2.2.1 Sample solutions preparation

0.1 g KSLP powder was added to 10 mL of 50 % ethanol, and ultrasonically extracted for 60 min. The solution was centrifuged at 15,000 g for 10 min, and the supernatant was used for UPLC/Q-Orbitrap-MS/MS qualitative analysis.

2.2.2 Standard solutions preparation

The standards of acteoside, isoacteoside, echinacoside, jionoside A1, ginsenoside Rb1, ginsenoside Rb2, ginsenoside Rc, ginsenoside Rd, ginsenoside Re, ginsenoside Rf, ginsenoside Rg1, ginsenoside Rg2 and ginsenoside Rg3 were weighed accurately and dissolved in 50 % ethanol to 1 mg/mL single standard solutions. 50 µL of each standard solutions were added to 350 µL of 50 % ethanol, and the mixed standard solution was collected after vortex mixing 1 min and the following centrifugation (12000 g, 10 min, 4℃).

2.2.3 Chromatographic and mass spectrographic conditions

The UPLC separation was performed on Ultimate 3000 UPLC system (Thermo Fisher Scientific, USA) with an ACQUITY UPLC HSS T3 (2.1 × 100 mm, 1.7 μm, Waters) maintained at 40℃. The mobile phase was acetonitrile (A) and water with 0.1 % formic acid (B) at a flow rate of 0.3 mL/min, and injection volume of samples was 4 μL. The gradient elution procedure was as follows: 0 ∼ 2 min, 3 % (A); 2 ∼ 5 min, 3 ∼ 21 % (A); 5 ∼ 7 min, 21 ∼ 22 % (A); 7 ∼ 9 min, 22 ∼ 30 % (A); 9 ∼ 13 min, 30 ∼ 33 % (A); 13 ∼ 18 min, 33 ∼ 34 % (A); 18 ∼ 23 min, 34 ∼ 55 % (A); 23 ∼ 28 min, 55 ∼ 70 % (A); 28 ∼ 33 min, 70 ∼ 98 % (A).

The mass spectrometry analysis was completed under Q-Orbitrap MS system (Thermo Fisher Scientific, USA). The optimized MS parameters were: ESI positive and negative (+/-) mode; spray voltage, 3.5kv; capillary temperature, 350℃; aux gas heater temperature, 350 ℃; sheath gas was nitrogen and its flow was 35 L/h; auxiliary gas was nitrogen and its flow was 10 L/h; full scan mode, m/z 100–1500.

2.3.1 Sample solutions preparation

In this study, the effects of four single factors (extraction method, extraction solution, extraction time and solid-to-liquid ratio) on the contents of compounds were investigated. The maximum total content of the 13 target compounds was used as the criterion to determine the optimal extraction process.

The levels of the four single factors were as follows: ultrasonic and reflux for extraction method; 30 % ethanol, 50 % ethanol, 70 % ethanol and 90 % ethanol for extraction solution; 30, 60, 90 and 120 min for extraction time; 1:20, 1:50, 1:100 and 1:200 g/mL for solid-to-liquid ratio. When the influence of any single factor was investigated on the total content of 13 target compounds, 50 % ethanol, ultrasonic, 90 min and 1:100 g/mL were selected for the other three factors.

The samples were extracted according to each extraction process, diluted with the corresponding solutions, and finally 100 µg/mL of sample solutions were obtained for UPLC/QQQ-MS/MS quantitative analysis.

2.3.2 Standard solutions preparation

The stock solutions of acteoside, isoacteoside, echinacoside, jionoside A1, ginsenoside Rb1, ginsenoside Rb2, ginsenoside Rc, ginsenoside Rd, ginsenoside Re, ginsenoside Rf, ginsenoside Rg1, ginsenoside Rg2 and ginsenoside Rg3 were prepared in 50 % ethanol. Working standard solutions containing each of the 13 compounds were prepared by diluting the stock solutions with 50 % ethanol to a series of proper concentrations. Similarly, the working solutions of quality control (QC) with low, medium and high concentrations were prepared. QC samples contained acteoside, isoacteoside, echinacoside, jionoside A1, ginsenoside Rg2 and ginsenoside Rg3 of 1, 10 and 80 ng/mL, ginsenoside Rb1, ginsenoside Rb2, ginsenoside Rc, ginsenoside Rd, ginsenoside Re, ginsenoside Rf and ginsenoside Rg1 of 10, 100 and 800 ng/mL.

2.3.3 Chromatographic and mass spectrographic conditions

The chromatographic separation was achieved on an ACQUITY UPLC Ultra Performance Liquid Chromatograph (Waters, American) with an ACQUITY UPLC HSS T3 (2.1 × 100 mm, 1.7 μm, Waters) maintained at 40℃. The mobile phase was acetonitrile (A) and water with 0.1 % formic acid (B) at a flow rate of 0.3 mL/min, and injection volume of samples was 4 μL. The gradient elution was performed as follows: 0 ∼ 2.5 min, 20 ∼ 30 % (A); 2.5 ∼ 10 min, 30 ∼ 60 % (A); 10 ∼ 10.5 min, 60 ∼ 98 % (A).

MS detection was carried on Waters Xevo TQ-S Triple Quadrupole Mass Spectrometer (Waters, American), and the mass spectrometer was operated in a MRM mode. The optimized MS parameters: capillary voltage, 3.0 KV (positive ion mode)/ 2.0 KV (negative ion mode); solvent removal temperature, 350℃. The remaining specific parameters, such as parents ion (Parents), daughters ion (Daughters), collision energy (CE) and cone voltage (CV), were optimized and summarized in Table S1.

2.3.4 Method validation

According to the above optimal analysis conditions and extraction conditions, the selectivity, linearity, limit of detection (LOD), limit of quantification (LOQ), accuracy, precision, stability and recovery of 13 compounds were determined.

The selectivity of the method was evaluated by comparing the chromatograms of each analyte in the blank solutions, standard solutions and sample solutions. Linearity was evaluated by plotting the calibration curves, and the curves were plotted with the concentration X (ng/mL) of the standard as the abscissa and the corresponding peak area Y of each standard as the ordinate. For each target compound, LOD and LOQ were determined at single to noise ratios (S/N) of 3 and 10 by continuous dilution of standard solutions. The QC samples of low, medium and high concentrations were analyzed for three consecutive days, accuracy was obtained from the relative error expressed as percentage (RE%), and precision was calculated using the relative standard deviation (RSD%). The stability of QC samples of low, medium and high concentrations was studied after being placed at room temperature for 0, 2, 4, 8, 12, 24 h. The test solutions and the standard solutions were added at a ratio of 1:1 to calculate the recovery.

2.4.1 KSLP preparation

KSLP powder was repeatedly extracted twice under the optimal extraction process conditions, and the extract was concentrated and freeze-dried to obtain crude components of KSLP. The equal crude components of KSLP were further eluted with water and different proportions of ethanol/ethanol/methanol by resin D101/ resin AB-8/ODS, the water-eluting fractions were discarded, other fractions were combined respectively, concentrated and freeze-dried to obtain refined components of KSLP.

0.1 g powders of KSLP, crude components of KSLP and refined components of KSLP were added to 10 mL of 50 % ethanol, and ultrasonically extracted for 90 min. Extracts of KSLP and its crude components were centrifuged and diluted to obtain 100 µg/mL sample solutions. Due to the large difference of the compound content in the refined components, 1000 µg/mL and 10 µg/mL of sample solutions were obtained, The UPLC/QQQ-MS/MS quantitative analysis of sample solutions was the same as in part 2.3. The refined components of KSLP obtained by the optimal refining process were used for pharmacokinetic studies.

2.4.2 Animals and experimental procedure

A total of 6 male Sprague-Dawley (SD) rats (200–220 g) were purchased from Beijing Huafukang Bio-Technology Co., Ltd. All of the rats were kept in pathogen-free animal laboratory in a 12 h light/dark cycle, with a feeding temperature of 26 ℃ and relative humidity of 50 %. Animal experiments were consistent with the Animal Ethics Committee of Tianjin University of Traditional Chinese Medicine, and the animal ethics approval number was TCM-LAEC2022124.

Rats were acclimatized for 7 days to laboratory environments before the experiment was directed. Diet was prohibited for 12 h before the experiment, but the water was freely available. Appropriate refined components of KSLP were dispersed in distilled water as a suspension with a concentration of 0.4 g/mL, and the oral administration dose was set at 10 mL/kg of rat weight. The blood samples were collected through canthus into heparinized tubes before administration and at 0.083, 0.25, 0.5, 1, 2, 4, 6, 8, 10, 12, 24, 36, 48, 60 and 72 h after oral administration of refined components of KSLP (4 g/kg). Plasma samples were obtained by centrifuging the blood samples immediately at 1600g for 10 min and stored at −80℃ until analysis.

2.4.3 Plasma sample preparation

100 μL of each sample was processed by adding 300 μL of acetonitrile, 100 μL of internal standard solution (IS) and 100 μL of 50 % acetonitrile. The supernatant was collected after vortex mixing 1 min and the following centrifugation (15000 g, 10 min, 4℃). Then, the supernatant was transferred and dried under a gentle stream of nitrogen gas at room temperature. The resulting residues were redissolved in 100 μL of 50 % acetonitrile, and 5 μL was injected into the UPLC/QQQ-MS/MS system.

2.4.4 Standard solutions preparation

The stock solutions of ginsenoside Rb1, ginsenoside Rb2, ginsenoside Rc, ginsenoside Rd, ginsenoside Re and ginsenoside Rg1 were prepared in 50 % acetonitrile, which were then diluted with 50 % acetonitrile to obtain mixed standard solutions of the 6 compounds at different concentrations. Digoxin was weighed accurately, dissolved in 50 % acetonitrile and diluted gradually to 100 ng/mL as the internal standard solution. 100 μL of blank plasma was processed by adding 300 μL of acetonitrile, 100 μL of IS and 100 μL of mixed standard solutions. Similar to post-administration plasma processing, a range of working standard solutions were obtained for analysis. The QC samples were prepared in a similar manner at three different concentrations (10, 100 and 800 ng/mL) of ginsenoside Rb1, ginsenoside Rb2, ginsenoside Rc, ginsenoside Rd, ginsenoside Re and ginsenoside Rg1.

2.4.5 Chromatographic and mass spectrographic conditions

The conditions of chromatographic and mass spectrographic were the same as in part 2.3.3, the remaining specific parameters of 7 compounds were shown in Table S2.

2.4.6 Method validation

According to the above optimal analysis conditions, the selectivity, linearity, lower limit of quantification (LLOQ), accuracy, precision, extraction recovery, matrix effect and stability were validated.

The chromatograms of blank plasma, standard spiked plasma and sample plasma were used to assess the selectivity of the method. Linear regression equations were obtained by least squares linear regression on the ratio Y of the compound peak area to IS peak area as the ordinate, the compound concentration X (ng/mL) in plasma as the abscissa, and LLOQ was the lowest concentration of linearity. Intra-day and inter-day accuracy and precision were estimated by analyzing a calibration curve and QC samples of low, medium and high concentrations on three days. The extraction recovery was evaluated through the ratio of the mean concentration between regularly prepared QC samples of low, medium and high concentrations and spike-after-extraction plasma samples. Similarly, the matrix effect was assessed through the ratio of concentration between post-extraction samples spiked with analytes and ultrapure water spiked with analytes at the same concentration. The stability of each analyte at different conditions (autosampler for 24 h and three freeze–thaw cycles from −80℃ to room temperature) was assessed by analyzing at QC levels.

3.1.1 Identification of ginsenosides in KSLP

Ginsenosides were the main active components of Ginseng radix et rhizoma rubra, which were mainly divided into protopanaxadiol (PPD), protopanaxatriol (PPT) and others according to their structural differences (Yang et al., 2014). The sapogenin fragments at m/z 459.38 (PPD) and m/z 475.38 (PPT) could be used as the diagnostic product ions to rapidly characterize ginsenosides of these two subtypes (Qiu et al., 2015, Li et al., 2021a, 2021b). Taking ginsenoside Rd as an example to illustrate the cleavage pathway of PPD, the excimer ion peak of m/z 991.5497 [M + COOH]⁻ was easily generated in the negative ion mode. Typical neutral losses (NL) of HCOOH (46 Da) and Glu (162 Da) produced fragments of m/z 945.5433 [M−H]⁻, m/z 783.4912 [M−H−Glu]⁻、m/z 621.4348 [M−H−2Glu]⁻ and m/z 459.3830 [M−H−3Glu]⁻. Taking the PPT type ginsenoside Re as an example, the quasimolecular ion peak of m/z 991.5497 [M + COOH]⁻ was easily generated in the negative ion mode. Due to NL of HCOOH (46 Da), Glu (162 Da) and Rha (146 Da), fragments of m/z 945.5433 [M−H]⁻, m/z 799.4824 [M−H−Rha]⁻、m/z 783.4907 [M−H−Glu]⁻ and m/z 637.4328 [M−H−Glu−Rha]⁻ and m/z 475.3794 [M−H−2Glu−Rha]⁻ were detected.

3.1.2 Identification of phenylethanol glycosides in KSLP

Phenylethanol glycosides, one of the bioactive components from Rehmanniae radix, possessed various pharmacological activities for human health. The fragmentation pathway was mainly related to breaking of ester bond or C-O bond, resulting in a series of degradation products. Taking acteoside for a witness, the excimer ion peak of m/z 623.1985 [M−H]⁻ was easily generated in the negative ion mode. Acteoside produced ions at m/z 461.1662 [M−H−caffeoyl]⁻ and m/z 315.1082 [M−H−caffeoyl−Rha]⁻ through the loss of caffeoyl and Rha moiety. The caffeoyl moiety was produced ion at m/z 161.0234 [Caffeoyl-H-H₂O]⁻ by the loss of H₂O (Qi et al., 2013).

3.2.1 Validation of analytical method

The chromatograms of blank solutions, standard solutions and sample solutions were displayed in Fig. S1. It could be seen from the chromatograms that 13 compounds had good peak shapes and no endogenous interference, indicating that the method was suitable. The linear regression equations, correlation coefficients (R²), linear ranges, LOD and LOQ of the 13 target compounds were listed in Table 2. The R² of analytes were greater than 0.999, indicating good linearity, the LOD and LOQ of the 13 target compounds were the range of 0.02 ∼ 0.6 ng/mL and 0.08 ∼ 2.0 ng/mL, indicating that the method had high sensitivity. The test results of accuracy, precision and stability were summarized in Table 3. Intra-day and inter-day accuracy (RE%) ranged from −4.78 % to 4.74 %, intra-day and inter-day precision (RSD%) were within 7.16 % and stability (RSD%) was less than 4.75 %, indicating that the method was reproducible and accurate for the determination of analytes. The sample recovery rate was showed in Table S6, and it was between 90.27 ∼ 108.22 % (RSD% less than 5.54 %), indicating that no significant loss of analyte occurred during the analysis.

3.2.2 Quantitative analysis of multi-components in KSLP

The effects of various extraction conditions on the yield of target compounds were represented in Table S3 and Table S4. The yield of crude components extracted by ultrasonic and reflux was not much different, and the total content of target compounds extracted by ultrasonic was slightly higher than that of reflux. With the increase of the ethanol content, the crude components yield showed a trend of first increasing and then decreasing. When the ethanol concentration reached 50 %, the total content of target compounds was higher than that of other concentration group. The yield of crude components increased slowly with increasing extraction time, eventually reached stability at 90 min, indicating that extraction time has a significant positive effect on yield when it is below 90 min. There was no significant change in the yield when extraction time was longer than 90 min. There was a gradual increase in the yield of crude components with increasing solid-to-liquid ratio. The yield increased slightly when the solid-to-liquid ratio was greater than 1:100 g/mL, however, a large solid-to-liquid ratio caused solvent wastage, 1:100 g/mL was selected as the optimal solid-to-liquid ratio. Therefore, 50 % ethanol, ultrasonic, 90 min and 1:100 g/mL were the optimal process for extracting crude components of KSLP based on the criterion of the maximum total content of the 13 target compounds, combined with economic considerations.

According to the established UPLC/QQQ-MS/MS quantitative analysis method and extraction process conditions, the content of 13 compounds in KSLP was determined. Among 13 analytes, the compounds with higher content (>1000 μg/g) were ginsenoside Rb1, ginsenoside Rb2, ginsenoside Rc, ginsenoside Re and ginsenoside Rg1.

3.3.1 Validation of analytical method

The chromatograms of blank plasma solutions, standard plasma solutions and sample plasma solutions were plotted in Fig. S2, all the peaks of the analytes and IS were detected with excellent resolutions as well as shapes. The endogenous substances in the plasma did not interfere with the determination of each compound and IS, suggesting that the method developed in this study had good selectivity. The 6 target compounds had a good linear relationship within the corresponding concentration range, and its LLOQ were 4.0 ng/mL. The statistical parameters of calibration were listed in Table S7. As exhibited in Table 4, intra-day and inter-day accuracy (RE%) was between −12.22 ∼ 11.89 %, intra-day and inter-day precision (RSD%) were within 7.22 %, the extraction recovery and matrix effect of 6 compounds ranged from 88.02 % to 109.78 %, it showed that the instrument had good precision, the method had high repeatability. The stability results in Table S8 indicated that the 6 compounds in rat plasma were stable with RSD < 14.61 % for autosampler for 24 h, three freeze–thaw cycles.

3.3.2 Determination of the content of KSLP in plasma and pharmacokinetic parameter fitting

The plasma drug concentration and time curve of refined components of KSLP after intragastric administration for 72 h with a single dose in rats was presented in Fig. 2, and the pharmacokinetic parameters were summarized in Table 5. The maximum plasma concentration (Cmax) of ginsenoside Rb1, ginsenoside Rb2, ginsenoside Rc and ginsenoside Rd was about 100 ng/mL, while the Cmax of ginsenoside Re and ginsenoside Rg1 was about 40 ng/mL. All compounds took long time to their elimination t_1/2, indicating that the elimination rate of the target compounds in vivo was slow. Also, the AUC_0∼∞ of ginsenoside Rb1, ginsenoside Rb2, ginsenoside Rc and ginsenoside Rd was greater than 4500 ng/ml·h, while the AUC_0∼∞ of ginsenoside Re and ginsenoside Rg1 was about 2500 ng/ml·h.

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Fig. 2

Plasma drug concentration and time curve of refined components of KSLP in rats after intragastric administration (n = 6).

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