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Metabolic profiles and pharmacokinetics of picroside I in rats by liquid chromatography combined with electrospray ionization tandem mass spectrometry
Journal of Chromatography B 1095 (2018) 157–165
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Journal of Chromatography B journal homepage: www.elsevier.com/locate/jchromb
Metabolic profiles and pharmacokinetics of picroside I in rats by liquid chromatography combined with electrospray ionization tandem mass spectrometry ⁎
Kai Xionga,b, Zhengcai Juc, Tong Zhanga,b, , Zhengtao Wangc, Han Hana,b,
T
⁎
a
Experiment Center for Teaching and Learning, Shanghai University of Traditional Chinese Medicine, Shanghai 201210, China School of Pharmacy, Shanghai University of Traditional Chinese Medicine, Shanghai 201210, China c Institute of Chinese Materia Medica, Shanghai University of Traditional Chinese Medicine, 1200 Cailun Road, Shanghai 201210, China b
A R T I C LE I N FO
A B S T R A C T
Keywords: Picroside I LC–MS/MS LC-Q/TOF-MS Pharmacokinetics Metabolism
Picroside I is an iridoid glycoside derived from Picrorhiza kurroa Royle ex Benth and Picrorhiza scrophulariiflora Pennell and characterized by many biological activities. In this study, a fast, selective, and sensitive UHPLC-MS/ MS method was developed and validated to determine picroside I in rat plasma. Analytes were separated by using an ACQUITY UPLC® BEH C18 (2.1 × 50 mm, 1.7 μm) column at a running time of 2 min. Selected reaction monitoring (SRM) transitions were m/z 491.1 → 147.1 for picroside I and m/z 511.1 → 235.1 for the internal standard in a negative ion mode. The established UHPLC-MS/MS method achieved good linearity for picroside I within the range of 0.1–500 ng/mL. The validated method was successfully applied for the pharmacokinetic analysis of picroside I in rats after oral administration. Fifteen metabolites of picroside I were tentatively identified through ultra-high-performance chromatography/tandem quadrupole time-of-flight mass spectrometry, and four metabolites were identified by comparing with the standards. Besides, nine of these metabolites were discovered for the first time. The proposed metabolic pathways of picroside I in vivo can be divided into four parts, namely, phase I reaction of picroside I, including hydroxylation and deoxygenation; phase II reaction of picroside I, including glucuronidation, sulfation, and methylation; phase I biotransformations of metabolites, such as reduction and hydroxylation; and phase II biotransformations of metabolites, such as glucuronidation and sulfation. These results could offer insights into the effectiveness and toxicity of picroside I.
1. Introduction Picroside I is an iridoid glycoside that can be isolated from Picrorhiza kurroa Royle ex Benth and Picrorhiza scrophulariiflora Pennell [1, 2]. Picroliv, which is a hepatic protectant found in India, contains approximately 60% picroside I and picroside II at a 1:1.5 ratio [3, 4]. Picroliv can protect the liver against galactosamine-, carbon tetrachloride-, thioacetamide-, and aflatoxin B1-induced damage [5–8]. The antioxidant and stabilizing actions on the cell membranes of hepatocytes are main reason for the hepatoprotective activity of picroliv [9, 10]. As the main constituent of picroliv, picroside I exhibits many biological activities, including anti-hepatotoxic [11], anti-oxidant [3], anti-inflammatory [12], anti-tumor [13], and P-glycoprotein stimulation [14]. Moreover, picroside I may enhance the neurite outgrowth stimulated by basic fibroblast growth factor, staurosporine, or dibutyryl cyclic adenosine monophosphate in PC12D cells through a mitogenactivated protein kinase signaling pathway [15]. Picroside I can be well ⁎
docked into the activity site of CYP3A4, which is an enzyme that metabolizes phase I drugs [16]. The main aspects of in vivo drug action are pharmacokinetics and drug metabolism, which play important roles in drug discovery and development [17]. Compared with pharmacodynamic investigations, limited efforts have been devoted to studying the pharmacokinetics and metabolism of picroside I. A few studies have applied liquid chromatography tandem-mass spectrometry (LC-MS/MS) method to determine picroside I in plasma; however, these studies have been hampered by either a complex sample pretreatment method or a long running time [18, 19]. Therefore, a fast and reliable method must be developed for pharmacokinetic studies on picroside I. Upadhyay et al. [20] found eight picroside I metabolites, including picroside I-glucuronide, -sulfate, -hydroxylated products, in rat liver microsomes and hepatocyte cultures. However, they utilized low-resolution mass spectrometry and thus failed to provide information on mass fragmentation because of technical limitations. Recent
Corresponding authors at: Experiment Center for Teaching and Learning, Shanghai University of Traditional Chinese Medicine, Shanghai 201210, China. E-mail addresses: [email protected] (T. Zhang), [email protected] (H. Han).
https://doi.org/10.1016/j.jchromb.2018.07.034 Received 15 June 2018; Received in revised form 22 July 2018; Accepted 26 July 2018 Available online 29 July 2018 1570-0232/ © 2018 Published by Elsevier B.V.
Journal of Chromatography B 1095 (2018) 157–165
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the plasma samples were stored at −80 °C until analysis. The serum, bile, urine, and fecal samples of six rats were collected for metabolic analysis. The rats were orally administrated with picroside I at a dose of 100 mg/kg. Blood samples (200 μL) were collected through coagulation-promoting tubes at 0, 0.167, 0.5, 1, 2, 4, 8, and 12 h post-dosing. The blood samples were then centrifuged at 5000 ×g for 10 min to obtain the serum. The urine and fecal samples were collected at 12 h pre-dosing and at 0–24 h post-dosing in metabolic cages. After the rats were anesthetized by 25% urethane (intraperitoneal injection, 4 mL/kg), the bile samples were collected at 6 h pre-dosing and at 0–12 h post-dosing. These samples were stored at −80 °C until analysis.
investigations on drug metabolism have widely involved an ultra-highperformance liquid chromatography tandem quadrupole time-of-flight mass spectrometry (UHPLC-Q/TOF-MS/MS) system, which can obtain the accurate tandem mass spectra of drug metabolites and is a useful tool for structural confirmation [21]. The high-resolution mass spectrometry component of this system allows a reliable identification of metabolites in vivo. In this study, a simple, fast, selective, and sensitive LC-MS/MS method was developed and validated to detect picroside I in rat plasma. The established method was successfully applied to pharmacokinetically analyze picroside I orally administered to rats. Picroside I was also subjected to metabolic studies in vivo through high-resolution mass spectrometry for the first time. Fifteen metabolites of picroside I were tentatively identified in rat bile, urine, serum, and feces through UHPLC-Q/TOF-MS/MS. The structures of these metabolites were confirmed by the obtained tandem mass spectra. The proposed metabolic pathways of picroside I in vivo contained phases I and II reaction metabolites of picroside I and product-related metabolites.
2.3. Pharmacokinetic analysis 2.3.1. LC-MS/MS conditions This study used an LC-MS/MS system, namely, a Nexera X2 UHPLC system (Shimadzu Co., Kyoto, Japan) coupled with an AB SCIEX QTRAP® 6500 mass spectrometer (Redwood City, CA, USA). The analytes were separated by using an ACQUITY UPLC® BEH C18 (2.1 × 50 mm, 1.7 μm) column (Waters, Dublin, Ireland) at 40 °C. The guard column (ACQUITY UPLC® BEH C18) was placed before the inlet of the analytical column. The mobile phases consisted of (A) water and (B) acetonitrile. The gradient elution was as follows: 18% → 28% B at 0–0.8 min, 28% → 60% B at 0.8–1.2 min, 60% → 90% B at 1.2–1.6 min, 90% → 2% B at 1.6–2 min. The flow rate was set at 0.4 mL/min. The auto-sampler tray was maintained at 10 °C, and the sample injection volume was 5 μL. The mass spectrometer was operated under negative electrospray ionization (ESI) mode. The MS conditions were set as follows: gas (nitrogen) temperature, 550 °C; curtain gas, 30 psi; ion source voltage, −4500 V; collisionally activated dissociation, medium level; and ion source gas 1 and 2, 55 psi. The SRM transitions (Table S1) were m/z 491.1 → 147.1 for picroside I and m/z 511.1 → 235.1 for the IS. Data were acquired and analyzed using Analyst 1.6.3 (AB SCIEX LLC.).
2. Materials and methods 2.1. Chemicals and reagents Picroside I and picroside II (internal standard [IS], structure shown in Fig. 1) with 98% purity were purchased from Nanjing GOREN BioTechnology Co., Ltd. (Nanjing, China). Catalpol (purity > 96%), cinnamic acid (purity > 99%), 3-phenylpropionic acid (purity > 99%) and p-coumaric acid (purity > 98%) were obtained from Sigma-Aldrich Co., LLC (St. Louis, MO, USA). LC-MS-grade acetonitrile, methanol, and formic acid were procured from Sigma-Aldrich Co., LLC. Water was acquired from a Milli-Q Advantage A10 purification system (Millipore Corporation, Billerica, MA, USA). All other chemicals were of analytical grade. 2.2. Animals and sampling
2.3.2. Sample pretreatment In this procedure, 50 μL of IS solution (100 ng/mL) was added to 50 μL of each plasma sample and mixed for 20 s. Afterward, 200 μL of acetonitrile was added to the mixture and vortexed for 1 min to precipitate the protein. The mixture was centrifuged at 15000 ×g for 10 min, and the clear supernatant was dried under N2 gas. The residue was re-dissolved in 50 μL of 20% acetonitrile and centrifuged at 15000 ×g for 10 min. Finally, 5 μL of the solution was injected into the LC-MS/MS system.
Male Wistar rats (7–8 weeks old, 240 ± 10 g) were obtained from the Laboratory Animal Center of Shanghai University of Traditional Chinese Medicine (Shanghai, China). The rats were kept under controlled conditions (room temperature, 22 ± 2 °C; relative humidity, 60 ± 5%) at a 12 h/12 h day/night cycle for 7 days before the experiments. All of the animal experiments were approved by the Institutional Animal Care and Use Committee of Shanghai University of Traditional Chinese Medicine. The rats were subjected to fasting overnight before the experiments and given free access to water. Six rats were used for pharmacokinetic analysis. After picroside I (dissolved in normal saline) was orally administered at a dose of 100 mg/kg, blood samples (0.2 mL) were collected in heparin tubes at 0, 0.083, 0.25, 0.5, 0.75, 1, 1.5, 2, 4, 6, 8, and 12 h. The blood samples were then centrifuged at 5000 ×g for 10 min to obtain the plasma, and
2.3.3. Method validation The established method was validated for selectivity, linearity, accuracy, precision, extraction recovery, matrix effects, and stability in accordance with the USFDA guidelines [22]. The selectivity of this method was assessed by comparing the chromatograms of blank plasma samples from six rats, blank plasma samples from rats spiked with picroside I and the IS, and plasma samples from rats orally administered with 100 mg/kg picroside I. Calibration curves were prepared by spiking the blank plasma samples with a standard solution to obtain the concentration curves equal to 0.1, 1, 5, 25, 50, 100, 200, and 500 ng/mL of picroside I and 100 ng/mL of the IS. The calibration curves were calculated by plotting the peak area ratio of the analyte to the IS (y) versus the nominal analyte concentrations (x) and evaluated by a weighted (1/x2) least square regression. The curves should have a correlation coefficient (r) of at least > 0.99. The LLOQ was defined as the lowest concentration of the analyte whose signal-to-noise (S/N) ratio was > 10:1, which should be determined at an accuracy of ± 20% and a precision of < 15%. Carryover was assessed by injecting blank samples after highest calibration standard in six replicates.
Fig. 1. Chemical structures of picroside I and picroside II (IS). 158
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20% methanol. The final solution was centrifuged at 15000 ×g at 4 °C for 10 min. Finally, 5 μL of the supernatant was injected into the UHPLC-Q/TOF-MS/MS system. The urine and bile samples were pretreated with the same procedure. A homogenized fecal sample (0.5 g) was added to 2 mL of 50% methanol and ultrasonically extracted in an ice bath for 30 min. The mixture was centrifuged at 15000 ×g at 4 °C for 10 min, and the supernatant was dried under nitrogen at room temperature. The residue was re-dissolved in 1 mL of 20% methanol and centrifuged at 15000 ×g at 4 °C for 10 min. Similarly, 5 μL of the supernatant was injected into the UHPLC-Q/TOF-MS/MS system. Enzymatic analysis was used to confirm the existence of glucuronidation product. 1.0 mL of drug-containing bile sample was added to 1.0 mL of glucuronidase (Sigma-Aldrich Co., LLC, 4000 U/mL, from Escherichia coli) and then incubated for 2 h at 37 °C.
The intra-day and inter-day accuracy and precision of the method were evaluated by analyzing the QC samples (0.2, 50, and 400 ng/mL) in six replicates on 3 validation days. Accuracy was expressed as a relative error, which should be within ± 15%, and precision was indicated by the RSD, which should not exceed 15%. The extraction recoveries of the analytes at three QC levels (0.2, 50, and 400 ng/mL; n = 6) were determined by comparing the peak area ratios of the analytes to the IS of the extracted samples with those of the extracted blank plasma spiked with the corresponding concentrations. Matrix effects were assessed by comparing the responses of the analytes that were spiked after plasma extraction with those of the standard solutions at equal concentrations. The blank plasmas were from six different lots of rats. The extraction recoveries and matrix effects of the IS were determined at 100 ng/mL by using the same procedure. The stability of picroside I in rat plasma was assessed by analyzing the QC samples (0.2, 50, and 400 ng/mL) in six replicates under different conditions. Freeze–thaw stability was tested by subjecting the QC samples to three freeze–thaw cycles from −80 °C to room temperature. Long-term stability was examined by evaluating the QC samples after storage at −80 °C for 2 months. Short-term stability was determined by analyzing the QC samples after storage at room temperature for 6 h. Auto-sampler stability was assessed by examining the QC samples after storage at 10 °C for 24 h.
2.4.3. Data analysis MetaboLynx™ XS Application Manager in MassLynx™ 4.1 was used to identify the metabolites of picroside I, which could employ numerous metabolic reactions, such as hydroxylation and methylation. This Application Manager generated an array of extracted ion chromatograms and eliminated the interference peak that existed in the sample and the control. Afterward, 5 mDa and 0.05 Da were set as the mass defect filter and mass window, respectively. The MS/MS spectrum could be used to further confirm the structure of the metabolites.
2.3.4. Data analysis The concentration of picroside I was obtained using Analyst® 1.6.3 according to the calibration curves. Pharmacokinetic parameters, including area under the plasma concentration-time curve (AUC), mean resident time (MRT), elimination half-life (t1/2z), time to reach the maximal plasma concentration (tmax), maximal plasma concentration (Cmax), volume of distribution (Vz/F), and clearance (CLz/F), were calculated through non-compartmental model analysis in DAS 3.1 (BioGuider Co., Shanghai, China).
3. Results and discussion 3.1. Pharmacokinetic study 3.1.1. Method development To obtain the optimum MS condition of picroside I and IS, we injected the standard solution into the ESI source and tested the positive and negative modes. We found that the response of picroside I at the negative mode was better than that at the positive mode. The SRM transitions of m/z 491.1 → 147.1 for picroside I and m/z 511.1 → 235.1 for IS were chosen because the abundance of the analytes was higher in these settings than in other settings. Table S1 shows the selected optimal dwell time, declustering potential, collision energy, and collision exit potential. Acetonitrile instead of methanol was chosen as the mobile phase because the elution range and sensitivity of the former are better than those of the latter. We found that the gradient mobile phase with acetonitrile and water could increase the response of picroside I compared with that of the mobile phase with acetonitrile and 0.1% formic acid. The ACQUITY UPLC® BEH C18 (2.1 mm × 50 mm, 1.7 μm) column contributed to the shortening of the retention time of picroside I and the IS. Picroside I and the IS achieved good separation with retention times of 1.5 and 1.1 min, respectively. The run time for each sample was 2 min, which was considerably shorter than that in previous studies [19, 20, 23, 24]. Pretreatment was a critical step before the sample was injected into the LC-MS/MS system. The plasma samples were pretreated with protein precipitation method, which involved simple procedures and achieved good sensitivity in LC-MS analysis.
2.4. Metabolism study 2.4.1. UHPLC-Q/TOF-MS conditions The metabolites were identified by using a UHPLC-Q/TOF-MS/MS system (Waters Corp., Milford, MA, USA). An ACQUITY UPLC® HSS T3 column (2.1 mm × 100 mm, 1.8 μm; Waters Corp.) maintained at 45 °C was utilized for the separation. The mobile phases consisted of (A) 0.1% formic acid and (B) acetonitrile under the following gradient conditions: 0–5 min, 2% → 8% B; 5–9 min, 8% → 18% B; 9–13 min, 18% → 28% B; 13–18 min, 28% → 60% B; 18–30 min, 60% → 90% B; 30–35 min, 90% → 2% B. The flow rate was set at 0.4 mL/min, the autosampler was maintained at 10 °C, and the injection volume was 5 μL. MS detection was performed by using an ACQUITY® Synapt G2 Q/ TOF tandem mass spectrometer (Waters Corp.) equipped with an ESI interface. The optimized ion source conditions were set as follows: capillary voltage, 3 kV; sampling cone voltage, 30 V; extraction cone voltage, 4 V; source temperature, 120 °C; desolvation temperature, 400 °C; cone gas flow, 50 L/h; and desolvation gas flow, 800 L/h. A HCOONa solution was used to calibrate the MS prior to the analysis. The MSE function parameters were as follows: 0.25 s scan time, 0.024 s inter-scan delay, and 6 V collision energy for Function 1 and 0.25 s scan time, 0.024 s inter-scan delay, and 15–25 V collision energy ramp for Function 2. Leucine enkephalin was used to generate the reference ions at m/z 556.2771 ([M + H]+) under positive mode and m/z 554.2615 ([M − H]−) under negative ion mode. Data were acquired from 50 Da to 1000 Da and processed in MassLynx® 4.1 (Waters Corp.).
3.1.2. Method validation Fig. 2 shows the representative SRM chromatograms of the plasma samples. The comparison of the blank plasma sample with the drugcontaining plasma sample revealed that no endogenous substance interfered at the retention time of picroside I and the IS. Good linearity was achieved within the range of 0.1–500 ng/mL for picroside I. The regression equation was y = 0.0981x + 0.0866 (r = 0.9957), where y represents the peak area ratio of picroside I to the IS, and x denotes the plasma concentration of picroside I. The RSD of slope of the regression equation was 3.7%. The back-calculated
2.4.2. Sample pretreatment A 200 μL aliquot of mixed serum samples and 1 mL of methanol were placed in a centrifuge tube, and the mixture was centrifuged at 15000 ×g for 10 min to precipitate the protein. The supernatant was dried under nitrogen at room temperature and re-dissolved in 100 μL of 159
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Fig. 3. Mean plasma concentration–time curves of picroside I after oral administration at a dose of 100 mg/kg. Vertical bars represent mean ± SD (n = 6).
Table 1 Pharmacokinetic parameters of picroside I in rat (n = 6) after oral administration at the dose of 100 mg/kg.
Fig. 2. Representative SRM chromatograms of picroside I and IS in the rat plasma sample: (A) a blank plasma sample; (B) a blank plasma sample spiked with picroside I at a lower limit of quantification and the IS; (C) a plasma sample at 4 h after oral administration of picroside I at the dose of 100 mg/kg (26 ng/mL).
Parameters
Units
Mean ± SD
AUC0-t AUC0-∞ MRT0-t MRT0-∞ t1/2z tmax Cmax Vz/F CLz/F
ng h/mL ng h/mL h h h h ng/mL L/kg L/h/kg
174.30 ± 20.35 196.56 ± 39.51 2.62 ± 0.24 4.67 ± 1.84 3.47 ± 0.60 0.50 ± 0.00 137.65 ± 22.12 3749.12 ± 1975.99 523.50 ± 110.84
of picroside I occurred quickly and reached the maximum plasma concentration of 137.65 ng/mL in 30 min. It was then eliminated rapidly with a t1/2z of 3.47 h. MRT0-t and MRT0-∞ of picroside I were 2.62 h and 4.67 h, respectively. These findings implied that picroside I was possibly metabolized rapidly in vivo, indicating the need to elucidate the metabolic pattern of picroside I in vivo. The pharmacokinetic parameters of picroside I were different from the previous studies. In our study, tmax was shorter and t1/2 was longer than that of the studies performed by Upadhyay et al. [20, 23]. These differences may be caused by different administration dosage, body weight and dosage forms [25]. The pharmacokinetics study in different dosage and species will be carried out in the future.
concentrations of every calibration curve points were within 96%–107% of the nominal concentrations (shown in Table S2). The LLOQ for picroside I was 0.1 ng/mL with a S/N ratio of > 10. The accuracy (relative error) and precision (RSD) at LLOQ were 12.7% and 10.5% respectively. As shown in Table S3, the intra-day and inter-day accuracies of picroside I at three concentration levels (0.2, 50, and 400 ng/mL) were within ± 9.7%, whereas the intra-day and inter-day precision were < 11.3%. There was also no carry-over effect in the method. The mean extraction recoveries of picroside I at the three QC concentrations were above 91.85%, and the recovery of IS was 93.50% (Table S4). The mean matrix effects of picroside I ranged from 89.63% to 94.04%, and the matrix effect of IS was 91.18%, indicating that the matrix did not remarkably influence the ionization of picroside I and the IS. The stability of picroside I in rat plasma is described in Table S5, which shows that the RSDs of the freeze–thaw, long-term, short-term, and auto-sampler stabilities of picroside I were below 12.2%. These results indicated that picroside I was stable in plasma under different conditions, suggesting that the established method could be applicable to pharmacokinetic studies.
3.2. Metabolic analysis 3.2.1. High-resolution mass fragmentation of picrosideI As a parent drug, picroside I was eluted at 12.92 min, and it showed deprotonated ion [M − H]− at m/z 491.1556 with a molecular formula of C24H27O11 and sodium adduct ions [M + Na]+ at m/z 515.1531 with a formula of C24H28O11Na. Fig. 4 presents the fragmentation information of picroside I in negative and positive ion mode. The MS/MS spectrum of picroside I in negative mode indicated that it produced characteristic fragment ions at m/z 199.0608, 169.0503, 147.0450, 103.0546, and 85.0288. The fragment ion at m/z 199.0608 ([M − H − 292]−) was an aglycone ion produced through the dissociation of glycoside at the C-1′ position, whereas the fragment ion at m/z 147.0450 ([M − H − 344]−) was formed through the cleavage of glycoside at the C-6′ position. The fragment ion at m/z 85.0288 was generated after a Retro–Diels–Alder (RDA) reaction and the loss of C20H22O9 (−406 Da). In positive mode, the fragment ions at m/z 333.0945 was formed by the loss of C9H10O4, and the fragment ions at m/z 431.1309 was derived from RDA reaction. This fragmentation information could serve as a basis for the structural determination of picroside I metabolites.
3.1.3. Pharmacokinetic study The developed LC-MS/MS method was successfully applied to pharmacokinetically analyze picroside I in rats. Fig. 3 illustrates the mean plasma concentration–time curves of picroside I after oral administration at a dose of 100 mg/kg, and Table 1 lists the pharmacokinetic parameters of picroside I. As shown in Fig. 3, the oral absorption 160
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Fig. 4. MS/MS spectrum of picroside I and its proposed fragmentation pathways in negative mode (A) and positive mode (B).
C9H12O5 (200 Da) and C15H16O7 (308 Da). The fragment ions at m/z 163.0401 and 119.0494 were produced by the loss of C6H8O4 and CO2 from the fragment ion at m/z 307.0814. The RDA cleavage product ion was detected at m/z 145.0504 from the fragment ion at m/z 199.0610. M01 could be identified as the hydroxylation product of picroside I and the hydroxy group may be on the benzene ring or the double bond in side chain. Metabolite M04 was eluted at 14.38 min with [M − H]− at m/z 683.1842 (C30H35O18, 2.8 ppm), which was 176 Da higher than that of M01, indicating the glucuronidation pathway of M01. M01 was found in the bile and fecal samples, whereas M04 was detected in the urine and serum samples. M02, M03, and M05 comprised the phase II biotransformations of picroside I. M02 was eluted at 13.72 min, and it had a deprotonated ion at m/z 667.1844 (C30H35O17, −4.5 ppm), which was 176 Da higher than that of picroside I. M03 was detected at 12.55 min, and it showed a deprotonated ion at m/z 571.1133 with a molecular formula of C24H27O14S (−1.6 ppm), which was 80 Da higher than that of picroside
3.2.2. Identification of metabolites We used the UHPLC-Q/TOF-MS/MS system to detect the serum, urine, bile, and fecal samples and to identify the metabolites of picroside I in vivo. Fifteen metabolites of picroside I were found using the MS data and structurally identified with the MS/MS data. M01–M04 were in a negative ion mode, and M05–M15 were in a positive mode. Table 2 provides the retention times, observed masses, fragmentation information, and sources of the metabolites. The maximum mass errors were expressed as parts per million (ppm), which should not exceed 5 ppm. Fig. 5 illustrates the typical MS/MS spectra and proposed fragmentation pathways of the selected metabolites (i.e., M01, M02, M06, M09, M10, and M15). Other MS/MS spectra and proposed fragmentation pathways of the metabolites are provided in Figs. S1–S9. M01 was eluted at a retention time of 11.32 min, and it had a deprotonated ion [M − H]− at m/z 507.1528 (C24H27O12, 4.9 ppm), which was 16 Da higher than that of picroside I. The MS/MS fragment ions at m/z 307.0814 and 199.0610 were formed by the neutral loss of 161
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Table 2 Metabolites of picroside I identified in rat serum, urine, bile and feces sample by UHPLC-Q/TOF-MS/MS system. Metabolite
tR (min)
Polarity
Molecular ions m/z
M01
11.32
Negative
507.1528
Fragment ions m/z
307.0814 199.0610 163.0401 145.0504 119.0494 M02
13.72
Negative
667.1844 491.1549 199.0611 147.0451 113.0242 85.0286
M03
12.55
Negative
571.1113 491.1539 199.0607 147.0452 85.0292
M04
14.38
Negative
683.1842 507.1490 307.0811 199.0602 163.0387 113.0244
M05
13.22
Positive
507.1873 359.1357 215.0929 101.0608
M06
14.15
Positive
477.1741 329.1246 185.0821 149.0612 105.0699
M07
11.14
Positive
638.1936 477.1739 185.0818 162.0232 149.0600 105.0699
M08
15.04
Positive
557.1352 477.1749 329.1252 185.0806 149.0598
M09
3.13
Positive
363.1308 201.0756 163.0612 87.0442
M10
8.26
Positive
149.0608 105.0709
M11
7.78
Positive
151.0753 107.0867
M12
3.02
Positive
165.0546 121.0658
M13
11.36
Positive
325.0936 177.0391 149.0600 115.0399 105.0708
M14
9.48
Positive
310.0762 162.0231 149.0597 105.0701
M15
14.98
Positive
163.0752 119.0866
Formula
Error (ppm)
Source
C24H27O12 C15H15O7 C9H11O5 C9H7O3 C6H9O4 C8H7O C30H35O17 C24H27O11 C9H11O5 C9H7O2 C5H5O3 C4H5O2 C24H27O14S C24H27O11 C9H11O5 C9H7O2 C4H5O2 C30H35O18 C24H27O12 C15H15O7 C9H11O5 C9H7O3 C5H5O3 C25H31O11 C16H23O9 C10H15O5 C5H9O2 C24H29O10 C15H21O8 C9H13O4 C9H9O2 C8H9 C29H36NO13S C24H29O10 C9H13O4 C5H8NO3S C9H9O2 C8H9 C24H29O13S C24H29O10 C15H21O8 C9H13O4 C9H9O2 C15H23O10 C9H13O5 C6H11O5 C4H7O2 C9H9O2 C8H9 C9H11O2 C8H11 C9H9O3 C8H9O C15H17O8 C6H9O6 C9H9O2 C5H7O3 C8H9 C14H16NO5S C5H8NO3S C9H9O2 C8H9 C10H11O2 C9H11
4.9 −1.3 2.0 3.7 2.1 −2.5 −4.5 −0.8 2.5 3.4 2.7 −4.7 −1.6 −2.9 0.5 4.1 2.4 2.8 −2.6 −2.3 −2.0 −4.9 4.4 1.4 4.2 4.6 4.9 −4.2 3.0 3.8 6.0 −4.8 4.5 −4.6 2.2 4.3 −2.0 −4.8 4.1 −2.5 4.9 −4.3 −3.4 4.7 −3.5 3.7 −4.6 3.4 4.8 −4.0 5.6 −3.6 4.1 4.0 −4.5 −2.0 3.5 3.8 4.2 3.7 −4.0 −2.9 −4.3 4.2
Bile, feces
Bile, feces
Bile, feces
Urine; serum
Bile, feces
Bile, feces
Bile, feces
Bile, feces
Bile, urine, serum, feces
Bile, urine, serum, feces Bile, urine, serum, feces Bile, urine, serum, feces Bile; feces
Bile; feces
Bile; feces
(C24H29O10, −4.2 ppm), which was 16 Da lower than that of picroside I. M06 was the deoxygenation metabolite of picroside I. M07 and M08 were observed at 11.14 and 15.04 min, respectively. M07 had a protonated ion at m/z 638.1936 (C29H36NO13S, 4.5 ppm), which was 161 Da higher than that of M06. M08 showed a [M + H]+ at m/z 557.1352 (C24H29O13S, 4.1 ppm), which was 80 Da higher than that of M06. M07 and M08 were the acetylcysteine conjugation and sulfation
I. M05 was eluted at 13.22 min, and it revealed a protonated ion [M + H]+ at m/z 507.1873 (C25H31O11, 1.4 ppm), which was 14 Da higher than that of picroside I. These results implied that M02, M03, and M05 were the glucuronidation, sulfation, and methylation products of picroside I. M02, M03, and M05 were observed in the bile and fecal samples. M06 was eluted at 14.15 min with [M + H]+ at m/z 477.1741 162
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Fig. 5. MS/MS spectra of M01, M02, M06, M09, M10, and M15 and their proposed fragmentation pathways.
Comparing with the chromatographic behavior of standard substance, M09 and M10 could be identified as catalpol and cinnamic acid (shown in Fig. S10), respectively. M09 and M10 were detected in the bile, urine, serum, and fecal samples. M11–M15 were found at 7.78, 3.02, 11.36, 9.48, and 14.98 min, and they had protonated ions at m/z 151.0753 (C9H11O2, −4.0 ppm), 165.0546 (C9H9O3, −3.6 ppm), 325.0936 (C15H17O8, 4.0 ppm), 310.0762 (C14H16NO5S, 4.2 ppm), and 163.0752 (C10H11O2,
products of M06. M06–M08 were found in the bile and fecal samples. M09 and M10 were the hydrolysis products of the parent drug. M09 was an iridoid glycoside eluted at 3.13 min, and it showed a protonated ion at m/z 363.1308 (C15H23O10, 4.7 ppm), which was 130 Da lower than that of picroside I. M10 was eluted at 8.26 min, and it displayed a protonated ion at m/z 149.0608 (C9H9O2, 3.4 ppm), which was 344 Da lower than that of picroside I. The fragment ion at m/z 105.0709 (C8H9) in the MS/MS spectrum of M10 was produced by the loss of CO2. 163
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Fig. 6. Proposed metabolic pathways of picroside I in rats.
−4.3 ppm), respectively, which were 2, 16, 176, 161, and 14 Da higher than that of M10, respectively. The MS/MS spectra of M11, M12, and M15 displayed characteristic fragment ions at m/z 107.0867, 121.0658, and 119.0866, which were derived from the loss of CO2. The fragment ion at m/z 149.0600 of M13 was formed through neutral loss of glucuronide moiety. M11–M15 were tentatively identified as the reduction, hydroxylation, glucuronidation, acetylcysteine conjugation, and methylation products of M10. In addition, M12 could be the hydrolysis product of M01. Through comparing the chromatographic behavior, we found that M11 was 3-phenylpropionic acid and M12 was p-coumaric
acid (shown in Fig. S10). M11 and M12 were detected in the bile, urine, serum, and fecal samples, whereas M13–M15 were detected in the bile and fecal samples. The enzymatic analysis proven that M02, M04, and M13 were glucuronidated metabolites. The peak intensity of M02, M04, and M13 weakened after co-incubating with glucuronidase. 3.2.3. Metabolic profiles Fig. 6 shows the proposed metabolic pathways of picroside I in rats. These metabolites can be divided into four parts. The first part consists 164
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of the phase I reaction metabolites of picroside I, including hydroxylation (M01), deoxygenation (M06), and hydrolysis (M09 and M10). The second part comprises the phase II reaction metabolites of picroside I (glucuronidation, sulfation, and methylation). The third part involves the phase I biotransformations of metabolites, such as the reduction (M11) and hydroxylation (M12) products of M10. The fourth part covers the phase II biotransformations of metabolites, which are as follows: glucuronidation (M04) product of M01; acetylcysteine conjugation (M07) and sulfation (M08) products of M06; glucuronidation (M13), acetylcysteine conjugation (M14), and methylation (M15) products of M10. M01–M04, M10, and M12 have been discovered in another study [20], but the hydroxylation and sulfation products of cinnamic acid were not found in current study.
[3] R. Chander, N.K. Kapoor, B.N. Dhawan, Picroliv, picroside-I and kutkoside from Picrorhiza-kurrooa are scavengers of superoxide anions, Biochem. Pharmacol. 44 (1992) 180–183. [4] P.P. Gupta, Picroliv - hepatoprotectant - immunomodulator, Drugs Future 26 (2001) 25–31. [5] Y. Dwivedi, R. Rastogi, N.K. Garg, B.N. Dhawan, Picroliv and its components kutkoside and picroside i protect liver against galactosamine-induced damage in Rats, Pharmacol. Toxicol. 71 (1992) 383–387. [6] Y. Dwivedi, R. Rastogi, R. Chander, S.K. Sharma, N.K. Kapoor, N.K. Garg, B.N. Dhawan, Hepatoprotective activity of picroliv against carbon tetrachlorideinduced liver damage in rats, Indian J. Med. Res. 92 (1990) 195–200. [7] B. Shukla, P.K.S. Visen, G.K. Patnaik, B.N. Dhawan, Reversal of thioacetamide induced cholestasis by picroliv in rodents, Phytother. Res. 6 (1992) 53–55. [8] R. Rastogi, A.K. Srivastava, A.K. Rastogi, Biochemical changes induced in liver and serum of aflatoxin b1-treated male Wistar rats: preventive effect of picroliv, Pharmacol. Toxicol. 88 (2001) 53–58. [9] C. Girish, S.C. Pradhan, Drug development for liver diseases: focus on picroliv, ellagic acid and curcumin, Fundam. Clin. Pharmacol. 22 (2008) 623–632. [10] P.C. Verma, V. Basu, V. Gupta, G. Saxena, L.U. Rahman, Pharmacology and chemistry of a potent hepatoprotective compound Picroliv isolated from the roots and rhizomes of Picrorhiza kurroa royle ex benth. (kutki), Curr. Pharm. Biotechnol. 10 (2009) 641–649. [11] Y. Kiso, Y. Kawakami, K. Kikuchi, H. Hikino, Assay method for antihepatotoxic activity using complement-mediated cytotoxicity in primary cultured hepatocytes, Planta Med. 53 (1987) 241–247. [12] G.B. Singh, S. Bani, S. Singh, A. Khajuria, M.L. Sharma, B.D. Gupta, S.K. Banerjee, Antiinflammatory activity of the iridoids kutkin, picroside-1 and kutkoside from Picrorhiza-kurrooa, Phytother. Res. 7 (1993) 402–407. [13] D. Rathee, M. Thanki, S. Bhuva, S. Anandjiwala, R. Agrawal, Iridoid glycosideskutkin, picroside I, and kutkoside from Picrorrhiza kurroa Benth inhibits the invasion and migration of MCF-7 breast cancer cells through the down regulation of matrix metalloproteinases, Arab. J. Chem. 6 (2013) 49–58. [14] I.A. Najar, B.S. Sachin, S.C. Sharma, N.K. Satti, K.A. Suri, R.K. Johri, Modulation of P-glycoprotein ATPase activity by some phytoconstituents, Phytother. Res. 24 (2010) 454–458. [15] P. Li, K. Matsunaga, T. Yamakuni, Y. Ohizumi, Picrosides I and II, selective enhancers of the mitogen-activated protein kinase-dependent signaling pathway in the action of neuritogenic substances on PC12D cells, Life Sci. 71 (2002) 1821–1835. [16] R. He, L. Kong, X.-Q. Meng, H.-Y. Sun, F.-H. Zhang, Z. Ma, Metabolic evaluation of anti-lung cancer drug picroside-I, Lat. Am. J. Pharm. 35 (2016) 609–611. [17] J.H. Lin, A.Y.H. Lu, Role of pharmacokinetics and metabolism in drug discovery and development, Pharmacol. Rev. 49 (1997) 403–449. [18] K. Vipul, M. Nitin, R.C. Gupta, A sensitive and selective LC-MS-MS method for simultaneous determination of picroside-I and kutkoside (active principles of herbal preparation picroliv) using solid phase extraction in rabbit plasma: application to pharmacokinetic study, J. Chromatogr. B Anal. Technol. Biomed. Life Sci. 820 (2005) 221–227. [19] J. Zhu, B. Xue, B. Ma, Q. Zhang, M. Liu, L. Liu, D. Yao, H. Qi, Y. Wang, H. Ying, Z. Wu, A pre-clinical pharmacokinetic study in rats of three naturally occurring iridoid glycosides, picroside-I, II and III, using a validated simultaneous HPLC-MS/ MS assay, J. Chromatogr. B Anal. Technol. Biomed. Life Sci. 993 (2015) 47–59. [20] D. Upadhyay, S. Anandjiwala, H. Padh, M. Nivsarkar, In vitro - in vivo metabolism and pharmacokinetics of picroside I and II using LC-ESI-MS method, Chem. Biol. Interact. 254 (2016) 83–92. [21] B. Wen, M.S. Zhu, Applications of mass spectrometry in drug metabolism: 50 years of progress, Drug Metab. Rev. 47 (2015) 71–87. [22] U.S. Department of Health and Human Services, Food and Drug Administration, Center for Drug Evaluation and Research, Center for Veterinary Medicine, Guidance for Industry, Bioanalytical Method Validation, Available at: https://www.fda.gov/ downloads/Drugs/Guidances/ucm368107.pdf. [23] D. Upadhyay, R.P. Dash, S. Anandjiwala, M. Nivsarkar, Comparative pharmacokinetic profiles of picrosides I and II from kutkin, Picrorhiza kurroa extract and its formulation in rats, Fitoterapia 85 (2013) 76–83. [24] S. Zahiruddin, W. Khan, R. Nehra, M.J. Alam, M.N. Mallick, R. Parveen, S. Ahmad, Pharmacokinetics and comparative metabolic profiling of iridoid enriched fraction of Picrorhiza kurroa - an Ayurvedic Herb, J. Ethnopharmacol. 197 (2017) 157–164. [25] M.A. Hedaya, Basic Pharmacokinetics, 2nd ed., Taylor & Francis/CRC Press, Boca Raton, 2012.
4. Conclusion In this study, a fast (2 min), selective, and sensitive (0.1 ng/mL) LCMS/MS method was developed and validated to detect picroside I in rat plasma. The established LC-MS/MS method was successfully applied to pharmacokinetically analyze picroside I orally administered to rats. Our results revealed that the oral absorption and elimination of picroside I occurred rapidly. Fifteen metabolites were found and tentatively identified in the bile, urine, serum, and fecal samples by using the UHPLCQ/TOF-MS/MS system, and four metabolites were identified by comparing with the standard substance. Of these metabolites, nine were discovered for the first time. Picroside I was extensively metabolized through phases I and II reactions in vivo. Our study elucidated the metabolic pathways of picroside I, and our findings could help explain the efficacy and safety of picroside I. Conflicts of interest We declare that there are no conflicts of interest. Acknowledgements This work was financially supported by National Natural Science Foundation of China (project No. 81503223) and “Xing lin” plan of Shanghai University of Traditional Chinese Medicine (SHUTCM2017). Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.jchromb.2018.07.034. References [1] H. Sood, R.S. Chauhan, Biosynthesis and accumulation of a medicinal compound, picroside-I, in cultures of Picrorhiza kurroa Royle ex Benth, Plant Cell Tissue Organ Cult. 100 (2010) 113–117. [2] F. Li, Y. Yu, H. Zhang, T. Liu, Y. Li, G. Duan, Infrared-assisted non-ionic surfactant extraction as a green analytical preparatory technique for the rapid extraction and pre-concentration of picroside I and picroside II from Picrorhiza scrophulariiflora Pennell, Anal. Methods 5 (2013) 3747–3753.
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Journal of Chromatography B journal homepage: www.elsevier.com/locate/jchromb
Metabolic profiles and pharmacokinetics of picroside I in rats by liquid chromatography combined with electrospray ionization tandem mass spectrometry ⁎
Kai Xionga,b, Zhengcai Juc, Tong Zhanga,b, , Zhengtao Wangc, Han Hana,b,
T
⁎
a
Experiment Center for Teaching and Learning, Shanghai University of Traditional Chinese Medicine, Shanghai 201210, China School of Pharmacy, Shanghai University of Traditional Chinese Medicine, Shanghai 201210, China c Institute of Chinese Materia Medica, Shanghai University of Traditional Chinese Medicine, 1200 Cailun Road, Shanghai 201210, China b
A R T I C LE I N FO
A B S T R A C T
Keywords: Picroside I LC–MS/MS LC-Q/TOF-MS Pharmacokinetics Metabolism
Picroside I is an iridoid glycoside derived from Picrorhiza kurroa Royle ex Benth and Picrorhiza scrophulariiflora Pennell and characterized by many biological activities. In this study, a fast, selective, and sensitive UHPLC-MS/ MS method was developed and validated to determine picroside I in rat plasma. Analytes were separated by using an ACQUITY UPLC® BEH C18 (2.1 × 50 mm, 1.7 μm) column at a running time of 2 min. Selected reaction monitoring (SRM) transitions were m/z 491.1 → 147.1 for picroside I and m/z 511.1 → 235.1 for the internal standard in a negative ion mode. The established UHPLC-MS/MS method achieved good linearity for picroside I within the range of 0.1–500 ng/mL. The validated method was successfully applied for the pharmacokinetic analysis of picroside I in rats after oral administration. Fifteen metabolites of picroside I were tentatively identified through ultra-high-performance chromatography/tandem quadrupole time-of-flight mass spectrometry, and four metabolites were identified by comparing with the standards. Besides, nine of these metabolites were discovered for the first time. The proposed metabolic pathways of picroside I in vivo can be divided into four parts, namely, phase I reaction of picroside I, including hydroxylation and deoxygenation; phase II reaction of picroside I, including glucuronidation, sulfation, and methylation; phase I biotransformations of metabolites, such as reduction and hydroxylation; and phase II biotransformations of metabolites, such as glucuronidation and sulfation. These results could offer insights into the effectiveness and toxicity of picroside I.
1. Introduction Picroside I is an iridoid glycoside that can be isolated from Picrorhiza kurroa Royle ex Benth and Picrorhiza scrophulariiflora Pennell [1, 2]. Picroliv, which is a hepatic protectant found in India, contains approximately 60% picroside I and picroside II at a 1:1.5 ratio [3, 4]. Picroliv can protect the liver against galactosamine-, carbon tetrachloride-, thioacetamide-, and aflatoxin B1-induced damage [5–8]. The antioxidant and stabilizing actions on the cell membranes of hepatocytes are main reason for the hepatoprotective activity of picroliv [9, 10]. As the main constituent of picroliv, picroside I exhibits many biological activities, including anti-hepatotoxic [11], anti-oxidant [3], anti-inflammatory [12], anti-tumor [13], and P-glycoprotein stimulation [14]. Moreover, picroside I may enhance the neurite outgrowth stimulated by basic fibroblast growth factor, staurosporine, or dibutyryl cyclic adenosine monophosphate in PC12D cells through a mitogenactivated protein kinase signaling pathway [15]. Picroside I can be well ⁎
docked into the activity site of CYP3A4, which is an enzyme that metabolizes phase I drugs [16]. The main aspects of in vivo drug action are pharmacokinetics and drug metabolism, which play important roles in drug discovery and development [17]. Compared with pharmacodynamic investigations, limited efforts have been devoted to studying the pharmacokinetics and metabolism of picroside I. A few studies have applied liquid chromatography tandem-mass spectrometry (LC-MS/MS) method to determine picroside I in plasma; however, these studies have been hampered by either a complex sample pretreatment method or a long running time [18, 19]. Therefore, a fast and reliable method must be developed for pharmacokinetic studies on picroside I. Upadhyay et al. [20] found eight picroside I metabolites, including picroside I-glucuronide, -sulfate, -hydroxylated products, in rat liver microsomes and hepatocyte cultures. However, they utilized low-resolution mass spectrometry and thus failed to provide information on mass fragmentation because of technical limitations. Recent
Corresponding authors at: Experiment Center for Teaching and Learning, Shanghai University of Traditional Chinese Medicine, Shanghai 201210, China. E-mail addresses: [email protected] (T. Zhang), [email protected] (H. Han).
https://doi.org/10.1016/j.jchromb.2018.07.034 Received 15 June 2018; Received in revised form 22 July 2018; Accepted 26 July 2018 Available online 29 July 2018 1570-0232/ © 2018 Published by Elsevier B.V.
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the plasma samples were stored at −80 °C until analysis. The serum, bile, urine, and fecal samples of six rats were collected for metabolic analysis. The rats were orally administrated with picroside I at a dose of 100 mg/kg. Blood samples (200 μL) were collected through coagulation-promoting tubes at 0, 0.167, 0.5, 1, 2, 4, 8, and 12 h post-dosing. The blood samples were then centrifuged at 5000 ×g for 10 min to obtain the serum. The urine and fecal samples were collected at 12 h pre-dosing and at 0–24 h post-dosing in metabolic cages. After the rats were anesthetized by 25% urethane (intraperitoneal injection, 4 mL/kg), the bile samples were collected at 6 h pre-dosing and at 0–12 h post-dosing. These samples were stored at −80 °C until analysis.
investigations on drug metabolism have widely involved an ultra-highperformance liquid chromatography tandem quadrupole time-of-flight mass spectrometry (UHPLC-Q/TOF-MS/MS) system, which can obtain the accurate tandem mass spectra of drug metabolites and is a useful tool for structural confirmation [21]. The high-resolution mass spectrometry component of this system allows a reliable identification of metabolites in vivo. In this study, a simple, fast, selective, and sensitive LC-MS/MS method was developed and validated to detect picroside I in rat plasma. The established method was successfully applied to pharmacokinetically analyze picroside I orally administered to rats. Picroside I was also subjected to metabolic studies in vivo through high-resolution mass spectrometry for the first time. Fifteen metabolites of picroside I were tentatively identified in rat bile, urine, serum, and feces through UHPLC-Q/TOF-MS/MS. The structures of these metabolites were confirmed by the obtained tandem mass spectra. The proposed metabolic pathways of picroside I in vivo contained phases I and II reaction metabolites of picroside I and product-related metabolites.
2.3. Pharmacokinetic analysis 2.3.1. LC-MS/MS conditions This study used an LC-MS/MS system, namely, a Nexera X2 UHPLC system (Shimadzu Co., Kyoto, Japan) coupled with an AB SCIEX QTRAP® 6500 mass spectrometer (Redwood City, CA, USA). The analytes were separated by using an ACQUITY UPLC® BEH C18 (2.1 × 50 mm, 1.7 μm) column (Waters, Dublin, Ireland) at 40 °C. The guard column (ACQUITY UPLC® BEH C18) was placed before the inlet of the analytical column. The mobile phases consisted of (A) water and (B) acetonitrile. The gradient elution was as follows: 18% → 28% B at 0–0.8 min, 28% → 60% B at 0.8–1.2 min, 60% → 90% B at 1.2–1.6 min, 90% → 2% B at 1.6–2 min. The flow rate was set at 0.4 mL/min. The auto-sampler tray was maintained at 10 °C, and the sample injection volume was 5 μL. The mass spectrometer was operated under negative electrospray ionization (ESI) mode. The MS conditions were set as follows: gas (nitrogen) temperature, 550 °C; curtain gas, 30 psi; ion source voltage, −4500 V; collisionally activated dissociation, medium level; and ion source gas 1 and 2, 55 psi. The SRM transitions (Table S1) were m/z 491.1 → 147.1 for picroside I and m/z 511.1 → 235.1 for the IS. Data were acquired and analyzed using Analyst 1.6.3 (AB SCIEX LLC.).
2. Materials and methods 2.1. Chemicals and reagents Picroside I and picroside II (internal standard [IS], structure shown in Fig. 1) with 98% purity were purchased from Nanjing GOREN BioTechnology Co., Ltd. (Nanjing, China). Catalpol (purity > 96%), cinnamic acid (purity > 99%), 3-phenylpropionic acid (purity > 99%) and p-coumaric acid (purity > 98%) were obtained from Sigma-Aldrich Co., LLC (St. Louis, MO, USA). LC-MS-grade acetonitrile, methanol, and formic acid were procured from Sigma-Aldrich Co., LLC. Water was acquired from a Milli-Q Advantage A10 purification system (Millipore Corporation, Billerica, MA, USA). All other chemicals were of analytical grade. 2.2. Animals and sampling
2.3.2. Sample pretreatment In this procedure, 50 μL of IS solution (100 ng/mL) was added to 50 μL of each plasma sample and mixed for 20 s. Afterward, 200 μL of acetonitrile was added to the mixture and vortexed for 1 min to precipitate the protein. The mixture was centrifuged at 15000 ×g for 10 min, and the clear supernatant was dried under N2 gas. The residue was re-dissolved in 50 μL of 20% acetonitrile and centrifuged at 15000 ×g for 10 min. Finally, 5 μL of the solution was injected into the LC-MS/MS system.
Male Wistar rats (7–8 weeks old, 240 ± 10 g) were obtained from the Laboratory Animal Center of Shanghai University of Traditional Chinese Medicine (Shanghai, China). The rats were kept under controlled conditions (room temperature, 22 ± 2 °C; relative humidity, 60 ± 5%) at a 12 h/12 h day/night cycle for 7 days before the experiments. All of the animal experiments were approved by the Institutional Animal Care and Use Committee of Shanghai University of Traditional Chinese Medicine. The rats were subjected to fasting overnight before the experiments and given free access to water. Six rats were used for pharmacokinetic analysis. After picroside I (dissolved in normal saline) was orally administered at a dose of 100 mg/kg, blood samples (0.2 mL) were collected in heparin tubes at 0, 0.083, 0.25, 0.5, 0.75, 1, 1.5, 2, 4, 6, 8, and 12 h. The blood samples were then centrifuged at 5000 ×g for 10 min to obtain the plasma, and
2.3.3. Method validation The established method was validated for selectivity, linearity, accuracy, precision, extraction recovery, matrix effects, and stability in accordance with the USFDA guidelines [22]. The selectivity of this method was assessed by comparing the chromatograms of blank plasma samples from six rats, blank plasma samples from rats spiked with picroside I and the IS, and plasma samples from rats orally administered with 100 mg/kg picroside I. Calibration curves were prepared by spiking the blank plasma samples with a standard solution to obtain the concentration curves equal to 0.1, 1, 5, 25, 50, 100, 200, and 500 ng/mL of picroside I and 100 ng/mL of the IS. The calibration curves were calculated by plotting the peak area ratio of the analyte to the IS (y) versus the nominal analyte concentrations (x) and evaluated by a weighted (1/x2) least square regression. The curves should have a correlation coefficient (r) of at least > 0.99. The LLOQ was defined as the lowest concentration of the analyte whose signal-to-noise (S/N) ratio was > 10:1, which should be determined at an accuracy of ± 20% and a precision of < 15%. Carryover was assessed by injecting blank samples after highest calibration standard in six replicates.
Fig. 1. Chemical structures of picroside I and picroside II (IS). 158
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20% methanol. The final solution was centrifuged at 15000 ×g at 4 °C for 10 min. Finally, 5 μL of the supernatant was injected into the UHPLC-Q/TOF-MS/MS system. The urine and bile samples were pretreated with the same procedure. A homogenized fecal sample (0.5 g) was added to 2 mL of 50% methanol and ultrasonically extracted in an ice bath for 30 min. The mixture was centrifuged at 15000 ×g at 4 °C for 10 min, and the supernatant was dried under nitrogen at room temperature. The residue was re-dissolved in 1 mL of 20% methanol and centrifuged at 15000 ×g at 4 °C for 10 min. Similarly, 5 μL of the supernatant was injected into the UHPLC-Q/TOF-MS/MS system. Enzymatic analysis was used to confirm the existence of glucuronidation product. 1.0 mL of drug-containing bile sample was added to 1.0 mL of glucuronidase (Sigma-Aldrich Co., LLC, 4000 U/mL, from Escherichia coli) and then incubated for 2 h at 37 °C.
The intra-day and inter-day accuracy and precision of the method were evaluated by analyzing the QC samples (0.2, 50, and 400 ng/mL) in six replicates on 3 validation days. Accuracy was expressed as a relative error, which should be within ± 15%, and precision was indicated by the RSD, which should not exceed 15%. The extraction recoveries of the analytes at three QC levels (0.2, 50, and 400 ng/mL; n = 6) were determined by comparing the peak area ratios of the analytes to the IS of the extracted samples with those of the extracted blank plasma spiked with the corresponding concentrations. Matrix effects were assessed by comparing the responses of the analytes that were spiked after plasma extraction with those of the standard solutions at equal concentrations. The blank plasmas were from six different lots of rats. The extraction recoveries and matrix effects of the IS were determined at 100 ng/mL by using the same procedure. The stability of picroside I in rat plasma was assessed by analyzing the QC samples (0.2, 50, and 400 ng/mL) in six replicates under different conditions. Freeze–thaw stability was tested by subjecting the QC samples to three freeze–thaw cycles from −80 °C to room temperature. Long-term stability was examined by evaluating the QC samples after storage at −80 °C for 2 months. Short-term stability was determined by analyzing the QC samples after storage at room temperature for 6 h. Auto-sampler stability was assessed by examining the QC samples after storage at 10 °C for 24 h.
2.4.3. Data analysis MetaboLynx™ XS Application Manager in MassLynx™ 4.1 was used to identify the metabolites of picroside I, which could employ numerous metabolic reactions, such as hydroxylation and methylation. This Application Manager generated an array of extracted ion chromatograms and eliminated the interference peak that existed in the sample and the control. Afterward, 5 mDa and 0.05 Da were set as the mass defect filter and mass window, respectively. The MS/MS spectrum could be used to further confirm the structure of the metabolites.
2.3.4. Data analysis The concentration of picroside I was obtained using Analyst® 1.6.3 according to the calibration curves. Pharmacokinetic parameters, including area under the plasma concentration-time curve (AUC), mean resident time (MRT), elimination half-life (t1/2z), time to reach the maximal plasma concentration (tmax), maximal plasma concentration (Cmax), volume of distribution (Vz/F), and clearance (CLz/F), were calculated through non-compartmental model analysis in DAS 3.1 (BioGuider Co., Shanghai, China).
3. Results and discussion 3.1. Pharmacokinetic study 3.1.1. Method development To obtain the optimum MS condition of picroside I and IS, we injected the standard solution into the ESI source and tested the positive and negative modes. We found that the response of picroside I at the negative mode was better than that at the positive mode. The SRM transitions of m/z 491.1 → 147.1 for picroside I and m/z 511.1 → 235.1 for IS were chosen because the abundance of the analytes was higher in these settings than in other settings. Table S1 shows the selected optimal dwell time, declustering potential, collision energy, and collision exit potential. Acetonitrile instead of methanol was chosen as the mobile phase because the elution range and sensitivity of the former are better than those of the latter. We found that the gradient mobile phase with acetonitrile and water could increase the response of picroside I compared with that of the mobile phase with acetonitrile and 0.1% formic acid. The ACQUITY UPLC® BEH C18 (2.1 mm × 50 mm, 1.7 μm) column contributed to the shortening of the retention time of picroside I and the IS. Picroside I and the IS achieved good separation with retention times of 1.5 and 1.1 min, respectively. The run time for each sample was 2 min, which was considerably shorter than that in previous studies [19, 20, 23, 24]. Pretreatment was a critical step before the sample was injected into the LC-MS/MS system. The plasma samples were pretreated with protein precipitation method, which involved simple procedures and achieved good sensitivity in LC-MS analysis.
2.4. Metabolism study 2.4.1. UHPLC-Q/TOF-MS conditions The metabolites were identified by using a UHPLC-Q/TOF-MS/MS system (Waters Corp., Milford, MA, USA). An ACQUITY UPLC® HSS T3 column (2.1 mm × 100 mm, 1.8 μm; Waters Corp.) maintained at 45 °C was utilized for the separation. The mobile phases consisted of (A) 0.1% formic acid and (B) acetonitrile under the following gradient conditions: 0–5 min, 2% → 8% B; 5–9 min, 8% → 18% B; 9–13 min, 18% → 28% B; 13–18 min, 28% → 60% B; 18–30 min, 60% → 90% B; 30–35 min, 90% → 2% B. The flow rate was set at 0.4 mL/min, the autosampler was maintained at 10 °C, and the injection volume was 5 μL. MS detection was performed by using an ACQUITY® Synapt G2 Q/ TOF tandem mass spectrometer (Waters Corp.) equipped with an ESI interface. The optimized ion source conditions were set as follows: capillary voltage, 3 kV; sampling cone voltage, 30 V; extraction cone voltage, 4 V; source temperature, 120 °C; desolvation temperature, 400 °C; cone gas flow, 50 L/h; and desolvation gas flow, 800 L/h. A HCOONa solution was used to calibrate the MS prior to the analysis. The MSE function parameters were as follows: 0.25 s scan time, 0.024 s inter-scan delay, and 6 V collision energy for Function 1 and 0.25 s scan time, 0.024 s inter-scan delay, and 15–25 V collision energy ramp for Function 2. Leucine enkephalin was used to generate the reference ions at m/z 556.2771 ([M + H]+) under positive mode and m/z 554.2615 ([M − H]−) under negative ion mode. Data were acquired from 50 Da to 1000 Da and processed in MassLynx® 4.1 (Waters Corp.).
3.1.2. Method validation Fig. 2 shows the representative SRM chromatograms of the plasma samples. The comparison of the blank plasma sample with the drugcontaining plasma sample revealed that no endogenous substance interfered at the retention time of picroside I and the IS. Good linearity was achieved within the range of 0.1–500 ng/mL for picroside I. The regression equation was y = 0.0981x + 0.0866 (r = 0.9957), where y represents the peak area ratio of picroside I to the IS, and x denotes the plasma concentration of picroside I. The RSD of slope of the regression equation was 3.7%. The back-calculated
2.4.2. Sample pretreatment A 200 μL aliquot of mixed serum samples and 1 mL of methanol were placed in a centrifuge tube, and the mixture was centrifuged at 15000 ×g for 10 min to precipitate the protein. The supernatant was dried under nitrogen at room temperature and re-dissolved in 100 μL of 159
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Fig. 3. Mean plasma concentration–time curves of picroside I after oral administration at a dose of 100 mg/kg. Vertical bars represent mean ± SD (n = 6).
Table 1 Pharmacokinetic parameters of picroside I in rat (n = 6) after oral administration at the dose of 100 mg/kg.
Fig. 2. Representative SRM chromatograms of picroside I and IS in the rat plasma sample: (A) a blank plasma sample; (B) a blank plasma sample spiked with picroside I at a lower limit of quantification and the IS; (C) a plasma sample at 4 h after oral administration of picroside I at the dose of 100 mg/kg (26 ng/mL).
Parameters
Units
Mean ± SD
AUC0-t AUC0-∞ MRT0-t MRT0-∞ t1/2z tmax Cmax Vz/F CLz/F
ng h/mL ng h/mL h h h h ng/mL L/kg L/h/kg
174.30 ± 20.35 196.56 ± 39.51 2.62 ± 0.24 4.67 ± 1.84 3.47 ± 0.60 0.50 ± 0.00 137.65 ± 22.12 3749.12 ± 1975.99 523.50 ± 110.84
of picroside I occurred quickly and reached the maximum plasma concentration of 137.65 ng/mL in 30 min. It was then eliminated rapidly with a t1/2z of 3.47 h. MRT0-t and MRT0-∞ of picroside I were 2.62 h and 4.67 h, respectively. These findings implied that picroside I was possibly metabolized rapidly in vivo, indicating the need to elucidate the metabolic pattern of picroside I in vivo. The pharmacokinetic parameters of picroside I were different from the previous studies. In our study, tmax was shorter and t1/2 was longer than that of the studies performed by Upadhyay et al. [20, 23]. These differences may be caused by different administration dosage, body weight and dosage forms [25]. The pharmacokinetics study in different dosage and species will be carried out in the future.
concentrations of every calibration curve points were within 96%–107% of the nominal concentrations (shown in Table S2). The LLOQ for picroside I was 0.1 ng/mL with a S/N ratio of > 10. The accuracy (relative error) and precision (RSD) at LLOQ were 12.7% and 10.5% respectively. As shown in Table S3, the intra-day and inter-day accuracies of picroside I at three concentration levels (0.2, 50, and 400 ng/mL) were within ± 9.7%, whereas the intra-day and inter-day precision were < 11.3%. There was also no carry-over effect in the method. The mean extraction recoveries of picroside I at the three QC concentrations were above 91.85%, and the recovery of IS was 93.50% (Table S4). The mean matrix effects of picroside I ranged from 89.63% to 94.04%, and the matrix effect of IS was 91.18%, indicating that the matrix did not remarkably influence the ionization of picroside I and the IS. The stability of picroside I in rat plasma is described in Table S5, which shows that the RSDs of the freeze–thaw, long-term, short-term, and auto-sampler stabilities of picroside I were below 12.2%. These results indicated that picroside I was stable in plasma under different conditions, suggesting that the established method could be applicable to pharmacokinetic studies.
3.2. Metabolic analysis 3.2.1. High-resolution mass fragmentation of picrosideI As a parent drug, picroside I was eluted at 12.92 min, and it showed deprotonated ion [M − H]− at m/z 491.1556 with a molecular formula of C24H27O11 and sodium adduct ions [M + Na]+ at m/z 515.1531 with a formula of C24H28O11Na. Fig. 4 presents the fragmentation information of picroside I in negative and positive ion mode. The MS/MS spectrum of picroside I in negative mode indicated that it produced characteristic fragment ions at m/z 199.0608, 169.0503, 147.0450, 103.0546, and 85.0288. The fragment ion at m/z 199.0608 ([M − H − 292]−) was an aglycone ion produced through the dissociation of glycoside at the C-1′ position, whereas the fragment ion at m/z 147.0450 ([M − H − 344]−) was formed through the cleavage of glycoside at the C-6′ position. The fragment ion at m/z 85.0288 was generated after a Retro–Diels–Alder (RDA) reaction and the loss of C20H22O9 (−406 Da). In positive mode, the fragment ions at m/z 333.0945 was formed by the loss of C9H10O4, and the fragment ions at m/z 431.1309 was derived from RDA reaction. This fragmentation information could serve as a basis for the structural determination of picroside I metabolites.
3.1.3. Pharmacokinetic study The developed LC-MS/MS method was successfully applied to pharmacokinetically analyze picroside I in rats. Fig. 3 illustrates the mean plasma concentration–time curves of picroside I after oral administration at a dose of 100 mg/kg, and Table 1 lists the pharmacokinetic parameters of picroside I. As shown in Fig. 3, the oral absorption 160
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Fig. 4. MS/MS spectrum of picroside I and its proposed fragmentation pathways in negative mode (A) and positive mode (B).
C9H12O5 (200 Da) and C15H16O7 (308 Da). The fragment ions at m/z 163.0401 and 119.0494 were produced by the loss of C6H8O4 and CO2 from the fragment ion at m/z 307.0814. The RDA cleavage product ion was detected at m/z 145.0504 from the fragment ion at m/z 199.0610. M01 could be identified as the hydroxylation product of picroside I and the hydroxy group may be on the benzene ring or the double bond in side chain. Metabolite M04 was eluted at 14.38 min with [M − H]− at m/z 683.1842 (C30H35O18, 2.8 ppm), which was 176 Da higher than that of M01, indicating the glucuronidation pathway of M01. M01 was found in the bile and fecal samples, whereas M04 was detected in the urine and serum samples. M02, M03, and M05 comprised the phase II biotransformations of picroside I. M02 was eluted at 13.72 min, and it had a deprotonated ion at m/z 667.1844 (C30H35O17, −4.5 ppm), which was 176 Da higher than that of picroside I. M03 was detected at 12.55 min, and it showed a deprotonated ion at m/z 571.1133 with a molecular formula of C24H27O14S (−1.6 ppm), which was 80 Da higher than that of picroside
3.2.2. Identification of metabolites We used the UHPLC-Q/TOF-MS/MS system to detect the serum, urine, bile, and fecal samples and to identify the metabolites of picroside I in vivo. Fifteen metabolites of picroside I were found using the MS data and structurally identified with the MS/MS data. M01–M04 were in a negative ion mode, and M05–M15 were in a positive mode. Table 2 provides the retention times, observed masses, fragmentation information, and sources of the metabolites. The maximum mass errors were expressed as parts per million (ppm), which should not exceed 5 ppm. Fig. 5 illustrates the typical MS/MS spectra and proposed fragmentation pathways of the selected metabolites (i.e., M01, M02, M06, M09, M10, and M15). Other MS/MS spectra and proposed fragmentation pathways of the metabolites are provided in Figs. S1–S9. M01 was eluted at a retention time of 11.32 min, and it had a deprotonated ion [M − H]− at m/z 507.1528 (C24H27O12, 4.9 ppm), which was 16 Da higher than that of picroside I. The MS/MS fragment ions at m/z 307.0814 and 199.0610 were formed by the neutral loss of 161
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Table 2 Metabolites of picroside I identified in rat serum, urine, bile and feces sample by UHPLC-Q/TOF-MS/MS system. Metabolite
tR (min)
Polarity
Molecular ions m/z
M01
11.32
Negative
507.1528
Fragment ions m/z
307.0814 199.0610 163.0401 145.0504 119.0494 M02
13.72
Negative
667.1844 491.1549 199.0611 147.0451 113.0242 85.0286
M03
12.55
Negative
571.1113 491.1539 199.0607 147.0452 85.0292
M04
14.38
Negative
683.1842 507.1490 307.0811 199.0602 163.0387 113.0244
M05
13.22
Positive
507.1873 359.1357 215.0929 101.0608
M06
14.15
Positive
477.1741 329.1246 185.0821 149.0612 105.0699
M07
11.14
Positive
638.1936 477.1739 185.0818 162.0232 149.0600 105.0699
M08
15.04
Positive
557.1352 477.1749 329.1252 185.0806 149.0598
M09
3.13
Positive
363.1308 201.0756 163.0612 87.0442
M10
8.26
Positive
149.0608 105.0709
M11
7.78
Positive
151.0753 107.0867
M12
3.02
Positive
165.0546 121.0658
M13
11.36
Positive
325.0936 177.0391 149.0600 115.0399 105.0708
M14
9.48
Positive
310.0762 162.0231 149.0597 105.0701
M15
14.98
Positive
163.0752 119.0866
Formula
Error (ppm)
Source
C24H27O12 C15H15O7 C9H11O5 C9H7O3 C6H9O4 C8H7O C30H35O17 C24H27O11 C9H11O5 C9H7O2 C5H5O3 C4H5O2 C24H27O14S C24H27O11 C9H11O5 C9H7O2 C4H5O2 C30H35O18 C24H27O12 C15H15O7 C9H11O5 C9H7O3 C5H5O3 C25H31O11 C16H23O9 C10H15O5 C5H9O2 C24H29O10 C15H21O8 C9H13O4 C9H9O2 C8H9 C29H36NO13S C24H29O10 C9H13O4 C5H8NO3S C9H9O2 C8H9 C24H29O13S C24H29O10 C15H21O8 C9H13O4 C9H9O2 C15H23O10 C9H13O5 C6H11O5 C4H7O2 C9H9O2 C8H9 C9H11O2 C8H11 C9H9O3 C8H9O C15H17O8 C6H9O6 C9H9O2 C5H7O3 C8H9 C14H16NO5S C5H8NO3S C9H9O2 C8H9 C10H11O2 C9H11
4.9 −1.3 2.0 3.7 2.1 −2.5 −4.5 −0.8 2.5 3.4 2.7 −4.7 −1.6 −2.9 0.5 4.1 2.4 2.8 −2.6 −2.3 −2.0 −4.9 4.4 1.4 4.2 4.6 4.9 −4.2 3.0 3.8 6.0 −4.8 4.5 −4.6 2.2 4.3 −2.0 −4.8 4.1 −2.5 4.9 −4.3 −3.4 4.7 −3.5 3.7 −4.6 3.4 4.8 −4.0 5.6 −3.6 4.1 4.0 −4.5 −2.0 3.5 3.8 4.2 3.7 −4.0 −2.9 −4.3 4.2
Bile, feces
Bile, feces
Bile, feces
Urine; serum
Bile, feces
Bile, feces
Bile, feces
Bile, feces
Bile, urine, serum, feces
Bile, urine, serum, feces Bile, urine, serum, feces Bile, urine, serum, feces Bile; feces
Bile; feces
Bile; feces
(C24H29O10, −4.2 ppm), which was 16 Da lower than that of picroside I. M06 was the deoxygenation metabolite of picroside I. M07 and M08 were observed at 11.14 and 15.04 min, respectively. M07 had a protonated ion at m/z 638.1936 (C29H36NO13S, 4.5 ppm), which was 161 Da higher than that of M06. M08 showed a [M + H]+ at m/z 557.1352 (C24H29O13S, 4.1 ppm), which was 80 Da higher than that of M06. M07 and M08 were the acetylcysteine conjugation and sulfation
I. M05 was eluted at 13.22 min, and it revealed a protonated ion [M + H]+ at m/z 507.1873 (C25H31O11, 1.4 ppm), which was 14 Da higher than that of picroside I. These results implied that M02, M03, and M05 were the glucuronidation, sulfation, and methylation products of picroside I. M02, M03, and M05 were observed in the bile and fecal samples. M06 was eluted at 14.15 min with [M + H]+ at m/z 477.1741 162
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Fig. 5. MS/MS spectra of M01, M02, M06, M09, M10, and M15 and their proposed fragmentation pathways.
Comparing with the chromatographic behavior of standard substance, M09 and M10 could be identified as catalpol and cinnamic acid (shown in Fig. S10), respectively. M09 and M10 were detected in the bile, urine, serum, and fecal samples. M11–M15 were found at 7.78, 3.02, 11.36, 9.48, and 14.98 min, and they had protonated ions at m/z 151.0753 (C9H11O2, −4.0 ppm), 165.0546 (C9H9O3, −3.6 ppm), 325.0936 (C15H17O8, 4.0 ppm), 310.0762 (C14H16NO5S, 4.2 ppm), and 163.0752 (C10H11O2,
products of M06. M06–M08 were found in the bile and fecal samples. M09 and M10 were the hydrolysis products of the parent drug. M09 was an iridoid glycoside eluted at 3.13 min, and it showed a protonated ion at m/z 363.1308 (C15H23O10, 4.7 ppm), which was 130 Da lower than that of picroside I. M10 was eluted at 8.26 min, and it displayed a protonated ion at m/z 149.0608 (C9H9O2, 3.4 ppm), which was 344 Da lower than that of picroside I. The fragment ion at m/z 105.0709 (C8H9) in the MS/MS spectrum of M10 was produced by the loss of CO2. 163
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Fig. 6. Proposed metabolic pathways of picroside I in rats.
−4.3 ppm), respectively, which were 2, 16, 176, 161, and 14 Da higher than that of M10, respectively. The MS/MS spectra of M11, M12, and M15 displayed characteristic fragment ions at m/z 107.0867, 121.0658, and 119.0866, which were derived from the loss of CO2. The fragment ion at m/z 149.0600 of M13 was formed through neutral loss of glucuronide moiety. M11–M15 were tentatively identified as the reduction, hydroxylation, glucuronidation, acetylcysteine conjugation, and methylation products of M10. In addition, M12 could be the hydrolysis product of M01. Through comparing the chromatographic behavior, we found that M11 was 3-phenylpropionic acid and M12 was p-coumaric
acid (shown in Fig. S10). M11 and M12 were detected in the bile, urine, serum, and fecal samples, whereas M13–M15 were detected in the bile and fecal samples. The enzymatic analysis proven that M02, M04, and M13 were glucuronidated metabolites. The peak intensity of M02, M04, and M13 weakened after co-incubating with glucuronidase. 3.2.3. Metabolic profiles Fig. 6 shows the proposed metabolic pathways of picroside I in rats. These metabolites can be divided into four parts. The first part consists 164
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of the phase I reaction metabolites of picroside I, including hydroxylation (M01), deoxygenation (M06), and hydrolysis (M09 and M10). The second part comprises the phase II reaction metabolites of picroside I (glucuronidation, sulfation, and methylation). The third part involves the phase I biotransformations of metabolites, such as the reduction (M11) and hydroxylation (M12) products of M10. The fourth part covers the phase II biotransformations of metabolites, which are as follows: glucuronidation (M04) product of M01; acetylcysteine conjugation (M07) and sulfation (M08) products of M06; glucuronidation (M13), acetylcysteine conjugation (M14), and methylation (M15) products of M10. M01–M04, M10, and M12 have been discovered in another study [20], but the hydroxylation and sulfation products of cinnamic acid were not found in current study.
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4. Conclusion In this study, a fast (2 min), selective, and sensitive (0.1 ng/mL) LCMS/MS method was developed and validated to detect picroside I in rat plasma. The established LC-MS/MS method was successfully applied to pharmacokinetically analyze picroside I orally administered to rats. Our results revealed that the oral absorption and elimination of picroside I occurred rapidly. Fifteen metabolites were found and tentatively identified in the bile, urine, serum, and fecal samples by using the UHPLCQ/TOF-MS/MS system, and four metabolites were identified by comparing with the standard substance. Of these metabolites, nine were discovered for the first time. Picroside I was extensively metabolized through phases I and II reactions in vivo. Our study elucidated the metabolic pathways of picroside I, and our findings could help explain the efficacy and safety of picroside I. Conflicts of interest We declare that there are no conflicts of interest. Acknowledgements This work was financially supported by National Natural Science Foundation of China (project No. 81503223) and “Xing lin” plan of Shanghai University of Traditional Chinese Medicine (SHUTCM2017). Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.jchromb.2018.07.034. References [1] H. Sood, R.S. Chauhan, Biosynthesis and accumulation of a medicinal compound, picroside-I, in cultures of Picrorhiza kurroa Royle ex Benth, Plant Cell Tissue Organ Cult. 100 (2010) 113–117. [2] F. Li, Y. Yu, H. Zhang, T. Liu, Y. Li, G. Duan, Infrared-assisted non-ionic surfactant extraction as a green analytical preparatory technique for the rapid extraction and pre-concentration of picroside I and picroside II from Picrorhiza scrophulariiflora Pennell, Anal. Methods 5 (2013) 3747–3753.
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