Determination of Tenacissoside G, Tenacissoside H, and Tenacissoside I in Rat Plasma by UPLC-MS/MS and Their Pharmacokinetics

An ultra-performance liquid chromatography/tandem mass spectrometry (UPLC-MS/MS) method was developed for the determination of tenacissoside G, tenacissoside H, and tenacissoside I in rat plasma. The rat plasma was treated with liquid-liquid extraction using ethyl acetate. The determination was performed on the UPLC HSS T3 column (50 mm × 2.1 mm, 1.8 μm) with a mobile phase consisting of acetonitrile-water (containing 0.1% formic acid) and gradient elution at a flow rate of 0.4 mL/min. Electrospray (ESI) positive ion mode detection and multireaction monitoring (MRM) quantitative analysis were performed. A total of 36 rats were given tenacissoside G, tenacissoside H, and tenacissoside I, respectively, orally (5 mg/kg) and intravenously (1 mg/kg), with 6 rats in each group, to evaluate the pharmacokinetic difference of tenacissoside G, tenacissoside H, and tenacissoside I in rats. The calibration curves showed good linearity in the range of 5–2000 ng/mL, where r was greater than 0.99. The results of precision, accuracy, recovery, matrix effect, and stability met the requirements of biological sample detection methods. The established UPLC-MS/MS method was successfully applied to pharmacokinetic studies of tenacissoside G, tenacissoside H, and tenacissoside I, and the bioavailability was 22.9%, 89.8%, and 9.4%, respectively.


Introduction
Marsdenia tenacissima, also known as glaucescent fssistigma root, Rosa banksiae f. lutea (Lindl.)Rehd, etc., is the dry stem and rattan of Marsdenia tenacissima (Roxb) Wight et Am., a plant belonging to the family Asclepiadaceae [1][2][3], which was originally published in the Herbal Medicines of Southern Yunnan and is now published in the Chinese Pharmacopoeia 2010 edition [4,5].It is mainly distributed in Guizhou, Yunnan, Sichuan, Guangxi, and other places.Marsdenia tenacissima tastes bitter and slightly cold.It has the efects of clearing heat and detoxifying, relieving cough, and asthma, dispersing knots and relieving pain, and fghting cancer.Te series of preparations made from its single medicinal material are widely used in clinics [6][7][8].
Te roots, stems, and leaves of Marsdenia tenacissima can be used as medicine.Its efective components are steroidal glycosides, alkaloids, and polysaccharides [9][10][11].Its active antitumor components are mainly steroidal components.It is reported that Marsdenia tenacissima polysaccharides and some fat-soluble components also have antitumor efects [12].At present, dozens of steroids have been isolated from this plant.Te C21 steroidal glycosides are mostly white crystals or powder, which mainly exist in the plants of Asclepiadaceae.Te main antitumor active component of Marsdenia tenacissima is C21 steroidal glycosides [13].Polysaccharide and some fat-soluble components also have antitumor efects.At present, more than 40 kinds of C21 steroidal glycosides have been isolated from the plant, which are a class of compounds formed by a class of steroidal derivatives of glycosides and 2-deoxysugars, and the sugar chain contains up to 6 sugars.Tese compounds contain a variety of aglycones with diferent structures [8,9], of which there are 6 main confguration aglycones.In vivo pharmacokinetics of tenacissoside H and tenacissoside I have been reported in literature [14][15][16], but bioavailability has not been reported.
High-performance liquid chromatography tandem mass spectrometry (LC-MS/MS) technology has the advantages of high sensitivity, low detection limit, and small sample consumption and is widely used in drug analysis of chemical composition, drug metabolism, and impurity identifcation [17,18].UPLC, which columns with small particle sizes and under ultra-high pressure, maintains the basic principles of a traditional HPLC system but demonstrates improved separation efciency and speed [19,20].
Terefore, this study was to establish an UPLC-MS/MS for the determination of tenacissoside G, tenacissoside H, and tenacissoside I in rat plasma and study the pharmacokinetics and bioavailability to provide a scientifc experimental basis for the basic research of clinical pharmacy.

Experimental
2.1.Reagents and Animals.Tenacissoside G, tenacissoside H, tenacissoside I, and astragaloside IV (internal standard) (purity ≥98%, Figure 1) were all purchased from Chengdu Master Pharmaceutical Co., Ltd.Acetonitrile and methanol in chromatographic purity were purchased from Merck.Ultra-pure water (resistance >18 mΩ) was prepared by the Milli-Q purifcation system in the United States.Sprague-Dawley (SD) rats (220-250 g) were from the Animal Experimental Center of Wenzhou Medical University.

Instrument Condition.
A Waters XEVO TQ-S microtriple quadrupole series mass spectrometer was used for the detection of tenacissoside G, tenacissoside H, and tenacissoside I.

Standard Curve.
Tenacissoside G, tenacissoside H, tenacissoside I, and astragaloside IV reserve solution (500 μg/mL) were prepared with methanol, respectively.Tenacissoside G, tenacissoside H, and tenacissoside I working solution was obtained by diluting the reserve solution with methanol.Both the reserve solution and working solution were stored at 4 °C.Appropriate amount of tenacissoside G, tenacissoside H, and tenacissoside I working solution was added to the blank rat plasma, and then the tenacissoside G, tenacissoside H, and tenacissoside I in rat plasma were 5, 10, 20, 50, 100, 200, 500, 1000, and 2000 ng/ mL.Tree quality control (QC) samples with plasma concentrations (8, 180, and 1800 ng/mL) were prepared by the same method.

Sample Handling.
A plasma sample of 100 μL was added to a 1.5 mL microcentrifuge tube, then 10 μL of astragaloside IV (1.0 μg/mL) and 1.0 mL ethyl acetate were added, vortex was mixed for 1.0 min, and it was centrifuged (13,000 rpm, 4 °C, 5 min).Te organic phase was transferred into another tube and evaporated to dryness at 40 °C under a gentle stream of nitrogen.Te residue was reconstituted in 100 μL methanol and centrifuged at 13,000 rpm for 5 min.Te supernatant was pipetted to an auto sampler vial, and 2 μL was injected into the UPLC-MS/MS for analysis.

Pharmacokinetic Study.
Tenacissoside G, tenacissoside H, and tenacissoside I were given sublingual intravenous administration (iv) of 1 mg/kg and oral administration (po) 5 mg/kg, respectively, with 6 rats in each group, for a total of 36 rats.All experimental procedures and protocols were approved by the Animal Care Committee of Wenzhou Medical University (xmsq 2023-0689).Te 0.4 mL blood was collected from the caudal vein at 0.083 3, 0.5, 1, 2, 3, 4, 6, and 8 h for tenacissoside G; 0.083 3, 0.5, 1, 2, 3, 4, 6, 8 and 12 h for tenacissoside H; and 0.083 3, 0.5, 1, 2, 3, 4, and 6 h for tenacissoside I, collected in heparinized test tubes and centrifuged at 13000 r/min for 10 min.100 μL of the plasma was then transferred to a new 1.5 mL microcentrifuge tube and held at −80 °C prior to analysis.Pharmacokinetic parameters were statistically calculated using the pharmacokinetic software (DAS 2.0 version).

Result
3.1.Selectivity.Te retention times of tenacissoside G, tenacissoside H, tenacissoside I, and astragaloside IV were 3.32, 3.42, 3.41, and 2.63 min, as shown in Figure 2, respectively.Te optimized gradient elution procedure was used to isolate tenacissoside G, tenacissoside H, and tenacissoside I, efectively, and no interference of endogenous 2 International Journal of Analytical Chemistry components was observed in the retention time of tenacissoside G, tenacissoside H, and tenacissoside I. Tis method has good selectivity.

Standard Curve.
Te calibration curves of tenacissoside G, tenacissoside H, and tenacissoside I in rat plasma showed good linearity in the range of 5-2000 ng/mL, with r greater than 0.99.Te typical regression equation of the tenacissoside G in rat plasma was as follows: y1 � 0.0045x1 + 0.0102 (r � 0.9976), x1 was the concentration of tenacissoside G in plasma, and y1 was the ratio of tenacissoside G peak area to the internal standard.Te typical regression equation for tenacissoside H in rat plasma was y2 � 0.0046x2 + 0.0022 (r � 0.9986), where x2 was the concentration of tenacissoside H in plasma, and y2 was the ratio of the tenacissoside H peak area to the internal standard.Te typical regression equation for tenacissoside I in rat plasma was y3 � 0.0020x3 + 0.0097 (r � 0.9977), x3 was the concentration of tenacissoside I in plasma, and y3 was the ratio of tenacissoside I peak area to internal standard.Te lower limit of quantitation of tenacissoside G, tenacissoside H, and tenacissoside I in rat plasma was 5 ng/mL, and the detection limit was 1.5 ng/mL.

Precision, Accuracy, Recovery, and Matrix Efect.
Te intraday and interday precision of tenacissoside G was within 10%, the accuracy was 90% to 111%, the recovery was over 92%, and the matrix efect was in the range of 94% to 109% (Table 1).Te intraday and interday precision of te nacissoside H was within 13%, the accuracy was 88% to 115%, the recovery was above 88%, and the matrix efect was in the range of 101% to 108% (Table 1).
Te intraday and interday precision of tenacissoside I was within 15%, the accuracy was 88% to 110%, the recovery was above 80%, and the matrix efect range was 91% to 99% (Table 1).

Stability.
Te plasma samples of rats were stored in an automatic injector for 2 h, pretreated, and placed at room temperature for 24 h.Te plasma samples underwent three freeze-thawing cycles, and the stability test was conducted at −20 °C for 30 days.Te accuracy of tenacissoside G was 88%-112%; RSD was within 13%.Te accuracy of tenacissoside H was 88%-111%; RSD was within 15%.Te accuracy of tenacissoside I was 88%-111%; RSD was within 15% (Table 2).Te results indicated that tenacissoside G, tenacissoside H, and tenacissoside I were stable.

Pharmacokinetic Study. Te concentration-time curves
of tenacissoside G, tenacissoside H, and tenacissoside I in rat plasma are shown in Figure 3. Te main pharmacokinetic parameters are listed in Table 3, and oral bioavailability was 22.9%, 89.8%, and 9.4%, respectively.

Discussion
In order to obtain the best mass spectrum conditions, the positive and negative ion modes were used for monitoring.Te responses of tenacissoside G, tenacissoside H, and tenacissoside I were higher in the positive ion mode than the negative ion modes.After optimizing various parameters, the mass spectrum parameters can meet the requirements of accurate quantifcation of efective substances in biological samples.Trough the standard sample, the capillary voltage and the collision energy were optimized.Te commonly used biological sample pretreatment methods were liquid-liquid extraction (LLE) [21][22][23][24], solidphase extraction (SPE) [25,26], and protein precipitation method (PPT).Te SPE method has complicated operation steps and a high extraction column price.LLE could efciently extract target substances, especially for lowconcentration samples, with higher extraction efciency  During quantitative analysis, it is necessary to add an internal standard substance of known concentration as a quantitative reference for the compounds to be measured in the sample.Te internal standard substance should have similar physical and chemical properties to the compound to be tested, and be stable in the sample, and be easy to detect and quantify.Commonly used internal standard substances include isotopelabeled compounds and structural analogues.Astragaloside IV has similar physical and chemical properties to tenacissoside, and it was selected as the internal standard.
Zhao et al. studied the plasma concentration and pharmacokinetic process of tenacissoside A in rats by the LC-MS/MS method [27].Medroxyprogesterone acetate was used as the internal standard.Te oral bioavailability of the drug was low (2.6%), the elimination was faster, and the frst-pass efect was obvious.Li established a LC-MS/MS method for simultaneous determination of garcinia extract in rat plasma samples of tenacissoside B, tenacissoside H, tenacissoside I, cafeic acid, cryptochlorogenic acid, chlorogenic acid, and neochlorogenic acid [16].Digoxin was used as an internal standard reference.Zeng et al. have developed a LC-MS/MS method for simultaneous determination of three isomerized gestrins (17β-tenacigenin B, tenacigenine B, and tenacigenine A) and their corresponding glycosides (tenacissoside A and tenacissoside B) in rat plasma [14].After dexamethasone acetate was added as an internal standard, a simple liquid-liquid extraction technique was used.Tis method was successfully applied to the pharmacokinetic study after intravenous injection of Xiao-Ai-Ping in rats.However, these methods did not study the bioavailability of tenacissoside B, tenacissoside H, and tenacissoside I.

Conclusion
In this study, the UPLC-MS/MS technique was established for the determination of tenacissoside G, tenacissoside H, and tenacissoside I in rat plasma in the range of 5-2000 ng/ mL.Te rat plasma was treated with liquid-liquid extraction using ethyl acetate, and astragaloside IV was used as internal standard.Te selectivity, linearity, precision, accuracy, recovery, and stability of this method have been verifed, and it has been applied to the pharmacokinetic study of tenacissoside G, tenacissoside H, and tenacissoside I in rats, and the bioavailability was calculated to be 22.9%, 89.8%, and 9.4%, respectively.

Table 1 :
Accuracy, precision, matrix efect, and recovery of tenacissoside G, tenacissoside H, and tenacissoside I in rat plasma.

Table 2 :
Stability of tenacissoside G, tenacissoside H, and tenacissoside I in rat plasma.