Journal of Chromatography B, 947–948 (2014) 151–155
Contents lists available at ScienceDirect
Journal of Chromatography B journal homepage: www.elsevier.com/locate/chromb
Pharmacokinetic evaluation of dipfluzine and its three metabolites in rat plasma using liquid chromatography–mass spectrometry Wei Guo a,b , Xiaowei Shi b , Wei Wang b , Chen Xiong a , Junxia Li a,∗ a Department of Pharmacology, Hebei Medical University, Key Laboratory of Pharmacology and Toxicology for New Drug, Hebei Province, Shijiazhuang, 050017, PR China b School of Pharmacy, Hebei Medical University, Shijiazhuang, 050017, PR China
a r t i c l e
i n f o
Article history: Received 3 July 2013 Received in revised form 10 December 2013 Accepted 19 December 2013 Available online 26 December 2013 Keywords: Dipfluzine Pharmacokinetics Metabolites LC–MS/MS
a b s t r a c t A validated LC–MS/MS method to determine the content of dipfluzine (Dip) and its three metabolites (M1, M2, and M5) simultaneously within rat plasma samples was developed. After a single liquid–liquid extraction, the assay was performed by using a C18 column and positive electrospray ionisation mode (ESI) in the multiple reaction monitoring (MRM) mode with transitions of m/z 417.3→167.3, 251.2→165.2, 199.1→121.3, and 183.2→105.1 for Dip, M1, M2, and M5, respectively. Sulfamethoxazole (SMZ) was used as internal standard (IS). The method was linear ranged from 0.5–518, 0.5–524, 1.0–1036, and 0.5–514 ng/ml for Dip, M1, M2, and M5, respectively and all correlation coefficients were greater than 0.9919. The intra- and inter-day precision values obtained were less than 11.5% and the accuracy was between −3.2 and 9.7% for each analyte. The extraction recoveries of their three concentrations for Dip and its three metabolites were all higher than 71.9%. The technique was successfully applied to a pharmacokinetic study of Dip and its metabolites after a single oral administration of Dip (20 mg/kg) to rats. The results indicated that the metabolite formation was rapid and generated M5 as the predominant metabolite, followed by M1 and M2. The maximum plasma concentrations (Cmax ) were 59 ± 7, 37 ± 4, 3 ± 0.2, and 55 ± 5 ng/ml; the time to maximum plasma concentration (Tmax ) were 65 ± 12, 95 ± 12, 190 ± 25, and 90 ± 0 min and the areas under the concentration–time curves (AUC0→∞ ) were 17573 ± 704, 8328 ± 355, 5602 ± 753, and 16101 ± 429 ng min/ml for Dip, M1, M2, and M5, respectively. These results suggested that Dip was extensively metabolized and rapidly absorbed. The half-life (t1/2 ) of Dip, M1, M2, and M5 were 329 ± 15, 767 ± 75, 2364 ± 434, and 378 ± 36 min, respectively, which indicated that Dip and M5 were eliminated quickly. M2 reached its Tmax later and exhibited a longer t1/2 than the other metabolites, which indicated that there might be some type of flip-flop mechanism at work in the pharmacokinetics of M2. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Dipfluzine hydrochloride (Dip), which is a diphenylpiperazine calcium channel blocker, is synthesized according to the characteristics of molecular formula of cinnarizine. Previous studies have demonstrated that Dip is a highly selective cerebral vasodilator and exerts protective effects against focused or global cerebral ischemic injury using multiple mechanisms [1–4]; these effects are more potent than the effects of Dip analogs, such as cinnarizine (CZ) or flunarizine (FZ) [1,5,6]. Therefore, Dip is a promising drug candidate for the treatment of cerebral vascular diseases.
∗ Corresponding author. Department of Pharmacology, Hebei Medical University, 361 East Zhongshan Road, Shijiazhuang 050017, PR China, Tel.: +86 311 86266432; fax: +86 311 86057291. E-mail address: [email protected] (J. Li). 1570-0232/$ – see front matter © 2014 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jchromb.2013.12.018
The recent guidelines regarding metabolites in safety testing (MIST) emphasizes the importance of monitoring the major metabolites during preclinical and clinical studies [7]. Therefore, to ensure the safety and efficacy during the new drug discovery process, the pharmacokinetic characteristics of Dip and its metabolites will be explored. First, it was necessary to develop a highly selective and sensitive analytical method to quantitatively determine the content of Dip and its metabolites in rat plasma, as well as to depict these compounds’ concentration–time curves. Previous studies demonstrated that Dip was extensively metabolized after oral administration to form five metabolites: M1, M2, M3, M4, and M5. These metabolites formed via multiple pathways, including N-dealkylation at 1- and 4-positions of the piperazine ring in rat urine [8]. The Dip metabolites in rat urine were identified using liquid chromatography–diode array detection–mass spectrometry (LC–DAD–MS) method. However, the quantitation research of Dip and its metabolites had not been done by this method. During the previous pharmacokinetic study using beagles
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dogs and the toxicokinetic research using rats with single intravenous doses, the concentrations of Dip in dog and rat plasma and tissues were determined using a high-performance liquid chromatography–ultraviolet detection (LC–UV) method [9]. In our preliminary experiment of pharmacokinetics, LC–UV was also used to determine Dip and its metabolites. However, the results showed that only Dip could be quantitated exactly, the concentrations of the metabolites were too low to be detected by LC–UV in the plasma samples. The objective of this study was to develop a simple, specific, rapid, and sensitive LC–MS/MS method to quantify Dip and its three metabolites in rat plasma simultaneously. 2. Materials and methods 2.1. Chemicals and materials Dipfluzine hydrochloride (Dip) and its metabolites 1-(4-fluorobenzene)-4-piperazine-butanone (M1), 4-hydroxy-benzophenone (M2), and benzophenone (M5) (>99% purity by HPLC) were synthesized by the Department of Medicinal Chemistry at Hebei Medical University. The internal standard (IS), which was sulfamethoxazole (SMZ, purity >99%) was purchased from Yanyu Chemical Corporation (Shanghai, China). The methanol and formic acid were HPLC grade and purchased from Dikma Company (Beijing, China). The ethyl acetate was analytical grade and purchased from Yongda Chemical Corporation (Tianjin, China). Sodium carboxymethylcellulose was purchased from Goodfriend Chemical Corporation (Nanjing, China) and sodium heparin (12500 I.U./ml) was obtained from was from Xinbao Corporation (Suzhou, China). Drug-free heparinized plasma was prepared in our laboratory from male Sprague-Dawley (SD) rats. All of the plasma samples were stored at −20 ◦ C. 2.2. Equipment and operating conditions 2.2.1. Equipment The LC–MS/MS system consisted of an Agilent 1200 Series (Agilent, USA) equipped with an autosampler and coupled to a triple quadrupole mass spectrometer API4000 (Applied Biosystems/MDS SCIEX; Foster City, CA, USA). Data acquisition and processing were performed using the Analyst software (version 1.4.2) from Applied Biosystems. 2.2.2. Liquid chromatography conditions The chromatographic separation was achieved on a Diamonsil C18 column (250 × 4.6 mm, 5 m, Dikma Technologies, China) equipped with a C18 guard column (4.0 × 3.0 mm, 5 m, Phenomenex, China) at room temperature using a linear gradient elution of A (methanol containing 0.05% formic acid) and B (water containing 0.05% formic acid) at a flow rate of 800 l/min. An 8-min linear gradient from 40% A to 95% A was applied, followed by a 5-min elution at 95% A and a 1-min wash to return to the starting conditions. The sample injection volume was 20 l. 2.2.3. Mass spectrometry conditions The eluent from the HPLC column was introduced directly into the API4000 using the ESI interface in the positive ion mode. The detection was operated in MRM mode. The precursor-toproduct ion pairs, declustering potential (DP), collision energy (CE), entrance potential (EP), ion spray voltage (IS), and cell exit potential (CXP) for each analyte are shown in Table 1. Nitrogen was used as collision gas, curtain gas, nebulizer, and heating gas. After optimization, the source parameters were set as follows: curtain gas, 25 psi; nebulizer gas and heater gas, 65 psi; and temperature, 600 ◦ C.
2.3. Preparation of standards and quality control (QC) samples Dip, M1, M2, and M5 were prepared in methanol and their concentrations were about 0.5 mg/ml. These five solutions were subsequently mixed containing 259 g/ml of Dip, 262 g/ml of M1, 518 g/ml of M2, and 257 g/ml of M5. And then, the mixed solution was successively diluted to construct the calibration plots ranging from 2.6–2590 ng/ml for Dip, 2.6–2620 ng/ml for M1, 5.2–5180 ng/ml for M2, and 2.6–2570 ng/ml for M5. Three concentrations were used as QC samples: 6.5, 1295, 1928 ng/ml for Dip; 6.6, 1310, 1965 ng/ml for M1; 13.0, 2590, 3885 ng/ml for M2 and 6.4, 1285, 1928 ng/ml for M5. SMZ acted as IS at a concentration of 279 ng/ml. All of the solutions were stored at −20 ◦ C. 2.4. Sample preparation To a 100 l aliquot of rat plasma, 20 l of the IS and 20 l of methanol (volume of the corresponding working solutions for calibration curve and QC samples) were added. This mixture was extracted with 1 ml ethyl acetate by shaking with a vortex-mixer for 5 min, and then centrifuged for 10 min at 10,000 g. An 850l portion of the organic layer was transferred to a clean tube and evaporated to dryness under a stream of nitrogen at 37 ◦ C from a nitrogen enrichment system (Yousheng, Beijing, China). The dried extracts were reconstituted in 100 l of 70% methanol, mixed on a vortex-mixer and centrifuged at 10,000 g for 5 min. A 20 l aliquot of the solution was injected into the LC–MS/MS system for analysis.
2.5. Method validation 2.5.1. Selectivity The selectivity of the assay was assessed by analyzing six blank rat plasma samples from different sources. The blank plasma samples without spiking the Dip, its metabolites or the IS working solutions were used to investigate whether any endogenous peak in the plasma may interfere with the peak of Dip, its four metabolites or IS in the MRM chromatograms.
2.5.2. Precision and accuracy Calibration standard solutions were prepared by adding 20 l of a series of working solutions and 20 l of the IS solutions to 100 l blank plasma. Calibration curves were constructed with plasma samples of 0.5–5180 ng/ml, 0.5–524 ng/ml, 1.0–1036 ng/ml, and 0.5–514 ng/ml for Dip, M1, M2, and M5, respectively. The calibration curve was generated by plotting the nominal concentrations of the analytes against the peak area ratio of the analyte to IS for the four analytes, followed by using the least-squares regression method with a weighting factor of 1/x2 . The correlation coefficient of the calibration curve, the relative standard deviation, and the relative error for each concentration were determined. The intra-day precision and accuracy for the method were determined by analyzing sextuplicates of three QC levels in addition to the LLOQ (lower limit of quantitation) level that was extracted from the sample batch on the same day. The QC concentrations were 1.3, 254, and 381 ng/ml for Dip; 1.0, 257, and 386 ng/ml for M1; 2.0, 506, and 759 ng/ml for M2; 1.3, 252, and 378 ng/ml for M5. For the inter-day precision and accuracy, six sets of QC and LLOQ samples were analyzed on three separate days. The precision was expressed as a coefficient of variation (%RSD) and the accuracy expressed as the percent relative error (%RE); both values were determined from each concentration of the replicated samples. The precision for quality controls must be within 15% and the accuracy was required to be within ±15%, except for the LLOQ.
W. Guo et al. / J. Chromatogr. B 947–948 (2014) 151–155
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Table 1 MRM transitions and compound specific parameters for: Dip, its three metabolites, and internal standard (IS). Analytes
Retention time(min)
Parent ions (m/z)
Product ions (m/z)
DP (V)
CE (V)
EP (V)
IS (V)
CXP (V)
Dip M1 M2 M5 IS
8.54 3.18 10.54 12.15 6.17
417.3 251.2 199.1 183.2 254.1
167 165.2 121.1 105.1 156
35 37 39 35 40
19 24 25 21 21
10.00 10.00 10.00 10.00 10.00
5500.00 5500.00 5500.00 5500.00 5500.00
3.00 3.00 3.00 3.00 3.00
LLOQ is defined as the lowest concentration that could be reliably measured, i.e., within 20% RSD and (100+/−20)% accuracy. 2.5.3. Matrix effect and extraction recovery Matrix effect was assessed by comparing the peak areas of analytes spiked into blank plasma after extraction to the corresponding peak areas of neat sample. The recovery was determined by comparing the mean peak areas of plasma samples spiked before and after extraction. The extraction process was the same as plasma sample preparation (Section 2.4). 2.5.4. Stability Stability of the analyte was tested by comparing the QC sample which had been maintained under different test conditions to the control at the nominal sample concentration and analyzed during the same analytical run. The conditions used during the stability
assessment included an evaluation of the QC samples held at room temperature for 24 h, at 4 ◦ C for 24 h in the autosampler, at −20 ◦ C for 12 weeks, and after three freeze-thaw cycles. 2.6. Application to pharmacokinetic study Male SD rats, which weighed 210–230 g, were purchased from the Laboratory Animals Center of Hebei Medical University (Shijiazhuang, China). All animal experimental procedures were conducted in accordance with the guidelines from the National Institutes of Health for the care and use of animals, as well as under the approval of the Laboratory Animals Center of Hebei Medical University. All rats were kept in an environmentally controlled breeding room for a week before beginning the experiments and were fed with standard laboratory food and water ad libitum. Before the experiment, rats fasted for 12 h but had free access
Fig. 1. Representative MRM chromatograms of Dip, M1, M2, M5, and IS in 60 min plasma sample after a single oral administration of Dip (20 mg/kg) to rats.
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Fig. 2. Mean plasma concentration–time curves of Dip, M1, M2, and M5 in rats after intragastric administration of 20 mg/kg Dip to six rats.
to drinking water. The suspension of Dip was prepared in a 0.5% sodium carboxymethylcellulose solution at a concentration of 2 mg/ml; the dose was administered orally to six rats at a dose of 20 mg/kg. The rats were anaesthetized with 40 mg/kg sodium pentobarbital via intra-peritoneal administration. Subsequently, 0.5 ml heparinized blood samples were collected from the ophthalmic veins using a sterile capillary tube under anesthesia at 15, 30, 60, 90, 120, 180, 240, 360, 480, 720, and 1440 min after Dip administration; the samples were gently mixed and centrifuged for 10 min to separate the plasma. These plasma samples were stored at −20 ◦ C until used in the assay. 3. Results and discussion 3.1. Method development We referred to the chromatographic conditions of the assay for Dip and its metabolites in the rat liver microsomes with LC–MS/MS [9]. By using the same chromatographic conditions as were used with the rat liver microsomes, the chromatographic peaks were separated well with good peak shape, and the contents of all of the analytes in the rat plasma could be determined in spite of the use of different sample matrices. Liquid–liquid extraction was chosen because this technique offers a simple operating procedure, pure extracted samples and high enough extraction rates were high enough to determine the analytes easily with LC–MS/MS analysis. Cyclohexane, isopropanol, ether, ethyl acetate, chloroform, and n-hexane were chosen as possible extraction solvents. Ethyl acetate displayed extraction rates almost 30% higher than ether for Dip, M1, and M5, as well as extraction rates 9.7∼19.7% higher than isopropanol for all analytes. Cyclohexane, n-hexane, and chloroform had the almost equal extraction recoveries for Dip and its metabolites, but chloroform was quite toxic; therefore, ethyl acetate was chosen as the extraction solvent. Vortex time also affected the extraction recoveries, and therefore times, such as 2, 3, 5, and 10 min, were surveyed. The extraction recoveries of Dip and its metabolites increased until 5 min was reached.
3.2. Method validation 3.2.1. Specificity The representative chromatograms obtained from the plasma sample at 60 min after oral administration of 20 mg/kg Dip are depicted in Fig. 1. The results revealed that there were no peaks interfering at the same retention times as Dip, its three metabolites or IS. 3.2.2. Calibration curve The %RSD at each level of the calibration analyses varied from 1.1 to 10.9 and the % RE varied from −14.4 to 13.7 for the four analytes. The R-squared for the five analytes were greater than 0.9919 during the daily runs. Based on the standard data presented here, it was concluded that the calibration curves used in this method were precise and accurate to measure the content of Dip and its three metabolites simultaneously in rat plasma. 3.2.3. Precision, accuracy, and LLOQ To assess the precision and accuracy of the method, QC and LLOQ samples were processed on the same day, as well as on three separate days of analysis. The intra- and inter-day precisions were less than 11.5 and 8.8%, 8.8 and 9.0%, 9.5 and 9.8%, and 8.2 and 8.2% for Dip, M1, M2, and M5, respectively. The intra- and inter-day accuracies measured were between −2.4 and 5.1%, 0.3 and 9.7%, −2.6 and 4.5%, and −3.2 and 4.1% for Dip, M1, M2, and M5, respectively. The LLOQ for the four analytes was reliably quantified with both precision 0.05). RSD values of all levels of Dip, M1, M2, and M5 varied from 2.1 to 14.7% and RE values were between −12.0 and 13.9%. The results suggested that the four analytes were stable under these conditions. 3.3. Application of analytical method in pharmacokinetic studies The plot describing the concentrations of Dip and its metabolites versus time and the principal pharmacokinetic parameters are illustrated in Fig. 2 and Table 2, respectively. Cmax of M1, M2, and M5 were 37 ± 4, 34 ± 0.2, and 55 ± 5 ng/ml, respectively. It could be concluded that M5 was the major metabolites of Dip formed in rat plasma, followed by M1 and M2. The results were not entirely in agreement with the data from the rat liver microsomes. For the rat liver microsomes, M1 was the predominant metabolite and the concentrations of M4 and M5 were almost equivalent. However, in both of the matrices, the concentrations of M2 were the lowest of the four metabolites. These results indicated that there were many differences between the in vitro and in vivo metabolic processes acting on Dip; we proposed that the liver was not the only main metabolic organ to metabolize Dip. Further research is needed to investigate the metabolism of Dip in greater detail. According to the concentration versus time profiles, Cmax and t1/2 of Dip were 59 ± 7 ng/ml and 329 ± 15 min, respectively, indicating that Dip was rapidly absorbed and quickly eliminated after oral administration. The three metabolites could be detected during the first sample point, which was 15 min after dosing; this result confirms that the metabolite formation was rapid. AUC0→∞ of Dip and its three metabolites M1, M2, and M5 were 17574 ± 704, 8329 ± 355, 5603 ± 753, and 16101 ± 429 ng min/ml, respectively.
These data indicated that Dip was extensively metabolized in rats and rapidly absorbed. T1/2 of Dip and its three metabolites M1, M2, and M5 were 329 ± 15, 767 ± 75, 2364 ± 434, and 378 ± 36 min, respectively, which indicated that Dip and M5 were eliminated rapidly. M2 reached its Tmax late and exhibited a longer t1/2 than the other metabolites, which indicated that the formation and elimination of M2 were much slower than the other metabolites. In other words, M2 was gradually and slowly transformed from the parent drug. It was proposed that there might be some type of flip-flop mechanism at work in the pharmacokinetics of M2. In the previous research, it was indicated that M1 was the active metabolite. However, the other two metabolites M2 and M5 did not show any activity. Conflict of interest statement The authors declare no conflict of interest. Acknowledgements This work was supported by the Natural Science Foundation of Hebei Province [Grant C2006001035] and the Technology Supporting Plan of Hebei Province [Grant 10276434]. We thank the Department of Medicinal Chemistry at Hebei Medical University for providing the reference standard samples of Dip and its metabolites. References Y.L. Wang, R.R. He, Acta Pharmacol. Sin. 14 (1993) 124–127. J. Bai, Y. Wang, Chin. Med. J. 82 (2002) 1130. G.H. Zhang, P. Lu, Y.L. Wang, Acta Pharm. Sin. 40 (2005) 1091. Y.J. Zhang, Y. Guo, Q.Z. Jia, Y.L. Wang, H.L. Zhang, Acta Pharm. Sin. 40 (2005) 97. Y.L. Wang, R.R. He, Acta Pharmacol. Sin. 15 (1994) 201. Y.H. Zhu, Y.L. Wang, X.P. Yang, Acta Pharmacol. Sin. 17 (1996) 321. U.S. FDA, Guidance for Industry, Safety Testing of Drug Metabolites, US Department of Health and Human Services, US FDA, Center for Drug Evaluation and Research, Rockville, MD, USA, 2008. [8] H.C. Liu, Y.M. Du, Y.L. Wang, Acta Pharm. Sin. 40 (2005) 168. [9] W. Guo, D.Z. Kong, Y.F. Du, X.W. Shi, Y.L. Wang, Pharmacology 89 (2012) 201. [1] [2] [3] [4] [5] [6] [7]
Contents lists available at ScienceDirect
Journal of Chromatography B journal homepage: www.elsevier.com/locate/chromb
Pharmacokinetic evaluation of dipfluzine and its three metabolites in rat plasma using liquid chromatography–mass spectrometry Wei Guo a,b , Xiaowei Shi b , Wei Wang b , Chen Xiong a , Junxia Li a,∗ a Department of Pharmacology, Hebei Medical University, Key Laboratory of Pharmacology and Toxicology for New Drug, Hebei Province, Shijiazhuang, 050017, PR China b School of Pharmacy, Hebei Medical University, Shijiazhuang, 050017, PR China
a r t i c l e
i n f o
Article history: Received 3 July 2013 Received in revised form 10 December 2013 Accepted 19 December 2013 Available online 26 December 2013 Keywords: Dipfluzine Pharmacokinetics Metabolites LC–MS/MS
a b s t r a c t A validated LC–MS/MS method to determine the content of dipfluzine (Dip) and its three metabolites (M1, M2, and M5) simultaneously within rat plasma samples was developed. After a single liquid–liquid extraction, the assay was performed by using a C18 column and positive electrospray ionisation mode (ESI) in the multiple reaction monitoring (MRM) mode with transitions of m/z 417.3→167.3, 251.2→165.2, 199.1→121.3, and 183.2→105.1 for Dip, M1, M2, and M5, respectively. Sulfamethoxazole (SMZ) was used as internal standard (IS). The method was linear ranged from 0.5–518, 0.5–524, 1.0–1036, and 0.5–514 ng/ml for Dip, M1, M2, and M5, respectively and all correlation coefficients were greater than 0.9919. The intra- and inter-day precision values obtained were less than 11.5% and the accuracy was between −3.2 and 9.7% for each analyte. The extraction recoveries of their three concentrations for Dip and its three metabolites were all higher than 71.9%. The technique was successfully applied to a pharmacokinetic study of Dip and its metabolites after a single oral administration of Dip (20 mg/kg) to rats. The results indicated that the metabolite formation was rapid and generated M5 as the predominant metabolite, followed by M1 and M2. The maximum plasma concentrations (Cmax ) were 59 ± 7, 37 ± 4, 3 ± 0.2, and 55 ± 5 ng/ml; the time to maximum plasma concentration (Tmax ) were 65 ± 12, 95 ± 12, 190 ± 25, and 90 ± 0 min and the areas under the concentration–time curves (AUC0→∞ ) were 17573 ± 704, 8328 ± 355, 5602 ± 753, and 16101 ± 429 ng min/ml for Dip, M1, M2, and M5, respectively. These results suggested that Dip was extensively metabolized and rapidly absorbed. The half-life (t1/2 ) of Dip, M1, M2, and M5 were 329 ± 15, 767 ± 75, 2364 ± 434, and 378 ± 36 min, respectively, which indicated that Dip and M5 were eliminated quickly. M2 reached its Tmax later and exhibited a longer t1/2 than the other metabolites, which indicated that there might be some type of flip-flop mechanism at work in the pharmacokinetics of M2. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Dipfluzine hydrochloride (Dip), which is a diphenylpiperazine calcium channel blocker, is synthesized according to the characteristics of molecular formula of cinnarizine. Previous studies have demonstrated that Dip is a highly selective cerebral vasodilator and exerts protective effects against focused or global cerebral ischemic injury using multiple mechanisms [1–4]; these effects are more potent than the effects of Dip analogs, such as cinnarizine (CZ) or flunarizine (FZ) [1,5,6]. Therefore, Dip is a promising drug candidate for the treatment of cerebral vascular diseases.
∗ Corresponding author. Department of Pharmacology, Hebei Medical University, 361 East Zhongshan Road, Shijiazhuang 050017, PR China, Tel.: +86 311 86266432; fax: +86 311 86057291. E-mail address: [email protected] (J. Li). 1570-0232/$ – see front matter © 2014 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jchromb.2013.12.018
The recent guidelines regarding metabolites in safety testing (MIST) emphasizes the importance of monitoring the major metabolites during preclinical and clinical studies [7]. Therefore, to ensure the safety and efficacy during the new drug discovery process, the pharmacokinetic characteristics of Dip and its metabolites will be explored. First, it was necessary to develop a highly selective and sensitive analytical method to quantitatively determine the content of Dip and its metabolites in rat plasma, as well as to depict these compounds’ concentration–time curves. Previous studies demonstrated that Dip was extensively metabolized after oral administration to form five metabolites: M1, M2, M3, M4, and M5. These metabolites formed via multiple pathways, including N-dealkylation at 1- and 4-positions of the piperazine ring in rat urine [8]. The Dip metabolites in rat urine were identified using liquid chromatography–diode array detection–mass spectrometry (LC–DAD–MS) method. However, the quantitation research of Dip and its metabolites had not been done by this method. During the previous pharmacokinetic study using beagles
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dogs and the toxicokinetic research using rats with single intravenous doses, the concentrations of Dip in dog and rat plasma and tissues were determined using a high-performance liquid chromatography–ultraviolet detection (LC–UV) method [9]. In our preliminary experiment of pharmacokinetics, LC–UV was also used to determine Dip and its metabolites. However, the results showed that only Dip could be quantitated exactly, the concentrations of the metabolites were too low to be detected by LC–UV in the plasma samples. The objective of this study was to develop a simple, specific, rapid, and sensitive LC–MS/MS method to quantify Dip and its three metabolites in rat plasma simultaneously. 2. Materials and methods 2.1. Chemicals and materials Dipfluzine hydrochloride (Dip) and its metabolites 1-(4-fluorobenzene)-4-piperazine-butanone (M1), 4-hydroxy-benzophenone (M2), and benzophenone (M5) (>99% purity by HPLC) were synthesized by the Department of Medicinal Chemistry at Hebei Medical University. The internal standard (IS), which was sulfamethoxazole (SMZ, purity >99%) was purchased from Yanyu Chemical Corporation (Shanghai, China). The methanol and formic acid were HPLC grade and purchased from Dikma Company (Beijing, China). The ethyl acetate was analytical grade and purchased from Yongda Chemical Corporation (Tianjin, China). Sodium carboxymethylcellulose was purchased from Goodfriend Chemical Corporation (Nanjing, China) and sodium heparin (12500 I.U./ml) was obtained from was from Xinbao Corporation (Suzhou, China). Drug-free heparinized plasma was prepared in our laboratory from male Sprague-Dawley (SD) rats. All of the plasma samples were stored at −20 ◦ C. 2.2. Equipment and operating conditions 2.2.1. Equipment The LC–MS/MS system consisted of an Agilent 1200 Series (Agilent, USA) equipped with an autosampler and coupled to a triple quadrupole mass spectrometer API4000 (Applied Biosystems/MDS SCIEX; Foster City, CA, USA). Data acquisition and processing were performed using the Analyst software (version 1.4.2) from Applied Biosystems. 2.2.2. Liquid chromatography conditions The chromatographic separation was achieved on a Diamonsil C18 column (250 × 4.6 mm, 5 m, Dikma Technologies, China) equipped with a C18 guard column (4.0 × 3.0 mm, 5 m, Phenomenex, China) at room temperature using a linear gradient elution of A (methanol containing 0.05% formic acid) and B (water containing 0.05% formic acid) at a flow rate of 800 l/min. An 8-min linear gradient from 40% A to 95% A was applied, followed by a 5-min elution at 95% A and a 1-min wash to return to the starting conditions. The sample injection volume was 20 l. 2.2.3. Mass spectrometry conditions The eluent from the HPLC column was introduced directly into the API4000 using the ESI interface in the positive ion mode. The detection was operated in MRM mode. The precursor-toproduct ion pairs, declustering potential (DP), collision energy (CE), entrance potential (EP), ion spray voltage (IS), and cell exit potential (CXP) for each analyte are shown in Table 1. Nitrogen was used as collision gas, curtain gas, nebulizer, and heating gas. After optimization, the source parameters were set as follows: curtain gas, 25 psi; nebulizer gas and heater gas, 65 psi; and temperature, 600 ◦ C.
2.3. Preparation of standards and quality control (QC) samples Dip, M1, M2, and M5 were prepared in methanol and their concentrations were about 0.5 mg/ml. These five solutions were subsequently mixed containing 259 g/ml of Dip, 262 g/ml of M1, 518 g/ml of M2, and 257 g/ml of M5. And then, the mixed solution was successively diluted to construct the calibration plots ranging from 2.6–2590 ng/ml for Dip, 2.6–2620 ng/ml for M1, 5.2–5180 ng/ml for M2, and 2.6–2570 ng/ml for M5. Three concentrations were used as QC samples: 6.5, 1295, 1928 ng/ml for Dip; 6.6, 1310, 1965 ng/ml for M1; 13.0, 2590, 3885 ng/ml for M2 and 6.4, 1285, 1928 ng/ml for M5. SMZ acted as IS at a concentration of 279 ng/ml. All of the solutions were stored at −20 ◦ C. 2.4. Sample preparation To a 100 l aliquot of rat plasma, 20 l of the IS and 20 l of methanol (volume of the corresponding working solutions for calibration curve and QC samples) were added. This mixture was extracted with 1 ml ethyl acetate by shaking with a vortex-mixer for 5 min, and then centrifuged for 10 min at 10,000 g. An 850l portion of the organic layer was transferred to a clean tube and evaporated to dryness under a stream of nitrogen at 37 ◦ C from a nitrogen enrichment system (Yousheng, Beijing, China). The dried extracts were reconstituted in 100 l of 70% methanol, mixed on a vortex-mixer and centrifuged at 10,000 g for 5 min. A 20 l aliquot of the solution was injected into the LC–MS/MS system for analysis.
2.5. Method validation 2.5.1. Selectivity The selectivity of the assay was assessed by analyzing six blank rat plasma samples from different sources. The blank plasma samples without spiking the Dip, its metabolites or the IS working solutions were used to investigate whether any endogenous peak in the plasma may interfere with the peak of Dip, its four metabolites or IS in the MRM chromatograms.
2.5.2. Precision and accuracy Calibration standard solutions were prepared by adding 20 l of a series of working solutions and 20 l of the IS solutions to 100 l blank plasma. Calibration curves were constructed with plasma samples of 0.5–5180 ng/ml, 0.5–524 ng/ml, 1.0–1036 ng/ml, and 0.5–514 ng/ml for Dip, M1, M2, and M5, respectively. The calibration curve was generated by plotting the nominal concentrations of the analytes against the peak area ratio of the analyte to IS for the four analytes, followed by using the least-squares regression method with a weighting factor of 1/x2 . The correlation coefficient of the calibration curve, the relative standard deviation, and the relative error for each concentration were determined. The intra-day precision and accuracy for the method were determined by analyzing sextuplicates of three QC levels in addition to the LLOQ (lower limit of quantitation) level that was extracted from the sample batch on the same day. The QC concentrations were 1.3, 254, and 381 ng/ml for Dip; 1.0, 257, and 386 ng/ml for M1; 2.0, 506, and 759 ng/ml for M2; 1.3, 252, and 378 ng/ml for M5. For the inter-day precision and accuracy, six sets of QC and LLOQ samples were analyzed on three separate days. The precision was expressed as a coefficient of variation (%RSD) and the accuracy expressed as the percent relative error (%RE); both values were determined from each concentration of the replicated samples. The precision for quality controls must be within 15% and the accuracy was required to be within ±15%, except for the LLOQ.
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Table 1 MRM transitions and compound specific parameters for: Dip, its three metabolites, and internal standard (IS). Analytes
Retention time(min)
Parent ions (m/z)
Product ions (m/z)
DP (V)
CE (V)
EP (V)
IS (V)
CXP (V)
Dip M1 M2 M5 IS
8.54 3.18 10.54 12.15 6.17
417.3 251.2 199.1 183.2 254.1
167 165.2 121.1 105.1 156
35 37 39 35 40
19 24 25 21 21
10.00 10.00 10.00 10.00 10.00
5500.00 5500.00 5500.00 5500.00 5500.00
3.00 3.00 3.00 3.00 3.00
LLOQ is defined as the lowest concentration that could be reliably measured, i.e., within 20% RSD and (100+/−20)% accuracy. 2.5.3. Matrix effect and extraction recovery Matrix effect was assessed by comparing the peak areas of analytes spiked into blank plasma after extraction to the corresponding peak areas of neat sample. The recovery was determined by comparing the mean peak areas of plasma samples spiked before and after extraction. The extraction process was the same as plasma sample preparation (Section 2.4). 2.5.4. Stability Stability of the analyte was tested by comparing the QC sample which had been maintained under different test conditions to the control at the nominal sample concentration and analyzed during the same analytical run. The conditions used during the stability
assessment included an evaluation of the QC samples held at room temperature for 24 h, at 4 ◦ C for 24 h in the autosampler, at −20 ◦ C for 12 weeks, and after three freeze-thaw cycles. 2.6. Application to pharmacokinetic study Male SD rats, which weighed 210–230 g, were purchased from the Laboratory Animals Center of Hebei Medical University (Shijiazhuang, China). All animal experimental procedures were conducted in accordance with the guidelines from the National Institutes of Health for the care and use of animals, as well as under the approval of the Laboratory Animals Center of Hebei Medical University. All rats were kept in an environmentally controlled breeding room for a week before beginning the experiments and were fed with standard laboratory food and water ad libitum. Before the experiment, rats fasted for 12 h but had free access
Fig. 1. Representative MRM chromatograms of Dip, M1, M2, M5, and IS in 60 min plasma sample after a single oral administration of Dip (20 mg/kg) to rats.
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Fig. 2. Mean plasma concentration–time curves of Dip, M1, M2, and M5 in rats after intragastric administration of 20 mg/kg Dip to six rats.
to drinking water. The suspension of Dip was prepared in a 0.5% sodium carboxymethylcellulose solution at a concentration of 2 mg/ml; the dose was administered orally to six rats at a dose of 20 mg/kg. The rats were anaesthetized with 40 mg/kg sodium pentobarbital via intra-peritoneal administration. Subsequently, 0.5 ml heparinized blood samples were collected from the ophthalmic veins using a sterile capillary tube under anesthesia at 15, 30, 60, 90, 120, 180, 240, 360, 480, 720, and 1440 min after Dip administration; the samples were gently mixed and centrifuged for 10 min to separate the plasma. These plasma samples were stored at −20 ◦ C until used in the assay. 3. Results and discussion 3.1. Method development We referred to the chromatographic conditions of the assay for Dip and its metabolites in the rat liver microsomes with LC–MS/MS [9]. By using the same chromatographic conditions as were used with the rat liver microsomes, the chromatographic peaks were separated well with good peak shape, and the contents of all of the analytes in the rat plasma could be determined in spite of the use of different sample matrices. Liquid–liquid extraction was chosen because this technique offers a simple operating procedure, pure extracted samples and high enough extraction rates were high enough to determine the analytes easily with LC–MS/MS analysis. Cyclohexane, isopropanol, ether, ethyl acetate, chloroform, and n-hexane were chosen as possible extraction solvents. Ethyl acetate displayed extraction rates almost 30% higher than ether for Dip, M1, and M5, as well as extraction rates 9.7∼19.7% higher than isopropanol for all analytes. Cyclohexane, n-hexane, and chloroform had the almost equal extraction recoveries for Dip and its metabolites, but chloroform was quite toxic; therefore, ethyl acetate was chosen as the extraction solvent. Vortex time also affected the extraction recoveries, and therefore times, such as 2, 3, 5, and 10 min, were surveyed. The extraction recoveries of Dip and its metabolites increased until 5 min was reached.
3.2. Method validation 3.2.1. Specificity The representative chromatograms obtained from the plasma sample at 60 min after oral administration of 20 mg/kg Dip are depicted in Fig. 1. The results revealed that there were no peaks interfering at the same retention times as Dip, its three metabolites or IS. 3.2.2. Calibration curve The %RSD at each level of the calibration analyses varied from 1.1 to 10.9 and the % RE varied from −14.4 to 13.7 for the four analytes. The R-squared for the five analytes were greater than 0.9919 during the daily runs. Based on the standard data presented here, it was concluded that the calibration curves used in this method were precise and accurate to measure the content of Dip and its three metabolites simultaneously in rat plasma. 3.2.3. Precision, accuracy, and LLOQ To assess the precision and accuracy of the method, QC and LLOQ samples were processed on the same day, as well as on three separate days of analysis. The intra- and inter-day precisions were less than 11.5 and 8.8%, 8.8 and 9.0%, 9.5 and 9.8%, and 8.2 and 8.2% for Dip, M1, M2, and M5, respectively. The intra- and inter-day accuracies measured were between −2.4 and 5.1%, 0.3 and 9.7%, −2.6 and 4.5%, and −3.2 and 4.1% for Dip, M1, M2, and M5, respectively. The LLOQ for the four analytes was reliably quantified with both precision 0.05). RSD values of all levels of Dip, M1, M2, and M5 varied from 2.1 to 14.7% and RE values were between −12.0 and 13.9%. The results suggested that the four analytes were stable under these conditions. 3.3. Application of analytical method in pharmacokinetic studies The plot describing the concentrations of Dip and its metabolites versus time and the principal pharmacokinetic parameters are illustrated in Fig. 2 and Table 2, respectively. Cmax of M1, M2, and M5 were 37 ± 4, 34 ± 0.2, and 55 ± 5 ng/ml, respectively. It could be concluded that M5 was the major metabolites of Dip formed in rat plasma, followed by M1 and M2. The results were not entirely in agreement with the data from the rat liver microsomes. For the rat liver microsomes, M1 was the predominant metabolite and the concentrations of M4 and M5 were almost equivalent. However, in both of the matrices, the concentrations of M2 were the lowest of the four metabolites. These results indicated that there were many differences between the in vitro and in vivo metabolic processes acting on Dip; we proposed that the liver was not the only main metabolic organ to metabolize Dip. Further research is needed to investigate the metabolism of Dip in greater detail. According to the concentration versus time profiles, Cmax and t1/2 of Dip were 59 ± 7 ng/ml and 329 ± 15 min, respectively, indicating that Dip was rapidly absorbed and quickly eliminated after oral administration. The three metabolites could be detected during the first sample point, which was 15 min after dosing; this result confirms that the metabolite formation was rapid. AUC0→∞ of Dip and its three metabolites M1, M2, and M5 were 17574 ± 704, 8329 ± 355, 5603 ± 753, and 16101 ± 429 ng min/ml, respectively.
These data indicated that Dip was extensively metabolized in rats and rapidly absorbed. T1/2 of Dip and its three metabolites M1, M2, and M5 were 329 ± 15, 767 ± 75, 2364 ± 434, and 378 ± 36 min, respectively, which indicated that Dip and M5 were eliminated rapidly. M2 reached its Tmax late and exhibited a longer t1/2 than the other metabolites, which indicated that the formation and elimination of M2 were much slower than the other metabolites. In other words, M2 was gradually and slowly transformed from the parent drug. It was proposed that there might be some type of flip-flop mechanism at work in the pharmacokinetics of M2. In the previous research, it was indicated that M1 was the active metabolite. However, the other two metabolites M2 and M5 did not show any activity. Conflict of interest statement The authors declare no conflict of interest. Acknowledgements This work was supported by the Natural Science Foundation of Hebei Province [Grant C2006001035] and the Technology Supporting Plan of Hebei Province [Grant 10276434]. We thank the Department of Medicinal Chemistry at Hebei Medical University for providing the reference standard samples of Dip and its metabolites. References Y.L. Wang, R.R. He, Acta Pharmacol. Sin. 14 (1993) 124–127. J. Bai, Y. Wang, Chin. Med. J. 82 (2002) 1130. G.H. Zhang, P. Lu, Y.L. Wang, Acta Pharm. Sin. 40 (2005) 1091. Y.J. Zhang, Y. Guo, Q.Z. Jia, Y.L. Wang, H.L. Zhang, Acta Pharm. Sin. 40 (2005) 97. Y.L. Wang, R.R. He, Acta Pharmacol. Sin. 15 (1994) 201. Y.H. Zhu, Y.L. Wang, X.P. Yang, Acta Pharmacol. Sin. 17 (1996) 321. U.S. FDA, Guidance for Industry, Safety Testing of Drug Metabolites, US Department of Health and Human Services, US FDA, Center for Drug Evaluation and Research, Rockville, MD, USA, 2008. [8] H.C. Liu, Y.M. Du, Y.L. Wang, Acta Pharm. Sin. 40 (2005) 168. [9] W. Guo, D.Z. Kong, Y.F. Du, X.W. Shi, Y.L. Wang, Pharmacology 89 (2012) 201. [1] [2] [3] [4] [5] [6] [7]
