Abstract
Flavonoids are the most abundant components in Salvia plebeia, with significant pharmacological antioxidant and hepatoprotective properties. Hispidulin and homoplantaginin are the main flavonoid components in S. Plebeia. In this study, we established an ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) to determine hispidulin and homoplantaginin in rat plasma samples, which were precipitated using acetonitrile-methanol (9:1, v/v). We used a UPLC HSS T3 (100 mm × 2.1 mm, 1.7 μm diameter) chromatographic column, an acetonitrile-water (containing 0.1% formic acid) mobile phase, and a gradient elution flow rate of 0.4 mL min−1 in an elution time of 4 min. We used electrospray ionization (ESI) detection in positive ion mode, and multiple reaction monitoring mode (MRM) for quantitative analysis: m/z 301 → 286 for hispidulin, m/z 463 → 301 for homoplantaginin, and m/z 465 → 303 for internal standard (IS). In pharmacokinetic studies, 24 rats were orally administered hispidulin and homoplantaginin (5 mg kg−1) and received sublingual intravenous injections (1 mg kg−1) at two different doses, four groups with six rats/group. Differences in hispidulin and homoplantaginin pharmacokinetics in rat plasma were evaluated. The calibration curve showed good linearity in the 0.5–1,000 ng mL−1 range, with r > 0.99. Precision, accuracy, recovery, matrix effects, and stability results all met standard biological sample detection requirements. Our pharmacokinetic studies showed hispidulin bioavailability was much higher than homoplantaginin at 17.8% and 0.1%, respectively.
Introduction
Salvia plebeia is also known as snow capsicum, fly grass, and holly, and comprises the whole grass component of S. plebeia R. Br [1–3]. It is cooling in nature and pungent to taste, effectively clears away heat and toxins, cools the blood, and dissipates blood stasis, diuresis, and swelling [4–7]. Previous pharmacological studies have shown that S. plebeia has anti-asthmatic, anti-bacterial, and anti-oxidant effects [8–12]. The plant mainly contains flavonoids, terpenes, phenolic acids, volatile oils, phytosterols, and other compounds [12, 13]. Flavonoids are the most abundant component in the plants and have significant pharmacological activities such as antioxidant and liver protective effects [14]. Hispidulin and homoplantaginin are the main flavonoid components in S. Plebeia. Homoplantaginin can be used to treat liver damage, improve endothelial insulin resistance, and inhibit tumor growth [15]. Hispidulin is the aglycone form of homoplantaginin; it has anti-osteoporosis properties, induces apoptosis in cancer cells, and is used to treat asthma [16, 17].
Previous studies reported that hispidulin and homoplantaginin levels in rat plasma could be determined using high performance liquid chromatography (HPLC) [18, 19], and their pharmacokinetics assessed after oral or intravenous (iv) intake. However, HPLC sensitivity is low, whereas ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) technology exhibits high sensitivity, low detection limits, low sample consumption, and is widely used for chemical composition, drug metabolism, and impurity identification analyses [20–23]. To the best of our knowledge, no study has reported the quantitation of hispidulin and homoplantaginin in plasma by UPLC-MS/MS. In this study, we established an UPLC-MS/MS method to determine hispidulin and homoplantaginin levels in rat plasma, investigated their pharmacokinetics, and calculated the absolute bioavailability of these compounds. The pharmacy basic research of hispidulin and homoplantaginin could provide scientific experimental basis.
Materials and methods
Reagents
Hispidulin (purity ≥98%, batch: MUST-21083002, Fig. 1a), homoplantaginin (purity ≥98%, batch: MUST-21010208, Fig. 1b), and spiraeoside (internal standard (IS), purity ≥98%, batch number: MUST-21042912, Fig. 1c) were purchased from Chengdu MUST Pharmaceutical Co., Ltd (Chengdu, China). Chromatographically pure acetonitrile and methanol were purchased from Merck Ltd (Darmstadt, Germany). Ultra-pure water (resistance >18 mΩ) was obtained from a Milli-Q purification system (Millipore, USA).
Chemical structure of hispidulin (a), homoplantaginin (b), and the internal standard (c)
Citation: Acta Chromatographica 35, 4; 10.1556/1326.2022.01082
Animals
Sprague Dawley rats (male, 220–250 g) were obtained from the Animal Experiment Center of Wenzhou Medical University (Wenzhou, China) and housed in separate cages with a 12-h light/12-h dark cycle for a week, under a temperature- and humidity-controlled laboratory conditions (23−25 °C and 50−70%, respectively). Diet was prohibited for 12 h prior to dosing of hispidulin and homoplantaginin while water was freely taken.
Instrument conditions
A Waters Acquity H-class UPLC and XEVO TQ-S micro-triple quadrupole tandem mass spectrometer was used to detect hispidulin and homoplantaginin (Milford, Ma, USA). Data acquisition and instrument control were performed using Masslynx 4.1 software (Waters Corp.).
Chromatographic conditions: chromatographic column was UPLC HSS T3 (100 mm × 2.1 mm, 1.7 μm) and column temperature was set at 40 °C. The mobile phase was acetonitrile-water (containing 0.1% formic acid), gradient elution flow rate was 0.4 mL min−1, and elution time was 4 min. Gradient elution procedure was: 0–0.2 min, acetonitrile 10%; 0.2–1.2 min, acetonitrile 10%–85%; 1.2–2.0 min, acetonitrile 85%; 2.0–2.5 min, acetonitrile 85%–10%; and 2.5–4.0 min, acetonitrile 10%.
Mass spectrometry conditions: nitrogen was used as a cone (50 L h−1) and desolvation gas (900 L h−1). Capillary voltage was 2.5 kV, ion source temperature was 150 °C, desolvation temperature was 450 °C, electrospray ionization (ESI) in positive ion mode detection, multiple reaction monitoring mode (MRM) was used for quantitative analysis, hispidulin m/z 301 → 286 (cone voltage 56 v, collision voltage 16 v), homoplantaginin m/z 463 → 301 (cone voltage 38 v, collision voltage 12 v), and IS m/z 465 → 303 (cone voltage 36 v, collision voltage 16 v) (Fig. 2).
Mass spectra of hispidulin (a), homoplantaginin (b), and the internal standard (c)
Citation: Acta Chromatographica 35, 4; 10.1556/1326.2022.01082
Standard curves
Stock solutions (500 μg mL−1) of hispidulin, homoplantaginin, and the IS, spiraeoside were prepared in methanol. Solutions were diluted in methanol to generate working solutions over a range of concentrations. All solutions were stored at 4 °C. Appropriate hispidulin and homoplantaginin working solution were added to blank rat plasma samples to generate 0.5, 2, 10, 20, 50, 100, 200, 500, and 1,000 ng mL−1 concentrations. The standard curve range was 0.5–1,000 ng mL−1. Quality control (QC) samples at three plasma concentrations (1, 90, and 900 ng mL−1) were similarly prepared.
Sample processing
To a tube, 50 μL plasma plus 150 μL acetonitrile-methanol (9:1, v/v) (containing 100 ng mL−1 IS) were added. Tubes were vortexed for 1 min and centrifuged (13,000 rpm at 4 °C for 10 min). Then, 100 μL supernatant was removed into the liner tube of an injection bottle. The injection volume for UPLC-MS/MS analysis was 2 μL.
Method validation
The selectivity was evaluated by analyzing a blank rat plasma, a blank rat plasma spiked with hispidulin, homoplantaginin, and the IS, and a rat plasma sample after oral administration of hispidulin and homoplantaginin spiked with IS.
A series of standard solutions of hispidulin and homoplantaginin (0.5–1,000 ng mL−1) with 100 ng mL−1 for IS were assayed under the same conditions as the plasma sample. The peak-area ratios of hispidulin and homoplantaginin to IS were detected, and the standard curve was developed by least squares linear fitting of the peak-area ratios to evaluate linearity.
The accuracy and precision were evaluated by determining six QC samples at the three levels of 1, 90, and 900 ng mL−1. The precision was expressed as relative standard deviation (RSD). The intra-day and inter-day precision of QC samples were calculated by measuring QC samples for three consecutive days. The accuracy of the intra-day and inter-day was then determined by the average value and the true value of QC samples.
The recovery was calculated by comparing the measured peak area of QC samples with that of the corresponding standard solution. The matrix effect was determined by comparing the measured peak area of the standard solution containing blank rat plasma after extraction with that of the corresponding standard solution.
Stability of hispidulin and homoplantaginin in plasma was investigated by analysis of QC samples at room temperature for 2 h, the frozen condition at −20 °C for 30 days and freeze/thaw cycles, respectively.
Pharmacokinetics
The sublingual iv. administration of hispidulin at 1 mg kg−1 and oral administration of 5 mg kg−1 was performed on six rats in two groups, respectively. The sublingual iv. administration of homoplantaginin at 1 mg kg−1 and oral administration of 5 mg kg−1 was performed on six rats in two groups, respectively. All experimental procedures and protocols were approved by the Animal Care Committee of Wenzhou Medical University (Wydw 2019-0982). At 0.0833, 0.25, 1, 2, 4, 6, 8, 12, and 24 h, we collected 0.2 mL tail vein blood into heparinized test tubes and centrifuged samples at 13,000 rpm for 10 min. Then, 50 μL plasma was transferred to a new tube and stored at −80 °C. Pharmacokinetic parameters were statistically calculated using pharmacokinetic software (DAS version 2.0).
Results
Selectivity
As indicated (Fig. 3), hispidulin, homoplantaginin, and IS retention time were 2.30, 1.98, and 1.92 min, respectively. They were effectively separated using optimized gradient elution procedures, and no interference from endogenous components was encountered during retention time.
Ultra-high performance liquid chromatography-tandem mass spectrometry of hispidulin, homoplantaginin, and the internal standard in rat plasma, (a) blank rat plasma, (b) blank rat plasma spiked with hispidulin, homoplantaginin, and internal standard, and (c) a rat plasma after oral administration of homoplantaginin
Citation: Acta Chromatographica 35, 4; 10.1556/1326.2022.01082
Standard curves
Hispidulin and homoplantaginin calibration curves in rat plasma showed good linearity in the 0.5–1,000 ng mL−1, with r > 0.99. The regression equation for hispidulin in rat plasma was: y1 = 0.0025 × 1 − 0.0018 (r = 0.9992), where ×1 was hispidulin concentration in plasma and y1 was the ratio of the hispidulin peak area to the IS. The regression equation for homoplantaginin in rat plasma was: y2 = 0.0019 × 2 + 0.0014 (r = 0.9984), where ×2 was homoplantaginin concentration in plasma and y2 was the ratio of the homoplantaginin peak area to the IS. The lower limit of hispidulin and homoplantaginin quantification in rat plasma was 0.5 ng mL−1 with the signal/noise ratio was 10, the detection limit was 0.1 ng mL−1 with the signal/noise ratio was 3.
Precision, accuracy, recovery, and matrix effects
The intra- and inter-day precision for hispidulin was within 13%, intra- and inter-day accuracies ranged from 90% to 104%, recovery was >87%, and matrix effects ranged from 88% to 100%. The intra- and inter-day precision for homoplantaginin was within 13%, intra- and inter-day accuracies ranged from 91% to 110%, recovery was >85%, and matrix effects ranged from 90% to 98% (Table 1).
Accuracy, precision, matrix effects and recovery of hispidulin and homoplantaginin in rat plasma
| Compound | Concentration (ng mL−1) | Accuracy (%) | Precision (RSD%) | Matrix effects (%) | Recovery (%) | ||
| Intra-day | Inter-day | Intra-day | Inter-day | ||||
| 0.5 | 93.1 | 90.2 | 12.8 | 11.6 | 88.0 | 89.6 | |
| Hispidulin | 1 | 95.8 | 101.4 | 8.9 | 5.7 | 95.6 | 91.7 |
| 90 | 98.2 | 103.3 | 8.3 | 6.0 | 99.7 | 87.8 | |
| 900 | 103.7 | 98.9 | 9.9 | 9.0 | 95.7 | 91.5 | |
| 0.5 | 91.1 | 109.2 | 12.6 | 13.0 | 93.0 | 92.9 | |
| Homoplantaginin | 1 | 96.0 | 105.2 | 7.1 | 10.7 | 90.6 | 91.9 |
| 90 | 103.5 | 94.9 | 5.3 | 9.9 | 97.3 | 85.6 | |
| 900 | 98.2 | 106.3 | 9.1 | 5.9 | 97.5 | 85.4 | |
Stability
Plasma samples were placed in an autosampler for 2 h, and were pretreated and placed at room temperature for 24 h. After three freeze-thaw cycles, stability tests were performed at −20 °C for 30 d. Accuracy of hispidulin was between 90% – 112% and the RSD was within 12%. Accuracy of homoplantaginin was between 88% – 113% and the RSD was within 10% (Table 2). These data indicated hispidulin and homoplantaginin displayed good stability.
Stability of hispidulin and homoplantaginin in rat plasma
| Compound | Concentration (ng mL−1) | Autosampler (4 °C, 12 h) | Ambient (2 h) | −20 °C (30 d) | Freeze-thawing | ||||
| Accuracy | RSD | Accuracy | RSD | Accuracy | RSD | Accuracy | RSD | ||
| 1 | 96.1 | 5.2 | 104.0 | 7.5 | 111.1 | 10.9 | 111.0 | 10.1 | |
| Hispidulin | 90 | 98.1 | 4.4 | 93.0 | 4.5 | 90.3 | 7.2 | 98.0 | 4.5 |
| 900 | 101.2 | 7.2 | 97.9 | 9.4 | 100.4 | 7.0 | 102.6 | 7.1 | |
| 1 | 104.5 | 4.2 | 107.1 | 8.4 | 112.6 | 7.7 | 95.5 | 8.2 | |
| Homoplantaginin | 90 | 103.7 | 7.7 | 104.1 | 8.9 | 91.3 | 9.7 | 108.5 | 7.8 |
| 900 | 97.3 | 2.0 | 99.8 | 6.9 | 94.1 | 9.0 | 88.2 | 8.1 | |
Pharmacokinetics
Concentration-time curves for hispidulin and homoplantaginin in rat plasma were shown in Fig. 4. The main pharmacokinetic parameters are listed in Table 3. The oral bioavailability of hispidulin and homoplantaginin was low, at 17.8% and 0.1%, respectively.
Concentration-time curves in rats after intravenous (iv, 1 mg kg−1) and oral (po, 5 mg kg−1) administration of hispidulin (a) and homoplantaginin (b)
Citation: Acta Chromatographica 35, 4; 10.1556/1326.2022.01082
Main pharmacokinetic parameters after intravenous (IV) and oral administration (PO) hispidulin and homoplantaginin in rats
| Compound | Groups | AUC(0–t) ng mL−1*h | AUC(0–∞) ng mL−1 *h | t1/2z h | CLz/F L/h/kg | Vz/F L kg−1 | Cmax ng mL−1 |
| Hispidulin | PO, 5 mg kg−1 | 33.1 ± 4.2 | 34.3 ± 4.5 | 2.3 ± 0.4 | 148.0 ± 22.7 | 486.5 ± 73.4 | 7.1 ± 0.9 |
| IV, 1 mg kg−1 | 37.2 ± 4.1 | 38.4 ± 4.0 | 2.6 ± 0.8 | 26.3 ± 2.5 | 97.1 ± 33.1 | 20.2 ± 3.1 | |
| Homoplantaginin | PO, 5 mg kg−1 | 2.7 ± 0.7 | 2.8 ± 0.7 | 1.4 ± 0.2 | 1868.5 ± 430.2 | 3736.4 ± 1281.9 | 1.3 ± 0.3 |
| IV, 1 mg kg−1 | 472.5 ± 68.2 | 510.0 ± 83.0 | 9.1 ± 2.1 | 2.0 ± 0.4 | 26.0 ± 5.2 | 553.8 ± 31.2 |
Discussion
To generate optimized UPLC-MS/MS conditions, both positive and negative ion modes were tested [24, 25]. Hispidulin and homoplantaginin displayed higher response in positive ion mode, mass conditions indicated better sensitivity and signal intensities. After this, mass parameters adequately met the requirements for the accurate quantification of substances in biological samples. Using standard samples, capillary voltage and energy were optimized, and finally, hispidulin m/z 301 → 286 (cone voltage 56 v, collision voltage 16 v), homoplantaginin m/z 463 → 301 (cone voltage = 38 v, collision voltage 12 v), and IS m/z 465 → 303 (cone voltage 36 v, collision voltage 16 v).
Chromatographic separation conditions greatly influenced retention time, peak shape, and response values. We also investigated different BEH C18 and HSS T3 chromatographic columns combined with different mobile phases, and found that HSS T3 (2.1 mm × 100 mm, 1.8 μm) and an acetonitrile-0.1% formic acid mobile phase displayed good chromatographic peaks. Elution gradients were also optimized to determine final elution conditions. Thus, our column and mobile conditions generated relatively good hispidulin, homoplantaginin, and IS separation.
For biological sample analyses, IS should display good solubility, have no chemical reactions with test samples, and peak positions should be close to analyte peak positions. We tested spiraeoside, isoscoparin, and flavanomarein, however, only spiraeoside generated good results. Hispidulin and homoplantaginin retention time were 2.30 and 1.98 min, respectively, while spiraeoside peaked at 1.92 min. Similarly, recoveries were stable. Therefore, spiraeoside was selected as an effective IS.
Commonly used biological sample pretreatment methods include: liquid-liquid extraction (LLE), solid phase extraction (SPE), and protein sedimentation (PPT). SPE has complicated operation steps, extraction cartridges were expensive, and the cost burden was high. LLE operations required specific professional skills. PPT was advantageous in terms of simple operation and good extraction recovery. In our study, the effect of different protein precipitants on extraction efficiency was investigated. Blank rat plasma was used to prepare plasma samples containing 100 ng mL−1 hispidulin and homoplantaginin. Acetonitrile, methanol, 10% trichloroacetic acid, and methanol-acetonitrile (1:1, v/v) precipitants were also investigated. Methanol-acetonitrile (1:9, v/v) had the highest extraction efficiency, so it was used for further analyses.
Flavonoids were mainly absorbed after hydrolysis to aglycones by β-D-glucuronidases in gastrointestinal flora, with most flavonoid glycosides bio-transformed by intestinal bacteria, thereby enhancing metabolite biological activity, improving water solubility, and increasing bioavailability [19]. Lee et al. observed that hesperidin produced during intestinal flora metabolism displayed strong anti-allergic effects when compared with normal hesperidin which had no such effects [26]. Gong et al. administered the same quantity of baicalein and baicalin to rats, and observed that the relative bioavailability of baicalein was 200.9% that of baicalin, suggesting baicalin was more easily absorbed after hydrolysis to aglycone baicalein [27]. In our study, after hispidulin and homoplantaginin gavage, hispidulin levels were approximately 10 times higher than homoplantaginin, suggesting hispidulin adsorption was much better than homoplantaginin. Whether post-drug efficacy was enhanced remains unclear, therefore more research is required in this area.
Conclusion
We established a UPLC-MS/MS method to determine hispidulin and homoplantaginin in rat plasma. Accuracy, precision, selectivity, and linearity were all verified and acceptable. The method was applied to pharmacokinetics in rat plasma and showed that bioavailability of hispidulin was much higher than homoplantaginin.
Conflict of interest statement
The author(s) declare(s) that there is no conflict of interest regarding the publication of this paper.
Data availability statement
The data used to support the findings of this study are included within the article.
Acknowledgements
This work was supported by the Ningbo Natural Science Foundation (202003N4265).
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