Arch. Pharm. Res. DOI 10.1007/s12272-014-0352-2
RESEARCH ARTICLE
Liquid chromatography–tandem mass spectrometry method for determining tolvaptan and its nine metabolites in rat serum: application to a pharmacokinetic study Masayuki Furukawa • Kenichi Miyata • Chie Kawasome • Yoshiko Himeda • Kenji Takeuchi Toshihisa Koga • Yukihiro Hirao • Ken Umehara
•
Received: 25 October 2013 / Accepted: 30 January 2014 Ó The Pharmaceutical Society of Korea 2014
Abstract Tolvaptan is a competitive vasopressin V2receptor antagonist that inhibits water reabsorption in the renal collecting ducts. A selective and sensitive liquid chromatography–tandem mass spectrometry method for determining tolvaptan and its nine metabolites in rat serum was developed and validated. An analogue of tolvaptan was used as an internal standard. Sample preparation involved protein precipitation following solid-phase extraction. Chromatographic separation was performed on a C18 reversed-phase column with a linear gradient elution. The flow rate was 0.25 mL/min, and total run time was 30 min. The analytes were detected by tandem mass spectrometry using an electrospray ionization interface in positive ion mode and multiple reaction monitoring. The calibration curve showed linearity over the concentration range from 5 to 1,000 ng/mL for each analyte. The lower limit of quantification using 0.1 mL of rat serum was 5 ng/mL for each analyte. Precision did not exceed 5.7 %, and accuracy as relative error were within ± 7.5 % for all analytes. The validated method was successfully applied to evaluate the pharmacokinetics of oral tolvaptan in rats, indicating the systemic exposure to tolvaptan in females eight times larger than that in males. Keywords Tolvaptan Metabolite Pharmacokinetics LC–MS/MS Serum Rat
M. Furukawa (&) C. Kawasome Y. Himeda K. Takeuchi T. Koga Y. Hirao K. Umehara Tokushima Research Institute, Otsuka Pharmaceutical Co., Ltd., 463-10 Kagasuno, Kawauchi-cho, Tokushima 771-0192, Japan e-mail: [email protected] K. Miyata Formulation Research Institute, Otsuka Pharmaceutical Co., Ltd., 224-18 Ebisuno Hiraishi, Kawauchi-cho, Tokushima 771-0182, Japan
Introduction Tolvaptan, N-{4-[(5RS)-7-chloro-5-hydroxy-2,3,4,5-tetrahydro-1H-benzo[b]azepin -1-carbonyl]-3-methylphenyl}2-methylbenzamide, is an orally-administered nonpeptide vasopressin V2 receptor antagonist developed by Otsuka Pharmaceutical Co., Ltd. (Tokyo, Japan) (Yamamura et al. 1998; Kondo et al. 1999; Miyazaki et al. 2007, 2013). Tolvaptan is used in the United States and Europe for the treatment of hypervolemic and euvolemic hyponatremia including patients with heart failure, cirrhosis, and syndrome of inappropriate antidiuretic hormone (Dixon and Lien 2008; Ghali et al. 2009). Tolvaptan was approved in the Japan for the treatment of excess water retention in patients with cardiac failure and liver cirrhosis when the treatment by other diuretics including loop diuretics is ineffective (Hori 2011; Sakaide et al. 2013). The determination of tolvaptan in rat serum has been reported using high-performance liquid chromatography (HPLC) (Furukawa et al. 2011). The HPLC method requires liquid–liquid extraction with an n-hexane:diethyl ether (1:1, v/v) solution. The liquid chromatography with tandem mass spectrometry (LC–MS/MS) method for determining tolvaptan in human plasma has been reported (Pei et al. 2013). None of the published LC–MS/MS methods fulfilled quantitative analysis of tolvaptan and its metabolites. Tolvaptan is metabolized primarily by dehydrogenation and hydroxylation. The proposed major metabolic pathways of tolvaptan are shown in Fig. 1. These pathways generate a number of metabolites. Structural characterization of the metabolites of tolvaptan revealed one hydroxide of the benzazepine ring (DM-4110, DM-4111, and DM4119), one oxide of the hydroxy group at the 50 position (MOP-21826) and its one hydroxide (DM-4121), and four metabolites with the benzazepine ring cleaved (DM-4103,
123
M. Furukawa et al. Table 1 LC–MS/MS parameters for ten analytes and internal standard Analyte
Precursor ion (m/z)
Product ion (m/z)
Collision energy (eV)
Tolvaptan
449.2
252
23
DM-4103
479.1
252
15
DM-4104 DM-4105, DM-4110,
467.1 465.2
252 252
23 27
DM-4107
481.1
252
27
DM-4121
463.1
252
29
MOP-21826
447.1
252
29
OPC-41100 (internal standard)
463.2
266
25
DM-4111, DM-4119
(HPMC), methanol (HPLC grade), acetonitrile (HPLC grade), 1/15 mol/L phosphate buffer at pH 7.0, and acetic acid (special grade) were purchased from Shin-Etsu Chemical Co. (Tokyo, Japan), Ltd., Merck Ltd, Japan (Tokyo, Japan), Thermo Fisher Scientific K.K. (Yokohama, Japan), Nacalai Tesque, Inc. (Kyoto, Japan), and SigmaAldrich Japan K.K. (Tokyo, Japan), respectively. Purified water was obtained from Milli-Q water purification system (Millipore, Billerica, USA). All other chemicals were of the highest purity commercially available. Fig. 1 Proposed major metabolic pathways of tolvaptan
DM-4104, DM-4105, and DM-4107). Sensitive and specific LC–MS/MS method was developed and validated for determining tolvaptan and its nine metabolites in rat serum. The validation was carried out by evaluating the selectivity, linearity, lower limit of quantification (LLOQ), precision, accuracy, recovery, dilution integrity, and stability. After the validation, the analytical method was applied to a pharmacokinetic study of tolvaptan in rats. This is the first reported paper on an analytical method for determining tolvaptan and its nine metabolites in rat serum using LC– MS/MS.
Materials and methods Chemicals, reagents, and animals Tolvaptan, DM-4103, DM-4104, DM-4105, DM-4107, DM-4110, DM-4111, DM-4119, DM-4121, MOP-21826, and OPC-41100, which was used an internal standard (IS), were supplied by Otsuka Pharmaceutical Co., Ltd. (Tokyo, Japan). Purity of individual standards was not less than 98 %. Blank rat serum and Sprague–Dawley rats were purchased from Kitayama Labes Co., Ltd. (Nagano, Japan) and Charles River Laboratories Japan Inc. (Yokohama, Japan), respectively. Hydroxypropyl methylcellulose 2,910
123
Instruments The Nanospace SI-2 HPLC system included 3001 pumps, 3033 autosampler, and 3004 column thermoregulation bath (Shiseido Co., Ltd., Tokyo, Japan). The HPLC elute was introduced directly into an API4000 triple-quadrupole mass spectrometer (Applied Biosystems/MDS SCIEX, Ontario, Canada) equipped with electrospray ionization (ESI) source. Data acquisition and processing were performed using Analyst version 1.2 (Applied Biosystems/MDS SCIEX). Chromatographic conditions Samples were separated on a chromatographic column CAPCELL PAK C18 UG80 (150 mm 9 2.0 mm, 5 lm; Shiseido Co., Ltd), held at 40 °C. The mobile phases consisted of A: purified water with 0.3 % acetic acid and B: acetonitrile with 0.3 % acetic acid. The flow rate was held constant at 0.25 mL/ min. A linear gradient elution was performed as follows: initial 65 % A; 10.0 min 55 % A; 18.0 min 30 % A; 18.1 min 5 % A; 23.0 min 5 % A; 23.1 min 65 % A; 30.0 min 65 % A. The autosampler tray was maintained at 4 °C. Mass spectrometry conditions The mass spectrometer was operated in the positive ion mode. Quantification was performed using multiple
Application to a pharmacokinetic study
reaction monitoring (MRM) with a scan time of 0.1 s per transition. The LC–MS/MS parameters are presented in Table 1. The detailed mass spectrometer conditions were as follows: ion spray voltage, 4,500 V; ion source gas 1 (air), 70 psi; ion source gas 2 (air), 50 psi; heated gas temperature, 450 °C; collision gas (nitrogen), 8; curtain gas (nitrogen), 10 psi. Preparation of calibration standard and quality control Analytes (tolvaptan, DM-4103, DM-4104, DM-4105, DM4107, DM-4110, DM-4111, DM-4119, DM-4121, and MOP-21826) and OPC-41100 dissolved in methanol at 1 mg/mL were mixed and followed by dilution in methanol
to prepare working standard solutions with concentration ranging from 50 to 10,000 ng/mL. The IS was dissolved in methanol at 100 lg/mL and then diluted in methanol to obtain a working solution at 2,000 ng/mL. All stock and working solutions were stored at or below 10 °C under protection from light. Calibration standards and quality control (QC) samples were prepared by spiking 10 lL of the working standard solutions to 0.1 mL of blank rat serum. The calibration standards comprised seven concentrations (5, 10, 30, 100, 300, 800, and 1,000 ng/mL). QC samples were prepared at the concentrations of 10, 100, and 800 ng/mL. For each analytical run, calibration standards and QC samples were freshly prepared prior to use.
Fig. 2 MS/MS spectra of tolvaptan (a) and internal standard OPC-41100 (b)
123
M. Furukawa et al.
Fig. 3 Chromatograms of blank rat serum (a) and spiked serum sample (b) at LLOQ
Sample preparation To 0.1 mL of serum sample, 10 lL of methanol or working solution, 10 lL of IS solution, and 0.15 mL of acetonitrile were added and mixed. The mixture was centrifuged at 12,000g for 5 min. The supernatant (0.2 mL) was mixed with 0.8 mL of 1/15 mol/L phosphate buffer at pH 7.0. The sample was applied to a solid phase extraction column (abselut NEXUS, 30 mg/1 mL, Varian Inc., Palo Alto, USA) equilibrated with 1 mL of methanol followed by 1 mL of purified water. The column was washed with 1 mL of purified water–methanol (70:30, v/v). Then, the analytes were eluted with 1 mL of acetonitrile. The eluate
123
was evaporated to dryness under a nitrogen gas stream at 40 °C. The residue was dissolved in 0.2 mL of purified water–methanol (50:50, v/v), and a 5 lL aliquot was injected into the LC–MS/MS system.
Method validation Selectivity The selectivity of the analytical method was assessed by comparing the chromatograms of six individual blank rat sera with the spiked serum at the LLOQ level. To be
Application to a pharmacokinetic study
acceptable, the peak area of an interfering peak should not exceed 20 % for analyte and 5 % for IS.
acceptable, intra-day and inter-day precision should not exceed 15 %, and intra-day and inter-day accuracy should be within ±15 %.
Linearity Recovery The linearity of calibration curves for each analyte was validated over eight different days. Calibration curve (y = ax ? b; y: peak area ratio of analyte to IS, x: nominal concentration) was calculated by least square linear regression using 1/x2 weighting. The correlation coefficient (r) was calculated from the calibration curve. The calibration concentrations were back-calculated from the peak response. To be acceptable, the back-calculated concentrations of the calibration standards should be within ±15.0 % of the nominal concentration, except for the LLOQ for which it should be within ±20 %. LLOQ The LLOQ was determined by the analysis of six individual rat serum samples spiked with the ten analytes at 5 ng/mL. Precision was expressed as the percent coefficient of variation, and accuracy was expressed as relative error of the calculated concentrations. To be acceptable, precision should not exceed 20 %, and accuracy should be within ± 20 %.
The extraction recoveries for the ten analytes at the concentrations of 10 and 800 ng/mL were assessed by comparing the peak area ratio of each analyte to IS obtained from serum samples (n = 3) spiked before extraction with those spiked after extraction. The extraction recovery for IS at a concentration of 200 ng/mL was assessed by comparing the peak area ratios of IS to tolvaptan. Dilution integrity The dilution integrities for tolvaptan, DM-4103, and DM4107 were evaluated by the analysis of five rat serum samples spiked with the three analytes at 50,000 ng/mL. The samples were diluted 1,000 times with blank rat serum prior to the sample preparation for the analysis. To be acceptable, precision of the back-calculated concentrations should not exceed 15 %, and accuracy should be within ±15 %.
Precision and accuracy
Stability
Precision and accuracy for the analytical method were evaluated by analysis of five replicates of QC samples at three concentration levels on three different days. To be
The stabilities of the ten analytes in rat serum under different storage conditions were evaluated by analyzing three replicates of QC samples at the concentrations of 10 and 800 ng/mL. The spiked sample was analyzed after three freeze–thaw cycles (at or below -15 °C), storage at room temperature for 42 h, at or below -15 °C for 2 or 6 weeks, and in the autosampler at 4 °C for 59 h after extraction. The ten analytes were considered stable when the measured concentration was within ±15 % of the initial concentration. In addition, the stabilities of the ten analytes in working solutions were evaluated after storage at or below 10 °C under protection from light for 22 days. The each analyte was considered stable when the variation was within ±15 % of the initial values.
Table 2 Accuracy of back-calculated concentrations in the calibration curves for tolvaptan and its metabolites in rat serum (mean, n = 8) Analyte
Accuracy (%) Nominal concentration (ng/mL) 5
10
30
100
300
800
1000
Tolvaptan
0.2
-0.4
-1.1
3.9
3.6
-1.3
-4.2
DM-4103 DM-4104
0.9 1.3
-1.6 -1.7
-1.3 -3.0
1.9 1.3
2.4 2.0
-0.1 0.8
-1.9 -0.5
DM-4105
1.1
-2.1
-1.3
2.7
2.5
-0.4
-2.5
DM-4107
2.1
-2.9
-4.3
1.2
2.1
1.2
0.9
DM-4110
0.9
-1.7
-1.3
2.8
3.9
-0.7
-3.5
DM-4111
0.4
-1.0
-0.7
3.3
3.8
-1.2
-4.3
DM-4119
0.7
-1.3
-1.3
2.1
2.7
-0.3
-2.5
DM-4121
0.5
-0.8
-1.7
2.5
2.6
-0.2
-2.6
-1.0
0.4
2.7
6.9
6.0
-5.1
-9.0
MOP-21826
Application of the method to a pharmacokinetic study Ethics statement The animal experimental protocol was approved by the Animal Care and Use Committee of Otsuka Pharmaceutical Co., Ltd.
123
M. Furukawa et al. Table 3 Intra- and inter-day precision and accuracy for tolvaptan and its metabolites
Analyte
Day
Nominal concentration (ng/mL) 10 Precision (%)
Tolvaptan
DM-4103
DM-4104
DM-4105
DM-4107
DM-4110
DM-4111
DM-4119
DM-4121
MOP21826
100 Accuracy (%)
Precision (%)
800 Accuracy (%)
Precision (%)
Accuracy (%)
1
2.8
2.5
1.6
3.4
1.1
2
1.7
-1.4
2.0
6.3
1.2
1.5
5
3 Total
2.6 2.9
2.1 1.0
1.9 2.2
3.2 4.3
1.4 1.5
-0.6 0.3
5 15
1
5.7
0.4
2.1
2.8
2.2
-0.3
5
2
4.6
3.6
2.5
5.7
2.8
2.4
5
3
2.3
3.8
1.6
-0.5
2.2
-0.7
5
Total
4.4
2.6
3.2
2.7
2.6
0.5
15
0.0
5
5
1
3.4
0.8
1.4
-0.1
1.2
1.0
2
1.5
-0.5
2.2
3.2
1.5
2.5
5
3
2.3
0.4
1.2
-0.4
1.7
-0.4
5
Total
2.4
0.3
2.3
0.9
1.8
1.0
15 5
1
1.5
-1.1
0.7
2.9
0.9
-0.7
2
3.3
-0.7
2.3
4.6
1.2
1.4
5
3
2.4
-0.2
0.8
0.1
1.8
-1.3
5
Total
2.3
-0.6
2.3
2.5
1.8
-0.2
15
1
3.5
0.1
1.6
0.9
1.8
1.1
5
2 3
2.6 3.7
-1.0 0.3
1.6 2.3
4.1 -3.0
1.5 1.9
2.5 -1.4
5 5
Total
3.1
-0.2
3.4
0.7
2.3
0.8
15
1
2.2
0.5
0.4
3.3
1.3
-1.2
5
2
2.9
-0.1
2.6
6.4
1.4
-0.6
5
3
2.4
-3.7
0.8
0.3
1.5
-1.8
5
Total
3.0
-1.1
2.9
3.3
1.4
-1.2
15
1
3.4
-0.9
1.1
2.9
1.4
-2.3
5
2
1.6
0.4
1.5
6.1
1.3
-1.6
5
3
2.9
0.8
0.8
-0.1
2.7
-4.1
5
Total
2.6
0.1
2.8
3.0
2.1
-2.7
15
1
3.0
-1.4
0.9
1.0
1.6
-0.8
5
2
1.7
-1.2
1.7
5.0
1.1
-0.1
5
3
2.7
-0.9
1.3
0.5
2.0
-1.5
5
Total
2.3
-1.1
2.4
2.2
1.6
-0.8
15
1
1.5
0.6
1.0
1.9
1.7
-0.1
5
2 3
5.5 2.6
2.1 -0.2
0.9 1.2
4.8 0.1
1.2 0.7
1.3 -1.3
5 5
Total
3.5
0.8
2.2
2.3
1.6
0.0
15
1
2.9
2.6
0.3
5.7
1.9
-5.8
5
2
1.5
-0.2
2.0
7.5
1.3
-2.3
5
3
2.8
-0.3
1.2
5.1
1.6
-5.3
5
Total
2.7
0.7
1.6
6.1
2.2
-4.5
15
Pharmacokinetic study in rats Tolvaptan was suspended in 1 % HPMC solution. Rats (Crlj:CD, specific pathogen-free) were housed individually
123
n
in a bracket cage (W222 9 D325 9 H180 mm) at a temperature of 22.7 to 23.1 °C and a relative humidity of 52 to 63 % with a 12-h light/dark cycle for acclimation period of 12 days. Food and tap water were supplied ad libitum.
Application to a pharmacokinetic study Table 4 Stability of tolvaptan and its metabolites in rat serum and extract (mean, n = 3) Analyte
Matrix
Storage conditions
Remaining (%) 10 ng/mL
Tolvaptan
DM-4103
DM-4104
DM-4105
DM-4107
DM-4110
DM-4111
DM-4119
DM-4121
MOP-21826
800 ng/mL
Serum
Three freeze–thaw cycles
98.0
96.2
Serum
Room temperature, 42 h
98.5
99.4
Serum
At or below -15 °C, 6 weeks
100.9
99.0
Extract
Autosampler, 4 °C, 59 h
101.4
98.4
Serum
Three freeze–thaw cycles
95.3
99.1
Serum
Room temperature, 42 h
92.0
99.0
Serum Extract
At or below -15 °C, 6 weeks Autosampler, 4 °C, 59 h
94.2 95.6
98.2 96.3
Serum
Three freeze–thaw cycles
95.7
97.4
Serum
Room temperature, 42 h
96.0
100.0
Serum
At or below -15 °C, 2 weeks
93.1
95.8
Extract
Autosampler, 4 °C, 59 h
100.4
100.7
Serum
Three freeze–thaw cycles
100.9
96.7
Serum
Room temperature, 42 h
102.0
100.3
Serum
At or below -15 °C, 2 weeks
103.4
97.5
Extract
Autosampler, 4 °C, 59 h
95.1
93.3
Serum
Three freeze–thaw cycles
89.4
96.4
Serum
Room temperature, 42 h
92.9
100.4
Serum
At or below -15 °C, 6 weeks
96.1
95.2
Extract
Autosampler, 4 °C, 59 h
101.3
101.0
Serum
Three freeze–thaw cycles
95.7
96.3
Serum Serum
Room temperature, 42 h At or below -15 °C, 2 weeks
98.0 94.4
97.6 97.4
Extract
Autosampler, 4 °C, 59 h
101.2
98.9
Serum
Three freeze–thaw cycles
99.3
95.0
Serum
Room temperature, 42 h
103.6
97.2
Serum
At or below -15 °C, 2 weeks
102.2
96.8
Extract
Autosampler, 4 °C, 59 h
100.9
97.6
Serum
Three freeze–thaw cycles
96.5
96.9
Serum
Room temperature, 42 h
95.1
99.6
Serum
At or below -15 °C, 2 weeks
95.9
96.4
Extract
Autosampler, 4 °C, 59 h
99.0
99.6
Serum
Three freeze–thaw cycles
95.8
95.5
Serum
Room temperature, 42 h
93.9
97.4
Serum
At or below -15 °C, 2 weeks
Extract
Autosampler, 4 °C, 59 h
Serum
Three freeze–thaw cycles
94.7
95.8
Serum Serum
Room temperature, 42 h At or below -15 °C, 2 weeks
98.3 98.5
98.4 99.3
Extract
Autosampler, 4 °C, 59 h
101.8
96.2
Blood samples were collected at 0.25, 0.5, 1, 2, 3, 4, 6, 8, 12, and 24 h after single oral administration of tolvaptan at a dose of 30 mg/kg to rats (age 6–7 weeks, body weight 212–242 g in males and 153–187 g in females) under fasting conditions. The serum was obtained by centrifugation at 1,630g for 10 min after blood coagulation. The
94.6
97.6
100.1
100.0
samples were stored at or below -15 °C until the quantification of tolvaptan and its nine metabolites in the rat serum. The pharmacokinetic parameters, peak serum concentration (Cmax), time to peak serum concentration (tmax), area under the concentration–time curve calculated to the last observable concentration at time t (AUCt), and
123
M. Furukawa et al.
Fig. 4 Serum concentrations of tolvaptan and its nine metabolites after single oral administration of tolvaptan at 30 mg/kg to male (a, b) and female (c, d) rats (mean ? SD, n = 3)
apparent elimination half-life (t1/2) were calculated based on the average concentration of each analyte in the serum (n = 3) by non-compartment analysis using WinNonlin Professional (Version 4.1, Pharsight Co., CA, USA).
Results and discussion Method validation for LC–MS/MS analysis Positive ESI mass spectrum of tolvaptan, DM-4103, DM4104, DM-4105, DM-4107, DM-4110, DM-4111, DM4119, DM-4121, MOP-21826, and IS revealed predominant protonated molecular ions [M ? H]? at m/z 449.2, 479.1, 467.1, 465.2, 481.1, 465.2, 465.2, 465.2, 463.1, 447.1 and 463.2, respectively. Figure 2 shows MS/MS spectra of [M ? H]? of the tolvaptan and IS. The most abundant fragment ion of each compound was selected for MRM. The MRM transitions and the other optimized MS/ MS conditions are described in the mass spectrometry conditions. Typical chromatograms of the blank serum and the spiked serum at the LLOQ level are shown in Fig. 3. There was no interference peak at the retention times of the ten analytes and IS.
123
The calibration curves of the ten analytes were linear over the concentration range of 5–1,000 ng/mL. Good linearity was obtained in this concentration range with a correlation coefficient of 0.9962 or more for all analytes. Accuracy of back-calculated concentrations in the calibration curves for tolvaptan and its metabolites in rat serum are shown in Table 2. The accuracy for each point in the calibration curves was -9.0 to 6.9 % for all analytes. The precision and accuracy in the LLOQ sample were 1.5 to 4.8 % and -1.4 to 4.1 % for all analytes, respectively. The results were within the criteria of the validation. The LLOQ of each analyte in this analytical method was determined to be 5 ng/mL. The results evaluated by the analysis of six individual rat serum samples spiked with the ten analytes indicated that the matrix effects on the ionization of tolvaptan and its metabolites were not observed. The intra-day and inter-day precision, and accuracy for all analytes are summarized in Table 3. The results were within the criteria of the validation. The extraction recoveries from rat serum were 65.7 to 87.4 % for all analytes and 73.0 % for IS. The precision and accuracy in the dilution integrity samples were 1.4–4.8 % and 6.5–10.5 % for tolvaptan, DM-4103, and DM-4107, respectively. The results were within the criteria of the validation, indicating that serum
Application to a pharmacokinetic study
samples with concentrations above the calibration curve range could be successfully diluted and analyzed. The stability results in rat serum and extract are shown in Table 4. The ten analytes were shown to remain stable in rat serum after three freeze–thaw cycles. The ten analytes were also found to be stable in rat serum for at least 42 h at room temperature, for at least 2 or 6 weeks at or below -15 °C, and for at least 59 h in processed samples at 4 °C. The ten analytes prepared in methanol (working solutions) were also found to be stable for 22 days at or below 10 °C (remaining 91.8–106.9 %). The analytical method was showing to be sufficiently sensitive, selective, precise, and accurate. Therefore, this method was considered to be reliable, reproducible, and useful to support pharmacokinetic and toxicokinetic studies with tolvaptan in rats.
Application of the method to a pharmacokinetic study Figure 4 shows representative courses of the serum concentration versus time of tolvaptan and its nine metabolites determined by the present LC–MS/MS method in male and female rats treated with oral tolvaptan at 30 mg/kg. The Pharmacokinetic parameters for tolvaptan and its nine Table 5 Pharmacokinetic parameters of tolvaptan and its metabolites in serum after single oral administration of tolvaptan at 30 mg/kg to rats Analyte Tolvaptan DM-4103 DM-4104 DM-4105 DM-4107 DM-4110
Gender
tmax (h)
Cmax (ng/mL)
AUCt (ng h/mL)
t1/2 (h)
Male
2
214
925
1.6
Female
4
1,759
7,437
2.5
Male
4
2,144
9,632
2.8
Female
4
864
6,493
3.7
Male
0.5
101
422
1.6
Female
4
383
1,700
3.8
Male
4
166
850
1.5
Female
4
133
811
3.4
Male
4
261
1,132
1.3
Female
4
524
3,064
2.7
Male
2
160
910
1.0 3.0
metabolites are summarized in Table 5. In the male rats, the Cmax, tmax, AUCt, and elimination t1/2 for tolvaptan were 214 ng/mL, 2 h, 925 ng h/mL, and 1.6 h, respectively. The rank order of the AUCt in the male rats were DM-4103 (1,041 %) [ DM-4107 (122 %) [ tolvaptan (100 %) [ DM-4110 (98 %) [ DM-4105 (92 %) [ DM-4119 (83 %) [ DM-4121 (69 %) [ DM-4104 (46 %) [ DM-4111 (43 %) [ MOP-21826 (0.49 %). In the female rats, the Cmax, tmax, AUCt, and elimination t1/2 for tolvaptan were 1,759 ng/mL, 4 h, 7,437 ng h/mL, and 2.5 h, respectively. The rank order of the AUCt in the female rats were as follows: tolvaptan (100 %) [ DM-4103 (87 %) [ DM-4119 (59 %) [ DM4110 (47 %) [ DM-4107 (41 %) [ DM-4121 (25 %) [ DM4104 and DM-4111 (23 %) [ DM-4105 (11 %) [ MOP-21826 (2.6 %). The oral pharmacokinetic study suggested that tolvaptan was rapidly absorbed in rats, and the Cmax and AUCt of tolvaptan in females were approximately eight times greater than those in the males. DM-4103 was prominent circulating metabolite in serum of male rats, while in female rats tolvaptan was metabolized to a lesser extent. In vitro metabolism studies of tolvaptan indicated that tolvaptan was eliminated primarily by metabolism via the CYP3A4 isozyme (Shoaf et al. 2011). The sex difference observed in this pharmacokinetic study was considered to be related to the sex difference in the metabolizing enzyme activities in the rat liver, since these activities were much higher in the male (Kato and Yamazoe 1992).
Conclusion We have developed and validated an LC–MS/MS method for determining of tolvaptan and its nine metabolites in rat serum. This method was demonstrated to be sensitive, selective, precise, and accurate. The validated method was successfully applied to the pharmacokinetic study of oral tolvaptan in rats. In male rats, the serum concentration of metabolite DM-4103 was higher than that of the unchanged compound, but in female rats, the serum concentration of the unchanged compound was higher than that of all metabolites. Tolvaptan was rapidly absorbed after oral administration and there was sex difference in the serum concentration–time profiles for tolvaptan and its metabolites in rats.
Female
4
625
3,473
DM-4111
Male
2
87
394
1.8
4 2
345 138
1,685 767
3.8 1.1
References
DM-4119
Female Male Female
4
887
4,389
3.3
Dixon, M.B., and Y.H. Lien. 2008. Tolvaptan and its potential in the treatment of hyponatremia. Therapeutics and Clinical Risk Management 4: 1149–1155. Ghali, J.K., B. Hamad, U. Yasothan, and P. Kirkpatrick. 2009. Tolvaptan. Nature Reviews Drug Discovery 8: 611–612. Furukawa, M., K. Umehara, and E. Kashiyama. 2011. Nonclinical pharmacokinetics of a new nonpeptide V2 receptor antagonist, tolvaptan. Cardiovascular Drugs and Therapy 25: S83–S89.
DM-4121 MOP-21826
Male
4
129
641
1.7
Female
4
249
1,859
3.1
Male
2
5
5
NC
Female
4
54
196
1.2
NC not calculated
123
M. Furukawa et al. Hori, M. 2011. Tolvaptan for heart failure patients with volume overload. Cardiovascular Drugs Therapy 25: S1–S4. Kato, R., and Y. Yamazoe. 1992. Sex-specific cytochrome P450 as a cause of sex- and species-related differences in drug toxicity. Toxicology Letters 64(65): 661–667. Kondo, K., H. Ogawa, H. Yamashita, H. Miyamoto, M. Tanaka, K. Nakaya, K. Kitano, Y. Yamamura, S. Nakamura, T. Onogawa, T. Mori, and M. Tominaga. 1999. 7-Chloro-5-hydroxy-1-[2methyl-4-(2-methylbenzoyl-amino)benzoyl]-2,3,4,5-tetrahydro1H-1benzazepine (OPC-41061): a potent, orally active nonpentide arginine vasopressin V2 receptor antagonist. Bioorganic and Medicinal Chemistry 7: 1743–1754. Miyazaki, T., H. Fujiki, Y. Yamamura, S. Nakamura, and T. Mori. 2007. Tolvaptan, an orally active vasopressin V(2)-receptor antagonist—Pharmacology and clinical trials. Cardiovascular Drug Reviews 25: 1–13. Miyazaki, T., H. Fujiki, and Y. Yamamura. 2013. Tolvaptan, an orally active non-peptide arginine vasopressin V2 receptor antagonist, reduces ascites in rats with chronic liver injury. Hepatology Research 43: 1224–1230. Pei, Q., B. Zhang, H. Tan, L. Liu, X. Peng, Z. Li, P. Huang, M. Luo, X. Zuo, C. Guo, and G. Yang. 2013. Development and validation
123
of an LC-MS/MS method for the determination of tolvaptan in human plasma and its application to a pharmacokinetic study. Journal of Chromatography B 913–914: 84–89. Sakaide, I., S. Yamashita, T. Kobayashi, M. Komatsu, T. Sakai, Y. Komorizono, M. Okada, and K. Okita. 2013. Efficacy and safety of a 14-day administration of tolvaptan in the treatment of patients with ascites in hepatic oedema. Journal of International Medical Research 41: 835–847. Shoaf, S.E., Y. Ohzone, S. Ninomiya, M. Furukawa, P. Bricmont, E. Kashiyama, and S. Mallikaarjun. 2011. In vitro P-glycoprotein interactions and steady-state pharmacokinetic interactions between tolvaptan and digoxin in healthy subjects. The Journal of Clinical Pharmacology 51: 761–769. Yamamura, Y., S. Nakamura, S. Itoh, T. Hirano, T. Onogawa, T. Yamashita, Y. Yamada, K. Tsujimae, M. Aoyama, K. Kotosai, H. Ogawa, H. Yamashita, K. Kondo, M. Tominaga, G. Tsujimoto, and T. Mori. 1998. OPC-41061, a highly potent human vasopressin V2-receptor antagonist: pharmacological profile and aquaretic effect by single and multiple oral dosing in rats. Journal of Pharmacology and Experimental Therapeutic 287: 860–867.
RESEARCH ARTICLE
Liquid chromatography–tandem mass spectrometry method for determining tolvaptan and its nine metabolites in rat serum: application to a pharmacokinetic study Masayuki Furukawa • Kenichi Miyata • Chie Kawasome • Yoshiko Himeda • Kenji Takeuchi Toshihisa Koga • Yukihiro Hirao • Ken Umehara
•
Received: 25 October 2013 / Accepted: 30 January 2014 Ó The Pharmaceutical Society of Korea 2014
Abstract Tolvaptan is a competitive vasopressin V2receptor antagonist that inhibits water reabsorption in the renal collecting ducts. A selective and sensitive liquid chromatography–tandem mass spectrometry method for determining tolvaptan and its nine metabolites in rat serum was developed and validated. An analogue of tolvaptan was used as an internal standard. Sample preparation involved protein precipitation following solid-phase extraction. Chromatographic separation was performed on a C18 reversed-phase column with a linear gradient elution. The flow rate was 0.25 mL/min, and total run time was 30 min. The analytes were detected by tandem mass spectrometry using an electrospray ionization interface in positive ion mode and multiple reaction monitoring. The calibration curve showed linearity over the concentration range from 5 to 1,000 ng/mL for each analyte. The lower limit of quantification using 0.1 mL of rat serum was 5 ng/mL for each analyte. Precision did not exceed 5.7 %, and accuracy as relative error were within ± 7.5 % for all analytes. The validated method was successfully applied to evaluate the pharmacokinetics of oral tolvaptan in rats, indicating the systemic exposure to tolvaptan in females eight times larger than that in males. Keywords Tolvaptan Metabolite Pharmacokinetics LC–MS/MS Serum Rat
M. Furukawa (&) C. Kawasome Y. Himeda K. Takeuchi T. Koga Y. Hirao K. Umehara Tokushima Research Institute, Otsuka Pharmaceutical Co., Ltd., 463-10 Kagasuno, Kawauchi-cho, Tokushima 771-0192, Japan e-mail: [email protected] K. Miyata Formulation Research Institute, Otsuka Pharmaceutical Co., Ltd., 224-18 Ebisuno Hiraishi, Kawauchi-cho, Tokushima 771-0182, Japan
Introduction Tolvaptan, N-{4-[(5RS)-7-chloro-5-hydroxy-2,3,4,5-tetrahydro-1H-benzo[b]azepin -1-carbonyl]-3-methylphenyl}2-methylbenzamide, is an orally-administered nonpeptide vasopressin V2 receptor antagonist developed by Otsuka Pharmaceutical Co., Ltd. (Tokyo, Japan) (Yamamura et al. 1998; Kondo et al. 1999; Miyazaki et al. 2007, 2013). Tolvaptan is used in the United States and Europe for the treatment of hypervolemic and euvolemic hyponatremia including patients with heart failure, cirrhosis, and syndrome of inappropriate antidiuretic hormone (Dixon and Lien 2008; Ghali et al. 2009). Tolvaptan was approved in the Japan for the treatment of excess water retention in patients with cardiac failure and liver cirrhosis when the treatment by other diuretics including loop diuretics is ineffective (Hori 2011; Sakaide et al. 2013). The determination of tolvaptan in rat serum has been reported using high-performance liquid chromatography (HPLC) (Furukawa et al. 2011). The HPLC method requires liquid–liquid extraction with an n-hexane:diethyl ether (1:1, v/v) solution. The liquid chromatography with tandem mass spectrometry (LC–MS/MS) method for determining tolvaptan in human plasma has been reported (Pei et al. 2013). None of the published LC–MS/MS methods fulfilled quantitative analysis of tolvaptan and its metabolites. Tolvaptan is metabolized primarily by dehydrogenation and hydroxylation. The proposed major metabolic pathways of tolvaptan are shown in Fig. 1. These pathways generate a number of metabolites. Structural characterization of the metabolites of tolvaptan revealed one hydroxide of the benzazepine ring (DM-4110, DM-4111, and DM4119), one oxide of the hydroxy group at the 50 position (MOP-21826) and its one hydroxide (DM-4121), and four metabolites with the benzazepine ring cleaved (DM-4103,
123
M. Furukawa et al. Table 1 LC–MS/MS parameters for ten analytes and internal standard Analyte
Precursor ion (m/z)
Product ion (m/z)
Collision energy (eV)
Tolvaptan
449.2
252
23
DM-4103
479.1
252
15
DM-4104 DM-4105, DM-4110,
467.1 465.2
252 252
23 27
DM-4107
481.1
252
27
DM-4121
463.1
252
29
MOP-21826
447.1
252
29
OPC-41100 (internal standard)
463.2
266
25
DM-4111, DM-4119
(HPMC), methanol (HPLC grade), acetonitrile (HPLC grade), 1/15 mol/L phosphate buffer at pH 7.0, and acetic acid (special grade) were purchased from Shin-Etsu Chemical Co. (Tokyo, Japan), Ltd., Merck Ltd, Japan (Tokyo, Japan), Thermo Fisher Scientific K.K. (Yokohama, Japan), Nacalai Tesque, Inc. (Kyoto, Japan), and SigmaAldrich Japan K.K. (Tokyo, Japan), respectively. Purified water was obtained from Milli-Q water purification system (Millipore, Billerica, USA). All other chemicals were of the highest purity commercially available. Fig. 1 Proposed major metabolic pathways of tolvaptan
DM-4104, DM-4105, and DM-4107). Sensitive and specific LC–MS/MS method was developed and validated for determining tolvaptan and its nine metabolites in rat serum. The validation was carried out by evaluating the selectivity, linearity, lower limit of quantification (LLOQ), precision, accuracy, recovery, dilution integrity, and stability. After the validation, the analytical method was applied to a pharmacokinetic study of tolvaptan in rats. This is the first reported paper on an analytical method for determining tolvaptan and its nine metabolites in rat serum using LC– MS/MS.
Materials and methods Chemicals, reagents, and animals Tolvaptan, DM-4103, DM-4104, DM-4105, DM-4107, DM-4110, DM-4111, DM-4119, DM-4121, MOP-21826, and OPC-41100, which was used an internal standard (IS), were supplied by Otsuka Pharmaceutical Co., Ltd. (Tokyo, Japan). Purity of individual standards was not less than 98 %. Blank rat serum and Sprague–Dawley rats were purchased from Kitayama Labes Co., Ltd. (Nagano, Japan) and Charles River Laboratories Japan Inc. (Yokohama, Japan), respectively. Hydroxypropyl methylcellulose 2,910
123
Instruments The Nanospace SI-2 HPLC system included 3001 pumps, 3033 autosampler, and 3004 column thermoregulation bath (Shiseido Co., Ltd., Tokyo, Japan). The HPLC elute was introduced directly into an API4000 triple-quadrupole mass spectrometer (Applied Biosystems/MDS SCIEX, Ontario, Canada) equipped with electrospray ionization (ESI) source. Data acquisition and processing were performed using Analyst version 1.2 (Applied Biosystems/MDS SCIEX). Chromatographic conditions Samples were separated on a chromatographic column CAPCELL PAK C18 UG80 (150 mm 9 2.0 mm, 5 lm; Shiseido Co., Ltd), held at 40 °C. The mobile phases consisted of A: purified water with 0.3 % acetic acid and B: acetonitrile with 0.3 % acetic acid. The flow rate was held constant at 0.25 mL/ min. A linear gradient elution was performed as follows: initial 65 % A; 10.0 min 55 % A; 18.0 min 30 % A; 18.1 min 5 % A; 23.0 min 5 % A; 23.1 min 65 % A; 30.0 min 65 % A. The autosampler tray was maintained at 4 °C. Mass spectrometry conditions The mass spectrometer was operated in the positive ion mode. Quantification was performed using multiple
Application to a pharmacokinetic study
reaction monitoring (MRM) with a scan time of 0.1 s per transition. The LC–MS/MS parameters are presented in Table 1. The detailed mass spectrometer conditions were as follows: ion spray voltage, 4,500 V; ion source gas 1 (air), 70 psi; ion source gas 2 (air), 50 psi; heated gas temperature, 450 °C; collision gas (nitrogen), 8; curtain gas (nitrogen), 10 psi. Preparation of calibration standard and quality control Analytes (tolvaptan, DM-4103, DM-4104, DM-4105, DM4107, DM-4110, DM-4111, DM-4119, DM-4121, and MOP-21826) and OPC-41100 dissolved in methanol at 1 mg/mL were mixed and followed by dilution in methanol
to prepare working standard solutions with concentration ranging from 50 to 10,000 ng/mL. The IS was dissolved in methanol at 100 lg/mL and then diluted in methanol to obtain a working solution at 2,000 ng/mL. All stock and working solutions were stored at or below 10 °C under protection from light. Calibration standards and quality control (QC) samples were prepared by spiking 10 lL of the working standard solutions to 0.1 mL of blank rat serum. The calibration standards comprised seven concentrations (5, 10, 30, 100, 300, 800, and 1,000 ng/mL). QC samples were prepared at the concentrations of 10, 100, and 800 ng/mL. For each analytical run, calibration standards and QC samples were freshly prepared prior to use.
Fig. 2 MS/MS spectra of tolvaptan (a) and internal standard OPC-41100 (b)
123
M. Furukawa et al.
Fig. 3 Chromatograms of blank rat serum (a) and spiked serum sample (b) at LLOQ
Sample preparation To 0.1 mL of serum sample, 10 lL of methanol or working solution, 10 lL of IS solution, and 0.15 mL of acetonitrile were added and mixed. The mixture was centrifuged at 12,000g for 5 min. The supernatant (0.2 mL) was mixed with 0.8 mL of 1/15 mol/L phosphate buffer at pH 7.0. The sample was applied to a solid phase extraction column (abselut NEXUS, 30 mg/1 mL, Varian Inc., Palo Alto, USA) equilibrated with 1 mL of methanol followed by 1 mL of purified water. The column was washed with 1 mL of purified water–methanol (70:30, v/v). Then, the analytes were eluted with 1 mL of acetonitrile. The eluate
123
was evaporated to dryness under a nitrogen gas stream at 40 °C. The residue was dissolved in 0.2 mL of purified water–methanol (50:50, v/v), and a 5 lL aliquot was injected into the LC–MS/MS system.
Method validation Selectivity The selectivity of the analytical method was assessed by comparing the chromatograms of six individual blank rat sera with the spiked serum at the LLOQ level. To be
Application to a pharmacokinetic study
acceptable, the peak area of an interfering peak should not exceed 20 % for analyte and 5 % for IS.
acceptable, intra-day and inter-day precision should not exceed 15 %, and intra-day and inter-day accuracy should be within ±15 %.
Linearity Recovery The linearity of calibration curves for each analyte was validated over eight different days. Calibration curve (y = ax ? b; y: peak area ratio of analyte to IS, x: nominal concentration) was calculated by least square linear regression using 1/x2 weighting. The correlation coefficient (r) was calculated from the calibration curve. The calibration concentrations were back-calculated from the peak response. To be acceptable, the back-calculated concentrations of the calibration standards should be within ±15.0 % of the nominal concentration, except for the LLOQ for which it should be within ±20 %. LLOQ The LLOQ was determined by the analysis of six individual rat serum samples spiked with the ten analytes at 5 ng/mL. Precision was expressed as the percent coefficient of variation, and accuracy was expressed as relative error of the calculated concentrations. To be acceptable, precision should not exceed 20 %, and accuracy should be within ± 20 %.
The extraction recoveries for the ten analytes at the concentrations of 10 and 800 ng/mL were assessed by comparing the peak area ratio of each analyte to IS obtained from serum samples (n = 3) spiked before extraction with those spiked after extraction. The extraction recovery for IS at a concentration of 200 ng/mL was assessed by comparing the peak area ratios of IS to tolvaptan. Dilution integrity The dilution integrities for tolvaptan, DM-4103, and DM4107 were evaluated by the analysis of five rat serum samples spiked with the three analytes at 50,000 ng/mL. The samples were diluted 1,000 times with blank rat serum prior to the sample preparation for the analysis. To be acceptable, precision of the back-calculated concentrations should not exceed 15 %, and accuracy should be within ±15 %.
Precision and accuracy
Stability
Precision and accuracy for the analytical method were evaluated by analysis of five replicates of QC samples at three concentration levels on three different days. To be
The stabilities of the ten analytes in rat serum under different storage conditions were evaluated by analyzing three replicates of QC samples at the concentrations of 10 and 800 ng/mL. The spiked sample was analyzed after three freeze–thaw cycles (at or below -15 °C), storage at room temperature for 42 h, at or below -15 °C for 2 or 6 weeks, and in the autosampler at 4 °C for 59 h after extraction. The ten analytes were considered stable when the measured concentration was within ±15 % of the initial concentration. In addition, the stabilities of the ten analytes in working solutions were evaluated after storage at or below 10 °C under protection from light for 22 days. The each analyte was considered stable when the variation was within ±15 % of the initial values.
Table 2 Accuracy of back-calculated concentrations in the calibration curves for tolvaptan and its metabolites in rat serum (mean, n = 8) Analyte
Accuracy (%) Nominal concentration (ng/mL) 5
10
30
100
300
800
1000
Tolvaptan
0.2
-0.4
-1.1
3.9
3.6
-1.3
-4.2
DM-4103 DM-4104
0.9 1.3
-1.6 -1.7
-1.3 -3.0
1.9 1.3
2.4 2.0
-0.1 0.8
-1.9 -0.5
DM-4105
1.1
-2.1
-1.3
2.7
2.5
-0.4
-2.5
DM-4107
2.1
-2.9
-4.3
1.2
2.1
1.2
0.9
DM-4110
0.9
-1.7
-1.3
2.8
3.9
-0.7
-3.5
DM-4111
0.4
-1.0
-0.7
3.3
3.8
-1.2
-4.3
DM-4119
0.7
-1.3
-1.3
2.1
2.7
-0.3
-2.5
DM-4121
0.5
-0.8
-1.7
2.5
2.6
-0.2
-2.6
-1.0
0.4
2.7
6.9
6.0
-5.1
-9.0
MOP-21826
Application of the method to a pharmacokinetic study Ethics statement The animal experimental protocol was approved by the Animal Care and Use Committee of Otsuka Pharmaceutical Co., Ltd.
123
M. Furukawa et al. Table 3 Intra- and inter-day precision and accuracy for tolvaptan and its metabolites
Analyte
Day
Nominal concentration (ng/mL) 10 Precision (%)
Tolvaptan
DM-4103
DM-4104
DM-4105
DM-4107
DM-4110
DM-4111
DM-4119
DM-4121
MOP21826
100 Accuracy (%)
Precision (%)
800 Accuracy (%)
Precision (%)
Accuracy (%)
1
2.8
2.5
1.6
3.4
1.1
2
1.7
-1.4
2.0
6.3
1.2
1.5
5
3 Total
2.6 2.9
2.1 1.0
1.9 2.2
3.2 4.3
1.4 1.5
-0.6 0.3
5 15
1
5.7
0.4
2.1
2.8
2.2
-0.3
5
2
4.6
3.6
2.5
5.7
2.8
2.4
5
3
2.3
3.8
1.6
-0.5
2.2
-0.7
5
Total
4.4
2.6
3.2
2.7
2.6
0.5
15
0.0
5
5
1
3.4
0.8
1.4
-0.1
1.2
1.0
2
1.5
-0.5
2.2
3.2
1.5
2.5
5
3
2.3
0.4
1.2
-0.4
1.7
-0.4
5
Total
2.4
0.3
2.3
0.9
1.8
1.0
15 5
1
1.5
-1.1
0.7
2.9
0.9
-0.7
2
3.3
-0.7
2.3
4.6
1.2
1.4
5
3
2.4
-0.2
0.8
0.1
1.8
-1.3
5
Total
2.3
-0.6
2.3
2.5
1.8
-0.2
15
1
3.5
0.1
1.6
0.9
1.8
1.1
5
2 3
2.6 3.7
-1.0 0.3
1.6 2.3
4.1 -3.0
1.5 1.9
2.5 -1.4
5 5
Total
3.1
-0.2
3.4
0.7
2.3
0.8
15
1
2.2
0.5
0.4
3.3
1.3
-1.2
5
2
2.9
-0.1
2.6
6.4
1.4
-0.6
5
3
2.4
-3.7
0.8
0.3
1.5
-1.8
5
Total
3.0
-1.1
2.9
3.3
1.4
-1.2
15
1
3.4
-0.9
1.1
2.9
1.4
-2.3
5
2
1.6
0.4
1.5
6.1
1.3
-1.6
5
3
2.9
0.8
0.8
-0.1
2.7
-4.1
5
Total
2.6
0.1
2.8
3.0
2.1
-2.7
15
1
3.0
-1.4
0.9
1.0
1.6
-0.8
5
2
1.7
-1.2
1.7
5.0
1.1
-0.1
5
3
2.7
-0.9
1.3
0.5
2.0
-1.5
5
Total
2.3
-1.1
2.4
2.2
1.6
-0.8
15
1
1.5
0.6
1.0
1.9
1.7
-0.1
5
2 3
5.5 2.6
2.1 -0.2
0.9 1.2
4.8 0.1
1.2 0.7
1.3 -1.3
5 5
Total
3.5
0.8
2.2
2.3
1.6
0.0
15
1
2.9
2.6
0.3
5.7
1.9
-5.8
5
2
1.5
-0.2
2.0
7.5
1.3
-2.3
5
3
2.8
-0.3
1.2
5.1
1.6
-5.3
5
Total
2.7
0.7
1.6
6.1
2.2
-4.5
15
Pharmacokinetic study in rats Tolvaptan was suspended in 1 % HPMC solution. Rats (Crlj:CD, specific pathogen-free) were housed individually
123
n
in a bracket cage (W222 9 D325 9 H180 mm) at a temperature of 22.7 to 23.1 °C and a relative humidity of 52 to 63 % with a 12-h light/dark cycle for acclimation period of 12 days. Food and tap water were supplied ad libitum.
Application to a pharmacokinetic study Table 4 Stability of tolvaptan and its metabolites in rat serum and extract (mean, n = 3) Analyte
Matrix
Storage conditions
Remaining (%) 10 ng/mL
Tolvaptan
DM-4103
DM-4104
DM-4105
DM-4107
DM-4110
DM-4111
DM-4119
DM-4121
MOP-21826
800 ng/mL
Serum
Three freeze–thaw cycles
98.0
96.2
Serum
Room temperature, 42 h
98.5
99.4
Serum
At or below -15 °C, 6 weeks
100.9
99.0
Extract
Autosampler, 4 °C, 59 h
101.4
98.4
Serum
Three freeze–thaw cycles
95.3
99.1
Serum
Room temperature, 42 h
92.0
99.0
Serum Extract
At or below -15 °C, 6 weeks Autosampler, 4 °C, 59 h
94.2 95.6
98.2 96.3
Serum
Three freeze–thaw cycles
95.7
97.4
Serum
Room temperature, 42 h
96.0
100.0
Serum
At or below -15 °C, 2 weeks
93.1
95.8
Extract
Autosampler, 4 °C, 59 h
100.4
100.7
Serum
Three freeze–thaw cycles
100.9
96.7
Serum
Room temperature, 42 h
102.0
100.3
Serum
At or below -15 °C, 2 weeks
103.4
97.5
Extract
Autosampler, 4 °C, 59 h
95.1
93.3
Serum
Three freeze–thaw cycles
89.4
96.4
Serum
Room temperature, 42 h
92.9
100.4
Serum
At or below -15 °C, 6 weeks
96.1
95.2
Extract
Autosampler, 4 °C, 59 h
101.3
101.0
Serum
Three freeze–thaw cycles
95.7
96.3
Serum Serum
Room temperature, 42 h At or below -15 °C, 2 weeks
98.0 94.4
97.6 97.4
Extract
Autosampler, 4 °C, 59 h
101.2
98.9
Serum
Three freeze–thaw cycles
99.3
95.0
Serum
Room temperature, 42 h
103.6
97.2
Serum
At or below -15 °C, 2 weeks
102.2
96.8
Extract
Autosampler, 4 °C, 59 h
100.9
97.6
Serum
Three freeze–thaw cycles
96.5
96.9
Serum
Room temperature, 42 h
95.1
99.6
Serum
At or below -15 °C, 2 weeks
95.9
96.4
Extract
Autosampler, 4 °C, 59 h
99.0
99.6
Serum
Three freeze–thaw cycles
95.8
95.5
Serum
Room temperature, 42 h
93.9
97.4
Serum
At or below -15 °C, 2 weeks
Extract
Autosampler, 4 °C, 59 h
Serum
Three freeze–thaw cycles
94.7
95.8
Serum Serum
Room temperature, 42 h At or below -15 °C, 2 weeks
98.3 98.5
98.4 99.3
Extract
Autosampler, 4 °C, 59 h
101.8
96.2
Blood samples were collected at 0.25, 0.5, 1, 2, 3, 4, 6, 8, 12, and 24 h after single oral administration of tolvaptan at a dose of 30 mg/kg to rats (age 6–7 weeks, body weight 212–242 g in males and 153–187 g in females) under fasting conditions. The serum was obtained by centrifugation at 1,630g for 10 min after blood coagulation. The
94.6
97.6
100.1
100.0
samples were stored at or below -15 °C until the quantification of tolvaptan and its nine metabolites in the rat serum. The pharmacokinetic parameters, peak serum concentration (Cmax), time to peak serum concentration (tmax), area under the concentration–time curve calculated to the last observable concentration at time t (AUCt), and
123
M. Furukawa et al.
Fig. 4 Serum concentrations of tolvaptan and its nine metabolites after single oral administration of tolvaptan at 30 mg/kg to male (a, b) and female (c, d) rats (mean ? SD, n = 3)
apparent elimination half-life (t1/2) were calculated based on the average concentration of each analyte in the serum (n = 3) by non-compartment analysis using WinNonlin Professional (Version 4.1, Pharsight Co., CA, USA).
Results and discussion Method validation for LC–MS/MS analysis Positive ESI mass spectrum of tolvaptan, DM-4103, DM4104, DM-4105, DM-4107, DM-4110, DM-4111, DM4119, DM-4121, MOP-21826, and IS revealed predominant protonated molecular ions [M ? H]? at m/z 449.2, 479.1, 467.1, 465.2, 481.1, 465.2, 465.2, 465.2, 463.1, 447.1 and 463.2, respectively. Figure 2 shows MS/MS spectra of [M ? H]? of the tolvaptan and IS. The most abundant fragment ion of each compound was selected for MRM. The MRM transitions and the other optimized MS/ MS conditions are described in the mass spectrometry conditions. Typical chromatograms of the blank serum and the spiked serum at the LLOQ level are shown in Fig. 3. There was no interference peak at the retention times of the ten analytes and IS.
123
The calibration curves of the ten analytes were linear over the concentration range of 5–1,000 ng/mL. Good linearity was obtained in this concentration range with a correlation coefficient of 0.9962 or more for all analytes. Accuracy of back-calculated concentrations in the calibration curves for tolvaptan and its metabolites in rat serum are shown in Table 2. The accuracy for each point in the calibration curves was -9.0 to 6.9 % for all analytes. The precision and accuracy in the LLOQ sample were 1.5 to 4.8 % and -1.4 to 4.1 % for all analytes, respectively. The results were within the criteria of the validation. The LLOQ of each analyte in this analytical method was determined to be 5 ng/mL. The results evaluated by the analysis of six individual rat serum samples spiked with the ten analytes indicated that the matrix effects on the ionization of tolvaptan and its metabolites were not observed. The intra-day and inter-day precision, and accuracy for all analytes are summarized in Table 3. The results were within the criteria of the validation. The extraction recoveries from rat serum were 65.7 to 87.4 % for all analytes and 73.0 % for IS. The precision and accuracy in the dilution integrity samples were 1.4–4.8 % and 6.5–10.5 % for tolvaptan, DM-4103, and DM-4107, respectively. The results were within the criteria of the validation, indicating that serum
Application to a pharmacokinetic study
samples with concentrations above the calibration curve range could be successfully diluted and analyzed. The stability results in rat serum and extract are shown in Table 4. The ten analytes were shown to remain stable in rat serum after three freeze–thaw cycles. The ten analytes were also found to be stable in rat serum for at least 42 h at room temperature, for at least 2 or 6 weeks at or below -15 °C, and for at least 59 h in processed samples at 4 °C. The ten analytes prepared in methanol (working solutions) were also found to be stable for 22 days at or below 10 °C (remaining 91.8–106.9 %). The analytical method was showing to be sufficiently sensitive, selective, precise, and accurate. Therefore, this method was considered to be reliable, reproducible, and useful to support pharmacokinetic and toxicokinetic studies with tolvaptan in rats.
Application of the method to a pharmacokinetic study Figure 4 shows representative courses of the serum concentration versus time of tolvaptan and its nine metabolites determined by the present LC–MS/MS method in male and female rats treated with oral tolvaptan at 30 mg/kg. The Pharmacokinetic parameters for tolvaptan and its nine Table 5 Pharmacokinetic parameters of tolvaptan and its metabolites in serum after single oral administration of tolvaptan at 30 mg/kg to rats Analyte Tolvaptan DM-4103 DM-4104 DM-4105 DM-4107 DM-4110
Gender
tmax (h)
Cmax (ng/mL)
AUCt (ng h/mL)
t1/2 (h)
Male
2
214
925
1.6
Female
4
1,759
7,437
2.5
Male
4
2,144
9,632
2.8
Female
4
864
6,493
3.7
Male
0.5
101
422
1.6
Female
4
383
1,700
3.8
Male
4
166
850
1.5
Female
4
133
811
3.4
Male
4
261
1,132
1.3
Female
4
524
3,064
2.7
Male
2
160
910
1.0 3.0
metabolites are summarized in Table 5. In the male rats, the Cmax, tmax, AUCt, and elimination t1/2 for tolvaptan were 214 ng/mL, 2 h, 925 ng h/mL, and 1.6 h, respectively. The rank order of the AUCt in the male rats were DM-4103 (1,041 %) [ DM-4107 (122 %) [ tolvaptan (100 %) [ DM-4110 (98 %) [ DM-4105 (92 %) [ DM-4119 (83 %) [ DM-4121 (69 %) [ DM-4104 (46 %) [ DM-4111 (43 %) [ MOP-21826 (0.49 %). In the female rats, the Cmax, tmax, AUCt, and elimination t1/2 for tolvaptan were 1,759 ng/mL, 4 h, 7,437 ng h/mL, and 2.5 h, respectively. The rank order of the AUCt in the female rats were as follows: tolvaptan (100 %) [ DM-4103 (87 %) [ DM-4119 (59 %) [ DM4110 (47 %) [ DM-4107 (41 %) [ DM-4121 (25 %) [ DM4104 and DM-4111 (23 %) [ DM-4105 (11 %) [ MOP-21826 (2.6 %). The oral pharmacokinetic study suggested that tolvaptan was rapidly absorbed in rats, and the Cmax and AUCt of tolvaptan in females were approximately eight times greater than those in the males. DM-4103 was prominent circulating metabolite in serum of male rats, while in female rats tolvaptan was metabolized to a lesser extent. In vitro metabolism studies of tolvaptan indicated that tolvaptan was eliminated primarily by metabolism via the CYP3A4 isozyme (Shoaf et al. 2011). The sex difference observed in this pharmacokinetic study was considered to be related to the sex difference in the metabolizing enzyme activities in the rat liver, since these activities were much higher in the male (Kato and Yamazoe 1992).
Conclusion We have developed and validated an LC–MS/MS method for determining of tolvaptan and its nine metabolites in rat serum. This method was demonstrated to be sensitive, selective, precise, and accurate. The validated method was successfully applied to the pharmacokinetic study of oral tolvaptan in rats. In male rats, the serum concentration of metabolite DM-4103 was higher than that of the unchanged compound, but in female rats, the serum concentration of the unchanged compound was higher than that of all metabolites. Tolvaptan was rapidly absorbed after oral administration and there was sex difference in the serum concentration–time profiles for tolvaptan and its metabolites in rats.
Female
4
625
3,473
DM-4111
Male
2
87
394
1.8
4 2
345 138
1,685 767
3.8 1.1
References
DM-4119
Female Male Female
4
887
4,389
3.3
Dixon, M.B., and Y.H. Lien. 2008. Tolvaptan and its potential in the treatment of hyponatremia. Therapeutics and Clinical Risk Management 4: 1149–1155. Ghali, J.K., B. Hamad, U. Yasothan, and P. Kirkpatrick. 2009. Tolvaptan. Nature Reviews Drug Discovery 8: 611–612. Furukawa, M., K. Umehara, and E. Kashiyama. 2011. Nonclinical pharmacokinetics of a new nonpeptide V2 receptor antagonist, tolvaptan. Cardiovascular Drugs and Therapy 25: S83–S89.
DM-4121 MOP-21826
Male
4
129
641
1.7
Female
4
249
1,859
3.1
Male
2
5
5
NC
Female
4
54
196
1.2
NC not calculated
123
M. Furukawa et al. Hori, M. 2011. Tolvaptan for heart failure patients with volume overload. Cardiovascular Drugs Therapy 25: S1–S4. Kato, R., and Y. Yamazoe. 1992. Sex-specific cytochrome P450 as a cause of sex- and species-related differences in drug toxicity. Toxicology Letters 64(65): 661–667. Kondo, K., H. Ogawa, H. Yamashita, H. Miyamoto, M. Tanaka, K. Nakaya, K. Kitano, Y. Yamamura, S. Nakamura, T. Onogawa, T. Mori, and M. Tominaga. 1999. 7-Chloro-5-hydroxy-1-[2methyl-4-(2-methylbenzoyl-amino)benzoyl]-2,3,4,5-tetrahydro1H-1benzazepine (OPC-41061): a potent, orally active nonpentide arginine vasopressin V2 receptor antagonist. Bioorganic and Medicinal Chemistry 7: 1743–1754. Miyazaki, T., H. Fujiki, Y. Yamamura, S. Nakamura, and T. Mori. 2007. Tolvaptan, an orally active vasopressin V(2)-receptor antagonist—Pharmacology and clinical trials. Cardiovascular Drug Reviews 25: 1–13. Miyazaki, T., H. Fujiki, and Y. Yamamura. 2013. Tolvaptan, an orally active non-peptide arginine vasopressin V2 receptor antagonist, reduces ascites in rats with chronic liver injury. Hepatology Research 43: 1224–1230. Pei, Q., B. Zhang, H. Tan, L. Liu, X. Peng, Z. Li, P. Huang, M. Luo, X. Zuo, C. Guo, and G. Yang. 2013. Development and validation
123
of an LC-MS/MS method for the determination of tolvaptan in human plasma and its application to a pharmacokinetic study. Journal of Chromatography B 913–914: 84–89. Sakaide, I., S. Yamashita, T. Kobayashi, M. Komatsu, T. Sakai, Y. Komorizono, M. Okada, and K. Okita. 2013. Efficacy and safety of a 14-day administration of tolvaptan in the treatment of patients with ascites in hepatic oedema. Journal of International Medical Research 41: 835–847. Shoaf, S.E., Y. Ohzone, S. Ninomiya, M. Furukawa, P. Bricmont, E. Kashiyama, and S. Mallikaarjun. 2011. In vitro P-glycoprotein interactions and steady-state pharmacokinetic interactions between tolvaptan and digoxin in healthy subjects. The Journal of Clinical Pharmacology 51: 761–769. Yamamura, Y., S. Nakamura, S. Itoh, T. Hirano, T. Onogawa, T. Yamashita, Y. Yamada, K. Tsujimae, M. Aoyama, K. Kotosai, H. Ogawa, H. Yamashita, K. Kondo, M. Tominaga, G. Tsujimoto, and T. Mori. 1998. OPC-41061, a highly potent human vasopressin V2-receptor antagonist: pharmacological profile and aquaretic effect by single and multiple oral dosing in rats. Journal of Pharmacology and Experimental Therapeutic 287: 860–867.
