Simultaneous determination of multiple components in rat plasma by UPLC-MS/MS for pharmacokinetic studies after oral administration of Pogostemon cablin extract

Introduction: Pogostemon cablin (PC) is used in traditional Chinese medicine and food, as it exerts pharmacological effects, such as immune-modulatory, antibacterial, antioxidant, antitumor, and antiviral. Currently, the pharmacokinetics (PK) studies of PC mainly focus on individual components. However, research on these individual components cannot reflect the actual PK characteristics of PC after administration. Therefore, the simultaneous determination of multiple components in rat plasma using UPLC-MS/MS was used for the pharmacokinetic study after oral administration of PC extract in this study, providing reference value for the clinical application of PC. Methods: In the present study, a reliable and sensitive ultra-high performance liquid chromatography/tandem mass spectrometry (UPLC-MS/MS) method was developed and validated for the simultaneous determination of 15 prototype components (vanillic acid, vitexin, verbascoside, isoacteoside, hyperoside, cosmosiin, apigenin, β-rhamnocitrin, acacetin, ombuin, pogostone, pachypodol, vicenin-2, retusin, and diosmetin-7-O-β-D-glucopyranoside) in rat plasma after oral administration of the PC extract. Plasma samples were prepared via protein precipitation using acetonitrile, and icariin was used as the internal standard (IS). Results: The intra-day and inter-day accuracies ranged from −12.0 to 14.3%, and the precision of the analytes was less than 11.3%. The extraction recovery rate of the analytes ranged from 70.6−104.5%, and the matrix effects ranged from 67.4−104.8%. Stability studies proved that the analytes were stable under the tested conditions, with a relative standard deviation lower than 14.1%. Conclusion: The developed method can be applied to evaluate the PK of 15 prototype components in PC extracts of rats after oral administration using UPLC-MS/MS, providing valuable information for the development and clinical safe, effective, and rational use of PC.

plays an important role in the determination of the drug's kinetics and bioavailability.Notably, PK is very important for evaluating the absorption properties of different active components of traditional Chinese medicine (TCM).Moreover, the development of sensitive and reliable biological sample analysis technology to simultaneously determine multiple active ingredients in vivo is a hotspot in the PK research of TCM extracts, due to the complex composition and significant differences in the content (Cao et al., 2021).Therefore, an accurate and selective bioanalytical method is urgently required for the simultaneous determination of multiple biological components in plasma to understand the characteristics and diversity of the PK properties of PC.

Chromatography and mass spectrometry (MS)
The analyte was separated and detected through the utilization of a UPLC-MS/MS system, consisting primarily of the Agilent-1290 high-performance liquid chromatography system (Agilent, United States) and the Agilent-6470 triple quadrupole tandem mass spectrometer (Agilent, United States).
Mass spectrometry analysis was conducted in the multiple reaction monitoring (MRM) mode with both positive and negative ionization (Zhu et al., 2022).The parameters for the mass spectrometer included drying gas (N 2 ) temperature, gas flow rate, atomizer pressure, and capillary voltage of 320 °C, 7 L/ min, 35 psi, and 3500 V, respectively.The detailed MRM parameters can be found in Supplementary Table S1.

Preparation of plasma sample
The 100 μL plasma sample was combined with 20 μL of ME, 20 μL of IS (500 ng/mL), and 2.5 μL of FA, followed by vortexing for 1 min.This mixture underwent extraction with 600 μL ACN through vortexing at room temperature for 3 min.Subsequent to centrifugation at 14,000 rpm for a period of 10 min, the resulting supernatant was moved to another tube and dried under a stream of nitrogen.The dried residue was dissolved in 100 μL ME-ACN (50:50 v/v), followed by vortexing for 5 min, and subsequent centrifugation at 14,000 rpm for 10 min.Finally, 20 μL of the supernatant was used for analysis.

Specificity
In order to assess specificity, chromatograms of rat plasma samples without any added analytes, plasma with added analytes, and post-dosing plasma samples from rats following oral ingestion of PC were analyzed.

Linearity and LLOQ
By graphing the correlation between the peak area ratios of each component to IS versus the concentration of those particular analytes to obtain calibration curves.The regression association was depicted utilizing a linear formula with a weighting factor of 1/ x 2 .The determination of LLOQ relied on the baseline noise, ensuring a signal-to-noise ratio of around ten.

Extraction recovery and matrix effect
Analyzing the peak areas of QC samples and the peak areas of the post-extraction mixed samples to measure the extraction recovery rates at three concentrations.Matrix effects were determined by comparing the peak areas of QC samples in post-extracted mixed samples to those in standard solutions, calculating the ratio for evaluation.

Stability
Plasma samples were retained for 12 h in an auto-sampler, exposed to room temperature for 4 h, undergoing three freezethaw cycles, and stored at −80 °C for a period of 1 week to assess the stability of all compounds.

PK study
Six male rats (220 ± 10 g) were utilized in this experiment and were not restricted from drinking water but fasted for 12 h before the investigation.A concentration of 0.5 g/mL of PC extract was obtained by dissolving in 0.5% CMC-Na aqueous solution.The PC suspension of the 4.0 g/kg dose was given to rats by single oral administration.Samples of blood (300 μL) were obtained from the orbital venous plexus prior to and following oral dosing at 0, 0.03, 0.08, 0.25, 0.5, 0.75, 1, 2, 6, 10, 12, 24, 36, and 48 h.Subsequently, the plasma collected underwent centrifugation at a speed of 7000 rpm for a duration of 10 min.It was then moved to clean tubes and kept at a temperature of −80 °C.

Data analysis
Data in this study was presented as the mean ± standard deviation (SD).The MassHunter Workstation software (version B.09.00, Agilent, United States) was utilized for determining the plasma levels of the 15 analytes.Additionally, PK parameters were analyzed using DAS 3.0 Software (Medical College of Wannan, China).

Optimization of chromatography and MS
To improve the separation of the 15 compounds, an investigation was conducted on the impact of different columns, including ACQUITY UPLC BEH C18 (2.1 × 100 mm, 1.7 µm) and ACQUITY UPLC HSS T3 (2.1 × 100 mm, 1.8 µm), on the chromatographic peaks and retention times.The findings revealed that the ACQUITY UPLC BEH C18 (2.1 × 100 mm, 1.7 µm) column provided superior separation capability for the 15 compounds.Different mobile phases were also evaluated, such as 0.1% FA/water-ACN, 0.1% FA/water-ME, water-ME, and water-ACN, to optimize the separation of all compounds.The findings demonstrated that the 0.1% FA/water-ACN proved to be superior in improving the separation and peak profiles of the compounds.All 15 components and IS were eluted successfully within a 7-min, and with no observed interference peaks.
Optimizing the primary parameters of the MS was crucial in enhancing the response of compounds.In positive ion mode, the

Sample preparation
The study evaluated four different extraction methods to determine the most effective method for preparing plasma samples, including protein precipitation using ME or ACN, ethyl acetate liquid-liquid extraction, and extraction using a mixture of ME and ACN (v/v = 1:4).The findings indicated that the protein precipitation using ACN showed higher extraction recovery rates for the 15 analytes tested.Additionally, different volumes (400, 600, 800, and 1,000 μL) of precipitated solvent were used to assess the extraction recovery and matrix effect.The outcomes demonstrated that the extraction recovery and matrix effects of the ACN-protein precipitation using 600 μL satisfied the criteria for analyzing biological specimens, with no interference from endogenous compounds.Additionally, the impacts of various reconstitution solvents such as ME, ACN, 50% ME, and ME-ACN (v/v = 1:1, 1:4, and 4:1) were assessed.The results indicated that the redissolution effectiveness was optimal with a ME-ACN mixture of (v/v = 1:1).

Specificity
The MRM chromatograms in Figure 1 displayed the rat plasma samples without any added analytes (A), plasma samples combined with the 15 compounds and IS (B), and post-dosing plasma samples from rats following oral ingestion of PC (C).The results suggested no noticeable interference peaks were noted at the retention time of the 15 compounds and IS.

Precision and accuracy
The precision and accuracy of QC samples at three concentrations are provided in Table 2.The RSD of intra-and inter-day were below 11.3%, the RE of intra-day ranged from −12.0%-14.3%, and the inter-day ranged from −6.5%-13.2%.These findings indicated that is accurate and precise to a satisfactory degree.

Extraction recovery and matrix effect
As listed in Table 3, the 15 analytes and the IS displayed extraction recovery and matrix effects within the ranges of 70.6%-104.5%,and 67.4%-104.8%,respectively.These findings revealed that the method's extraction recovery and matrix effects were accurate and satisfactory.

Stability
As shown in Table 4, the RSD values for the stability of all analytes tested were less than 14.1%, indicating that they were adequately stable across different conditions.This further suggested that the established UPLC-MS/MS method could effectively measure the 15 components in rat plasma.

PK study
Following oral administration of the PC extract, 15 plasma constituents were analyzed using the validated method.Unfortunately, certain analytes such as vitexin, hyperoside, acacetin, and diosmetin-7-O-β-D-glucopyranoside were only detected in the initial plasma samples, leading to difficulties in obtaining a complete PK profile.As a result, these analytes were not included in our analysis.The mean plasma concentration- Frontiers in Pharmacology frontiersin.org09 Zhu et al. 10.3389/fphar.2024.1293464time curves for the remaining 11 analytes are displayed in Figure 2, with corresponding PK parameters detailed in Table 5.
The concentration maximum (T max ) of vanillic acid, verbascoside, isoacteoside, cosmosiin, apigenin, βrhamnocitrin, ombuin, pogostone, pachypodol, vicenin-2, and retusin were 1.4, 0.4, 0.5, 0.5, 0.3, 0.5, 0.6, 1.9, 0.8, 0.3 and 0.9 h, respectively.The T max of all components was within 2 h, indicating that the absorption of these compounds happened quickly.Pogostone was rapidly absorbed in rats after oral administration, potentially attributed to its low polarity profile and small molecule size (Liu et al., 2012;Chen et al., 2013).Verbascoside and isoacteoside could be rapidly absorbed in vivo, which might be related to their relatively large polarity, and previous research has shown comparable PK characteristics.(Zheng et al., 2015).The plasma half-life (T 1/2 ) of pogostone was 0.8 ± 0.5 h, which was relatively short compared to other compounds.It is speculated that this might be due to its distribution and elimination rapidly in rats (Zheng et al., 2015).The area-under-the-curve (AUC) for pogostone significantly exceeded that of the other constituents, suggesting that pogostone exhibited a higher plasma exposure level, correlating with its abundant PC content.Additionally, the concentration maximum (C max ) of pogostone was 37204.0 ± 10,901.0ng/mL, coupled with its substantial exposure in vivo, suggesting that pogostone may be the mian active component in the PC extract (Li et al., 2021).Furthermore, the maximum concentration (Cmax) of pogostone was measured at 37204.0 ± 10,901.0ng/mL.
The apigenin, β-rhamnocitrin, and pachypodol have a double-peak phenomenon in Figure 2, which is likely due to enterohepatic circulation (Wang et al., 2014).Additionally, the absorption of drugs in the gastrointestinal tract is a complex process that is influenced by many physical, chemical, and physiological factors.There are multiple absorption sites in different parts of the gastrointestinal tract, but due to the different permeability of the inner membrane of the cavity to drugs at different sites, the absorption rates and times differ following oral administration.Consequently, absorbed drugs overlap in the blood, creating a bimodal phenomenon (Zhou, 2003).
PK is an indispensable strategy for understanding the behavior in vivo after drug administration, which is of great significance in elucidating the mechanism of action, reducing toxic and side effects, optimizing the drug administration program, and guiding the clinical application of drugs (Jin et al., 2019;Liu et al., 2021).Currently, the PK studies of PC mainly focus on a few components, such as pogostone and verbascoside (Li et al., 2012;Chen et al., 2013;Huo et al., 2016).However, research on these individual components cannot reflect the actual PK characteristics of PC after administration.Therefore, in this study, the simultaneous determination of multiple components in rat plasma and PK study by UPLC-MS/MS, which is crucial in elucidating the pharmacological substance basis, mechanism of action, and optimal dosing regimen of PC, and may provide a reference for the clinical application of PC.
Notably, most of the current PK studies are in normal animals, and there are fewer in model animals.Numerous studies have shown that disease states may cause significant alterations in PK parameters (Chen et al., 2022;Liu et al., 2022;Xiong et al., 2023).Since the drugs are mainly used in the pathological state of the body, it is more meaningful to study the PK of TCM in pathological states (Luo et al., 2014).Moreover, PK and pharmacodynamics (PD) are two important interrelated and inseparable aspects in the field of pharmacological research of TCM.PK/PD modeling is extensively utilized in both preclinical and clinical drug research, which aids in gaining a comprehensive and precise Frontiers in Pharmacology frontiersin.orgunderstanding of drug effectiveness over time and plasma concentration.Additionally, it provides a valuable reference for optimizing clinical dosage, improving efficacy, and minimizing adverse effects.Therefore, it is vital to explore the ideas and methods of PK-PD modeling of TCM to elucidate the nature and laws (Zhang Z. et al., 2016;Zhu et al., 2023).Only normal rats were investigated in this study, while the PK of PC in model animals was lacking.In the future, The PK research of PC should focus on studies in disease states and develop PK-PD models to elucidate the pharmacodynamic basis and mechanism of action of PC.

Conclusion
In this study, a UPLC-MS/MS method was developed to simultaneously measure 15 components in rat plasma following the oral administration of PC extract.The method has the benefits of uncomplicated sample preparation and the simultaneous analysis of multiple components within a brief timeframe.Furthermore, the method is specific, stable, and reliable.Moreover, PK results are crucial in elucidating the pharmacodynamic material basis, mechanism of action, and optimal dosing regimen of PC, and may provide guidance for the future advancement and clinical application of PC.Mean plasma concentration-time profiles in rats after oral administration of PC extract.

TABLE 1
Standard curve regression equation, linear range and LLOQ of 15 components.

TABLE 2
Accuracy and precision of 15 components in rat plasma (n = 6).

TABLE 2 (
Continued) Accuracy and precision of 15 components in rat plasma (n = 6).

TABLE 3
Extraction recovery and matrix effects of 15 components in rat plasma (n = 6).

TABLE 3 (
Continued) Extraction recovery and matrix effects of 15 components in rat plasma (n = 6).