1. Introduction
Alnustone, a nonphenolic diarylheptanoid with a typical chemical structure of an aryl-C7-aryl skeleton, was first isolated from the male flower of
Alnus pendula (Betulaceae) [
1,
2,
3]. Then, it was also found in the seeds of
Alpinia katsumadai Hayata (Zingiberaceae) [
4,
5,
6,
7,
8,
9,
10], the rhizomes of
Curcuma xanthorrhiza Roxb [
11,
12] and
Curcuma comosa Roxb (Zingiberaceae) [
13]. It is reported that diphenylheptanes have a wide range of pharmacological activities, such as anti-inflammatory, hepatoprotective, antioxidant and anti-tumor effects [
14,
15,
16]. Diphenylhexane natural drugs account for 5% of the market share of neurosinosidase inhibitors [
17]. Alnustone reportedly exhibits a variety of activities, including antihepatotoxic [
18], anti-inflammatory [
11], antibacterial [
4], antiemetic [
5,
8,
9] and weak estrogenic [
13]. Recently, Grienke et al. revealed that alnustone showed neuraminidase inhibitory activity, and it was concluded that the compound may be employed as an antiviral agent [
6]. Additionally, the chemical compounds from
Alpinia katsumadai Hayata seeds have been evaluated for their antitumor activities in vitro. Among the isolated compounds, alnustone was found to exhibit significant antitumor activity against the Bel-7402 (human hepatocellular carcinoma cells) and LO-2 (human normal liver cells) cell lines [
19]. As a good drug candidate, several methods have been developed to prepare alnustone, including the purification from plants [
16] and synthesis through an organic method [
20,
21].
Although the bioactivities have been investigated extensively, a few analytical methods have been reported. Currently, there are only three articles concerning the analytical methods for the determination of alnustone in natural medical plants or Chinese patent medicine, including HPLC [
22,
23], semi-preparative liquid chromatography [
24]. As far as the authors know, the pharmacokinetics of this compound still remains unknown. It is generally accepted that the study of pharmacokinetics and tissue distribution plays an important role in the drug development because it helps to predict and explain the various issues associated with drug efficacy and toxicity [
25,
26]. Therefore, it is necessary to establish an effective method to investigate the pharmacokinetic characteristics of alnustone so as to better understand its mechanism of action.
In the present study, a liquid chromatography tandem mass spectrometry (LC-MS/MS) method was developed and validated for the determination of alnustone in rat plasma. The pharmacokinetic behavior and tissue distribution of the intravenous injection of alnustone in rats was subsequently investigated by this method. To the authors’ knowledge, this is the first report on the pharmacokinetics of alnustone.
3. Materials and Methods
3.1. Chemicals and Reagents
Alnustone 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).
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, 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 agent 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/x2) 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].