂O₃₂), have Glc-(1 → 3)-Glc and Glc-(1 → 6)-Glc, respectively. Thus, the polygalacin D2 must be eluted later than platycoside G3. Finally, in the BPI chromatogram of m/z 1415.63 [M−H]⁻, the two peaks were determined as polygalacin D2 (RT: 9.63) and platycoside G3 (RT: 6.34).

Fig. 2

Separation and identification of two isomers, platycodin D2 (RT: 9.23 min) and platycodin D3 (RT: 5.53 min), and their molecular structures

Next, in the data for m/z 1295.59 [M+HCOO]⁻, two peaks having the same formula (C₅₉H₉₄O₂₈) might be 3′-O-acetyl-polygalacin D and 2′-O-acetyl-polygalacin D. The structural difference of these two compounds is only the position of an acetyl group (Ac) attached to α-l-rhamnopyranose (Rha), such as Rha (2′-O-Ac) and Rha (3′-O-Ac). As it is limiting to use these standards, we only relied on a previous report showed that 2′-O-acetyl-polygalacin D is eluted later than 3′-O-acetyl-polygalacin D [25]. This indicated that Rha (2′-O-Ac) platycosides have longer RT than Rha (3′-O-Ac) platycosides. Thus, the two isomers of formula (C₅₉H₉₄O₂₈) were determined as 2′-O-acetyl-polygalacin D (RT: 10.79) and 3′-O-acetyl-polygalacin D (RT: 8.97) (Fig. 3). We also determined the other isomeric pairs of platycosides as having Rha (2-O-Ac) or Rha (3-O-Ac). In the data of m/z 1428.64 [M−H]⁻, two peaks having the same formula (C₆₅H₁₀₄O₃₄) might be 3′-O-acetyl-platycodin D2 and 2′-O-acetyl-platycodin D2. 3′-O-acetyl-platycodin D2 has Rha (3-O-Ac) and 2′-O-acetyl-platycodin D2 has Rha (2-O-Ac). Thus, the two isomers were determined as 3′-O-acetyl-platycodin D2 (RT: 9.66) and 2′-O-acetyl-platycodin D2 (RT: 11.84).

Fig. 3

Separation and identification of two isomers, 3′-O-acetyl-polygalacin D (RT: 8.97 min) and 2′-O-acetyl-polygalacin D (RT: 10.79 min), and their molecular structures

Data analysis of four Platycodi Radix samples

Finally, we identified a total of 32 platycosides, including the six pairs of isomers in Platycodi Radix (Table 1). They include the name of the compound, molecular formula, and observed m/z, RT, adducts, and mass accuracy (ppm). This information can be used as database for platycosides profiling. The proposed method based on UPLC-QTOF/MS with an in-house library was used to profile various platycosides from four Platycodi Radix samples. The numbers of individual samples were as follows: FB (n = 3), AP (n = 3), AB (n = 4), and JB (n = 3). And then, all the processed data were exported to the EZinfo software for PCA. PCA is an effective tool to visualize the clustering trends among individual samples for finding the similarities or differences between the metabolite data of samples. In the score plot, four groups of FB, AP, AB, and JB samples were well separated (Fig. 4A). Moreover, in the loading plot, we identified the main variables that contribute to differentiate the four groups in the score plot. Each point represented the m/z–RT pairs of molecules. Based on the in-house library, the eight variables of loading plot were identified as platycosides (Fig. 4B). This indicated that each Platycodi Radix cultivars showed different contents of platycosides. In the data processing, various platycosides were identified from FB (n = 25), AP (n = 19), AB (n = 26), and JB (n = 26). Next, in the MS data, the peak intensities of various platycosides (Mean ± SD) were calculated for the relative quantification of high- and low-abundance platycosides (Table 2). Unfortunately, as it is limiting to use all the platycoside standards, the absolute quantification was not performed. However, this method can be used to compare the relative amount of each platycoside from four different Platycodi Radix cultivars. The developed method was applied to the simultaneous quantification of six major in Platycodi Radix cultivars, respectively. All calibration curves showed good linearity (r² > 0.998) within the test ranges and their contents were listed in Table 3. The results showed that platycosides were the major components in Platycodi Radix and detected in all its samples, but polygalacin D was not found in Fuji Blue and Astra Pink cultivars. Actually, platycosides contribute to the biological activities of Platycodi Radix. Therefore, the platycosides should be considered as the markers for component breeding of Platycodi Radix.

Table 1 The list of various platycosides analyzed in Platycodi Radix (ESI−)

Fig. 4

The principal component analysis (PCA) score plot (A) and loading plot (B) of four Platycodi Radix samples (i.e. Astra Pink, Astra Blue, Jangbaek, Fuji Blue). (a) platyconic acid B, (b) platycoside E, (c) methyl-2-O-methylplatycogenate A, (d) 2′-O-acetyl-platycodin D2, (e) 3′-O-acetyl-platycodin D2, (f) platycodin C, (g) platycodin D, (h) platyconic acid A

Table 2 Relative quantification of platycosides from four Platycodi Radix cultivars

Table 3 Contents of six major platycosides in four Platycodi Radix cultivars