2.1 Chemicals, reagents, and plant materials

Scopolamine, anisodamine hydrobromide, hyoscyamine, protocatechuic acid, fabiatrin, scopolin, 4-hydroxybenzoic acid, caffeic acid, tropic acid, scopoletin, isoquercitrin, scoparone and coptisine (internal standard [IS], purity ≥ 98 %) were purchased from Chengdu Desite Biotechnology Co., Ltd. (Chengdu, China). Fig. 1 shows the chemical structures of the twelve analytes and IS. Fisher Scientific (Fair Lawn, NJ, USA) provided the methanol and acetonitrile (chromatographic purity) used in this study. Chromatographic purity formic acid was prepared from ROE (St. Louis, MO, USA). The demineralized water was obtained from Millipore's Milli-Q water purification system (Milford, MA, USA). PR was collected from Shaanxi province (China), and preserved at Tianjin University of Traditional Chinese Medicine, China.

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Fig. 1

Chemical structures of twelve compounds and IS.

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2.2 Instruments and conditions

In UHPLC-MS/MS, an Agilent 1290 ultra-high performance liquid chromatography system along with an Agilent 6470 series triple quadrupole mass spectrometer were used. ACQUITY UPLC CSH C18 column was employed for the chromatographic separation, and the column temperature of 30 °C was maintained. As mobile phases, 0.1 % formic acid in water (A) and acetonitrile (B) were used with the following gradient elution method: 0 – 7 min, 10 – 55 % B; 7 – 8 min, 55 – 61 % B, post run time of 4 min. The flow rate and injection volume were 0.3 mL/min and 2 μL, respectively. Multiple reaction monitoring (MRM) mode was applied for analysis in both positive and negative ionization mode simultaneously. The instrumental parameters were set as follows: gas temperature at 350 °C, gas flow rate at 11 L/min, and nebulizer pressure at 30 psig. The quantitative parameters of twelve components and IS were listed in Table 1. The mass spectroscopy characterization of twelve compounds were shown in Figure S1.

2.3 PR extract preparation

PR (700.0 g) was weighed accurately, and ten times 85 % ethanol (v/v) was extracted under hot reflux for two times, each time for two hours. Extract was mixed and concentrated by evaporation at reduced pressure. The dried PR extract was crushed and stored. The contents of scopolamine, anisodamine, hyoscyamine, protocatechuic acid, fabiatrin, scopolin, 4-hydroxybenzoic acid, caffeic acid, tropic acid, scopoletin, isoquercitrin, and scoparone in PR extract were listed in Table 2.

2.4 Preparation of standard solutions, calibration standards and quality control samples

Scopolamine, anisodamine, hyoscyamine, protocatechuic acid, fabiatrin, scopolin, 4-hydroxybenzoic acid, caffeic acid, tropic acid, scopoletin, isoquercitrin, scoparone and coptisine (IS) were separately weighed and dissolved with methanol at a concentration of 1.0 mg/mL. The calibration solutions were obtained by adding appropriate volumes of mixture working solution and 20 μL of IS into 100 μL blank rat plasma, resulting in concentrations: 1, 2, 4, 10, 20, 40, 80, 160, and 320 ng/mL for scopolin, 4-hydroxybenzoic acid, isoquercitrin, protocatechuic acid, and caffeic acid; 2, 4, 8, 20, 40, 80, 160, 320, and 640 ng/mL for scopoletin, fabiatrin, anisodamine, and hyoscyamine; 0.3, 0.6, 1.2, 3, 6, 12, 24, 48, and 96 ng/mL for scopolamine and scoparone; 10, 20, 40, 100, 200, 400, 800, 1600, and 3200 ng/mL for tropic acid. Three levels (low, medium, and high concentrations) of quality control (QC) samples were prepared in the same way.

2.5 Plasma sample preparation

20 μL of methanol, 20 μL of IS (coptisine, 1 μg/mL) were added to 100 μL of plasma sample and then vortex-mixed. The mixture was extracted with 600 μL methanol for 5 min. After centrifuging at 14,000 g for 10 min, the upper purified liquid was collected and evaporated under a stream of nitrogen until dry. The residue was redissolved in 100 μL of 50 % methanol. Finally, 2 μL of upper purifying solution was injected into the UHPLC-MS/MS system for analysis.

2.6 Method validation

The method was evaluated for specificity, linearity, sensitivity, precision and accuracy, recovery, matrix effect, and stability to ensure that it meets the U.S. Food and Drug Administration (FDA) guideline for accurate quantitation. (Tang et al., 2021; Xu et al., 2019; Zhang et al., 2022).

2.7 Pharmacokinetic study

Six SD rats (SPF, 220 ± 10 g, Male) were prepared from HuaFuKang Bioscience Co., Inc. (Beijing, China). Rats were allowed to drink freely and fasted for 12 h prior to the study. The PR extract was dissolved to a concentration of 62 mg/mL with 0.5 % CMC-Na aqueous solution. A suspension of 0.62 g/kg was administered orally to rats and roughly 300 μL of blood were collected before and at 0, 0.03, 0.08, 0.17, 0.25, 0.5, 0.75, 1, 2, 4, 6, 8, 10, 12, 24, and 36 h after oral administration. After centrifugation at 7000 rpm for 10 min, the plasma is frozen at −80 °C until analysis. Pharmacokinetic parameters were calculated using “Drug and Statistics 3.0” (DAS 3.0) (Medical College of Wannan, China).

3.1 Optimization of LC and MS/MS conditions

In the paper, the analytes were separated by UHPLC and characterized by mass spectrometry (QQQ-MS/MS). The UHPLC conditions, including column stationary phase type, mobile phase and additives, were optimized to obtain better separation. The stationary phase is crucial for obtaining satisfactory separation results. Main chemical components of most traditional Chinese medicines are readily absorbed by reversed-phase stationary phases, and three commonly used reversed-phase stationary phases were employed in this study: ACQUITY UPLC BEH C18 (2.1 × 100 mm, 1.7 µm), CORTECS UPLC C18 (2.1 mm × 100 mm, 1.6 μm), and ACQUITY UPLC CSH C18 (2.1 × 100 mm, 1.7 μm) to optimize the separation of twelve analytes. In comparison, the ACQUITY UPLC CSH C18 (2.1 × 100 mm, 1.7 µm) column provided better retention and shorter analysis time. Moreover, different mobile phases (ACN/H2O, ACN/0.1 %FA-H2O, ME/H2O, and ME/0.1 %FA-H2O) were compared and the results showed that ACN/0.1 %FA-H2O had higher sensitivity for analytes and IS. The twelve analytes and IS were eluted within eight min without any interference peaks (Fig. 2).

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Fig. 2

MRM chromatograms of twelve analytes and IS. Blank plasma sample (A); Blank plasma spiked with twelve analytes and IS (B); Plasma samples after oral administration of PR extract (C). 1. scopolamine, 2. anisodamine, 3. hyoscyamine, 4. protocatechuic acid, 5. fabiatrin, 6. scopolin, 7. 4-hydroxybenzoic acid, 8. caffeic acid, 9. tropic acid, 10. scopoletin, 11. isoquercitrin, 12. scoparone, and 13. coptisine (IS).

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For QQQ-MS/MS conditions, key parameters of the mass spectrometry that may affect the ion response were optimized. Three main ion source parameters, including gas flow rate (5–11 L/min), gas temperature (350–380 °C), and nebulizer pressure (20–50 psig), were optimized using a single factor experiment. Figure S2 shows the histogram of the trend of ion response for these twelve compositions at different parameter levels. The final optimized MS/MS conditions were as follows: gas temperature of 350 °C, gas flow rate of 11 L/min and nebulizer pressure of 30 psig.

3.2 Sample preparation

Sample preparation by appropriate methods is a key step in pharmacokinetic study. Three methods, ethyl acetate liquid–liquid extraction, acetonitrile precipitated protein, and methanol precipitated protein were compared for plasma sample preparation. Results revealed that methanol precipitated protein method has higher extraction efficiency, lower matrix effect, and simpler operation procedure. In order to meet the demands of this experiment for biological sample determination, methanol precipitated protein method was adopted for sample preparation in this study.

3.3 Method validation

3.4 Pharmacokinetic study

In this study, a high sensitivity UHPLC-MS/MS method was developed to determine twelve compounds in rat plasma after administration of PR extract. However, the plasma concentrations of some compounds were too low to attain a complete pharmacokinetic curve, such as protocatechuic acid, caffeic acid, and isoquercitrin. This may be caused by their low content in PR extract. Eventually, a total of nine detectable compounds were fitted with pharmacokinetic parameters. The mean plasma concentration–time curves of the other nine components are shown in Fig. 3. The main pharmacokinetic parameters, elimination half-life (T_1/2), time to reach maximum concentration (T_max), maximum concentration (C_max), and area under the curve (AUC), are summarized in Table 7.

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Fig. 3

Mean plasma concentration–time curves of scopolamine, anisodamine, hyoscyamine, fabiatrin, scopolin, 4-hydroxybenzoic acid, tropic acid, scopoletin, and scoparone after oral administration of PR extract (mean ± SD, n = 6).

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As shown in Table 7, the AUC_(0-t) and AUC_(0-∞) values of all nine compounds were close, indicating that the monitoring time of this study was appropriate. The AUC_(0-t) value of tropic acid was larger than the other analytes, indicating that this component had a higher level of plasma exposure. The C_max value of tropic acid was 1553.51 ± 736.52 ng/mL, and the blood concentration was higher than other compounds, which was related to the fact that scopolamine and anisodamine were metabolized to tropic acid in vivo (Chen et al., 2008; Chen et al., 2005a, Chen et al., 2005b). Meanwhile, fabiatrin also had a higher level of plasma exposure, which might be attributed to its higher content in PR extract.

The T_max values of scopolamine, anisodamine, hyoscyamine, fabiatrin, scopolin, 4-hydroxybenzoic acid, tropic acid, scopoletin, and scoparone were 0.08, 0.75, 0.88, 0.81, 0.21, 0.05, 0.70, 0.08, and 0.08 h. These results showed that these nine components were absorbed quickly in vivo. The T_max values of scopolamine, scopolin, scopoletin, and scoparone were similar to those previously reported (Zhang et al., 2014; Zhang et al., 2020; Li et al., 2019; Yin et al., 2012). The T_1/2 values of scopolamine, anisodamine, hyoscyamine, 4-hydroxybenzoic acid, and scoparone were 2.99, 3.15, 2.08, 0.41 and 4.22 h, respectively, indicating that these five analytes are eliminated shortly after oral administration of PR extract. The T_1/2 values of fabiatrin, scopolin, 4-hydroxybenzoic acid, and tropic acid were 6.45, 6.16, 9.87, and 7.12 h, respectively, which revealed that these four analytes are present in vivo for a relatively longer period of time and may result in a more durable effect.