Standards of Tac (Lot: S31618) (purity>98%), deoxyschizandrin (Lot:20210612) (purity>98%), and bifendatatum (Lot: Y06O7C22268) (internal standard, IS) (purity>98%) were all provided by Shanghai Yuanye Bio-Technology CO., Ltd (Shanghai, China), and the Wuzhi capsule was purchased from Sichuan Hezheng Pharmaceutical Co., Ltd. Acetonitrile and methanol in mass spectrometry grade were purchased from Merck Company (Kenilworth, United States of America). Formic acid in analytical grade was obtained from Tedia Company (Fairfield, United States of America). HPLC-grade isopropanol was obtained from Shanghai Titan Technology CO., Ltd. (Shanghai, China), and distilled water was supplied by Watsons Distilled Water CO., Ltd. (Shenzhen, China).

All analyses were performed with a model 1,290 series UHPLC system equipped with an Agilent G1969-80230 online degasser, a G4220A binary pump, a G4226A autosampler, and a G1316C column oven, which was interfaced with an Agilent G6460A triple-quadrupole mass spectrometer and the mass spectrometer was equipped with electrospray ionization source (Agilent Technologies, Santa Clara, United States of America). All raw data were acquired and analyzed using Agilent Mass Hunter data processing software (version B.06.00). A VX230 vortex-mixer was purchased from Labnet (Edison, New Jersey, United States of America); a 5810R low temperature high speed centrifuge and single lane pipette are products of Eppendorf Company (Eppendorf, Hamburg, Germany). The SK7200H ultrasound instrument is provided by Shanghai Kedao Ultrasonic Instrument Co., Ltd (Shanghai, China). The standards were weighed using an electronic balance provided by Sartorius Company (CPA225D 1/ppm, Gottingen, Germany).

The chromatographic separation of two analytes was achieved in an Agilent ZORBAX SB-C18 column (2.1 mm * 100 mm, 3.5 μm, Agilent, MA, United States of America) at a flow rate of 0.3 mL/min with column temperature set at 35°C. The mobile phase A was acetonitrile, and mobile phase B was 0.1% formic acid in water, and the initial mobile phase of gradient elution program was consisted of 60% phase A and 40% phase B. The sample injection volume was set at 10 μL. Gradient elution program was supplied in Supplementary Table S1.

The mass spectrometry detection was achieved on an Agilent 6460A mass spectrometer with ESI source. Data acquisition was operated in multiple reaction monitoring (MRM) mode, and the source temperature was 105°C. The settings of the mass spectrometer source were as follows: capillary voltage 4,000 V, sheath gas flow 12 L/min, temperature 400°C, dry gas (nitrogen) flow 10 L/min and temperature 350°C, nebulizer pressure 45 psi. All analytes were detected in positive ionization mode. The optimized MRM parameters are shown in Table 1. Collision gas (high purity liquid nitrogen) was set at 0.25 MPa. The structures of both two analytes and IS are display in Figure 1.

To prepare the stock solution, approximately 2.00 mg of Tac, deoxyschizandrin and IS were accurately weighed and dissolved in methanol, respectively. The stock solutions of Tac and deoxyschizandrin with a final concentration of 1.00 mg/mL was obtained, and the IS stock solution was diluted to 100.00 ng/mL using methanol before use. The stock solutions were aliquoted and stored at −80°C. Combined working solutions were prepared by diluting the stock solution using 10% aqueous methanol at the following concentrations: 5.00, 10.00, 20.00, 50.00, 100.00, 200.00, 500.00, 1,000.00, and 2,000.00 ng/mL. Blank rat plasma was used to prepare the calibration standards. Quality-control (QC) samples of two analytes were prepared separately in the same manner at three concentrations, namely, 10 (low concentration), 100 (middle), 1,000 ng/mL (high), and all QC samples were stored at −80°C.

The pretreatment was performed for the plasma sample by one-step protein precipitation, Briefly, 100 μL sample was transferred into a 1.5 mL EP tube prior to adding with 300 μL methanol(containing 200 ng/mL IS). The mixture was vortex-mixed for 30 s before centrifugation at 17,400 g for 10 min at room temperature, and 100 μL of the supernatant was diluted with 100 μL of acetonitrile (v/v, 1:1) Then, a 10 μL aliquoted of diluted solution was injected into the UHPLC-MS/MS system for analysis.

The methodology validation was completed according to FDA guidelines and Chinese Pharmacopoeia (2015 edition), and the linearity, inter- and intra-precision and accuracy, matrix effect and extraction recovery, stability and specificity of this method were validated according to previous study (Lu et al., 2022; Wang et al., 2022; Shu et al., 2016).

The protocol of the animal study was approved by the Experimental Animal Ethics Committee of the Shanghai University of Traditional Chinese Medicine. Specific Pathogen Free (SPF) grade SD rats, male, 8 weeks old, 200–250 g, were obtained from the animal experiment center of Shanghai University of Traditional Chinese Medicine. After passing quarantine, the rats were adaptive fed for 2 days. Rats were fasted for 12–14 h, followed by the initiation of pharmacokinetic study of Tac and deoxyschizandrin. This animal study was carried out in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory animals.

In this study, 42 rats were divided equally into seven groups of six rats each, numbered NG2201-NG2207. Among them, the NG2201 and NG2202 group was dosed with 150 mg/kg deoxyschizandrin alone to study the PK of deoxyschizandrin in rats after single and multiple doses; the NG2203 group was administrate dosed with 1.0 mg/kg of Tac; the NG2204-NG2206 groups were dosed with 75, 150, and 300 mg/kg of deoxyschizandrin plus 1.0 mg/kg Tac, respectively, and the NG2207 group was dosed with the Wuzhi capsule (approximately equal to 150 mg/kg of deoxyschizandrin) plus 1.0 mg/kg of Tac (Teng et al., 2022). The detailed dosing regimen is shown in Table 2, and all dose in this experiment were calculated according to clinical dose. Blood samples (approximately 300 μL for each) for PK analysis were collected into lithium–heparin tubes. The plasma sampling schedules are outlined below: Blood samples were drawn pre-dose, at 5, 10, 20 and 30 min, and at 1, 1.5, 2, 4, 8 and 24 h after drug administration. The blood samples were centrifuged at 5,000 g for 15 min at 4°C to collect plasma samples, which were placed −80 refrigerator.

The rat plasma collected from each group was pretreated according to the method in section 2.5, and exposure levels of Tac and deoxyschizandrin were determined using the above established UHPLC-MS/MS method and the PKs were calculated using DAS software using non-compartment model (version 2.0, China Pharmacological Society). The data were presented as mean ± SD.

Specifically. The ionization mode tested in positive and negative ionization modes showed that no precursor ions were found in the negative ionization mode, whereas the [M + H]+peak of the analytes were observed in the positive ionization mode with high responses. In addition, several common columns were tested, such as Agilent ZORBAX SB-C18 (2.1 × 100 mm, 3.5 µm), Agilent ZORBAX SB-C8 (2.1 × 150 mm, 5 µm), and Waters XBridge^® C18 (2.1 × 100 mm, 3.5 µm) and so on, and finally the ZORBAX SB-C18 column (2.1 mm × 100 mm, 3.5 μm) was selected to be the most appropriate for two analytes and IS separation. By comparing the common mobile phases, such as methanol, acetonitrile, and their mixture and others, the best mobile phase was determined to be acetonitrile and 0.1% formic acid in water. Besides, we tried both isocratic and gradient elution programs, and the retention times of two analytes and IS were 3.5 min by isocratic elution with 60% acetonitrile, while the retention times of analytes were reduced to less than 2 min with gradient elution with acetonitrile. The acetonitrile and 0.1% formic acid in water delivered in gradient program were the best mobile phases for the Tac, deoxyschizandrin, and IS. Previous studies have established UHPLC-MS/MS methods, which contain deoxyschizandrin and Tac (Wei et al., 2013), but the methods were time-consuming and inefficient, with complex pre-treatment methods for plasma samples (Wei et al., 2010). In this study, we improved the UHPLC-MS/MS method based on previously reported, and established a modified method that can be used for rapid and accurate detection of deoxyschizandrin and Tac. The mobile phases in this study were suitable for the separation and ionization of deoxyschizandrin, Tac and IS, as it provided good peak shapes and resolutions.

Plasma is a complex matrix containing a variety of compounds, including proteins, lipids, soluble inorganic salts, and small polar molecules, etc., and these compounds can cause ion interference in LC-MS/MS analyses (Peng et al., 2016). Thus, pretreatment of plasma samples is an effective way to reduce the matrix effect, remove potential interferences, and improve the resolution and sensitivity of mass spectrometry detection (Hyötyläinen, 2009). The protein precipitation method was tested with methanol and acetonitrile with consideration of simplicity and cost. For this purpose, plasma samples were mixed with methanol (1:3, V/V), and the recovery of Tac reached about 92.36% and matrix effect of Tac was approximately 99.23%, the recovery of deoxyschizandrin reached 104.03% and matrix effect of deoxyschizandrin was approximately 106.49%, showing advantages compared to acetonitrile as precipitant. In addition, the dilution of supernatant with different solvents, for example, methanol, acetonitrile and initial mobile phase in different ratios were further assessed, and the 100 μL acetonitrile added into 100 μL supernatant brought better and more stable effect, and the matrix effects of both Tac and deoxyschizandrin were optimized to 99.23% and 106.49%.

An UHPLC-MS/MS method was established and validated for determining Tac and deoxyschizandrin, which was then applied to a pharmacokinetic study of them. A total of 420 plasma samples were collected from all rats at 10 time points according to the protocol. This method was applied to determine the contents of Tac and deoxyschizandrin in 420 plasma samples.

NG2201 and NG2202 groups were designed to study the PKs of deoxyschizandrin after single and multiple dosing, in which group NG2201 was administered 150 mg/kg deoxyschizandrin and plasma samples were collected on the first day, and group NG2202 was collected after 12 days of continuous administration of 150 mg/kg deoxyschizandrin. No adverse reactions occurred in the rats, indicating the safety of long-term oral administration of deoxyschizandrin. In addition, pharmacokinetic results showed that the C _max of deoxyschizandrin in rats increased from 148.27 ± 23.20 to 229.13 ± 54.77 ng/mL after multiple administrations, and the AUC_0–t elevated from 785.77 ± 173.66 to 1806.48 ± 707.19 h/ng/mL (Table 5), and the other parameters such as AUC_0→∞, MRT, and T _1/2 were also significantly enhanced (Figure 3A). These results suggests that multiple administrations increased the concentration of deoxyschizandrin in rats, which may exert their pharmacological effects for a longer period of time and obtain better efficacy.

The basic PKs of Tac in rats was calculated in NG2203 group, and plasma samples were gathered from rats after administration of Tac at a dose of 1 mg/kg. The analysis data showed that the C _max of Tac was 14.26 ± 4.73 ng/mL and varied widely between rats in the group; the AUC_0–t was 95.10 ± 32.61 h/ng/mL; the AUC_0→∞ was 105.20 ± 31.28 h/ng/mL; the MRT was 6.54 h, and the T _1/2 was 7.3 ± 3.06 h (Table 6).

In this study, the influence of three different doses of deoxyschizandrin on the PKs of Tac were assessed. NG2204-NG2206 groups were administered 75, 150, and 300 mg/kg deoxyschizandrin plus 1 mg/kg Tac, respectively. Apparently, the C _max, AUC_0–t, and AUC_0→∞ of Tac in rats significantly improved as the dose of deoxyschizandrin increased. For example, the C _max of Tac increased 3-4-fold and plasma concentrations were more stable after co-administered the deoxyschizandrin; the T _max of Tac was shortening along with increasing dose of deoxyschizandrin. Besides, the other PKs fluctuated in a small range, independent of the dose variation of deoxyschizandrin (Figure 3B). Taking group NG2205 as an example, after co-administrated with 150 mg/mL deoxyschizandrin, the C _max of Tac in rats increased from 14.26 ± 4.73 to 54.48 ± 14.37 ng/mL, and the AUC_0–t from 95.10 ± 32.61 to 315.23 ± 92.22 ng/mL (Table 6).

The results showed that the deoxyschizandrin could remarkably increase the plasma concentration of Tac, narrow the inter-individual concentration fluctation, and shorten T _max in rats. The combination of deoxyschizandrin with Tac may solve the problems of low bioavailability and unstable exposure of Tac.

To compare the influences of deoxyschizandrin and Wuzhi capsule on Tac, after administration of Wuzhi capsule (equivalent to 150 mg/kg deoxyschizandrin) and 1.0 mg/kg of Tac in rats, the plasma concentrations of Tac at different time points were measured and the PKs were calculated. The analysis data showed that the C _max of Tac was 43.25 ± 21.86 ng/mL and the AUC_0–t was 340.40 ± 193.26 h/ng/mL; the AUC_0→∞ was 388.49 ± 216.74 h/ng/mL; the MRT was 7.23 ± 1.04 h, and the T _1/2 was 8.27 ± 2.20 h (Table 6). Obviously, compared with administration of deoxyschizandrin, the T _max of Tac was later with administration of Wuzhi capsule, and although the mean C _max was only slightly lower by 11.23 ng/mL, the fluctuation of Tac concentration was significantly larger (Figure 3C). These results suggest that the PKs of Tac co-administered with deoxyschizandrin are more stable and effective.