Chemicals

Panasenoside (purity >98%, Figure 1a) and naringin (IS (internal standard), purity >98%, Figure 1b) were obtained from Chengdu Mansite Pharmaceutical Co. Ltd. (Chengdu, China). Chromatography grade methanol, acetonitrile, and formic acid were obtained from Merck Company (Darmstadt, Germany). Ultrapure water (resistance >18 MΩ) was prepared by Milli-Q purification system (Bedford, USA).

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Chemical structure of panasenoside (A) and naringin (IS, B)

Citation: Acta Chromatographica Acta Chromatographica 31, 2; 10.1556/1326.2018.00415

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Instrumentation and Conditions

XEVO TQS-micro triple quadrupole mass spectrometer and ACQUITY I-Class UPLC, equipped with electrospray ionization (ESI) interface, were from Waters Corp. (Milford, MA, USA). Masslynx 4.1 software (Milford, MA, USA) was used for instrument control and data acquisition.

Panasenoside and IS were chromatographic separation achieved on a UPLC BEH C18 column (2.1 mm × 50 mm, 1.7 μm). The mobile phase consisted of 0.1% formic acid and acetonitrile, the flow rate was set at 0.4 mL/min, and column temperature was set at 40 °C. The gradient elution condition was as follows: 0–0.2 min, acetonitrile 10%; 0.2–1.5 min, acetonitrile 10%–80%; 1.5–2.0 min, acetonitrile 80%; 2.0–2.5 min, acetonitrile 80%–10%; and 2.5–3.5 min, acetonitrile 10%.

The mass parameters were listed as follows: capillary voltage of 2.0 kV, desolvation temperature of 400 °C, desolvation gas (nitrogen) of 800 L/h, and cone gas of 50 L/h. MRM modes of m/z 609.2 → 284.1 for panasenoside and m/z 579.2 → 271.1 for IS were used for quantitative analysis in negative mode (Figure 2).

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Mass spectrum of panasenoside (A) and naringin (IS, B)

Citation: Acta Chromatographica Acta Chromatographica 31, 2; 10.1556/1326.2018.00415

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Calibration Standards

The stock solutions of panasenoside (1.0 mg/mL) were prepared in methanol. The stock solutions of IS (0.5 mg/mL) were prepared in methanol. The 0.5 μg/mL IS working solution was prepared from the stock solution by dilution with methanol. The stock solutions and working solution were stored at 4 °C.

Panasenoside calibration standards were prepared by spiking working solutions of incremental concentration into blank rat plasma. Calibration plots were offset to range between 2 and 2000 ng/mL for panasenoside in rat plasma (2, 6, 20, 100, 200, 400, 800, 1500, and 2000 ng/mL) and between 2 and 3000 ng/mL in mouse tissue (2, 10, 50, 200, 500, 1000, 2000, and 3000 ng/mL), each by adding 5 μL of the appropriate working solution to 50 μL of blank rat plasma or 50 mg of mouse tissue, and then mixed by brief vortexing. Quality-control (QC) samples were prepared in three concentrations of rat plasma (3, 250, and 1800 ng/mL) or mouse liver (3, 400, and 2500 ng/mL). The proteins of calibration standards and QC samples were precipitated by methanol before UPLC–MS/MS analysis.

Sample Preparation

The 5 μL IS working solution (0.5 μg/mL) was added to 50 μL plasma sample in a 1.5 mL centrifuge tube, and 150 μL methanol was added, then vortex-mixed for 1.0 min, and centrifuged at 14,900 g for 10 min; 2 μL of the supernatant was injected into the UPLC–MS/MS system.

The mouse tissues (heart, liver, spleen, lung, kidney, and brain) were respectively weighed accurately 50 mg and placed in 1.5 mL centrifuge tubes, and then filled with 200 μL methanol and 5 μL IS working solution (0.5 μg/mL). The mixtures were stored at −80 °C for 10 min and then grinded for 3 min by SCIENTZ-48 Tissue Grinder (64 Hz and 1800 r/s). The tubes were vortex-mixed for 1.0 min and centrifuged at 14,900 g for 10 min, and 2 μL of the supernatant was injected into the UPLC–MS/MS system.

Method Validation

Method validation was conducted according to the guidelines set by the European Medicines Agency (EMA). Validation runs were conducted on three consecutive days, with each run consisted of six replicates of QC samples and one set of calibration standards [8–10].

The selectivity of the method was evaluated by analyzing six lots of different sources of blank rat plasma or mouse liver, blank plasma or mouse liver spiked with panasenoside and IS, and a rat plasma sample or a mouse liver sample.

Calibration curves were obtained by analyzing spiked calibration samples on three separate days. Peak area ratios of panasenoside-to-IS were plotted against panasenoside concentrations. Standard curves were well fitted to the equations by linear regression, with a weighting factor of the reciprocal of the concentration (1/x) in the concentration range of 2–2000 ng/mL for rat plasma and 2–3000 ng/mL for mouse tissues. The lower limit of quantitation (LLOQ) was defined as the lowest concentration on the calibration curves.

To evaluate the matrix effect, blank rat plasma was extracted and then spiked with the analyte at 3, 250, and 1800 ng/mL (n = 6), and blank mouse tissues (heart, liver, spleen, lung, kidney, and brain) were extracted and then spiked with the analyte at 3, 400, and 2500 ng/mL. The corresponding peak areas were then compared to those of neat standard solutions at equivalent concentrations, and this peak area ratio is defined as the matrix effect. The matrix effect of the IS was evaluated at a concentration of 50 ng/mL in the similar manner.

Accuracy and precision were evaluated by the determination of QC samples at three concentration levels (3, 250, and 1800 ng/mL for rat plasma; 3, 400, and 2500 ng/mL for mouse tissue) in six replicates over three validation. The precision is expressed by relative standard deviation (RSD).

The recovery of panasenoside was evaluated by comparing the peak area of extracted QC samples with those of reference QC solutions reconstituted in blank plasma or blank mouse tissue extracts (n = 6). The recovery of the IS was determined in the same way [11, 12].

Stability of panasenoside in rat plasma and mouse liver was evaluated by analyzing three replicates of plasma samples (3, 250, and 1800 ng/mL) and liver sample (3, 400, and 2500 ng/mL) that are exposed to different conditions. These results were compared with that of freshly prepared plasma or liver samples. Short-term stability was determined after the exposure of the spiked samples to room temperature for 2 h and the ready-to-inject samples (after protein precipitation) in the HPLC autosampler at room temperature for 12 h. Freeze–thaw stability was evaluated after three complete freeze–thaw cycles (−20 to 25 °C) on consecutive days. Long-term stability was assessed after storage of the standard spiked plasma samples at −20 °C for 20 days. The stability of the IS (50 ng/mL) was also evaluated with the similar method [13, 14].

Pharmacokinetic Study

All twelve male Sprague-Dawley rats (200–220 g) were obtained from the Laboratory Animal Center of Wenzhou Medical University. Diet was prohibited for 12 h before the experiment while water was freely available. Blood samples (0.2 mL) were collected from the tail vein into heparinized 1.5 mL centrifuge tubes at 0.0333, 0.25, 0.5, 1, 2, 3, 4, 6, and 8 h after intravenous (2 or 10 mg/kg) administration of panasenoside, six rats for each dose, and then centrifuged at 3000 g for 10 min. The freshly obtained plasma was stored at −20 °C until analysis. Plasma panasenoside concentration versus time was analyzed by version 2.0 DAS (Drug and Statistics) software (China Pharmaceutical University).

Tissue Distribution Study

Thirty mice were randomly divided into six groups (five mice for each group, one group for each time point) and received 20 mg/kg of panasenoside by intraperitoneal administration. The mice were euthanized by decapitation at 0 (blank group), 0.25, 0.5, 2, 4, and 6 h after given panasenoside. Tissues were removed and washed with saline then stored at −20 °C.

Method Development

In comparison with conventional analytical techniques, UPLC–MS/MS has been proven to improve sensitivity, selectivity, and specificity in determination of the active compound of herbal drug in biological samples. Consequently, we established a UPLC–MS/MS method for the determination of panasenoside in rat plasma and mouse tissue, and then successfully applied the method to the pharmacokinetic study in rats and tissue distribution study in mice.

The feasibility of ESI in negative and positive ion modes of detection was evaluated [15–17], and ESI with negative ion detection resulted in a better signal-to-noise ratio for both panasenoside and IS.

The mobile phase was also optimized for UPLC–MS/MS. We explored the mobile phase with methanol, acetonitrile, water, 0.1% formic acid, and 0.1% ammonia in combinations and concluded with acetonitrile–0.1% formic acid to be the optimal choice as this combination provided relatively clean chromatographic peaks and acceptable sensitivity. The gradient elution mode was chosen in our work for its thorough cleaning of the impurity in the column and the protection of the chromatographic system [18–21].

UPLC–MS/MS analysis requires a rapid, simple, and convenient sample treatment method with acceptable recovery and matrix effect [22–26]. We tried plasma (50 μL) with acetonitrile, methanol, ethyl acetate, and ether in our study. The result indicated that methanol has the highest extraction efficiency with acceptable matrix effect, and thus, methanol was used in protein precipitation for plasma and tissues in this work.

We further investigated the performance of naringin as IS. As naringin and panasenoside exhibited similar chromatographic retentions and both were suitable for analysis in the negative ESI interface, naringin was used as IS in our study.

Selectivity and Matrix Effect

Figure 3 showed typical chromatograms of a blank plasma sample, a blank plasma sample spiked with panasenoside and IS, and a plasma sample from a rat after intravenous administration of panasenoside. Figure 4 showed typical chromatograms of a blank mouse liver, a blank mouse liver sample spiked with panasenoside and IS, and a mouse liver sample after intraperitoneal administration of panasenoside in mouse. There were no interfering endogenous substances observed at the retention time of the panasenoside and IS.

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Representative UPLC–MS/MS chromatograms of panasenoside and naringin (IS) in rat plasma. A, blank plasma; B, blank plasma spiked with panasenoside (20 ng/mL) and IS (50 ng/mL); C, a rat plasma sample after intravenous administration of single dosage 2 mg/kg panasenoside

Citation: Acta Chromatographica Acta Chromatographica 31, 2; 10.1556/1326.2018.00415

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Representative UPLC–MS/MS chromatograms of panasenoside and naringin (IS) in mouse liver. A, blank mouse liver; B, blank mouse liver spiked with panasenoside (20 ng/mL) and IS (50 ng/mL); C, a mouse liver sample after intraperitoneal administration of 20 mg/kg panasenoside in mouse

Citation: Acta Chromatographica Acta Chromatographica 31, 2; 10.1556/1326.2018.00415

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The matrix effect of panasenoside at concentrations of 3, 250, and 1800 ng/mL in rat plasma was measured to be between 91.7% and 94.5% (n = 6) and at 3, 400, and 2500 ng/mL in mouse tissues was measured to be between 85.1% and 98.8% (n = 6) (Table 1). The matrix effect of IS (50 ng/mL) was measured to be 95.6% (n = 6) in rat plasma and 90.4% (n = 6) in mouse liver. The result of matrix effect from plasma and mouse tissues was acceptable.

Calibration Curve and Sensitivity

Linear regressions were fitted over the 2–2000 ng/mL for panasenoside in rat plasma. The calibration curve was as follows: y = 0.005037x − 0.007529, r = 0.9985, where y represents the ratios of panasenoside peak area to that of IS and x represents the plasma concentration. The LLOQ of panasenoside in plasma was 2 ng/mL. The precision and accuracy were 13.5% and 88.5% at LLOQ, respectively.

Linear regressions were fitted over the range of 2–3000 ng/mL for panasenoside in mouse tissue. The calibration curve was y = 0.012663x − 0.004953, r = 0.9978 for heart; y = 0.005746x − 0.001557, r = 0.9978 for liver; y = 0.005191x − 0.003484, r = 0.9957 for spleen; y = 0.010151x + 0.005098, r = 0.9989 for lung; y = 0.002603x − 0.001299, r = 0.9966 for kidney; and y = 0.005523x − 0.004665, r = 0.9983 for brain. The LLOQ of panasenoside in tissues was 2 ng/mL. The precision at LLOQ was between 8.2% and 17.5%, and accuracy was between 83.6% and 117.4%, respectively.

Precision, accuracy and recovery

The inter-day and intra-day precision at each QC level was 14% or less and 13% or less, respectively, while the accuracy was between 89.6% and 110.9% (Table 1). Mean recoveries ranged from 84.2% to 94.6% in rat plasma and from 84.3% to 99.4% in mouse tissues. The recovery of the IS (50 ng/mL) was 85.8% in rat plasma and 82.7% in mouse liver.

Stability of Rat Plasma and Mouse Liver

The panasenoside in rat plasma and mouse liver was stable under short-term room temperature, freeze–thaw cycling, and long-term (20 days) storage conditions, as the bias in concentrations was within ±12% of their nominal values, and precision was less than 12% (Table 2).

Pharmacokinetic Study

The main pharmacokinetic parameters were listed in Table 3, based on non-compartment model analysis. The plasma concentration–time curves after intravenous (2 and 10 mg/kg) administration of panasenoside in rats were shown in Figure 5. The AUC_(0–t) was 160.8 ± 13.0 and 404.9 ± 78.0 ng/mL*h; t_1/2, 3.2 ± 1.2 and 4.6 ± 1.7 h; and CL, 10.0 ± 2.0 and 21.4 ± 2.0 L/h/kg after intravenous administration of 2 mg/kg and 10 mg/kg of panasenoside, respectively.

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Mean plasma concentration time profile after intravenous (2 and 10 mg/kg) administration of panasenoside in rats

Citation: Acta Chromatographica Acta Chromatographica 31, 2; 10.1556/1326.2018.00415

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We have done pre-experimental oral administration of 10 mg/kg panasenoside, but the panasenoside in rat plasma is not detected, indicating that the oral absorption is poor and there is low bioavailability. The pharmacokinetics of panasenoside metabolism right after intravenous administration and the pharmacokinetic profile of panasenoside in rats were characterized for the first time, which contributed to a deeper understanding of the pharmacological features of panasenoside.

Tissue Distribution

The concentration–time curves of panasenoside in mouse plasma, heart, liver, spleen, lung, kidney, and brain after intraperitoneal administration of 15 mg/kg were shown in Figure 6.

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Mean concentration of panasenoside in tissues at different time (0.25, 0.5, 2, 4, and 6 h) after intraperitoneal administration of 20 mg/kg panasenoside in mouse

Citation: Acta Chromatographica Acta Chromatographica 31, 2; 10.1556/1326.2018.00415

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The tissue distribution results indicated that the panasenoside diffuses rapidly and widely into major organs. The level of panasenoside was highest in mouse liver, followed by kidney, lung, and spleen. The level of panasenoside in brain remains considerably low, which indicated that blood–brain barrier may effectively prevent panasenoside from moving across. The overwhelming accumulation in liver indicated that liver was responsible for the extensive metabolism of panasenoside.