Materials and chemicals
Menispermi Rhizoma was obtained from Anguo Chinese medicine wholesale market, Hebei Province, China (place of origin: Liaoning Province), and authenticated by Prof. Tianxiang Li (Department of Pharmacognosy, Tianjin University of Traditional Chinese Medicine). A voucher specimen (No. 20190701) has been deposited in the Department of Pharmaceutical Analysis, Tianjin University of Traditional Chinese Medicine. The standard of DMP was isolated in the author’s laboratory. The procedure for isolation, purification, and characterization of DMP as well as the corresponding spectral and physico-chemical data (MS, 1H-NMR, and 13C-NMR) have been published by the author (Shao et al., 2019; Wei et al., 2016). Based on these data as well as the literature report (Li et al., 2013), the structure of DMP was accurately characterized. HPLC-DAD and HPLC-MS analysis showed that the purity was 98.10%. Curcumin (HPLC purity 99.00%) was provided by China Food and Drug Inspection and Research Institute (Beijing, China). Analytical grade acetonitrile and methanol were provided by Tianjin Concord Technology Co., Ltd., China. Formic acid, acetic acid, hydrochloric acid, and tetrahydrofuran (HPLC-grade) were obtained from Kermel Chemical Reagent Co., Ltd. (Tianjin, China). Dimethyl sulfoxide (DMSO) and polyethylene glycol 400 (PEG-400) of analytical grade were obtained from Sigma-Aldrich (St. Louis, MO, USA). Water (HPLC-grade) was purified by SZ97 ultrapure water preparation device (Shanghai Yarong Biochemical Instrument Factory, China).
The quantification of DMP in biological samples was conducted on a JASCO HPLC system (JASCO Corporation, Japan) involving two JASCO-PU-2080 plus pumps, an auto-sampler, and an Agilent 1100 fluorescence detector (Agilent Technologies, USA). A Capcell Pak C18 column (150 mm × 4.6 mm i.d., 5 μm) was used with an isocratic mobile phase (delivered at 1.0 mL/min) consisting of acetonitrile and water (68:32, v/v, containing 1% formic acid). The column temperature was set at 25 °C. Injection volume was 20 μL, and the maximum response for DMP was obtained through a fluorescence detector with 426-nm excitation and 514-nm emission wavelengths. The excitation and emission wavelengths for curcumin (IS) are the same as those of DMP.
Preparation of MR extract and determination of DMP in MR extract
Ten volumes of extracting solvent were added to 100 g MR powder. The mixture was extracted by refluxing twice with ethanol-water (95:5, v/v) and twice with ethanol-water (75:25, v/v) (1 h per time). And the extracts were filtered, combined, and further evaporated to dryness under reduced pressure. An external standard method with the chromatographic condition as described above was used for the quantification of DMP in MR extract. Two hundred milligrams of MR extract was accurately weighted and dissolved in methanol in a 10-mL volumetric flask. After filtration using a 0.45-μm membrane filter, the solution was injected into HPLC-FLD for analysis.
Preparation of solutions
The primary solution of DMP was prepared in 5 mL of tetrahydrofuran and diluted with methanol to the desired concentration of 0.1 mg/mL. The DMP working solutions with concentrations that ranged from 10 to 2500 ng/mL were harvested by diluting a solution of DMP with methanol. Curcumin (IS) stock solution with the concentration of 0.1 mg/mL was obtained in a similar manner, then it was diluted with methanol to get 50 μg/mL IS working solution.
Standard curves of DMP were obtained by evaporating 100 μL of working solution to dryness with a stream of N2 at 35 °C and mixing with 100 μL blank biomatrices. The final concentrations were 10, 25, 100, 250, 500, 1500, and 2500 ng/mL. QC samples were independently obtained at a final concentration of 10 (LLOQ), 30 (low-QC), 200 (mid-QC), and 2000 ng/mL (high-QC).
Owing to the relatively high lipid solubility, DMP and MR extract were extremely difficult to dissolve in water or 0.5% carboxymethyl cellulose sodium solution (CMC-Na). By searching relevant literatures, a method for the preparation of dosage form of DMP and MR extract was confirmed (Shen et al., 2019; Wang et al., 2013; Yoon et al., 2020). DMP and MR extract were firstly dissolved in DMSO, then diluted with a mixture of PEG-400 and water, respectively. Finally, the volume ratio of the three solvents is 5:45:50 (DMSO:PEG-400:water).
Processing of samples
Twenty microliters of IS solution (50 μg/mL) was transferred into a 10-mL clean glass tube and dried with a stream of N2 at 35 °C, and then 100 μL plasma and 20 μL 0.5 mol/L hydrochloric acid solution were added. After vortexing for 1 min, 3 mL ethyl acetate was added to the extracted analyte from plasma. The mixture was shaken again for 3 min and centrifugated at 12,000×g for 10 min, then the supernatant was collected and dried in a water bath (35 °C) under N2. The dried residue was reconstituted with 200 μL 50% acetonitrile, then vortexed for 1 min, and centrifugated for 10 min at 12,000×g, and 20 μL supernatant was analyzed.
According to the guidelines for Industry on Bioanalytical Method Validation (U.S. Food and Drug Administration , 2018), validation of the method was conducted.
In order to evaluate the specificity of the method, blank plasma from six different batches, QC samples at LLOQ, and the actual sample obtained following intragastric dosing of MR extract were analyzed to exclude the interference peaks. Carryover was estimated by injecting blank plasma in six replicates following injection of an ULOQ sample.
Linearity and sensitivity
In order to evaluate linearity, a 7-point calibration curve was run in duplicate during each of the 3 days of the validation. Using weighted least-squares linear regression (1/x2 as the weighting factor), the calibration curve was described by analyzing raw data (plot DMP-to-IS ratio versus DMP concentrations). Limit of detection (LOD) was defined as signal-to-noise above 3. LLOQ was defined as signal-to-noise above 10, which is the lowest concentration of calibration curve with an acceptable precision < 20% and accuracy within ± 20%.
Accuracy and precision
Simulated biological samples were analyzed on the same day and on three consecutive days to assess intra-assay and inter-assay accuracy and precision, which were conducted following the standard calibration curve. Each analytical run consisted of LLOQ and three QC samples in six replicates, and the concentrations for DMP were determined using the calibration curve. The percentage relative standard deviation (RSD%) was measured to evaluate the precision, which was expected to be less than 15%. Accuracy was expressed by relative error (RE%), the criteria of which was within ± 15%. For LLOQ, the RSD% and RE% should not exceed ± 20%.
Extraction recovery and matrix effect
The recovery of DMP was estimated by comparing the responses of DMP in the extracted QC samples with that of samples spiked post-extraction at corresponding concentration. Matrix effect was estimated by the ratio of the responses of DMP in post-extracted blank matrices to that of pure DMP standard solutions at corresponding concentration. The above two values of IS were estimated in the same way. All analyses of recovery and matrix effect were carried out at three QC levels in six replicates.
Three aliquots of QC samples were used to estimate the experimental stability of DMP under various conditions. Different storage conditions included three freeze-thawing cycles, storage at ambient temperature for 8 h (QC samples), QC samples kept at − 20 °C for at least 2 weeks, and post-preparative samples stored in the auto-sampler at 4 °C for 8 h. RSD within 15% was considered reliable. The stability of standard solutions was evaluated by comparing the concentration of freshly prepared with that of solution stored at 4 °C for 2 weeks.
When the concentrations of actual samples exceed ULOQ, it is necessary to dilute with blank plasma. In order to validate the dilution process, five- and twenty-times dilution of plasma samples were assessed in six duplicates. The simulated plasma biosamples with concentrations of 1000 and 4000 ng/mL of DMP were diluted with blank plasma matrices to get samples with concentrations of 200 ng/mL.
The validated HPLC-FLD method was examined by exploring the pharmacokinetic characteristics of DMP in rats. Twelve SPF-grade male Sprague-Dawley rats weighing 180–220 g were housed for 1 week under an air-conditioned environment with a temperature at 23–27 °C and relative humidity of 40–60%. This research was approved by the Tianjin University of Traditional Chinese Medicine Animal Ethics Committee (Tianjin, China) and carried out according to the ethical guidelines. Animals were fasted for at least 12 h prior to oral administration of DMP and MR extract, with free access to water. Twelve animals were randomly separated into two groups for oral administration of DMP and MR extract, respectively. After oral dosing of DMP (1 mg/kg) and MR extract (1.605 g/kg, a dose equivalent to 1 mg/kg DMP), 0.25 mL of blood samples from the suborbital vein was collected into heparinized 0.5-mL tubes at 0, 5, 10, 20, and 45 min and then at 1, 2, 3, 4, 5, 8, 12, 24, 36, and 48 h. The plasma sample (100 μL) was achieved after centrifugation at 12,000×g for 8 min and frozen at − 20 °C before analysis.
Drug and statistics (DAS) 3.2 and SPSS 16.0 software were employed to calculate and compare the pharmacokinetic parameters harvested following oral dosing of DMP neat substance and MR extract. A non-compartmental model was employed to calculate parameters as described previously (Balla et al., 2018). An independent sample t test was used to compare the Cmax (peak plasma concentration), AUC0–∞, AUC0–t (area under the plasma concentration-time curve). And, the t1/2 (terminal elimination half-life), MRT0–t, MRT0–∞ (mean residence time), Clz/F (apparent oral clearance), and Tmax (time to reach peak plasma concentration) were analyzed via a Mann-Whitney nonparametric statistical test (Huo et al., 2013; Zhao et al., 2011). It was considered to be statistically significant when the p value was below 0.05.