Journal of Pharmaceutical and Biomedical Analysis 114 (2015) 200–207
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UPLC–MS method for quantification of pterostilbene and its application to comparative study of bioavailability and tissue distribution in normal and Lewis lung carcinoma bearing mice Li Deng a , Yongzhi Li b , Xinshi Zhang c , Bo Chen a , Yulin Deng a , Yujuan Li a,∗ a
School of Life Science, Beijing Institute of Technology, 5 South Street Zhongguancun, Beijing 100081, China China Astronaut Research and Training Centre, Beijing 100094, China c Hebei North University, 11 Zuanshi South Road, Zhangjiakou 075000, China b
a r t i c l e
i n f o
Article history: Received 31 October 2014 Received in revised form 10 April 2015 Accepted 26 April 2015 Available online 29 May 2015 Keywords: Pterostilbene UPLC–MS Bioavailability Tissue distribution
a b s t r a c t A UPLC–MS method was developed for determination of pterostilbene (PTS) in plasma and tissues of mice. PTS was separated on Agilent Zorbax XDB-C18 column (50 × 2.1 mm, 1.8 m) with gradient mobile phase at the flow rate of 0.2 ml/min. The detection was performed by negative ion electrospray ionization in multiple reaction monitoring mode. The linear calibration curve of PTS in mouse plasma and tissues ranged from 1.0 to 5000 and 0.50 to 500 ng/ml (r2 > 0.9979), respectively, with lowest limits of quantification (LLOQ) were between 0.5 and 2.0 ng/ml, respectively. The accuracy and precision of the assay were satisfactory. The validated method was applied to the study of bioavailability and tissue distribution of PTS in normal and Lewis lung carcinoma (LLC) bearing mice. The bioavailability of PTS (dose 14, 28 and 56 mg/kg) in normal mice were 11.9%, 13.9% and 26.4%, respectively; and the maximum level (82.1 ± 14.2 g/g) was found in stomach (dose 28 mg/kg). The bioavailability, peak concentration (Cmax ), time to peak concentration (Tmax ) of PTS in LLC mice was increased compared with normal mice. The results indicated the UPLC–MS method is reliable and bioavailability and tissue distribution of PTS in normal and LLC mice were dramatically different. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Pterostilbene (trans-3,5-dimethoxy-4-hydroxystilbene, PTS, Fig. 1A) is a naturally occurring stilbenoid compound which has been found in many dietary plants such as grapes, berries [1–3], and medicinal herbs including Pterocarpus indicus, Pterocarpus marsupium, Dracaena cochinchinensis [4–7]. Recently, PTS has drawn more and more attention because of various health-promoting activities, such as antioxidant, anti-inflammatory, anti-diabetic, and prevention of age-related disease [8–14]. PTS possess anticancer properties against breast, colon, leukemia, liver, prostate, and lung cancers [15–20]. Although PTS shows various pharmacological activities, the information of PTS in vivo is still limited. Therefore, further in vivo research is necessary to understand the health-promoting activities of PTS. The plasma pharmacokinetics, oral bioavailability of PTS in normal rats using high performance liquid chromatography (HPLC) with ultraviolet detection (UV) or mass spectrometry (MS)
∗ Corresponding author. Tel.: +86 10 68914607; fax: +86 10 68914907. http://dx.doi.org/10.1016/j.jpba.2015.04.045 0731-7085/© 2015 Elsevier B.V. All rights reserved.
have been reported [21–24]. The effect of aqueous solubility and fasting on PTS bioavailability in rats with HPLC–UV method has been described [25]. The lowest limits of quantification (LLOQ) in HPLC–UV methods [21–23] were between 10 and 50 ng/ml, which might not be sensitive enough for quantification of PTS because of relatively low PTS level in tissues, urine or feces of mice. In the reported HPLC–MS method [24], the method validation is incomplete (data on recovery, stability or matrix effect are unavailable), and there is no internal standard (it is an external method). LLOQ of PTS is 5 ng/ml in rat plasma. It is known that internal standard and full validation are indispensible during bio-analytical method development. Obviously, a more reliable, highly sensitive and fully validated UPLC–MS method is essential for assay of PTS in biosamples. To our knowledge, no liquid chromatography LC–MS method for determination of PTS in urine, feces or tissues of experimental animals is available. Studies evaluating tissue distribution or identifying tissue metabolites of PTS with LC–MS method in experimental animals have not been reported. In addition, reports on the pharmacokinetics, bioavailability or excretion of PTS in tumor bearing animals are not available. Further, there have been no comparative
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Fig. 1. Chemical structures of pterostilbene (A) and loureirin B (B).
studies on pharmacokinetics, bioavailability or tissue distribution between tumor bearing and normal animals. Therefore, the major aim of present study is to develop a reliable and sensitive ultra-performance liquid chromatography (UPLC)–MS method for quantification of PTS in mouse plasma and main tissues, and then apply it to investigate oral bioavailability and tissue distribution of PTS in normal and Lewis lung carcinoma (LLC) bearing C57 BL/6 mice, and identify any possible metabolites. It is expected that the present findings would be helpful to explore the association between in vivo behavior and anticancer effect of PTS in LLC mice.
2. Experimental 2.1. Chemicals Pterostilbene (purity > 99.0%) and loureirin B (used as internal standard, IS, purity > 98.0%) were obtained from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). Their chemical structures were shown in Fig. 1. Distilled water was prepared by a Milli-Q water purification system (Millipore, Mississauga, Canada). Acetonitrile and methanol were of chromatographic grade from Thermo Fisher Scientific (Waltham, Massachusetts, USA). Analytical grade ammonium acetate and methyl tert-butyl ether (MTBE) were purchased from Beijing Chemical Manufacturer (Beijing, China). 2.2. HPLC–MS conditions Chromatographic separation was achieved using an Agilent 1290HPLC system equipped with a binary Infinity Bin G4220 pump, a G1316C column oven and a G2426A infinity auto-sampler (Agilent Technologies, Palo Alto, California, USA). The analytes were separated on an Agilent Zorbax XDB-C18 column (50 × 2.1 mm, 1.8 m, Santa Clara, California, USA). The mobile phase consisted of solvent A (0.1% ammonium acetate in water) and solvent B (0.1% ammonium acetate in acetonitrile). The gradient program was as following: 0 min 30% B, 0.5 min 30% B, 2.0 min 80% B, 3.5 min 80% B, 5.0 min 30% B, 6.0 min 30% B, 6.5 min stop. The flow rate was 0.2 ml/min and the temperature was maintained at 25 ◦ C. The injection volume was 10 l. The MS system consisted of an Agilent 1290-6460 tandem quadrupole mass spectrometer (Agilent Technologies, Palo Alto, California, USA) equipped with electrospray ionization (ESI) interface. Agilent masshunter workstation (version B.04.00) was used for data acquisition and processing. The mass spectrometer was operated in the negative ion detection mode. Nitrogen was used as the nebulizer, drying gas, heater gas as well as the collision gas. The main parameters were as follows: drying gas temperature 350 ◦ C; drying gas flow 7 l/min; nebulizer gas 45 psi; capillary voltage 3000 V; fragmentor 138 and 111 V for PTS and IS, respectively; collision energy –40 eV for PTS and IS. PTS was quantified by multiple-reaction monitoring (MRM) mode using the selected
ion transitions of each compound. The precursor to product ion transitions was m/z 255.1 → 240.1 for PTS and 315.1 → 134.1 for IS. 2.3. Sample preparation 2.3.1. Preparation of calibration standards and quality control samples PTS stock solution was prepared by dissolving 15 mg of PTS in 15 ml of methanol and then diluted with methanol to give the final concentration of 100 g/ml. Working solutions were prepared by appropriate dilution of the stock solution with 20% methanol–water (v:v). A stock solution of IS at 100 g/ml was also prepared in methanol and then diluted with 20% methanol–water (v:v) to obtain a working solution of 100 ng/ml. All the solutions were stored at 4 ◦ C before use. The calibration standards and quality control (QC) samples were prepared by spiking blank mouse plasma and all tissues with standard working solutions. The calibration samples of PTS in all tissues ranged from 0.10 to 5000 ng/ml. The calibration samples of PTS in mouse plasma were made at the ranges of 1.00–5000 ng/ml. QC samples was prepared at three different levels (low, medium and high concentration) for method validation. For plasma samples, QC samples were 1.00, 500, and 4000 ng/ml, respectively. For tissues, QC samples were made by lowest limit of quantification (LLOQ), the medium and 80% of the highest concentration level of respective calibration curves. 2.3.2. Preparation of plasma and tissue samples After thawing of plasma samples at room temperature, 15 l of plasma was mixed with 10 l of IS solution and 150 l of MTBE. The samples were centrifuged at 5000 rpm for 10 min. 150 l of supernatant was transferred to new vials and evaporated to dryness under a stream of nitrogen at room temperature. The residuals were dissolved in 150 l of methanol and water (1:1, v/v). Tissues samples were homogenized with saline (ten-fold of tissue weight) and then centrifuged at 5000 rpm for 10 min to get the homogenates.10 l of IS solution and 200 l of MTBE were added to 20 l of tissue homogenates. Each vial was mixed thoroughly by vortexing for 60 s. The samples were centrifuged at 5000 rpm for 10 min. 200 l of supernatant was transferred to new vials and evaporated to dryness under a stream of nitrogen at room temperature. The residuals were dissolved in 200 l of methanol and water (1:1, v/v). 2.4. Method validation According to US Food and Drug Administration Guidance for Industry – Bioanalytical Method Validation [27], the method was fully validated for the selectivity, linearity, accuracy and precision, recovery and stability. Matrix effect (ME) was also evaluated according to published procedures [28]. Selectivity was tested by comparison of blank matrices from six individual mice with corresponding spiked matrices. The calibra-
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tion standards of PTS (1.00–5000 ng/ml for plasma, 0.10–500 ng/ml for tissues) were prepared by spiking blank plasma or tissues with appropriate amounts of working solutions. A calibration curve for PTS were obtained by plotting the peak area ratio (y) of the analyte over IS against the concentrations (x) of PTS with weighting function 1/x2 . LLOQ is the lowest concentration of analyte on the calibration curve, which should be quantified reliably with an acceptable accuracy and precision (less than or equal to 20%) [27]. QC samples were prepared with blank matrices at low, medium, and high concentration levels of PTS for precision and accuracy assessment. QC samples at three concentrations in six replicates were analyzed during the same day using the same calibration curve to determine the intra-day precision. QC samples were analyzed for three continuous days to define inter-day precision. Accuracy was expressed as a percentage of the measured concentration over the nominal concentration of the QC samples. The recovery of PTS and IS was determined by comparing the peak areas of the PTS and IS in extracted QC samples with those in post-extraction blank samples spiked at the corresponding concentrations. Matrix effect (ME) was evaluated by the method reported in literature [28]. Peak areas of PTS in spiked plasma, bile, urine and tissues post extraction (B) were compared with those of PTS neat solutions at QC levels (A). ME was defined as the ratio of B/A × 100%. ME values > 100% mean ionization enhancement, whereas ME values < 100% indicate ionization suppression. The stability of PTS in plasma and tissues after long-term (−20 ◦ C for 30 days), short-term storage (room temperature for 8 h), and after going through three freeze and thaw cycles were evaluated with respective QC samples. Stability for standard stock solution of PTS and IS kept at 4 ◦ C for 30 days was tested. 2.5. Animal treatment Normal C57 BL/6 mice (body weight 20 ± 2 g, 8 weeks) were purchased from Beijing Military Medical Sciences Experimental Animal Co., Ltd. (Beijing, China). An LLC cell line (mouse) was supplied by Bogu Biotechonology Company (Shanghai, China). Normal mice were inoculated with LLC cell to obtain tumor bearing mice with reference to published methods [26]. Briefly, LLC cells (4 × 106 , 0.1 ml of cell suspension) were inoculated into the right upper limbs of mice subcutaneously. Normal mice were treated with saline in the same way. Two weeks after treatment, mice were administered with PTS orally or intravenously. All the mice were housed in an air-conditioned room with the temperature of 24 ± 2 ◦ C, relative humidity of 55 ± 10% and alternating 12-h light/dark cycle. 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). The experimental procedures were approved by Beijing Institute of Technology Committee on Animal Care and Use (Beijing, China. Permission Number: SYXK (Jing) 2012-0035). 2.6. Oral bioavailability For six oral administration groups (n = 10 for each time point), all animals were fasted for 12 h prior to administration with free access to water. PTS suspended in 5% (w/v) carboxy-mehtylcellulose sodium (CMC-Na) solution was administrated by gastric gavage (14, 28 and 56 mg/kg to LLC and normal mice, respectively). In two intravenous administration groups (n = 10 for each time point), PTS dissolved in saline (containing 0.4% Tween 80 and 30% PEG300) was given via the caudal vein (10 mg/kg to LLC and normal mice separately). Blood sample (0.2 ml) was collected from orbital vein into heparinized tubes at appropriate intervals (5, 10, 15, 20, 30, 45, 60, 90, 180, 360, and 540 min) after dosing. Blank plasma sample was collected before dosing. All the blood samples were centrifuged at
5000 rpm for 10 min and then plasma samples were stored at –20 ◦ C until analysis.
2.7. Tissue distribution after oral administration In tissue distribution experiment, normal and LLC mice (n = 10 for each time point) were fasted for 12 h with free access to water. After mice were orally administered of 28 mg/kg PTS, main tissues (heart, lung, intestine, liver, brain, kidney, stomach, spleen, skeleton muscle, and testis) were collected at 20, 45, and 90 min post dosing and then weighted. Blank tissues samples were collected separately before ingestion. Tissues were homogenized with saline (ten-fold of tissue weight) and then centrifuged at 5000 rpm for 10 min to get the homogenates. The homogenate samples were stored at –20 ◦ C until use.
2.8. Data analysis Main pharmacokinetic parameters including elimination rate constant (Ke ), elimination half-life (T1/2 ), time to peak concentrtion (Tmax ), peak concentration (Cmax ), area under concentration–time curve (AUC0−t and AUC0-∞ ), clearance (CL) of PTS were analyzed by non-compartmental method using DAS Version 2.0 (Drug and Statistics, Mathematical Pharmacology Professional Committee of China, Beijing, China). The data were expressed as mean ± SD (standard deviation). The concentrations of PTS in tissue samples were calculated with the respective calibration curves.
3. Results and discussion 3.1. Method validation Selectivity was tested by comparison of blank matrices from six individual mice with corresponding spiked matrices. Typical chromatograms for determination of PTS in rat plasma and representative tissue (brain, liver and intestine) were presented in Fig. 2. Gradient separation of PTS from endogenous interference was achieved in the mobile phase. No obvious interference was observed under present chromatographic condition. The retention times of PTS and IS were 3.0 and 2.8 min, respectively. Typical regression equations, correlation coefficients (r2 ), and LLOQ were listed in Table 1. The calibration curves of PTS in mouse plasma and tissues were linear from 1.00 to 5000 ng/ml, and 0.10 to 500 ng/ml, respectively. The correlation coefficients for all samples were greater than 0.9954. The results of precision and accuracy in mouse plasma and three representative tissues (liver, brain and intestine) were shown in Table 2. Accuracy for plasma samples ranged from –4.8% to 7.1% RE (relative error). For brain samples, accuracy was between –6.7% and 2.1% RE. Accuracy for liver and intestine was in the range of –5.7% and 9.4% RE. The intra-day and inter-day precision for plasma, feces, liver and intestine was below 9.3% RSD (relative standard deviation). Accuracy and precision for all the other tissues were satisfactory with both RE and RSD less than ±15% and 15%, respectively (data not listed here). Mean recoveries of PTS in all matrices were higher than 97.2 ± 3.4%. Matrix effect data for PTS in all matrices was between 92.1% and 105.7%. Usually, a 15% deviation on a matrix effect could be a meaningful limit [28]. It can be concluded that no significant signal suppression or enhancement were observed in the present study. PTS in different matrices was quite stable under three different conditions with RE% ranging from –4.9% to 9.1%. Stabilities for standard stock solution of PTS and IS kept at 4 ◦ C for 30 days were between –1.8% and 3.1% RE.
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Fig. 2. Typical MRM chromatograms of (A) blank plasma; (B) blank plasma spiked with PTS (50 ng/ml) and IS (100 ng/ml); (C) a real normal mouse plasma sample at 90 min post dosing; (D) a real normal mouse liver sample at 45 min post dosing; (E) a real normal mouse intestine sample at 90 min post dosing; (F) a real normal mouse brain sample at 45 min post dosing. Peak I, PTS; Peak II, IS.
Table 1 Calibration range, equation, correlation coefficient and LLOQ of PTS in all biological matrices. Matrix
Calibration range (ng/ml)
Calibration equation
Correlation coefficient (r)
LLOQ (ng/ml)
Plasma Heart Liver Spleen Lung Kidney Stomach Brain Testis Intestine s.m.
1.00–5000 0.50–50.0 1.0–2000 0.50–50.0 1.00–500 1.0–2000 2.0–5000 1.00–1000 1.00–1000 1.00–2000 0.50–50.0
y = 0.029x + 0.037 y = 0.011x + 0.0029 y = 0.014x + 0.048 y = 0.008x + 0.012 y = 0.023x + 0.002 y = 0.018x − 0.027 y = 0.011x + 0.13 y = 0.003x + 0.022 y = 0.009x + 0.077 y = 0.013x + 0.019 y = 0.019x + 0.11
0.9980 0.9976 0.9959 0.9957 0.9989 0.9963 0.9954 0.9980 0.9979 0.9979 0.9982
1.00 0.50 1.00 0.50 1.00 1.00 2.00 1.00 1.00 1.00 0.50
LLOQ: lowest limit of quantification; s.m.: skeleton muscle.
3.2. Oral bioavailability PTS (14, 28 and 56 mg/kg) was observed to exert anticancer activity in LLC mice in our previous study (data not published).
Therefore, the three PTS doses were used in the present study. The plasma PTS concentration versus time curves after oral (14, 28, and 56 mg/kg) administration to mice was shown in Fig. 3.The corresponding pharmacokinetic parameters are summarized in Table 3.
Table 2 Precision and accuracy for determination of PTS in mouse plasma, brain, liver and intestine by LC–MS/MS Method (n = 6). Matrix
Added concentration (ng/ml)
Calculated concentration (ng/ml)
Accuracy RE (%)
Intra-day RSD (%)
Inter-day RSD (%)
Plasma
1.00 500 4000
1.125 535.5 3808
2.5 7.1 −4.8
2.4 9.1 3.8
5.6 3.2 6.1
Brain
1.00 400.0 800.0
0.976 373.2 816.8
−2.4 −6.7 2.1
8.2 3.0 9.3
4.7 2.8 1.2
Liver
1.00 400.0 1600
1.094 381.6 1508
9.4 −4.6 −5.8
3.9 9.3 2.7
5.6 8.1 2.4
Intestine
1.00 500.0 1600
0.970 513.5 1744
−3.0 2.7 0.9
5.4 1.9 6.7
2.6 5.4 5.6
RE, relative error; RSD, relative standard deviation; Note, accuracy and precision for other tissues were not listed here.
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Table 3 Main pharmacokinetic parameters of PTS after administration to micea (n = 10). Parameters
AUC(0−T) (mg h/l) AUC(0−∞) (mg h/l) Ke (1/min) Tmax (min) Cmax (mg/l) T1/2 (min) Vc (l/kg) CL (l/min/kg) BA
Normal mice
LLC mice
14 mg/kg, (po)
28 mg/kg, (po)
56 mg/kg, (po)
10 mg/kg, (iv)
14 mg/kg, (po)
28 mg/kg, (po)
56 mg/kg, (po)
10 mg/kg, (iv)
4.34 ± 1.7 4.43 ± 2.0 0.005 ± 0.003 23.3 ± 6.5 10.1 ± 3.2 102 ± 19.2 4.9 ± 1.9 0.027 ± 0.008 11.9%
10.1 ± 2.0 11.9 ± 2.1 0.009 ± 0.003 18.8 ± 2.5 21.8 ± 6.9 87.8 ± 29.2 3.76 ± 0.85 0.012 ± 0.008 13.9%
38.3 ± 8.5 39.4 ± 9.7 0.0125 ± 0.003 41.3 ± 13.9 32.7 ± 3.4 56.9 ± 13.9 1.57 ± 0.27 0.030 ± 0.005 26.4%
25.9 ± 2.8 26.7 ± 8.2 0.02 ± 0.01 – – 34.5 ± 1.0 0.674 ± 0.12 0.014 ± 0.003
10.8 ± 2.0 11.5 ± 2.1 0.016 ± 0.003 31.6 ± 8.8 17.4 ± 6.9 43.9 ± 12.1 1.16 ± 0.85 0.018 ± 0.008 20.7%
22.1 ± 4.6* 24.8 ± 5.1 0.008 ± 0.003 56.2 ± 7.5* 27.7 ± 7.3 101.1 ± 37.6 1.40 ± 0.14* 0.010 ± 0.002 21.2%*
55.8 ± 5.0# 57.5 ± 5.2# 0.002 ± 0.006 48.8 ± 8.5 69.0 ± 10.8# 51.3 ± 18.4 1.51 ± 0.55 0.021 ± 0.003 26.7%
37.3 ± 10.6 39.9 ± 9.7 0.006 ± 0.003 – – 49.0 ± 6.7 0.489 ± 0.09 0.012 ± 0.004
a Data were expressed as Mean ± SD. po, per oral; iv, intravenous; Tmax , peak concentration; Cmax , peak concentration; Ke , elimination rate constant; T1/2 , elimination half-life; AUC(0−Tand0−∞) , area under curve; Vc, apparent distribution volume; CL, clearance. P < 0.05, compared with normal mice (14 mg/kg); *P < 0.05, compared with normal mice (28 mg/kg); # P < 0.05, compared with normal mice (56 mg//kg).
Fig. 3. Mean plasma concentration-time curve of PTS after oral administration to mice ((A) 14 mg/kg; (B) 28 mg/kg; (C) 56 mg/kg, po). Data is expressed as mean ± SD (n = 10 for each time point).
When administered orally with the same dose, PTS demonstrates substantially greater plasma level in LLC mice than that in normal mice regardless of dose or route of administration. System exposure to PTS in LLC mice was several folds greater than in normal mice based on their Cmax and AUC. The plasma PTS concentration versus time curves showed double peaks in LLC mice (28, 56 mg/kg) and normal mice (56 mg/kg). From Table 3, it could be seen that AUC, Cmax , and Tmax of PTS in LLC groups were increased, while Ke , Vc and CL decreased with comparison of normal mice. Following oral and IV dosing, Vc of PTS in normal mice exceeded that of LLC mice, suggesting more extensive distribution of PTS in normal groups. PTS (14, 28 and 56 mg/kg) was absorbed quickly in normal mice with Tmax of 23.3 ± 6.5, 18.8 ± 2.5 and 41.3 ± 13.9 min. Cmax and CL were slightly increased with dose levels from 14 to 56 mg/kg. The Cmax in normal mice were 17.4 ± 6.9 (14 mg/kg); 27.7 ± 7.3 g/ml (28 mg/kg); 69.0 ± 10.8 g/ml (56 mg/kg). They dramatically increased in LLC mice by 70% (14 mg/kg), 27% (28 mg/kg), and 110% (56 mg/kg), respectively. The Tmax and T1/2 were 56.2 ± 7.5 min and 101.1 ± 37.6 min, extending significantly by about 2.6 and 1.1 fold of normal mice (28 mg/kg), respectively. A small decrease in Ke (0.008 ± 0.003 1/min) and CL (0.010 ± 0.002 l/min/kg) and a significant decrease in Vc (1.4 ± 0.14 l/kg) were observed in LLC mice administered of 28 mg/kg of PTS. At 56 mg/kg dose, the same trend of PK parameters except T1/2 was found in both normal and LLC mice as that at 28 mg/kg. T1/2 (51.3 ± 18.4 min) of PTS in LLC mice was slightly decreased than that of normal mice (56.9 ± 13.9 min). These results suggest the same dose of PTS has different PK pattern between normal and LLC mice. The AUC0−T for normal mice was 25.9 ± 2.8 mg h/l for the intravenous group (10 mg/kg). AUCs for three oral groups were 4.34 ± 1.7 (14 mg/kg), 10.1 ± 2.0 (28 mg/kg) and 36.3 ± 8.5 mg h/l (56 mg/kg), respectively. According to the equation (AUCpo × doseiv )/(AUCiv × dosepo ), oral bioavailability (BA) is calculated by dividing the average AUC of the po group with that of the iv group corrected by dose. The BAs of PTS in normal mice were 11.9% (14 mg/kg), 13.9% (28 mg/kg) and 26.4% (56 mg/kg), respectively. BAs of PTS in LLC mice were 20.7%, 21.2% and 26.7%, respectively. These differences suggest that BAs of PTS in LLC mice are much greater than that in normal mice. AUCs normalized by the doses in normal mice indicate a non-linear PK behavior during the dose range, while the BA appeared to be independent of the dose in LLC mice. Compared with normal mice, Table 3 indicated that BAs (28 mg/kg) in LLC mice were increased by 53%. No dramatic difference in BA between normal and LLC mice at the dose of 56 mg/kg. Plasma concentration of PTS in LLC mice was greater than that in normal mice at three levels evaluated in the present study. A trend of increasing BA and decreasing Ke and CL was observed in
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140.0
20 min
A
45 min
120.0 Tissue concentration (ug/g)
205
90 min
100.0 80.0 60.0 40.0 20.0 0.0
PTS level in normal mice
140.0
20 min
B
45 min
Tissue concentration (ug/g)
120.0
90 min
100.0 80.0 60.0 40.0 20.0 0.0
PTS level in LLC mice Fig. 4. Levels of PTS in main tissue of normal (A) and LLC (B) mice after administration (28 mg/kg, po). s.m., skeleton muscle. *P < 0.05, **P < 0.01, compared with normal mice (20 min); # P < 0.05, ## P < 0.05, compared with normal mice (45 min); P < 0.05, (90 min).
LLC mice, which might be useful for anti-cancer effect of PTS. These differences mainly stem from altered physiological conditions. It is known that BA is easily affected by several physiological factors, including gastrointestinal transit, regional blood flow, metabolism, intestinal flora, diseases of impacting absorption [29]. BA or pharmacokinetic characteristic of PTS in normal mice was significantly different from that observed in LLC mice, suggesting the changed physiological factors in LLC mice might have an effect on in vivo kinetic process of PTS. 3.3. Tissue distribution PTS levels in heart, liver, spleen, lung, kidney, brain, stomach, intestine, skeleton muscle (s.m.), and testis of mice at 20, 45, and 90 min post oral administration of PTS (28 mg/kg) were shown in Fig. 4. For normal mice, PTS was found in most tissues, which indicated that it was distributed extensively in mice. PTS was accumulated especially in stomach, kidney, liver, testis and intestine.
P < 0.05, compared with normal mice
Stomach exhibited the highest PTS concentration (82.1 ± 14.2 g/g) at 20 min post dosing, followed by liver (26.4 ± 5.1 g/g) and testis (19.5 ± 5.8 g/g). The lowest level of PTS was observed in mice heart (0.26 ± 0.04 g/g). PTS was detected in mice brain, which indicated PTS might easily pass through the blood–brain barrier. The highest level of PTS in brain was found at 45 min (10.3 ± 3.2 g/g). For most of tissues, PTS showed continuous decrease with sampling points (from 20 to 90 min), while PTS in brain and spleen reached the maximum level at 45 min. For LLC mice, PTS distribution pattern was dramatically different from that observed in normal mice. The maximum PTS level was found in stomach (115.4 ± 16.1 g/g) at 45 min post dosing, which showed approximately 1.7-fold increase compared with normal mice. PTS in spleen showed remarkably higher level (53.2 ± 15.2 g/g, approximately 50-fold) than that in normal mice at 20 min after administration. For most of tissues, PTS level was continuously decreased from 20 to 90 min in compared with normal mice. PTS in intestine and stomach showed peak level at 45 min
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Fig. 5. Typical MRM chromatograms for identification of sulfate conjugate of PTS in mouse plasma after administration (28 mg/kg, po). Ion channel A (m/z 315.1 → 134.1), B (m/z 255.1 → 240.1), C (m/z 431.1 → 240.1), D (m/z 431.1 → 255.1), E (m/z 335.1 → 240.1), F (m/z 335.1 → 255.1).
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and then decreased till 90 min. PTS in intestine of LLC mice was 0.94 ± 0.08 g/g at 20 min post dosing, accounting for only 7.4% of normal mice. Dramatic decrease of PTS in lung of LLC mice was also observed. The different distribution characteristics might result from different affinity between PTS and respective tissues, and blood flow [29]. 3.4. Preliminary study of PTS metabolite in mouse plasma and tissue Researchers have reported that glucuronide and sulfate metabolite of PTS in rat plasma are the primary metabolites [23]. However, no metabolic information in mouse tissues or plasma was available until now. Under present MRM detection mode with ion transitions m/z (255.1 → 240.1), another chromatographic peak (with retention time of 2.0 min approximately, marked as M) was observed in mouse plasma and most of tissues (such as liver and intestine, shown in Fig. 3A–C). It was not found in brain and testis. There is one phenol hydroxyl group in PTS, which might be easy to form glucuronide or sulfate conjugates in vivo. In order to identify whether M was glucuronide or sulfate conjugate of PTS, the other four ion transitions of PTS, including m/z 431.1 (PTS glucuronide) → 240.1, 431.1 → 255.1, 335.1(PTS sulfate) → 240.1, 335.1 → 255.1 were selected under MRM mode. The results (shown in Fig. 5 from plasma) indicated figures from ion channel (m/z 335.1 → 240.1, 335.1 → 255.1) were identical with that of m/z 255.1 → 240.1. Based on these results, M was preliminarily estimated as sulfate conjugate of PTS (PTS-Sul). PTS-Sul was observed in mouse plasma, intestine, liver, spleen, kidney, heart, and skeleton muscle except brain and testis. However, it needs further confirmation with reference standard of PTS-Sul. Because the reference standard of PTS-Sul is not commercially available, its levels in tissues or plasma were not quantified. No PTS glucuronide was observed in all samples in present study. It could be seen from Fig. 3 that there were double peaks (approximately at 15 and 45 min for LLC mice at 28 mg/kg; 20 and 45 min for LLC mice at 56 mg/kg; 15 and 45 min for normal mice at 56 mg/kg, respectively) of PTS in mouse plasma. The phenomena might be attributed to several reasons, such as enterohepatic circulation, two intestine absorption sites, gastric emptying process, and physicochemical nature of the compound [29]. It has been proposed that Phase II metabolites (such as glucuronide and sulfate metabolite) could serve as storage pools and might be related with enterohepatic recirculation of the parent drugs [30]. However, association between PTS-Sul and double peaks of PTS in plasma still needs further study. 4. Conclusions In summary, a sensitive and rapid UPLC–MS method has been developed for determination of PTS in mouse plasma and tissues. Significant difference in BA and distribution of PTS was observed between LLC and normal mice. Possible sulfate metabolite was observed in plasma and main tissues of mice except brain and testis. These results will be helpful for clinical application of PTS. Acknowledgement This research was financially supported by the National Natural Science Foundation of China (Grant No. 81202996). References ˜ N.M. Davies, Pharmacometrics of [1] K.A. Roupe, C.M. Remsberg, J.A. Yánez, stilbenes: seguing towards the clinic, Curr. Clin. Pharmacol. 1 (2006) 81–101. [2] H.S. Lin, B.D. Yue, P.C. Ho, Determination of pterostilbene in rat plasma by a simple HPLC–UV method and its application in pre-clinical pharmacokinetic study, Biomed. Chromatogr. 23 (2009) 1308–1315.
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Journal of Pharmaceutical and Biomedical Analysis journal homepage: www.elsevier.com/locate/jpba
UPLC–MS method for quantification of pterostilbene and its application to comparative study of bioavailability and tissue distribution in normal and Lewis lung carcinoma bearing mice Li Deng a , Yongzhi Li b , Xinshi Zhang c , Bo Chen a , Yulin Deng a , Yujuan Li a,∗ a
School of Life Science, Beijing Institute of Technology, 5 South Street Zhongguancun, Beijing 100081, China China Astronaut Research and Training Centre, Beijing 100094, China c Hebei North University, 11 Zuanshi South Road, Zhangjiakou 075000, China b
a r t i c l e
i n f o
Article history: Received 31 October 2014 Received in revised form 10 April 2015 Accepted 26 April 2015 Available online 29 May 2015 Keywords: Pterostilbene UPLC–MS Bioavailability Tissue distribution
a b s t r a c t A UPLC–MS method was developed for determination of pterostilbene (PTS) in plasma and tissues of mice. PTS was separated on Agilent Zorbax XDB-C18 column (50 × 2.1 mm, 1.8 m) with gradient mobile phase at the flow rate of 0.2 ml/min. The detection was performed by negative ion electrospray ionization in multiple reaction monitoring mode. The linear calibration curve of PTS in mouse plasma and tissues ranged from 1.0 to 5000 and 0.50 to 500 ng/ml (r2 > 0.9979), respectively, with lowest limits of quantification (LLOQ) were between 0.5 and 2.0 ng/ml, respectively. The accuracy and precision of the assay were satisfactory. The validated method was applied to the study of bioavailability and tissue distribution of PTS in normal and Lewis lung carcinoma (LLC) bearing mice. The bioavailability of PTS (dose 14, 28 and 56 mg/kg) in normal mice were 11.9%, 13.9% and 26.4%, respectively; and the maximum level (82.1 ± 14.2 g/g) was found in stomach (dose 28 mg/kg). The bioavailability, peak concentration (Cmax ), time to peak concentration (Tmax ) of PTS in LLC mice was increased compared with normal mice. The results indicated the UPLC–MS method is reliable and bioavailability and tissue distribution of PTS in normal and LLC mice were dramatically different. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Pterostilbene (trans-3,5-dimethoxy-4-hydroxystilbene, PTS, Fig. 1A) is a naturally occurring stilbenoid compound which has been found in many dietary plants such as grapes, berries [1–3], and medicinal herbs including Pterocarpus indicus, Pterocarpus marsupium, Dracaena cochinchinensis [4–7]. Recently, PTS has drawn more and more attention because of various health-promoting activities, such as antioxidant, anti-inflammatory, anti-diabetic, and prevention of age-related disease [8–14]. PTS possess anticancer properties against breast, colon, leukemia, liver, prostate, and lung cancers [15–20]. Although PTS shows various pharmacological activities, the information of PTS in vivo is still limited. Therefore, further in vivo research is necessary to understand the health-promoting activities of PTS. The plasma pharmacokinetics, oral bioavailability of PTS in normal rats using high performance liquid chromatography (HPLC) with ultraviolet detection (UV) or mass spectrometry (MS)
∗ Corresponding author. Tel.: +86 10 68914607; fax: +86 10 68914907. http://dx.doi.org/10.1016/j.jpba.2015.04.045 0731-7085/© 2015 Elsevier B.V. All rights reserved.
have been reported [21–24]. The effect of aqueous solubility and fasting on PTS bioavailability in rats with HPLC–UV method has been described [25]. The lowest limits of quantification (LLOQ) in HPLC–UV methods [21–23] were between 10 and 50 ng/ml, which might not be sensitive enough for quantification of PTS because of relatively low PTS level in tissues, urine or feces of mice. In the reported HPLC–MS method [24], the method validation is incomplete (data on recovery, stability or matrix effect are unavailable), and there is no internal standard (it is an external method). LLOQ of PTS is 5 ng/ml in rat plasma. It is known that internal standard and full validation are indispensible during bio-analytical method development. Obviously, a more reliable, highly sensitive and fully validated UPLC–MS method is essential for assay of PTS in biosamples. To our knowledge, no liquid chromatography LC–MS method for determination of PTS in urine, feces or tissues of experimental animals is available. Studies evaluating tissue distribution or identifying tissue metabolites of PTS with LC–MS method in experimental animals have not been reported. In addition, reports on the pharmacokinetics, bioavailability or excretion of PTS in tumor bearing animals are not available. Further, there have been no comparative
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Fig. 1. Chemical structures of pterostilbene (A) and loureirin B (B).
studies on pharmacokinetics, bioavailability or tissue distribution between tumor bearing and normal animals. Therefore, the major aim of present study is to develop a reliable and sensitive ultra-performance liquid chromatography (UPLC)–MS method for quantification of PTS in mouse plasma and main tissues, and then apply it to investigate oral bioavailability and tissue distribution of PTS in normal and Lewis lung carcinoma (LLC) bearing C57 BL/6 mice, and identify any possible metabolites. It is expected that the present findings would be helpful to explore the association between in vivo behavior and anticancer effect of PTS in LLC mice.
2. Experimental 2.1. Chemicals Pterostilbene (purity > 99.0%) and loureirin B (used as internal standard, IS, purity > 98.0%) were obtained from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). Their chemical structures were shown in Fig. 1. Distilled water was prepared by a Milli-Q water purification system (Millipore, Mississauga, Canada). Acetonitrile and methanol were of chromatographic grade from Thermo Fisher Scientific (Waltham, Massachusetts, USA). Analytical grade ammonium acetate and methyl tert-butyl ether (MTBE) were purchased from Beijing Chemical Manufacturer (Beijing, China). 2.2. HPLC–MS conditions Chromatographic separation was achieved using an Agilent 1290HPLC system equipped with a binary Infinity Bin G4220 pump, a G1316C column oven and a G2426A infinity auto-sampler (Agilent Technologies, Palo Alto, California, USA). The analytes were separated on an Agilent Zorbax XDB-C18 column (50 × 2.1 mm, 1.8 m, Santa Clara, California, USA). The mobile phase consisted of solvent A (0.1% ammonium acetate in water) and solvent B (0.1% ammonium acetate in acetonitrile). The gradient program was as following: 0 min 30% B, 0.5 min 30% B, 2.0 min 80% B, 3.5 min 80% B, 5.0 min 30% B, 6.0 min 30% B, 6.5 min stop. The flow rate was 0.2 ml/min and the temperature was maintained at 25 ◦ C. The injection volume was 10 l. The MS system consisted of an Agilent 1290-6460 tandem quadrupole mass spectrometer (Agilent Technologies, Palo Alto, California, USA) equipped with electrospray ionization (ESI) interface. Agilent masshunter workstation (version B.04.00) was used for data acquisition and processing. The mass spectrometer was operated in the negative ion detection mode. Nitrogen was used as the nebulizer, drying gas, heater gas as well as the collision gas. The main parameters were as follows: drying gas temperature 350 ◦ C; drying gas flow 7 l/min; nebulizer gas 45 psi; capillary voltage 3000 V; fragmentor 138 and 111 V for PTS and IS, respectively; collision energy –40 eV for PTS and IS. PTS was quantified by multiple-reaction monitoring (MRM) mode using the selected
ion transitions of each compound. The precursor to product ion transitions was m/z 255.1 → 240.1 for PTS and 315.1 → 134.1 for IS. 2.3. Sample preparation 2.3.1. Preparation of calibration standards and quality control samples PTS stock solution was prepared by dissolving 15 mg of PTS in 15 ml of methanol and then diluted with methanol to give the final concentration of 100 g/ml. Working solutions were prepared by appropriate dilution of the stock solution with 20% methanol–water (v:v). A stock solution of IS at 100 g/ml was also prepared in methanol and then diluted with 20% methanol–water (v:v) to obtain a working solution of 100 ng/ml. All the solutions were stored at 4 ◦ C before use. The calibration standards and quality control (QC) samples were prepared by spiking blank mouse plasma and all tissues with standard working solutions. The calibration samples of PTS in all tissues ranged from 0.10 to 5000 ng/ml. The calibration samples of PTS in mouse plasma were made at the ranges of 1.00–5000 ng/ml. QC samples was prepared at three different levels (low, medium and high concentration) for method validation. For plasma samples, QC samples were 1.00, 500, and 4000 ng/ml, respectively. For tissues, QC samples were made by lowest limit of quantification (LLOQ), the medium and 80% of the highest concentration level of respective calibration curves. 2.3.2. Preparation of plasma and tissue samples After thawing of plasma samples at room temperature, 15 l of plasma was mixed with 10 l of IS solution and 150 l of MTBE. The samples were centrifuged at 5000 rpm for 10 min. 150 l of supernatant was transferred to new vials and evaporated to dryness under a stream of nitrogen at room temperature. The residuals were dissolved in 150 l of methanol and water (1:1, v/v). Tissues samples were homogenized with saline (ten-fold of tissue weight) and then centrifuged at 5000 rpm for 10 min to get the homogenates.10 l of IS solution and 200 l of MTBE were added to 20 l of tissue homogenates. Each vial was mixed thoroughly by vortexing for 60 s. The samples were centrifuged at 5000 rpm for 10 min. 200 l of supernatant was transferred to new vials and evaporated to dryness under a stream of nitrogen at room temperature. The residuals were dissolved in 200 l of methanol and water (1:1, v/v). 2.4. Method validation According to US Food and Drug Administration Guidance for Industry – Bioanalytical Method Validation [27], the method was fully validated for the selectivity, linearity, accuracy and precision, recovery and stability. Matrix effect (ME) was also evaluated according to published procedures [28]. Selectivity was tested by comparison of blank matrices from six individual mice with corresponding spiked matrices. The calibra-
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tion standards of PTS (1.00–5000 ng/ml for plasma, 0.10–500 ng/ml for tissues) were prepared by spiking blank plasma or tissues with appropriate amounts of working solutions. A calibration curve for PTS were obtained by plotting the peak area ratio (y) of the analyte over IS against the concentrations (x) of PTS with weighting function 1/x2 . LLOQ is the lowest concentration of analyte on the calibration curve, which should be quantified reliably with an acceptable accuracy and precision (less than or equal to 20%) [27]. QC samples were prepared with blank matrices at low, medium, and high concentration levels of PTS for precision and accuracy assessment. QC samples at three concentrations in six replicates were analyzed during the same day using the same calibration curve to determine the intra-day precision. QC samples were analyzed for three continuous days to define inter-day precision. Accuracy was expressed as a percentage of the measured concentration over the nominal concentration of the QC samples. The recovery of PTS and IS was determined by comparing the peak areas of the PTS and IS in extracted QC samples with those in post-extraction blank samples spiked at the corresponding concentrations. Matrix effect (ME) was evaluated by the method reported in literature [28]. Peak areas of PTS in spiked plasma, bile, urine and tissues post extraction (B) were compared with those of PTS neat solutions at QC levels (A). ME was defined as the ratio of B/A × 100%. ME values > 100% mean ionization enhancement, whereas ME values < 100% indicate ionization suppression. The stability of PTS in plasma and tissues after long-term (−20 ◦ C for 30 days), short-term storage (room temperature for 8 h), and after going through three freeze and thaw cycles were evaluated with respective QC samples. Stability for standard stock solution of PTS and IS kept at 4 ◦ C for 30 days was tested. 2.5. Animal treatment Normal C57 BL/6 mice (body weight 20 ± 2 g, 8 weeks) were purchased from Beijing Military Medical Sciences Experimental Animal Co., Ltd. (Beijing, China). An LLC cell line (mouse) was supplied by Bogu Biotechonology Company (Shanghai, China). Normal mice were inoculated with LLC cell to obtain tumor bearing mice with reference to published methods [26]. Briefly, LLC cells (4 × 106 , 0.1 ml of cell suspension) were inoculated into the right upper limbs of mice subcutaneously. Normal mice were treated with saline in the same way. Two weeks after treatment, mice were administered with PTS orally or intravenously. All the mice were housed in an air-conditioned room with the temperature of 24 ± 2 ◦ C, relative humidity of 55 ± 10% and alternating 12-h light/dark cycle. 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). The experimental procedures were approved by Beijing Institute of Technology Committee on Animal Care and Use (Beijing, China. Permission Number: SYXK (Jing) 2012-0035). 2.6. Oral bioavailability For six oral administration groups (n = 10 for each time point), all animals were fasted for 12 h prior to administration with free access to water. PTS suspended in 5% (w/v) carboxy-mehtylcellulose sodium (CMC-Na) solution was administrated by gastric gavage (14, 28 and 56 mg/kg to LLC and normal mice, respectively). In two intravenous administration groups (n = 10 for each time point), PTS dissolved in saline (containing 0.4% Tween 80 and 30% PEG300) was given via the caudal vein (10 mg/kg to LLC and normal mice separately). Blood sample (0.2 ml) was collected from orbital vein into heparinized tubes at appropriate intervals (5, 10, 15, 20, 30, 45, 60, 90, 180, 360, and 540 min) after dosing. Blank plasma sample was collected before dosing. All the blood samples were centrifuged at
5000 rpm for 10 min and then plasma samples were stored at –20 ◦ C until analysis.
2.7. Tissue distribution after oral administration In tissue distribution experiment, normal and LLC mice (n = 10 for each time point) were fasted for 12 h with free access to water. After mice were orally administered of 28 mg/kg PTS, main tissues (heart, lung, intestine, liver, brain, kidney, stomach, spleen, skeleton muscle, and testis) were collected at 20, 45, and 90 min post dosing and then weighted. Blank tissues samples were collected separately before ingestion. Tissues were homogenized with saline (ten-fold of tissue weight) and then centrifuged at 5000 rpm for 10 min to get the homogenates. The homogenate samples were stored at –20 ◦ C until use.
2.8. Data analysis Main pharmacokinetic parameters including elimination rate constant (Ke ), elimination half-life (T1/2 ), time to peak concentrtion (Tmax ), peak concentration (Cmax ), area under concentration–time curve (AUC0−t and AUC0-∞ ), clearance (CL) of PTS were analyzed by non-compartmental method using DAS Version 2.0 (Drug and Statistics, Mathematical Pharmacology Professional Committee of China, Beijing, China). The data were expressed as mean ± SD (standard deviation). The concentrations of PTS in tissue samples were calculated with the respective calibration curves.
3. Results and discussion 3.1. Method validation Selectivity was tested by comparison of blank matrices from six individual mice with corresponding spiked matrices. Typical chromatograms for determination of PTS in rat plasma and representative tissue (brain, liver and intestine) were presented in Fig. 2. Gradient separation of PTS from endogenous interference was achieved in the mobile phase. No obvious interference was observed under present chromatographic condition. The retention times of PTS and IS were 3.0 and 2.8 min, respectively. Typical regression equations, correlation coefficients (r2 ), and LLOQ were listed in Table 1. The calibration curves of PTS in mouse plasma and tissues were linear from 1.00 to 5000 ng/ml, and 0.10 to 500 ng/ml, respectively. The correlation coefficients for all samples were greater than 0.9954. The results of precision and accuracy in mouse plasma and three representative tissues (liver, brain and intestine) were shown in Table 2. Accuracy for plasma samples ranged from –4.8% to 7.1% RE (relative error). For brain samples, accuracy was between –6.7% and 2.1% RE. Accuracy for liver and intestine was in the range of –5.7% and 9.4% RE. The intra-day and inter-day precision for plasma, feces, liver and intestine was below 9.3% RSD (relative standard deviation). Accuracy and precision for all the other tissues were satisfactory with both RE and RSD less than ±15% and 15%, respectively (data not listed here). Mean recoveries of PTS in all matrices were higher than 97.2 ± 3.4%. Matrix effect data for PTS in all matrices was between 92.1% and 105.7%. Usually, a 15% deviation on a matrix effect could be a meaningful limit [28]. It can be concluded that no significant signal suppression or enhancement were observed in the present study. PTS in different matrices was quite stable under three different conditions with RE% ranging from –4.9% to 9.1%. Stabilities for standard stock solution of PTS and IS kept at 4 ◦ C for 30 days were between –1.8% and 3.1% RE.
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Fig. 2. Typical MRM chromatograms of (A) blank plasma; (B) blank plasma spiked with PTS (50 ng/ml) and IS (100 ng/ml); (C) a real normal mouse plasma sample at 90 min post dosing; (D) a real normal mouse liver sample at 45 min post dosing; (E) a real normal mouse intestine sample at 90 min post dosing; (F) a real normal mouse brain sample at 45 min post dosing. Peak I, PTS; Peak II, IS.
Table 1 Calibration range, equation, correlation coefficient and LLOQ of PTS in all biological matrices. Matrix
Calibration range (ng/ml)
Calibration equation
Correlation coefficient (r)
LLOQ (ng/ml)
Plasma Heart Liver Spleen Lung Kidney Stomach Brain Testis Intestine s.m.
1.00–5000 0.50–50.0 1.0–2000 0.50–50.0 1.00–500 1.0–2000 2.0–5000 1.00–1000 1.00–1000 1.00–2000 0.50–50.0
y = 0.029x + 0.037 y = 0.011x + 0.0029 y = 0.014x + 0.048 y = 0.008x + 0.012 y = 0.023x + 0.002 y = 0.018x − 0.027 y = 0.011x + 0.13 y = 0.003x + 0.022 y = 0.009x + 0.077 y = 0.013x + 0.019 y = 0.019x + 0.11
0.9980 0.9976 0.9959 0.9957 0.9989 0.9963 0.9954 0.9980 0.9979 0.9979 0.9982
1.00 0.50 1.00 0.50 1.00 1.00 2.00 1.00 1.00 1.00 0.50
LLOQ: lowest limit of quantification; s.m.: skeleton muscle.
3.2. Oral bioavailability PTS (14, 28 and 56 mg/kg) was observed to exert anticancer activity in LLC mice in our previous study (data not published).
Therefore, the three PTS doses were used in the present study. The plasma PTS concentration versus time curves after oral (14, 28, and 56 mg/kg) administration to mice was shown in Fig. 3.The corresponding pharmacokinetic parameters are summarized in Table 3.
Table 2 Precision and accuracy for determination of PTS in mouse plasma, brain, liver and intestine by LC–MS/MS Method (n = 6). Matrix
Added concentration (ng/ml)
Calculated concentration (ng/ml)
Accuracy RE (%)
Intra-day RSD (%)
Inter-day RSD (%)
Plasma
1.00 500 4000
1.125 535.5 3808
2.5 7.1 −4.8
2.4 9.1 3.8
5.6 3.2 6.1
Brain
1.00 400.0 800.0
0.976 373.2 816.8
−2.4 −6.7 2.1
8.2 3.0 9.3
4.7 2.8 1.2
Liver
1.00 400.0 1600
1.094 381.6 1508
9.4 −4.6 −5.8
3.9 9.3 2.7
5.6 8.1 2.4
Intestine
1.00 500.0 1600
0.970 513.5 1744
−3.0 2.7 0.9
5.4 1.9 6.7
2.6 5.4 5.6
RE, relative error; RSD, relative standard deviation; Note, accuracy and precision for other tissues were not listed here.
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Table 3 Main pharmacokinetic parameters of PTS after administration to micea (n = 10). Parameters
AUC(0−T) (mg h/l) AUC(0−∞) (mg h/l) Ke (1/min) Tmax (min) Cmax (mg/l) T1/2 (min) Vc (l/kg) CL (l/min/kg) BA
Normal mice
LLC mice
14 mg/kg, (po)
28 mg/kg, (po)
56 mg/kg, (po)
10 mg/kg, (iv)
14 mg/kg, (po)
28 mg/kg, (po)
56 mg/kg, (po)
10 mg/kg, (iv)
4.34 ± 1.7 4.43 ± 2.0 0.005 ± 0.003 23.3 ± 6.5 10.1 ± 3.2 102 ± 19.2 4.9 ± 1.9 0.027 ± 0.008 11.9%
10.1 ± 2.0 11.9 ± 2.1 0.009 ± 0.003 18.8 ± 2.5 21.8 ± 6.9 87.8 ± 29.2 3.76 ± 0.85 0.012 ± 0.008 13.9%
38.3 ± 8.5 39.4 ± 9.7 0.0125 ± 0.003 41.3 ± 13.9 32.7 ± 3.4 56.9 ± 13.9 1.57 ± 0.27 0.030 ± 0.005 26.4%
25.9 ± 2.8 26.7 ± 8.2 0.02 ± 0.01 – – 34.5 ± 1.0 0.674 ± 0.12 0.014 ± 0.003
10.8 ± 2.0 11.5 ± 2.1 0.016 ± 0.003 31.6 ± 8.8 17.4 ± 6.9 43.9 ± 12.1 1.16 ± 0.85 0.018 ± 0.008 20.7%
22.1 ± 4.6* 24.8 ± 5.1 0.008 ± 0.003 56.2 ± 7.5* 27.7 ± 7.3 101.1 ± 37.6 1.40 ± 0.14* 0.010 ± 0.002 21.2%*
55.8 ± 5.0# 57.5 ± 5.2# 0.002 ± 0.006 48.8 ± 8.5 69.0 ± 10.8# 51.3 ± 18.4 1.51 ± 0.55 0.021 ± 0.003 26.7%
37.3 ± 10.6 39.9 ± 9.7 0.006 ± 0.003 – – 49.0 ± 6.7 0.489 ± 0.09 0.012 ± 0.004
a Data were expressed as Mean ± SD. po, per oral; iv, intravenous; Tmax , peak concentration; Cmax , peak concentration; Ke , elimination rate constant; T1/2 , elimination half-life; AUC(0−Tand0−∞) , area under curve; Vc, apparent distribution volume; CL, clearance. P < 0.05, compared with normal mice (14 mg/kg); *P < 0.05, compared with normal mice (28 mg/kg); # P < 0.05, compared with normal mice (56 mg//kg).
Fig. 3. Mean plasma concentration-time curve of PTS after oral administration to mice ((A) 14 mg/kg; (B) 28 mg/kg; (C) 56 mg/kg, po). Data is expressed as mean ± SD (n = 10 for each time point).
When administered orally with the same dose, PTS demonstrates substantially greater plasma level in LLC mice than that in normal mice regardless of dose or route of administration. System exposure to PTS in LLC mice was several folds greater than in normal mice based on their Cmax and AUC. The plasma PTS concentration versus time curves showed double peaks in LLC mice (28, 56 mg/kg) and normal mice (56 mg/kg). From Table 3, it could be seen that AUC, Cmax , and Tmax of PTS in LLC groups were increased, while Ke , Vc and CL decreased with comparison of normal mice. Following oral and IV dosing, Vc of PTS in normal mice exceeded that of LLC mice, suggesting more extensive distribution of PTS in normal groups. PTS (14, 28 and 56 mg/kg) was absorbed quickly in normal mice with Tmax of 23.3 ± 6.5, 18.8 ± 2.5 and 41.3 ± 13.9 min. Cmax and CL were slightly increased with dose levels from 14 to 56 mg/kg. The Cmax in normal mice were 17.4 ± 6.9 (14 mg/kg); 27.7 ± 7.3 g/ml (28 mg/kg); 69.0 ± 10.8 g/ml (56 mg/kg). They dramatically increased in LLC mice by 70% (14 mg/kg), 27% (28 mg/kg), and 110% (56 mg/kg), respectively. The Tmax and T1/2 were 56.2 ± 7.5 min and 101.1 ± 37.6 min, extending significantly by about 2.6 and 1.1 fold of normal mice (28 mg/kg), respectively. A small decrease in Ke (0.008 ± 0.003 1/min) and CL (0.010 ± 0.002 l/min/kg) and a significant decrease in Vc (1.4 ± 0.14 l/kg) were observed in LLC mice administered of 28 mg/kg of PTS. At 56 mg/kg dose, the same trend of PK parameters except T1/2 was found in both normal and LLC mice as that at 28 mg/kg. T1/2 (51.3 ± 18.4 min) of PTS in LLC mice was slightly decreased than that of normal mice (56.9 ± 13.9 min). These results suggest the same dose of PTS has different PK pattern between normal and LLC mice. The AUC0−T for normal mice was 25.9 ± 2.8 mg h/l for the intravenous group (10 mg/kg). AUCs for three oral groups were 4.34 ± 1.7 (14 mg/kg), 10.1 ± 2.0 (28 mg/kg) and 36.3 ± 8.5 mg h/l (56 mg/kg), respectively. According to the equation (AUCpo × doseiv )/(AUCiv × dosepo ), oral bioavailability (BA) is calculated by dividing the average AUC of the po group with that of the iv group corrected by dose. The BAs of PTS in normal mice were 11.9% (14 mg/kg), 13.9% (28 mg/kg) and 26.4% (56 mg/kg), respectively. BAs of PTS in LLC mice were 20.7%, 21.2% and 26.7%, respectively. These differences suggest that BAs of PTS in LLC mice are much greater than that in normal mice. AUCs normalized by the doses in normal mice indicate a non-linear PK behavior during the dose range, while the BA appeared to be independent of the dose in LLC mice. Compared with normal mice, Table 3 indicated that BAs (28 mg/kg) in LLC mice were increased by 53%. No dramatic difference in BA between normal and LLC mice at the dose of 56 mg/kg. Plasma concentration of PTS in LLC mice was greater than that in normal mice at three levels evaluated in the present study. A trend of increasing BA and decreasing Ke and CL was observed in
L. Deng et al. / Journal of Pharmaceutical and Biomedical Analysis 114 (2015) 200–207
140.0
20 min
A
45 min
120.0 Tissue concentration (ug/g)
205
90 min
100.0 80.0 60.0 40.0 20.0 0.0
PTS level in normal mice
140.0
20 min
B
45 min
Tissue concentration (ug/g)
120.0
90 min
100.0 80.0 60.0 40.0 20.0 0.0
PTS level in LLC mice Fig. 4. Levels of PTS in main tissue of normal (A) and LLC (B) mice after administration (28 mg/kg, po). s.m., skeleton muscle. *P < 0.05, **P < 0.01, compared with normal mice (20 min); # P < 0.05, ## P < 0.05, compared with normal mice (45 min); P < 0.05, (90 min).
LLC mice, which might be useful for anti-cancer effect of PTS. These differences mainly stem from altered physiological conditions. It is known that BA is easily affected by several physiological factors, including gastrointestinal transit, regional blood flow, metabolism, intestinal flora, diseases of impacting absorption [29]. BA or pharmacokinetic characteristic of PTS in normal mice was significantly different from that observed in LLC mice, suggesting the changed physiological factors in LLC mice might have an effect on in vivo kinetic process of PTS. 3.3. Tissue distribution PTS levels in heart, liver, spleen, lung, kidney, brain, stomach, intestine, skeleton muscle (s.m.), and testis of mice at 20, 45, and 90 min post oral administration of PTS (28 mg/kg) were shown in Fig. 4. For normal mice, PTS was found in most tissues, which indicated that it was distributed extensively in mice. PTS was accumulated especially in stomach, kidney, liver, testis and intestine.
P < 0.05, compared with normal mice
Stomach exhibited the highest PTS concentration (82.1 ± 14.2 g/g) at 20 min post dosing, followed by liver (26.4 ± 5.1 g/g) and testis (19.5 ± 5.8 g/g). The lowest level of PTS was observed in mice heart (0.26 ± 0.04 g/g). PTS was detected in mice brain, which indicated PTS might easily pass through the blood–brain barrier. The highest level of PTS in brain was found at 45 min (10.3 ± 3.2 g/g). For most of tissues, PTS showed continuous decrease with sampling points (from 20 to 90 min), while PTS in brain and spleen reached the maximum level at 45 min. For LLC mice, PTS distribution pattern was dramatically different from that observed in normal mice. The maximum PTS level was found in stomach (115.4 ± 16.1 g/g) at 45 min post dosing, which showed approximately 1.7-fold increase compared with normal mice. PTS in spleen showed remarkably higher level (53.2 ± 15.2 g/g, approximately 50-fold) than that in normal mice at 20 min after administration. For most of tissues, PTS level was continuously decreased from 20 to 90 min in compared with normal mice. PTS in intestine and stomach showed peak level at 45 min
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Fig. 5. Typical MRM chromatograms for identification of sulfate conjugate of PTS in mouse plasma after administration (28 mg/kg, po). Ion channel A (m/z 315.1 → 134.1), B (m/z 255.1 → 240.1), C (m/z 431.1 → 240.1), D (m/z 431.1 → 255.1), E (m/z 335.1 → 240.1), F (m/z 335.1 → 255.1).
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and then decreased till 90 min. PTS in intestine of LLC mice was 0.94 ± 0.08 g/g at 20 min post dosing, accounting for only 7.4% of normal mice. Dramatic decrease of PTS in lung of LLC mice was also observed. The different distribution characteristics might result from different affinity between PTS and respective tissues, and blood flow [29]. 3.4. Preliminary study of PTS metabolite in mouse plasma and tissue Researchers have reported that glucuronide and sulfate metabolite of PTS in rat plasma are the primary metabolites [23]. However, no metabolic information in mouse tissues or plasma was available until now. Under present MRM detection mode with ion transitions m/z (255.1 → 240.1), another chromatographic peak (with retention time of 2.0 min approximately, marked as M) was observed in mouse plasma and most of tissues (such as liver and intestine, shown in Fig. 3A–C). It was not found in brain and testis. There is one phenol hydroxyl group in PTS, which might be easy to form glucuronide or sulfate conjugates in vivo. In order to identify whether M was glucuronide or sulfate conjugate of PTS, the other four ion transitions of PTS, including m/z 431.1 (PTS glucuronide) → 240.1, 431.1 → 255.1, 335.1(PTS sulfate) → 240.1, 335.1 → 255.1 were selected under MRM mode. The results (shown in Fig. 5 from plasma) indicated figures from ion channel (m/z 335.1 → 240.1, 335.1 → 255.1) were identical with that of m/z 255.1 → 240.1. Based on these results, M was preliminarily estimated as sulfate conjugate of PTS (PTS-Sul). PTS-Sul was observed in mouse plasma, intestine, liver, spleen, kidney, heart, and skeleton muscle except brain and testis. However, it needs further confirmation with reference standard of PTS-Sul. Because the reference standard of PTS-Sul is not commercially available, its levels in tissues or plasma were not quantified. No PTS glucuronide was observed in all samples in present study. It could be seen from Fig. 3 that there were double peaks (approximately at 15 and 45 min for LLC mice at 28 mg/kg; 20 and 45 min for LLC mice at 56 mg/kg; 15 and 45 min for normal mice at 56 mg/kg, respectively) of PTS in mouse plasma. The phenomena might be attributed to several reasons, such as enterohepatic circulation, two intestine absorption sites, gastric emptying process, and physicochemical nature of the compound [29]. It has been proposed that Phase II metabolites (such as glucuronide and sulfate metabolite) could serve as storage pools and might be related with enterohepatic recirculation of the parent drugs [30]. However, association between PTS-Sul and double peaks of PTS in plasma still needs further study. 4. Conclusions In summary, a sensitive and rapid UPLC–MS method has been developed for determination of PTS in mouse plasma and tissues. Significant difference in BA and distribution of PTS was observed between LLC and normal mice. Possible sulfate metabolite was observed in plasma and main tissues of mice except brain and testis. These results will be helpful for clinical application of PTS. Acknowledgement This research was financially supported by the National Natural Science Foundation of China (Grant No. 81202996). References ˜ N.M. Davies, Pharmacometrics of [1] K.A. Roupe, C.M. Remsberg, J.A. Yánez, stilbenes: seguing towards the clinic, Curr. Clin. Pharmacol. 1 (2006) 81–101. [2] H.S. Lin, B.D. Yue, P.C. Ho, Determination of pterostilbene in rat plasma by a simple HPLC–UV method and its application in pre-clinical pharmacokinetic study, Biomed. Chromatogr. 23 (2009) 1308–1315.
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