Atorvastatin (purity $>$98%) was purchased from Aladdin (Aladdin, Shanghai, China). Sodium danshensu and simvastatin (IS) were provided by Beijing Sunflower and Technology Development Co., Ltd. (Beijing, China). HPLC-grade formic acid was purchased from Sigma-Aldrich (St. Louis, MO, USA). Chromatography grade acetonitrile and methanol were provided by Fisher Scientific Co. (Fair Lawn, NJ, USA). Rat blank plasma samples were obtained from blank rats of the Experimental Animal Center of Wenzhou Medical University. All other chemicals and reagents were of analytical grade.

The determination of atorvastatin was performed by an Acquity UPLC Xevo TQD triple quadrupole mass spectrometer (Waters Co., Milford, MA, USA). Chromatography was carried out using a CORTECST UPLC C18 column (2.1 $\times$ 1.5 mm, 1.6 µm) at a constant temperature of 40 °C. The mobile phase consisted of acetonitrile and water at a rate of 0.4 mL/min. The gradient was as follows: 0.0–0.4 min (20% A), 0.4–1 min (rapidly rose from 20% A to 95% A), 1–2 min (maintained at 95% A), 2–2.5 min (reduced to 20% A). The total running time was 3 min. At the end of each injection, the syringe would be washed with a different ratio of methanol-water (50/50, V/V; 10/90, V/V).

The settings for the multi-reaction monitoring (MRM) scan mode were 150 °C for the source, 500 °C for desolvation, 40 V for the cone voltage, and 20 V and 10 V for collision energy for atorvastatin and IS, respectively. The production for MRM detection was m/z 559.25$\to$440.17 for atorvastatin and m/z 419.22$\to$199.17 for internal standard simvastatin (Fig. 1). All data was processed by Masslynx 4.1 software (Waters Corp., Milford, MA, USA).

Fig. 1.

Mass spectra of atorvastatin (A) and simvastatin (IS) (B).

The stock solution of atorvastatin (1 mg/mL) was formulated in methanol. A set of standard working solutions was prepared by diluting the atorvastatin stock solution with methanol. Similarly, the stock solution of simvastatin was prepared in the same way and finally diluted to 2 µg/mL before use. The calibration standard samples for atorvastatin (100, 50, 25, 10, 5, 2.5, 1 ng/mL) were generated by adding 20 µL of the working standard solution to 100 µL of blank rat plasma. Low quality control (LQC) (2 ng/mL), middle quality control (MQC) (8 ng/mL), and higher quality control (HQC) (80 ng/mL) samples were prepared in the same manner. All solutions and samples were stored in a refrigerator at 4 °C until use.

A 10 µL aliquot of IS solution (500 ng/mL simvastatin) and 100 µL of acetonitrile was added to 50 µL plasma sample. The mixture was vortex-mixed for 30 sec, and centrifuged at 13,000 rpm for 5 min. Then 2 µL aliquot of the supernatant was injected into the UPLC-MS/MS system for analysis.

Methodological validation was performed under the guidance of the FDA and EMA literature [37, 38], including selectivity, linearity and the lower limit of quantification (LLOQ), linearity, accuracy, precision, matrix effect, extraction recovery and stability. Each set of validations need to be determined for three consecutive days including five sets of four different concentrations of QC samples (LLOQ, LQC, MQC, HQC).

To evaluate the specificity, we analyzed five blank biological samples, five biological samples spiked with atorvastatin and IS, and five biological samples following administration of atorvastatin orally or intravenously.

The standard curve for atorvastatin was within 1 to 100 ng/mL for plasma samples. The reciprocal of concentration (1/X) was used as the weighting factor to evaluate calibration curves using linear regression analysis. LLOQ was defined as the lowest concentration quantified on the scale of calibration curves, where relative standard deviation (RSD) for precision and RE for accuracy were less than 20%.

Five replicate samples at four concentrations (LLOQ, LQC, MQC, HQC) of 1, 2, 8, and 80 ng/mL, respectively, were determined on one day to calculate accuracy and precision. The validation procedure was carried out over three consecutive days. RSD and relative error (RE) were employed for evaluating precision and accuracy, respectively. Intra-day and inter-day precision were verified by measuring concentration changes over the same day and for three consecutive days. Accuracy was achieved by comparing the results to the prepared concentration, with a lower than 15% error and a variation within $\pm$ 15%, respectively.

The recovery rate is the ratio of the response value of the test substance recovered from the sample to the response value of the standard substance. Four different concentrations of LLOQ, LQC, MQC, HQC (1, 2, 8, 80 ng/mL, respectively) were prepared according to the method of the sample preparation, and each concentration was made in five parallel groups and the corresponding peak area of atorvastatin was measured as A1. 500 µL of blank plasma and 1 mL of acetonitrile were added to a 2 mL centrifuge tube, vortexed for 30 s, and centrifuged for 5 min (13,000 rpm). Unextracted atorvastatin samples were prepared from 145 µL of supernatant, IS and atorvastatin standard solutions to obtain the average A2 of the corresponding peak area of atorvastatin and calculated the recovery rate by A1/A2.

Significant interferences frequently occur throughout the analyte analysis procedure, impacting the accuracy of the results. These influences and disturbances are referred to as matrix effects. The standard solution and IS were added to 45 µL aliquots of methanol to make four different concentrations of atorvastatin solution (1, 2, 8, 80 ng/mL, respectively). The matrix effect was calculated as A2/A3 based on the average value of the area A3.

Long-term stability and short-term stability indicate the reliability and reproducibility of results. Five sets of parallel experiments were carried out for each of the four sample concentrations of LLOQ, LQC, MQC and HQC (1, 2, 8, 80 ng/mL, respectively).

Sprague-Dawley (SD) male rats (280 $\pm$ 20 g) from the Experimental Animal Center of Wenzhou Medical University (Wenzhou, China) were housed in a laboratory with appropriate temperature and humidity (Laboratory Animal Center of Wenzhou Medical University). Rats were acclimated to the laboratory environment for approximately 10 days to minimize any potential animal suffering before the experiment commenced. All experimental procedures were approved by the Institutional Animal Ethics Committee of Wenzhou Medical University and conducted in accordance with the Guidelines for the Care and Use of Laboratory Animals (ID No: xmsq2021-0408). According to the AVMA Guidelines for the Euthanasia of Animals (2020), all animals were humanely euthanized at the end of the blood sampling period by 200 mg/mL pentobarbital sodium, 60 mg/kg, i.p, deep anesthesia was confirmed by loss of pedal withdrawal and corneal reflexes. After confirming deep anesthesia, thoracotomy (open-chest procedure) was performed as the terminal procedure to ensure rapid and irreversible cessation of cardiac function. Death was confirmed by absence of respiration, heartbeat, and reflexes.

Twelve SD rats were randomly allocated into two groups: the experimental group (Group A, n = 6, receiving a dosage of 150 mg/kg/day of danshensu for two weeks) and the control group (Group B, n = 6, administered 0.5% CMC-Na). Subsequent to the last administration of danshensu or 0.5% CMC-Na (control group), both cohorts were administered a single oral dosage of atorvastatin at 10 mg/kg. The rats underwent a 12-hour fasting period before to injection. The 100 µL blood samples were obtained by the tail vein at intervals of 0.083, 0.25, 0.5, 1, 1.5, 2, 3, 4, 6, 8, and 12 hours into a 1.5 mL centrifuge tube. The samples underwent centrifugation at 4000 rpm for 10 minutes and were promptly separated. The plasma samples were subsequently transferred to a different centrifuge tube and frozen at –80 °C until analysis, following the removal of the supernatant.

Electrospray ionization (ESI) was the most common method, which enhanced the sensitivities and reproducibility of UPLC-MS/MS. The second impact of the analysis results was to take into account the type of column and the choice of mobile phase. The CORTECST UPLC C18 column (2.1 $\times$ 1.5 mm, 1.6 µm) was selected for this column due to its smoother chromatogram and more satisfactory peak shape. Acetonitrile water was chosen for elution due to its appropriate retention time. The retention times of atorvastatin and simvastatin were 1.24 and 1.49 min, respectively. The removal of proteins and biological samples was a critical step before performing LC-MS analysis. This study demonstrated that protein precipitation using acetonitrile yielded superior matrix effects and enhanced recovery compared to liquid-liquid extraction.

Standard chromatograms for blanks, pooled samples, and individual biological samples are shown in Fig. 2. The outcomes demonstrated that atorvastatin and IS were not hampered by any endogenous molecules.

Fig. 2.

Typical ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) chromatograms of atorvastatin and simvastatin (IS) include: (A) blank plasma; (B) blank plasma spiked with IS (2 µg/mL) and atorvastatin (1 ng/mL); and (C) rat plasma samples obtained orally two hours after receiving a dosage of 10 mg/kg atorvastatin.

The standard curve of atorvastatin in rat plasma had good linearity, ranging from 1–100 ng/mL, with the linear regression equation of y = 0.0370389 $\times$ x + 0.0616107, R² = 0.9964. The LLOQ of atorvastatin in plasma was determined to be at least 1 ng/mL. The RSD of precision and accuracy at LLOQ was less than 20%, suggesting that this method was rather sensitive to our analysis.

Table 1 shows the results of precision, accuracy, extraction recovery, and matrix effects of atorvastatin in rat plasma. The results showed that the precision and accuracy were within 15% as required by the FDA biological sample analysis method. The extraction recovery rate ranged from 85.28% to 95.36%, and the matrix effect ranged from 87.06% to 91.87%. The analytical results meet the requirements. The results of the matrix effect also indicated that plasma has no significant impact on the matrix. Fig. 2 illustrates the representative chromatograms of a blank plasma sample, a plasma sample spiked with atorvastatin and internal standard, and a plasma sample collected 2 hours post-oral administration of 10 mg/kg atorvastatin.

The results of Table 2 indicated the stability of atorvastatin LLOQ, LQC, MQC and HQC (1, 2, 8, 80 ng/mL, respectively) in rat plasma. There was no difference in stability between samples, whether they were stored at room temperature for 24 hours in a refrigerator at 4 °C for 6 hours or at –20 °C for repeated freeze-thaw cycles three times.

The pharmacokinetic character of atorvastatin in combination with danshensu (150 mg/kg) were profiled by DAS software (Version 3.2.8, The People’s Hospital of Lishui, China) and the statistical results were shown in Table 3. When the two drugs were administered simultaneously, it may significantly affect the atorvastatin pharmacokinetic parameters including AUC, C_max, t_1/2 and CL_z/F (p 0.05). The highest blood concentration of atorvastatin in rats was reached about 0.35 hours after oral administration. The changes in AUC, C_max, t_1/2 and CL_z/F in rats after the combination treatment are sufficient to demonstrate an interaction between the two types of treatment. The AUC of the experimental group was about 3-fold that of the control group, and the C_max and t_1/2 of the experimental group were about 2-fold that of the control group. The results showed that combined with drugs, atorvastatin slowed down elimination in rats and inhibited metabolism, resulting in significantly increased drug accumulation. CL_z/F was three times higher in the control group than in the experimental group. The lower clearance rate for the experimental group may also be related to its slower metabolism and longer residence time in the body, resulting in a slower clearance of the drug. Therefore, the results show that danshensu and atorvastatin interact in rats. The mean plasma concentration-time curve for atorvastatin was shown in Fig. 3. The peak plasma concentration of the experimental group was higher than that of the control group, but its peak time was slower. This phenomenon may be caused by the interaction of danshensu and atorvastatin, which increases the accumulation of atorvastatin in the body and inhibits its metabolism.

Fig. 3.

Mean plasma concentration-time curve of atorvastatin in rats (n = 6, mean $\mathrm{\pm }$ SD).

Danshensu has been reported to act as a competitive inhibitor of CYP2C9, while it does not significantly affect CYP3A4-mediated metabolism [31]. Atorvastatin is metabolized primarily by CYP3A4, while P-gp and OATP1B1 modulate its hepatic uptake and excretion [20, 22]. Danshensu has also been reported to inhibit OATP1B1-mediated transport of rosuvastatin, providing a mechanistic basis for the pharmacokinetic interactions observed in vivo [39]. Therefore, the in vivo increase in atorvastatin exposure observed in the presence of danshensu may result from reduced efflux via P-gp and/or altered hepatic uptake via OATP1B1, rather than altered CYP3A4 metabolism. Understanding these transporter-mediated interactions is important for predicting potential herb–drug interactions and optimizing individualized therapy. Both danshensu and atorvastatin are used for the regulation of blood lipids and the treatment of cardiovascular diseases. In this study, a sensitive and reproducible UPLC-MS/MS method was developed to simultaneously quantify danshensu and atorvastatin in rat plasma. The findings provide a foundation for future studies to evaluate the efficacy and safety of the combined use of these two drugs in cardiovascular therapy.