ŘǯŘǯŘǯȱ¢ȱ

ȱȱȱȱȱǰȱȱȱȱȱ ȱȱ¢ȱȱȱ ȱǯ ȱȱȱȱȱȱǰȱȱȱȱȱȱ ȱȱȱ ȱȱȱȱŗǯȱȱȱȱȱǻ*r*Ǽȱȱȱȱȱ

In the chromatograms of blank samples injected immediately after the HQC samples were injected, no peak was observed at the retention time of alnustone and IS, indicating that the carry-over effect in the sample analysis could be ignored.

#### 2.2.2. Linearity

For blood samples and tissue samples, the linear calibration curves were calculated by a linear regression model. The linear equations of the calibration curves, the linear regression coefficients and the linear ranges were given in Table 1. Each coefficient of determination (*r*) in all validation batches were all greater than 0.99.


**Table 1.** Calibration curves, correlation coefficients, linear ranges and lower limit of the quantification(LLOQ) of alnustone in different biological matrices.

 SES—Standard error of the slope (*n* = 6). # SEI—Standard error of the intercept (*n* = 6).

#### 2.2.3. Accuracy and Precision

\*

Table 2. showed the results of intra- and inter-day accuracy and the precision of alnustone in the biological matrices of rats. The *RE* values of intra- and inter-day accuracies ranged from −10.3% to 9.9%, and the *RSD* values of the precision assays were all not more than 9.0%, indicating that the precision and accuracy results of this method were within the acceptable criteria, proving that this method was reliable and reproducible to quantitatively analyze alnustone in rat biological samples.


**Table 2.** Intra- and inter-day accuracy and precision of alnustone in the plasma and tissue homogenates of rats (*n* = 5).


**Table 2.** *Cont*.

#### 2.2.4. Recovery and Matrix E ffect

As shown in Table 3, the extraction recoveries were within the range of 86.3–110.4%, indicating that the optimized pretreatment method of direct protein precipitation could o ffer good extraction efficiency for alnustone in these matrices. The matrix e ffects of alnustone ranged from 89.5% to 114.4%, showing that the matrix had a little co-eluting endogenous substance that could influence the ionization of alnustone.


**Table 3.** The matrix e ffect and recovery of alnustone in di fferent samples (*n* = 5).


#### 2.2.5. Stability

The results of the stability studies were shown in Table 4, indicating that there was no stability issue occurred.

**Table 4.** Short-term, post-preparative storage, freeze-thaw and long-term stability of alnustone in different samples (*n* = 5).



#### *2.3. Pharmacokinetic Study and Tissue Distribution*

The validated LC-ESI-MS/MS method was successfully applied to a pharmacokinetics and tissue distribution of alnustone after the intravenous administration at a single dose of 5 mg/kg. The intravenous administration dose was generally determined based on 1/20–1/50 of the LD50 value or 1/2–1/3 of the maximum tolerated dose of the reference MTD [27–29]. The LD50 obtained by our previous pharmacological experiment was 200 mg/kg and therefore the selected i.v. dose was 5 mg/kg. Meanwhile, it was reported that the structural analog of alnustone was given to the rat i.v. at a dose of 4.5 mg/kg for a pharmacokinetic study [30]. The dosage of another piece of literature was 5.45 mg/kg for the pharmacological activity study [31]. Above all, the dosage of the drug administration in our pharmacokinetics experiment was determined to be 5 mg/kg. Figure 3 showed the mean plasma concentration-time profile of alnustone after administration**.** Table 5 summarized the pharmacokinetic parameters based on the non-compartmental method. Upon intravenous administration of 5 mg/kg alnustone, the *Tmax* was 2 min and the *Cmax* was 7066.36 ± 820.62 ng/mL, indicating that alnustone could be quickly detected in the plasma. The pharmacokinetic results have demonstrated the area under the curve up to 10 h (*AUC0*−*<sup>t</sup>*) and the infinite time (*AUC0*−∞) of 6009.79 ± 567.30 and 6032.45 ± 472.50 ng/mL·h, respectively. The total mean residence time up to 10 h (*MRT0-t*) was found to be 1.60 ± 0.22 h. Alnustone had a relatively short *t1*/*2* of 1.31 ± 0.19 h, an apparent *Vd* of 1.57 ± 0.18 L/kg, and *CL* of 0.83 ± 0.09 L/h/kg. The short elimination half-life of alnustone in vivo after a single administration indicated that alnustone would not tend to accumulate in rat plasma during long-term use.

**Table 5.** Non-compartmental plasma pharmacokinetic parameters following a single intravenous administration of alnustone (5 mg/kg) to rats (*n* = 12).


*Molecules* **2019**, *24*, 3183 ǻ*AUCŖ t*Ǽ ǻ*AUCŖLj*Ǽ ƹȱśŜŝǯřŖȱȱŜŖřŘǯŚśȱƹȱŚŝŘǯśŖȱȦL·hǰȱ¢ǯȱȱȱȱȱȱȱȱŗŖȱȱǻ*MRT0- t*Ǽȱ ȱȱȱȱŗǯŜŖȱƹȱŖǯŘŘȱǯȱȱȱȱ¢ȱȱ*t1/2*ȱȱŗǯřŗȱƹȱŖǯŗşȱǰȱȱȱ*Vd* ȱ ŗǯśŝȱƹȱŖǯŗŞȱȦǰȱȱ*CL*ȱȱŖǯŞřȱƹȱŖǯŖşȱȦȦǯȱȱȱȱȬȱȱȱȱȱȱ ȱȱȱȱȱȱ ȱȱȱȱȱȱȱȱȱ Ȭȱǯȱ

ŜŖŖşǯŝş

ŗŖ

**ȱ řǯȱ** ȱ ȱ ȱ Ȭȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱȱȱȱȱȱȱśȱȦȱǻ*<sup>n</sup>*ȱƽȱŗŘǰȱȱƹȱǼǯȱ **Figure 3.** The mean plasma concentration-time curves of alnustone after the intravenous administration of alnustone at a dose of 5 mg/kg (*n* = 12, mean ± SD).

**ȱ śǯȱ** Ȭȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱȱȱǻśȱȦǼȱȱȱǻ*<sup>n</sup>*ȱƽȱŗŘǼǯȱ **ȱȱ ȱȱǯǯȱȱǻśȱȦǼȱ** ¡ǻȦǼ ŝŖŜŜǯřŜȱƹȱŞŘŖǯŜŘȱ *t1/2* **ǻǼ** ŗǯřŗȱƹȱŖǯŗşȱ *<sup>0</sup>***Ȯ***<sup>t</sup>***ǻ**Ȧ·**Ǽȱ** ŜŖŖşǯŝşȱƹȱśŜŝǯřŖȱ The changing trend of alnustone concentration in different tissues at different time points of 0.5, 1, 2 and 4 h after administration was investigated. Figure 4 revealed that alnustone distributed rapidly and widely in different tissues. In the tissues of brain, intestine, lung, and spleen, the maximum concentration appeared at 1 h, while in heart, kidney, liver and stomach, the maximum concentration of alnustone appeared earlier at 0.5 h to 1h. Among all the tissues, the highest mean concentration was found in the lung (5685.40 ng/g), followed by the liver (3199.49 ng/g), spleen (2077.14 ng/g), stomach (1588.90 ng/g), kidney (1450.43 ng/g), intestine (1099.14 ng/g), heart (900.97 ng/g), and brain (344.27 ng/g). The high level in the lung and liver might be related to the bioaccumulation of alnustone in these two organs. In the liver, an increase of drug exposure was observed at 4 h. Similar situations also occurred in the lung and stomach. The most possible reason was that alnustone might be reabsorbed in these organs. Alustone is fat-soluble, so it might bind to the fat after the first absorption. When the drug reached a certain accumulation in the fat, it would be reabsorbed into the liver, lung and stomach, which have good affinity with the drug. In addition, hepatointestinal circulation may also lead to an increase in the liver at 4 h. Thus, in the liver, lung and stomach, alustone maintained a high concentration and underwent a slow elimination and accumulation. They are highly likely to be the target organs for the efficacy of alnustone, which needs to be studied systematically in the future. In addition, the metabolic clearance rate of alnustone in different tissues was quite different. For example, in the spleen, intestine, and kidney, the elimination rate of alnustone was relatively fast. For 4 h after administration, the concentration of alnustone in these tissues had decreased to less than half of the corresponding maximum concentration. In the other tissues, the elimination rate of alnustone was relatively slow, and the tissue concentrations were either kept at the same level during the measured period or even higher within 4 h after administration. In contrast, due to the rapid elimination and less drug accumulation, alnustone was unlikely to cause drug accumulation and immunological side effects in the spleen, intestine, and kidney. At the same time, the concentration of alnustone in the brain did not change with time, and reached a certain level (567.0 ng/mL, 4 h), suggesting that it could enter the brain through the blood-brain barrier. The tissue distribution experiment provided a reference for the study of the distribution characteristics of drugs in vivo and also provided a theoretical basis for the development of drugs in the later stage.

**ȱŚǯȱ**ȱȱȱȱȱȱȱȱǻȦǼȱȱŖǯśǰȱŗǰȱŘȱȱŚȱȱȱȱ ȱȱȱśȱȦȱȱȱȱȱǻȱƹȱǰȱ*<sup>n</sup>*ȱƽȱśǼǯȱ **Figure 4.** The mean concentration of alnustone in different tissues (ng/g) at 0.5, 1, 2 and 4 h after an intravenous administration of 5 mg/kg alnustone to the rats (Mean ± SD, *n* = 5).

ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ ȱ Śȱ ȱ ȱ ǯǯȱ ȱ ȱ ȱ ȱ śȱȦǯȱȱ ȱ ȱȱȱ ȱ ǻ
Ǽȱ ȱ ȱ ȱȱȱŚȱǯȱȱ
ȱȱȱȱȱȱȱȱ£ȱȱȱŜǯȱȱ ȱ
ȱ ȱȱȱȱȱǻŗśǯŞŚȱƹȱŗǯřřǼǰȱ ȱ¢ȱȱȱȱȱǻśǯŚŞȱƹȱŖǯŜŜǼǰȱȱ ǻřǯŜŜȱƹȱŖǯŘŝǼȱǰȱȱ¢ȱǻŗǯŞŜȱƹȱŖǯŗŞǼǯȱȱȱ
ȱȱȱȱȱȱ ȱȱȱȱȱȱȱȱȱȱǯȱ The tissue distribution characteristics of alnustone were determined at 4 h after i.v. administration of alnustone at 5 mg/kg. The tissue to plasma partition coefficients (Kp) shows an upward trend in 4 h. The Kp values of alnustone in iv administration are summarized in Table 6. The highest Kp was observed in the lung (15.84 ± 1.33), followed by that in the liver (5.48 ± 0.66), stomach (3.66 ± 0.27) tissue, and kidney (1.86 ± 0.18). The highest Kp values and concentrations of alnustone in the lung and liver indicated the possible bioaccumulation of alnustone.

**ȱŜǯȱ**ȱȱȱȱȱǻ
Ǽȱȱȱȱǯǯȱȱȱȱ ǻśȱȦǼȱȱȱȱǻȱƹȱǰȱ*<sup>n</sup>*ȱƽȱśǼǯȱ **Table 6.** Plasma to tissue partition coefficients (Kp) of alnustone after i.v. administration of alnustone (5 mg/kg) to the rats (Mean ± SD, *n* = 5).


**řǯȱȱȱȱ** *3.1. Chemicals and Reagents*  ȱȱȱǻ¢ȱȱşŞƖǼȱ ȱȱ¢ȱȱȱǯȱǻǼȱAlnustone and caffeine (purity over 98%) were provided by Target Molecule Corp. (TargetMol) (Boston, MA, USA). Acetonitrile and methanol, both are LC-MS-grade, were purchased from Merck KGaA Company (Darmstadt, Germany). The formic acid (HCOOH, HPLC-grade) was obtained from Dikma Technologies Inc. (Lake Forest, CA 92630, USA). The purified water was provided using a Millipore Milli-Q system (Millipore, Bedford, MA, USA).

 ǻǰȱǰȱǼǯȱȱȱǰȱȱȱȬȬǰȱ ȱȱȱȱ ȱ¢ȱǻǰȱ ¢Ǽǯȱȱȱȱǻ ǰȱ ȬǼȱ ȱȱȱ *3.2. Instrumentations*

ȱȱ ǯȱ ǻȱǰȱȱşŘŜřŖǰȱǼǯȱȱȱ ȱ ȱȱȱȱȱȬȱ¢ȱǻǰȱǰȱǰȱǼǯȱ *3.2. Instrumentations*  The UHPLC-MS/MS method was performed on 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. The separation process was performed on an ACQUITY UPLC BEH C18 Column (100 mm × 2.1 mm,

ȱ ȬȦȱȱ ȱȱȱȱȱȱŗŘşŖȱ ȱ¢ȱǻȱ ǰȱ ȱ ǰȱ ǰȱ Ǽǰȱ ȱ ȱ ȱ ȱ ȱ ȱ řśŖŖȱ ȱ ȱ ȱ ȱ ǻȱ ¡ǰȱ ǰȱ ǰȱ Ǽȱ ȱ ȱ ¢ȱ £ȱ ǻǼȱ ǯȱ ȱ 1.7 μm, Agilent Technologies, Santa Clara, CA, USA).

#### *3.3. Animals*

Further, thirty two Sprague-Dawley rats (male, 200–220 g body weight) were purchased from the Experimental Animal Research Center, China Medical University, China. The protocol for this study (protocol number # CMU2019194) was approved by the Institutional Animal Care and Use Committee at China Medical University. The study complied with guidelines for the Care and Use of Laboratory Animals (published by the National Institutes of Health, NIH publication no. 85–23).

#### *3.4. Preparation of Calibration Standards and Quality Control Samples*

A 10.0 mg of alnustone was dissolved in 10.0 mL of methanol to give the stock solution with the concentration of 1.0 mg/mL. The working solutions were prepared by serial dilution of the stock solution with the initial mobile phase (methanol—0.1% fomic acid water, 20:80, *v*/*v*). A 10.0 mg of caffeine (IS) was dissolved in 10.0 mL of methanol to give the IS stock solution with the concentration of 1.0 mg/mL. All of the solutions were stored at 4 ◦C before use.

The calibration standards were prepared by spiking a certain volume of blank plasma or blank tissue homogenates with appropriate amounts of working solutions to yield a final concentration range from 1 to 2000 ng/mL. The effective concentrations of alnustone were 1, 5, 10, 40, 160, 200, 800, 2000 ng/mL for the plasma samples and tissue homogenates samples (heart, liver, spleen, lung, kidney, brain, stomach, intestine). The quality control (QC) samples were prepared in the same manner at three concentration levels (low, mid, high) of 5, 100, 1600 ng/mL for the plasma and tissue homogenates.

#### *3.5. Preparation of Plasma and Tissues Samples*

In this study, a direct protein precipitation method was applied to extract alnustone and IS from the biological matrix. An aliquot 100 μL of plasma, 50 μL of IS solution (1 μg/mL) and 200 μL of precipitate agen<sup>t</sup> acetonitrile with 0.5% formic acid was added into a 1.5 mL Eppendorf tube. The mixture was vortex-mixed for 30 s at room temperature, and centrifuged at 12,000× *g* for 10 min. Then, a 200 μL aliquot of the supernatant was carefully removed and transferred to a new 1.5 mL Eppendorf tube and evaporated to dryness at 40 ◦C under a slight stream of nitrogen. The residuals were reconstituted in 100 μL of methanol—0.1% fomic acid water (20:80, *v*/*v*) by vortex mixing for 30 s. After centrifuging at 12,000× *g* for 10 min, a 10 μL aliquot of the supernatant was used for the UHPLC-MS/MS analysis.

The rat was sacrificed quickly by decapitation on the ice. The various tissues (heart, liver, spleen, lung, kidney, brain, stomach, intestine) were harvested and rinsed with ice-cold 0.9% NaCl to remove the superficial blood. After being blotted dry with filter paper, each tissue sample was weighed on ice and physiological saline added (1:2, *w*/*v*) to homogenize. Then, a 100 μL (equivalent to 50 mg) of tissue homogenate was taken and processed using the same method as the plasma samples processing method as shown in the above steps.

#### *3.6. Chromatographic and Mass Conditions*

The mobile phase consisted of methanol and 0.1% formic acid water using a gradient dilution at a flow rate of 0.3 mL/min. The column temperature was maintained at 30 ◦C. Alnustone was quantitatively determined with MRM in the positive ion mode, and nitrogen was used to assist nebulization in the ESI source. The MS parameters were set as follows: The ionspray voltage (IS) 5500 V; nebulizer gas (gas 1) 19 arbitrary units; curtain gas (CUR) 10 arbitrary units; collision cell exit potential (CXP) 7.0 V; entrance potential (EP) 10.0 V. The optimization of the MS transitions using the multiple-reaction monitoring (MRM) mode were accomplished as alnustone *m*/*z* 262.9→105.2 and IS (caffeine) *m*/*z* 195.2→138.0, respectively.

#### *3.7. Method Validation*

The method was fully validated according to the Bioanalytical Method Validation Guidance for Industry (FDA, 2018) [32].

#### 3.7.1. Selectivity

Selectivity was investigated by comparing chromatograms of blank rat plasma obtained from six individual rats with those of the corresponding standard biosample spiked with alnustone at LLOQ and IS and the actual plasma sample 2h after an intravenous administration of alnustone (5 mg/kg).

#### 3.7.2. Linearity and LLOQ

The calibration curves for alnustone in the plasma or tissue homogenates were generated by plotting the peak area ratios (*y*) of alnustone to IS versus those nominal concentrations (*x*) in standard plasma or tissue homogenates using weighted (1/*x<sup>2</sup>*) least squares linear regression. The LLOQ was defined as the lowest concentration of the calibration curve at which the precision did not exceed 20%, the accuracy was within ±20% and the signal-to-noise ratio (S/N) was at least 10.

#### 3.7.3. Accuracy and Precision

The intra- and inter-day accuracy and precision were assessed by analyzing QC samples at three levels of alnustone (5, 100, 1600 ng/mL for plasma and tissue homogenates) on three consecutive days with five replicates at each concentration. The accuracy and precision were depicted as the relative error (*RE*) and the relative standard deviation (*RSD*), respectively. The *RE* was within ±15% and the *RSD* could not exceed 15%.

#### 3.7.4. Recovery and Matrix Effect

The recovery was determined by comparing the peak areas of alnustone or IS in processed QC samples with the mean peak areas of alnustone or IS in the spike-after-extraction samples (blank plasma or tissue homogenates extracted then spiked with QC standards). (*n* = 5)

The matrix effect was evaluated by comparing the peak areas of alnustone or IS in the spike-after-extraction samples (blank plasma or tissue homogenates extracted then spiked with QC standards) with the mean peak areas of alnustone or IS dissolved with the mobile phase at high, medium and low levels, respectively. (*n* = 5). It was generally considered that the matrix effect was obvious if the ratio was <85% or >115%.

#### 3.7.5. Stability

The stability tests involved the following four conditions. The short-term stability study was examined by analyzing samples at room temperature for 4 h. The long-term stability study was performed by analyzing samples stored at −75 ◦C for 1 month. For the freeze-thaw stability study, the samples were analyzed after three freeze/thaw cycles (−75 ◦C to 25 ◦C). The post-preparative storage stability was assessed by analyzing the samples left in autosampler vials at 4 ◦C for 24 h. The stability analysis was performed using three aliquots of each QC samples at three different concentrations. The samples were considered stable if the assay values were within the acceptable limits of accuracy (±15% *RE*) and precision (≤15% *RSD*).

#### *3.8. Drug Administration and Sampling*

This study was approved by the Animals Experimental Ethical Committee of China Medical University (Liaoning, China). Alnustone was dissolved in 0.5% (*v*/*v*) DMSO saline to give the injection solution at the dose of 5 mg/kg for intravenous administration.

For the pharmacokinetic study, 12 SD rats fasted for 12 h with free access to water prior to administration. After the rats were tail intravenously administrated of alnustone (5 mg/kg), a blood sample (0.4 mL) was collected from the orbital vein into heparinized tubes at appropriate time intervals (2 min, 5 min, 10 min, 15 min, 30 min, 45 min, 1 h, 1.5 h, 2 h, 2.5 h, 3 h, 4 h, 8 h, 10 h). The blank plasma samples were prepared before dosing. All blood samples were immediately centrifuged at 12,000× *g* for 10 min and stored at –75 ◦C until analysis.

For the tissue distribution study, 20 SD rats were randomly divided into four groups (5 rats for each time point) and fasted for 12 h with free access to water prior to administration. After the rats were tail intravenously administrated of alnustone (5 mg/kg), the tissue specimens (heart, liver, spleen, lung, kidney, brain, stomach and intestine) were collected at 0.5, 1, 2, and 4 h post-dosing, respectively. The blank tissues were prepared separately using drug-free SD rats. The tissue harvesting and homogenizing method were given in Section 3.5. For the plasma and tissue homogenate sample preparation method, also see Section 3.5.

#### *3.9. Statistical Analysis*

The pharmacokinetic parameters were evaluated using DAS 3.2.8 pharmacokinetic program [33].

#### **4. Conclusions**

In this study, a rapid, simple and sensitive UHPLC–ESI MS/MS method was established and successfully applied to the study of pharmacokinetics and tissue distribution of alnustone after a single intravenous administration with 5 mg/kg alnustone in rats. The method has been validated for the first time in this paper and proved to be able to detect a low concentration of 1 ng/mL for alnustone with one direct protein precipitation method. The elimination half-life of alnustone was short implying the residence time of alnustone in vivo was not long and it eliminated quickly from the rat plasma. According to the investigation of tissue distribution, alnustone was mainly distributed in the lung and liver tissues within 1 h, and the drug in each tissue increased slightly at 4 h, which indicated that the drug was mainly distributed in the tissues with large blood flow, and the lung and liver might be the target organs for the curative e ffect of alnustone. The developed and validated LC-MS/MS method and the pharmacokinetic study of alnustone in the present paper might provide helpful information for its future preclinical applications and theoretical basis for its further exploitation.

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

**Funding:** This project was financially supported 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); 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); and Scientific Research Project of Department of Education of Liaoning Province (ZF2019036).

**Acknowledgments:** We are very grateful to Heran Li and Xiaolan Deng 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.
