Reagents
The aerial parts of T. triquetrum plant were collected from the Lingshui, Hainan province of China. The plant was identified by Professor Niankai Zeng (School of Pharmaceutical Science, Hainan Medical University, Hainan, China). Tadehaginoside (the purity is over 98%) was separated and purified from T. triquetrum by our research group. HYD (the purity is over 98%) was from Beijing Bailingwei Technology Co., Ltd. (Beijing, China). Quercetin, purchased from the National Institute for Food and Drug Control (Beijing, China), was used as the internal standard (IS, purity = 99.1%) for LC-MS/MS analysis. Ascorbic acid, CH3OH, and HCOOH were of HPLC grade and were obtained from Aladdin Reagents (Shanghai, China). Deionized water was sourced from Hangzhou Wahaha (Hangzhou, China).
Instruments and LC-MS/MS conditions
The HPLC system was equipped with a SIL-20ACXR autosampler, two LC-20ADXR pumps, an online degasser, and a CTO-20A column oven, and they were all purchased from Shimadzu (Kyoto, Japan). The chromatographic column was Synergi™ Fusion-RP 80 Å C18 (4 μm, 2.10 mm i.d × 50 mm, Phenomenex, Torrance, CA, USA), the temperature was maintained at 40°C during analysis. The aqueous solution containing 0.1% formic acid and methanol with 0.1% formic acid made up the mobile phase. The gradient elution was 10% B at 0-0.29 min, 90% B at 0.30-3.00 min, and 10% B at 3.01-4.00 min. The flow rate was set at 0.5 mL/min and the injection volume was 5 μL.
An AB Sciex Triple Quad™ 5500 system (Applied Biosystems, Foster City, CA, USA) was operated in the electrospray negative ionization mode (ESI-). To separate and determine tadehaginoside, HYD, and IS efficiently, the MS analysis detection was optimized when the collision energy at -22 V for tadehaginoside, -18 V for HYD and -29 V for IS, respectively. The optimized declustering potential was -120 V for tadehaginoside, 120 V for HYD, -120 V for IS. Temperature, 550°C; curtain gas, 25 psi; nebulizer gas, 55 psi; heater gas, 50 psi; ion spray voltage, -4500 V. The ion pairs of m/z 433.3→125.2 (tadehaginoside), m/z 162.8→119.0 (HYD) and m/z 301.1→151.0 (IS) were used for the quantitative analysis while undergoing multiple reactions monitoring (MRM).
Animals
Animal studies were conducted with 180-240 g male Sprague-Dawley rats, which were purchased from Hunan Slack Jingda Experimental Animals (Hunan, China; approval number: SCXK (Xiang) 2016-0002). All rats were placed in cages in a room with a relative humidity of 50% and temperature at 23 ± 2°C and were exposed to a 12 h light-dark cycle for a week before experiments. Animals were fasted for 12 h before drug administration, and water could be obtained ad libitum. Experimental procedures on animals were undertaken following the National Guidelines and were approved by the animal ethics committee of Hainan Medical University (reg. no. 201506017/HMU).
Preparation of IS and samples
Preparation of stock solutions and working solutions
A certain amount of tadehaginoside, HYD, and IS was dissolved in methanol to obtain the stock solution at a concentration of 1 mg/mL. The working solution of IS was further handled by dilution with methanol of stock solution to a final concentration of 10 μg/mL. Next, a linear concentration gradient (1, 5, 10, 100, 1000, 5000, 10000, 20000 ng/mL) of tadehaginoside stock working solution was serially diluted with methanol for pharmacokinetic studies and 50, 100, 1000, 5000, 10000 and 20000 ng/mL for tissue-distribution studies. Moreover, the concentrations of working solutions in plasma were 10, 50, 100, 500, 1000, 5000, 10000, 20000 ng/mL for HYD. All solutions were kept refrigerated at 4°C.
Preparation of calibration standards and quality control (QC) samples
Calibration standards and QC samples were prepared by mixing blank plasma or tissues with the working solutions as mentioned in section 2.4.1. The concentrations of calibration standards were ranged from 1 ng/mL to 2000 ng/mL (1, 10, 100, 500, 1000 and 2000 ng/mL) for tadehaginoside in plasma, and 5 ng/mL to 2000 ng/mL (5, 10, 100, 500, 1000 and 2000 ng/mL) in tissues. Similarly, calibration curve was prepared in the range of 10 ng/mL to 2000 ng/mL at six concentration levels (10, 50, 100, 500, 1000, 2000 ng/mL) for HYD in plasma. The final concentrations of QC samples were 3, 120, 1500 ng/mL for tadehaginoside in plasma samples, and 12, 120, 1500 ng/mL for tissue samples. In the same manner, three QC samples were set at 30, 120, 1500 ng/mL for HYD.
Preparation of sample solutions
The solution of ascorbic acid (5 μL) was transferred to 50 μL of rat plasma and then vortex-mixed for 15 s. Next, the sample was mixed with IS solution (5 μL, 10 μg/mL in methanol) and methanol (150 μL) and mixed for 1 min. After centrifugation (13,000 × g, 10 min), 5 μL of the supernatant was injected into the apparatus.
To investigate its tissue-distribution, each weighed tissue was homogenized in 0.9% NaCl (1:2, w/v) after thawing. Thereafter, 100 μL of the tissue homogenate and 10 μL of the ascorbic acid-saturated solution were added to a glass tube, and mixed for 15 s. The IS working solution (5 μL, 10 μg/mL in methanol) and methanol (300 μL) were added to it in turn. After vortex-mixing for 1 min, then it was centrifuged at 13,000 × g for 10 min under 4°C. The subsequent steps were conducted according to the procedure applied above.
Method validation
The method in the present study was validated according to the FDA and other related guidelines [13, 14]. The rat plasma and all of the target tissues or organs were analyzed to assess the specificity. Plasma and blank homogenates of the livers and kidneys as representative samples were screened for linearity, precision, accuracy, recovery, matrix effect, and stability.
Specificity
Specificity was determined by testing blank rat plasma and tissue homogenates from different rats, tadehaginoside, HYD and IS mixed with biological samples, and biological samples collected after treatment with tadehaginoside, respectively.
Linearity and LLOQ
The plasma samples and homogenates were handled as depicted in section 2.4.3, separately. 5 μL of working solutions were added into 45 μL of blank rat plasma to prepare the standard plasma samples. After spiking different concentrations of working solution (10 μL) to blank tissue homogenates (90 μL), the tissue standard solutions were obtained.
Calibration curves were constructed according to the previous report. Briefly, the least-squares linear regression method with 1/ x2 weighting was used to generate the slope, intercept, and correlation coefficient of each linear regression equation. The lowest concentrations (LLOQ) of tadehaginoside and HYD in the calibration curve were detected with an acceptable precision ≤20% and accuracy within ±20%.
Accuracy and Precision
The accuracy and precision of the method in within-run and between-run conditions were evaluated using three consecutive batches and on more than two days at low, medium, and high QC levels (n = 6). The relative error (RE%) was applied to express the accuracy and the relative standard deviation (RSD%) was applied to express the precision.
Stability
Stability was assayed by quintuplicate determinations of QC samples for each concentration. The following conditions were applied to test the stability of tadehaginoside and its metabolite: (i) after 4 h at room temperature (samples which had undergone a protein-precipitation procedure); (ii) after 2 h at room temperature (samples which had not undergone a protein-precipitation procedure); (iii) after 6 h in the autosampler (15°C); (iv) after 24 h at 2-8°C; (v) after three freeze-thaw cycles; (vi) after 7 days of storage at -20°C.
Matrix effect
Blank plasma and tissues were processed, the QC samples were added, and the matrix effect in samples was analyzed in three levels (low, medium, and high). Next, the mean peak area of the analyte or IS in post-extracted spiked plasma/tissue homogenates was compared against the neat sample at the corresponding concentration.
The matrix factor (MF) of analytes (or IS) and IS-normalized MF were evaluated using Eq. (1) and Eq. (2) [11, 15].
Recovery
Recovery experiments were calculated via the determination of six replicates from the QC samples. The extraction recoveries were obtained by comparing the response of analytes from the extracted samples with the response of the same concentration of analytes spiked into the solution extracted from blank biological samples.
Pharmacokinetic study
Two groups (n = 5 per group) were set up by randomly dividing the ten male Sprague-Dawley rats. One group was given tadehaginoside intravenously at a dose of 5 mg/kg. The other group was orally administered tadehaginoside at doses of 25 mg/kg. Blood samples (0.2 mL) were gathered immediately from the suborbital vein and placed in heparinized 1.5-mL polythene tubes at 0, 5.0, 10.0, 15.0, 20.0, 30.0, 45.0, 60.0, 90.0, 120.0, 240.0, and 360.0 min following intravenous and oral administration. Then, each blood sample was immediately centrifuged immediately at 2000 × g for 10 min at 4°C, and plasma was harvested and stored at -20℃ until further treatment.
Tissue-distribution study
The tissue-distribution investigation was conducted on twenty Sprague-Dawley rats which were divided randomly into five groups. Rats were intravenous administration at a dose of 5 mg/kg and sacrificed by overdose of pentobarbital sodium (100 mg/kg) intraperitoneally for each time point (30.0, 60.0, 120.0, and 240.0 min). To remove superficial blood and contents, tissues (brain, heart, liver, spleen, lungs, kidneys, stomach, small intestine, skeletal muscle, body fat, and testes) were harvested and rinsed with ice-cold physiologic (0.9%) NaCl. Next, tissues were blotted with filter paper, weighed accurately, and homogenized in 0.9% NaCl (1:2, m/v). The obtained tissue homogenates were immediately stored at -20℃ until analysis.
Statistical analysis
To calculate pharmacokinetic parameters, DAS 3.2.8 (Mathematical Pharmacology Professional Committee of China, Shanghai, China) was applied as a non-compartmental model. The half-life, area under the curve, clearance rate and mean residual time were calculated. Results are all expressed as the mean ± standard deviation (SD).