*3.1. Chemicals and Reagents*

DOX (purity over 99%) was obtained from Dalian Meilun Biotech Co., Ltd. (Dalian, China). HER and tetrandrine (purity over 99%) were purchased from Chengdu Biopurity Phytochemicals Ltd. (Chengdu, China). Ammonium acetate, HPLC-grade, was purchased from Dikma Company (Lake Forest, CA 92630, USA). Acetonitrile and methanol, LC-MS-grade, were purchased from Merck KGaA Company (Darmstadt, Germany). Ultra-pure water was provied using a Millipore 52

#### **3. Experimental**

#### *3.1. Chemicals and Reagents*

DOX (purity over 99%) was obtained from Dalian Meilun Biotech Co., Ltd. (Dalian, China). HER and tetrandrine (purity over 99%) were purchased from Chengdu Biopurity Phytochemicals Ltd. (Chengdu, China). Ammonium acetate, HPLC-grade, was purchased from Dikma Company (Lake Forest, CA 92630, USA). Acetonitrile and methanol, LC-MS-grade, were purchased from Merck KGaA Company (Darmstadt, Germany). Ultra-pure water was provied using a Millipore Milli-Q system (Millipore, Bedford, MA, USA). Other chemical reagents were of analytical grade.

#### *3.2. Animals*

Sprague–Dawley rats (male, 250 ± 20 g) were supplied by the Experimental Animal Research Center, China Medical University, China. The rats were raised in a temperature-controlled room at 24 ± 2 ◦C for free feeding and water intake, and the light/dark cycle was 12 h. The rats were fed for 2 weeks to adapt to the laboratory environment. All the rats fasted for 12 h before the experiment, but with water supplied freely. The protocol for animal care and use in our study (protocol number # CMU2019194) was approved by the Institutional Animal Care and Use Committee at China Medical University.

Eighteen rats were randomly divided into three groups (six rats in each group) and given intravenous treatment with different drugs: group A, DOX (5 mg/kg); group B, HER (5 mg/kg); group C, DOX + HER (5.0 mg/kg, respectively). The injection was prepared in normal saline with 0.5% *v*/*v* DMSO.

#### *3.3. Instrumentation and Conditions*

The biological samples were analyzed with an Agilent series 1290 UHPLC system (Agilent Technologies, Santa Clara, CA, USA), which was coupled to an AB 3500 triple quadrupole mass spectrometer (AB Sciex, Ontario, ON, Canada) with an electrospray ionization (ESI) source. Data acquisition and instrument control were performed using the 1.6.3 version Analyst software package (AB Sciex, ON, Canada).

The separation process was performed on an ACQUITY UPLC BEH C18 Column (100 mm × 2.1 mm, 1.7 μm, Agilent Technologies, Santa Clara, CA, USA). The column temperature was set at 40 ◦C. The mobile phase was composed of acetonitrile and 10 mM ammonium acetate aqueous solution (70:30, *v*/*v*) at the flow rate of 0.3 mL/min in an isocratic elution manner. The injection volume was set at 10 μL.

DOX and HER were quantitatively determined with MRM in the positive ion mode. The MS condition was as follows: the ion spray voltage (IS) was set at 5500 V, the turbo spray temperature (TEM) was set at 500 ◦C, and the nebulizer gas and heater gas were set at 50 and 50 arbitrary units, respectively. The curtain gas (CUR) was kept at 40 arbitrary units, and the interface heater was on. The collision cell exit potential (CXP) and entrance potential (EP) were set at 7.0 V and 10.0 V, respectively. The declustering potentials (DPs) of DOX, HER, and IS were set at 160 V, 218 V, and 87 V; the collision energies (CEs) were 60 eV, 60 eV, and 27 eV, respectively. Nitrogen was used in all cases. The optimization of the MS transitions for quantification were accomplished as DOX *m*/*z* 544.2→379.1, HER *m*/*z* 653.4→411.2, and IS (tetrandrine) *m*/*z* 623.3→381.3, respectively. Moreover, the qualifier ions for DOX, HER, and IS were set at *m*/*z* 321.1, *m*/*z* 191.1, and *m*/*z* 174.1, respectively.

#### *3.4. Preparation of Stock Solutions, Working Solutions, Calibration Standards, and Quality Control Samples*

The standard substances of the analytes were accurately weighed and dissolved in methanol to prepare the DOX and HER stock solution with the concentration of 1.0 mg/mL, respectively. The working solution for preparing calibration standards and QC samples was obtained by diluting the stock solution with acetonitrile–water (50:50, *v*/*v*). The IS working solution with a concentration of 200 ng/mL was also prepared. The stock solution and working solution were placed under 4 ◦C dark condition and brought to room temperature before use.

The calibration standards were prepared by spiking 50 μL of rat blank plasma (or blank myocardial tissue homogenate) with 20 μL of the working solution. The concentration of DOX in rat plasma and myocardial tissue homogenate ranged from 32 to 8000 ng/mL, and HER ranged from 20 to 4000 ng/mL. Low, medium, and high quality control (QC) samples were prepared in the same way as above (40.0, 400, and 3200 ng/mL for DOX; 80.0, 800, and 4000 ng/mL for HER) in both rat plasma and myocardial tissue homogenate. Each concentration needed three replicates.

#### *3.5. Sample Preparation*

In this study, DOX, HER, and IS were extracted from the biological matrix (plasma and myocardial tissue homogenate) by routine step protein precipitation. Detailed steps were as follows: take 50 μL of biological matrix, add 20 μL of acetonitrile–water (50:50, *v*/*v*), and 20 μL of the IS solution, and add 200 μL of precipitation reagen<sup>t</sup> acetonitrile, placed in a 1.5-mL EP tube. Vortex for 1 min, followed by centrifuging at 14,000 rpm for 10 min. Transfer 200 μL of supernatant to another clean 1.5-mL EP tube and centrifuge at 14,000 rpm for another 3 min. Then, an aliquot of 10 μL of the supernatant was injected into the LC-MS system for analysis.

#### *3.6. Pharmacokinetic Study*

The method was used to determine the concentration–time profiles of DOX and HER in the plasma of rats after the intravenous administration of DOX (5.0 mg/kg), HER (5.0 mg/kg), and the mixture of DOX and HER (5.0 mg/kg, respectively). Blood samples (250 μL) were taken from the orbital vein at 5 min, 10 min, 15 min, 30 min, 45 min, 1 h, 1.5 h, 2 h, 2.5 h, 3 h, 4 h, 6 h, and 8 h, respectively, and were injected into heparinized 1.5-mL EP tubes. Heparin (2 mg/mL blood volume) was used as an anticoagulant for this study, and blood samples were immediately centrifuged at 14,000 rpm for 10 min at room temperature, followed by a supernatant plasma layer collected and stored at −80 ◦C for analysis.

After the last blood sample was taken, the rats were sacrificed for cervical dislocation. The heart was removed and rinsed with cold saline to remove the superficial blood. Then, it was blotted dry with filter paper and weighed accurately. After that, the heart was homogenized with normal saline to prepare a homogenate (0.2 g/mL). All samples were stored at −80 ◦C for analysis.

Plasma concentration–time plots were plotted, and the PK parameters were evaluated by means of non-compartmental pharmacokinetic analysis using DAS 3.2.8 pharmacokinetic program [27]. The PK parameters concerned include half-life (t1/2), mean residence time (MRT), area under the plasma concentration–time curve (AUC), clearance (CL), etc. Data was expressed as mean ± SD. The pharmacokinetic parameters were compared using Student's t-test. Differences were considered to be significant at a level of *p* < 0.05.

#### **4. Conclusions**

An LC-MS method for the simultaneous determination of DOX and HER in rat plasma was established. The method is sensitive, accurate, easy to follow, and suitable for the pharmacokinetic study. This analytical method has been successfully applied to the pharmacokinetic study of DOX and HER in rats.

The results of this study showed that there were significant differences in the pharmacokinetic parameters of DOX and HER after the intravenous administration of a single dose of DOX, single dose of HER, and a combination of the two. This result might help to explain the influence of DOX and HER interaction on pharmacokinetics and provide a basis for guiding clinical medication.

**Author Contributions:** The experiments were conceived and designed by X.-S.F. The experiments were equally performed by Y.S. and Y.Z., Chromatographic analysis of plasma and biosamples were performed by W.-P.Z., B.-Z.Z., Analysis of pharmacokinetic behavior of alnustone was finished by K.-F.W. All authors read and approved the final manuscript.

**Funding:** This research was funded by Key Program of the Natural Science Foundation of Liaoning Province of China (No. 20170541027); Liaoning planning Program of philosophy and social science (No. L17BGL034); and Major subject in education research founded by Chinese Medical Association Medical Education Branch and China Association of Higher Education Medical Education Professional Committee (2018A-N19012).

**Acknowledgments:** We are very grateful to Q.C. and J.C. for making a major contribution to the revision of the article. The authors are grateful to every person who helped in this work.

**Conflicts of Interest:** The authors declare no conflicts of interest.
