Establishment of UPLC/MS method for ginsenoside analysis
Owing to the complexity of metabolites, many components may be coeluted during analyses. To develop a sensitive and accurate UPLC/MS method for determining the active compounds in ginseng, we used a triple quadruple mass spectrometer equipped with ESI, one of the most useful tools available for simultaneous quantification of ginsenosides, to analyze the compounds in ginseng. To quantify the analytes using the MRM mode, we evaluated full scan and product ion spectra of the analytes. First, in tests of positive and negative ion detection modes, the positive ESI achieved higher sensitivities than the negative. With positive ESI, Rd, Rf, Rh1, Rh2, and Rg3 formed predominately protonated molecules, [M + H]+, at m/z 964.1, 801.7, 639.2, 623.2, and 786.1, respectively, in the full-scan spectra. In addition, Rg1, Re, Rb1, Rb2, and Rc predominantly formed sodium adduct ions, [M + Na]+, at m/z of 824.0, 970.0, 1131.1, 1101.8, and 1101.2 with the response approximately 5 times higher than with [M + H]+. The [M + H]+ and [M + Na]+ ions were therefore chosen as the precursor ions to obtain their major fragment ions for MRM analysis. The fragment ions at m/z values of 643.9, 789.9, 767.2, 334.4, 621.1, 605.2, 789.2, 335.0, 325.0, and 424.2, were present in the highest abundance for Rg1, Re, Rd, Rc, Rh1, Rh2, Rb1, Rb2, Rg3, Rf, respectively (Additional file 1: Figure S1). The instrument was tuned to yield the maximum product ion for each compound.
In optimizing the UPLC system to detect the 10 ginsenosides, chromatographic separation was tested on several C18 columns to achieve the best efficiency and peak shape. The ACQUITY UPLC BEH C18 column (1.7 μm, 2.1 mm × 50 mm) gave good peak shape when ultra-pure was used as the mobile phase. Different mobile phases were evaluated to improve LC separation and enhance MS sensitivity. When methanol and acetonitrile were tested as the organic modifier, acetonitrile was superior for ionizing most ginsenosides. In tests on the isocratic and gradient systems, the gradient system achieved better peak shape than the isocratic system; thus, a gradient elution with a mobile phase consisting of acetonitrile and no acid in water gave optimal peak shape and mass spectral response for the analytes.
The representative total ion current (TIC) spectra in 10 min was obtained from the analysis (Fig. 2). The retention time of Re, Rg1, Rf, Rb1, Rh1, Rc, Rb2, Rd, Rg3, and Rh2 was 0.76, 0.78, 2.32, 2.97, 3.48, 3.69, 4.94, 5.83, 6.69, and 7.34 min, respectively. No endogenous interference was detected at these retention times for the 10 analytes; therefore, high, very acceptable selectivity was achieved by this method. Regression equations and a linear range for calibration curves were also obtained. To avoid bias to the low concentrations of the standard curve caused by the high concentrations, the calibration curves were separated for different ranges. Within the linear range, the calibration curve had good linearity (r
2 > 0.999) for each analyte (Additional file 2: Table S1). In addition, the proposed method delivers reliable accuracy and good reproducibility for the simultaneous separation and detection of the metabolites using UPLC/MS (Additional file 2: Table S2, 3). The optimized method was employed for the determination of the ginsenosides by comparing the area calculated for each peak to the standard curves obtained from the authentic ginsenoside standards (Additional file 2: Table S1). Calibration curves were plotted using five concentrations of each ginsenoside standard according to the peak area.
Specific accumulation of ginsenosides in various parts of P. ginseng
To investigate the accumulation of ginsenosides in different part of P. ginseng, samples from five different parts were collected, including leaf, petiole, stem, lateral root, and main root. For each part, samples from different growth stage were obtained. The profile for the main ginsenosides was visualized using a hierarchical cluster analysis (HCA; Fig. 3). Accumulation of the ginsenosides displayed a clear phenotypic variation in terms of their abundance in different parts of the same age. Roots contained the highest levels of most ginsenosides, followed by lateral roots, stems, and petioles; leaves had the lowest. In addition to the greatest accumulation of ginsenosides, roots also had the most complex profile for ginsenoside composition in the different ages of the parts.
Based on their part- and growth stage-specific accumulation patterns, ginsenosides could be clearly grouped into five stages (Fig. 3a). Ginsenosides in the first year had higher levels in lateral roots than in other parts and were mainly represented by PPD-type ginsenosides, including some of the major ginsenosides such as Rc, Rb1, and Rd. In the second year, the PPD-type such as Rc, Rb2, Rg3, and Rd predominated, again with higher levels in lateral roots. Moreover, in the same stage, other ginsenosides had their highest levels in the main roots and were mainly represented by PPT-type, Re, Rg1, and Rf. Major PPD- (Rc, Rb2, Rd, and Rg3) and PPT-types (Re and Rg1) were tightly grouped in lateral roots and main roots, respectively, during the third year. Three PPD-types, including Rc, Rh2 and Rg3, in the fourth year were significantly higher in lateral roots than in leaves, petioles, stems and main roots. Furthermore, one PPD-(Rb1) and two PPT-types (Rg1 and Rf) significantly accumulated in the main roots. Re and Rg1 during the fifth year had highest levels in the main roots and were mainly represented by PPT, which have been characterized for their medicinal value. However, the PPD-type Rh2 had higher levels in lateral roots, followed by stems, petioles, main roots and leaves. In addition, PPD-types Rb1, Rb2 and Rg3 showed the same accumulation pattern as the PPT-types Re and Rg1, suggesting highest accumulation in the main roots than in other parts during the fifth year.
In addition, the “Q” score of the principal component is an indicator of a comprehensive, scientific evaluation of objective phenomenon, which has no practical significance. In the Fig. 3b, the comprehensive PCA Q value result revealed that the overall accumulation of ginsenosides in different parts and stages followed a relatively stable distribution. The above- and belowground parts could be clearly differentiated, except for the roots of 1-year olds. Leaves contained the lowest levels of ginsenosides, followed by petioles and stems, and second year and fifth year parts shared this uniform tendency, which these ginsenosides in leaves had lower levels than in the petioles and stems. Contrasting the aboveground parts ginsenosides changing, we observed that the belowground parts (lateral roots and main roots) increasing their concentration in main roots is higher than those increasing their concentration in lateral roots of 2- and 3-year-old plants. In addition, an increase of the number of Q value which accumulate higher in lateral roots than main roots during 4 to 5-year-old samples.
Ginsenoside accumulation patterns during development
To further clarify ginsenoside accumulation patterns during different developmental stages, five parts were sampled at five stages, and the 10 major ginsenosides in each part were quantified. Ginsenosides with different modifications accumulated differently at different developmental stages. The various forms of ginsenosides were subsequently quantified, and the major ginsenosides were selected to determine the range of variations observed in leaves, petioles, stems, lateral root and main roots during each stage (Fig. 4).
Most PPD-types such Rb1, Rb2, Rc and Rg3 significantly increased in the roots during years 1–5 (R1–R5) (Fig. 4a). The levels of Rb1 and Rb2, showed marked cross-change during the lateral root stage (LR2–LR5), sharply increasing at early stages (P1–P2). Levels of Rb2 decreased over time in stems (S1–S5). Rb1 and Rd sharply decreased and Rb2 and Rh2 levels increased in leaves during later stages (L2–L5). For most of the PPT-types such as Rg1, Re and Rh, accumulation in roots significantly increased during R1 to R5 (Fig. 4b). In lateral roots, Rg1 and Re sharply increased during LR1 to LR3. However, Rg1 sharply decreased during LR4 to LR5 and for Re during LR3–LR5. In addition, the level of Rg1 and Re had an inverse relationship during all petiole stages (P1–P5). Re and Rf increased in a similar way in stems (S1–S5). In contrast, a discrepant synthesis of Re and Rh1 during leaf development (L1–L5) suggested that their accumulation was developmentally controlled.
Differential accumulation of ginsenosides between P. ginseng and P. quinquefolius
Differences in genetic background and geographical distribution between P. ginseng and P. quinquefolius may lead to differing accumulation and composition of ginsenosides in these two species. For each part sample (leaf, petiole, stem, lateral root, main root), the relative concentration of each ginsenoside of interest was determined for P. ginseng and P. quinquefolius (Fig. 5).
In the main roots, the levels of seven ginsenosides differed significantly between the two species. Among them, four PPD-types (Rb1, Rb2, Rc, Rg3) were higher in P. ginseng than in P. quinquefolius. Three major PPT-types (Rg1, Re, Rh1) were higher in P. ginseng than in P. quinquefolius (Fig. 5a), but Rg3, Rg1, and Rh2 in lateral roots were lower in P. ginseng than in P. quinquefolius (Fig. 5b). In stems, major PPD-types Rb1, Rb2, Rd, and Rg3 were significantly elevated levels in P. quinquefolius compared with P. ginseng (Fig. 5c). In petioles, Rg3 was 80 times higher and Rh1 was 3 times higher in P. ginseng than in P. quinquefolius (Fig. 5d). Similarly, Rb2, Rc, Rf, and Rh2 levels in leaves were higher in P. ginseng than in P. quinquefolius, whereas Rb1, Rg3, Rg1 and Re were significantly higher in P. quinquefolius than in P. ginseng (Fig. 5e). No significant difference was found between the two species for Rd and Rh1. Furthermore, a two-dimensional PCA score plot (Fig. 5f) was able to discriminate the differential levels in parts between P. ginseng and P. quinquefolius, thus simplifying data management. The above results suggested that the levels of ginsenosides in the two Panax species could be determined through UPLC coupled with PCA and the values of ginsenosides.