Journal of Pharmaceutical and Biomedical Analysis 97 (2014) 33–38
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Journal of Pharmaceutical and Biomedical Analysis journal homepage: www.elsevier.com/locate/jpba
Short communication
Development and validation of UFLC–MS/MS method for determination of bosentan in rat plasma Alptug Atila a,∗ , Murat Ozturk b , Yucel Kadioglu a , Zekai Halici c , Didar Turkan a , Muhammed Yayla c , Harun Un d a
Ataturk University, Department of Analytical Chemistry, Faculty of Pharmacy, Erzurum 25240, Turkey Turkish Public Health Association, Directorate of Erzurum, Erzurum 25100, Turkey c Ataturk University, Department of Pharmacology, Faculty of Medicine, Erzurum 25240, Turkey d Ataturk University, Department of Biochemistry, Faculty of Pharmacy, Erzurum 25240, Turkey b
a r t i c l e
i n f o
Article history: Received 29 October 2013 Received in revised form 20 March 2014 Accepted 24 March 2014 Available online 4 April 2014 Keywords: Bosentan UFLC–MS/MS Rat plasma Validation Liquid-liquid extraction
a b s t r a c t A rapid, simple and sensitive UFLC–MS/MS method was developed and validated for the determination of bosentan in rat plasma using etodolac as an internal standard (IS) after liquid–liquid extraction with diethyl ether–chloroform (4:1, v/v). Bosentan and IS were detected using electrospray ionization in positive ion multiple reaction monitoring mode by monitoring the transitions m/z 551.90 → 201.90 and 288.20 → 172.00, respectively. Chromatographic separation was performed on the inertsil ODS-4 column with a gradient mobile phase, which consisted of 0.1% acetic acid with 5 mM ammonium acetate in water for solvent A and 5 mM ammonium acetate in acetonitrile–methanol (50:50, v/v) for solvent B at a flow rate of 0.3 mL/min. The method was sensitive with 0.5 ng/mL as the lower limit of quantitation (LLOQ) and the standard calibration curve for bosentan was linear (r > 0.997) over the studied concentration range (0.5–2000 ng/mL). The proposed method was fully validated by determining specificity, linearity, LLOQ, precision and accuracy, recovery, matrix effect and stability. The validated method was successfully applied to plasma samples obtained from rats. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Bosentan, [(4-tert-butyl-N-[6-(2-hydroxyethoxy)-5-(2methoxyphenoxy)-2-(pyrimidin-2-yl) pyrimidin-4-yl], is a competitive oral dual endothelin receptor antagonist which is non-selective for endothelin A and B receptors (Fig. 1). Bosentan is used as an oral drug for the treatment of pulmonary arterial hypertension [1,2]. It has a high protein binding (98%), especially to albumin and is rapidly absorbed after oral administration. Its bioavailability is 45–50%. The peak plasma concentration occurs within 3–5 h [3,4]. Bosentan is eliminated mainly by hepatic metabolism; renal elimination occurs for only 0.9% of the administered dose [5]. Few studies of high performance liquid chromatography with UV detection (HPLC-UV) [6,7] and liquid chromatography-tandem mass spectrometry (LC–MS/MS) methods have been presented for the determination of bosentan in biological matrices [8–14]. HPLC methods were long chromatographic run time, low sensitivity and, usually had large volumes of biological samples. The analytical time of each run in these reports were 24 min [6] and 16 min [7]. ∗ Corresponding author. Tel.: +90 442 2315216; fax: +90 442 2360962. E-mail address: [email protected] (A. Atila). http://dx.doi.org/10.1016/j.jpba.2014.03.039 0731-7085/© 2014 Elsevier B.V. All rights reserved.
The relatively long chromatographic run time could not satisfy the requirement of high throughput determination of bosentan in plasma. Therefore, mass spectrometric detection coupled to an ultra fast liquid chromatography (UFLC) method has been considered as very important to performing bioanalytical analysis with speed, selectivity and sensitivity. Therefore, we developed UFLC–MS/MS method for determination of bosentan in rat plasma. Then, the developed method was validated by using linearity, stability, precision, accuracy and sensitivity parameters according to Food and Drug Administration (FDA) guidelines [15]. Finally, plasma samples obtained from rats after oral administration of bosentan were analyzed in order to demonstrate the applicability of the method. 2. Experimental 2.1. Chemicals and reagents The reference standard of bosentan (purity > 99%) was obtained from Actelion Pharmaceuticals (Allschwil, Switzerland). Etodolac (IS, purity > 99%) was generously supplied by Novagenix Company (Ankara – Turkey). High-purity grade acetonitrile, methanol, acetic
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A. Atila et al. / Journal of Pharmaceutical and Biomedical Analysis 97 (2014) 33–38
phase. All the solutions were stored at −20 ◦ C until analysis and were brought to room temperature before use. The standard and QC samples were extracted on each analysis run along with the procedure described below. 2.4. Plasma sample preparation
acid, ammonium acetate and all other reagents were purchased from Merck (Darmstadt, Germany) and were used without further purification.
An aliquot of 250 L plasma sample was transferred to a polypropylene tube; 20 L of IS solution (100 ng/mL) were added and vortex was mixed for 30 s. 100 L of methanol and 500 L of 1 M HCl were added to the mixture. The mixture was vortexed for 30 s and extracted with 5 mL of diethyl ether–chloroform (4:1, v/v) by vortexing for another 60 s. After centrifugation at 3500 rpm for 8 min, the upper organic layer (4.9 mL) was then transferred into another clean polypropylene tube and evaporated to dryness at 40 ◦ C under a gentle stream of nitrogen. The residue was reconstituted with mobile phase solvent A–solvent B (20:80, v/v), and transferred into an autosampler vial. An aliquot of 1 L was injected into the UFLC–MS/MS system for analysis.
2.2. Instrumentation and operation conditions
2.5. Selection of internal standard
The Shimadzu UFLC-XR-MS/MS system consisted of an 8030 LC-MS/MS system, a CBM-20ALite system controller, two LC20ADXR pumps with a micro gradient mixer and DGU-20A3R degasser, one SIL-20ACXR autosampler with a cooling function and, one CTO-20AC column oven (Shimadzu Co., Kyoto/Japan). All of the operations and analysis of data obtained were controlled by lab solutions Main software. Inertsil ODS-4 column (3 m, 2.1 mm × 50 mm; GL Science, Japan) was employed for the separation at 40 ◦ C. The mobile phase was composed of 0.1% acetic acid with 5 mM ammonium acetate in water for solvent A and 5 mM ammonium acetate in acetonitrile–methanol (50:50, v/v) for solvent B. The gradient was 30% B (0–3.50 min), 95% B (3.51–6.5 min) and 30% B (6.51–10 min). Efficient and symmetrical peaks were obtained at a flow rate of 0.3 mL/min and injection volume of 10 L. Mass spectrometric detection was performed using ESI ion source in the positive ionization mode; the nebulizing gas, and drying gas flow rates and the ESI voltage were 3 L/min, 15 L/min and 4500 V, respectively. The collision gas was argon and had a pressure of 230 kPa. The gas used for nebulizing and drying was high pure nitrogen. MS data acquisition was conducted with the MRM mode in order to quantify and identify the investigated analytes. Detailed information for MS-parameters is represented in Table 1.
To meet the internal standard, short analysis time, good extraction recovery, chromatographic and mass spectrometric behaviour similar to the analytes is required. Also, analytes should be readily available. We evaluated different analytes such as etodolac, nimesulide, meloxicam, indomethacin, lidocaine, prilocaine and amlodipine as internal standards. Etodolac was selected as the internal standard for its similarity in retention time, mass conditions and extraction efficiency to those of the analytes.
Fig. 1. Chemical structure of bosentan.
2.3. Preparation of standards and quality control samples Stock solutions of bosentan and IS were prepared separately in the mobile phase solvent A–solvent B (20:80, v/v) at concentrations of 100 g/mL. Working solutions of bosentan were prepared by serial dilution of the stock solution in the mobile phase. Calibration standards were prepared by spiking 20 L of the appropriate standard solutions to 250 L of blank plasma giving concentrations of 0.5, 5, 25, 100, 500, 1000 and 2000 ng/mL. Quality control (QC) samples were prepared in the same way as the calibration standards, to achieve low, medium and high concentrations of 3, 600 and 1800 ng/mL. IS working solution (1 g/mL) for routine use was freshly prepared by diluting IS stock solution in the mobile
2.6. Method validation This method was fully validated for selectivity, linearity, precision and accuracy, recovery, matrix effect and stability according to FDA guidance for validation of bioanalytical methods [15]. 2.6.1. Selectivity and specificity The samples were prepared to determine whether endogenous matrix constituents interfere with the mass transitions chosen for bosentan and IS. From six different batches of blank rat plasma, blank (spiked with IS) and the LLOQ (at concentration of 0.5 ng/mL) samples were tested for interferences using the proposed liquid phase extraction procedure and UFLC–MS/MS conditions. 2.6.2. Linearity and sensitivity The seven-point calibration curve over the concentration range of 0.5–2000 ng/mL was constructed by plotting the peak area ratio of BOS/IS against the nominal concentration of calibration standards in blank rat plasma. Calibration curves of peak area ratio as a function of nominal concentration were linear using weighted (1/×2) linear regression. The LLOQ was defined as the lowest concentration on the calibration curve. It has an acceptable accuracy (relative error, RE) within ±15% and a precision (relative standard deviation, RSD) below 15% can be obtained. 2.6.3. Precision and accuracy The intra- and inter-day precision and accuracy in rat plasma were evaluated using three QC samples on the same day and on
Table 1 Detailed information of MS parameters for bosentan and IS. Analyte
Precursor
Product
Dwell time
q1 pre-bias (V)
Collision energy (V)
q3 pre-bias (V)
Bosentan IS
551.90 288.20
201.90 172.00
100.00 100.00
−26.00 −15.00
−38.00 −14.00
−13.00 −18.00
A. Atila et al. / Journal of Pharmaceutical and Biomedical Analysis 97 (2014) 33–38
three consecutive days. Each run consisted with six replicates of each concentration level (low, medium and high) sample. The precision was expressed by relative standard deviation (RSD) and accuracy by relative error (RE). The precision determined at each concentration level should not exceed 15% of the RSD and accuracy must be within 15% relative error of the actual values. 2.6.4. Extraction recovery and matrix effect Extraction recovery was obtained by comparing the peak areas six replicates of samples at three QC concentration levels after extraction with those of the processed blank plasma spiked with standard solutions at corresponding concentrations. Matrix effect was defined as the direct or indirect alteration or interference in response due to the presence of unintended analytes or other interfering substances in the sample [15]. The matrix effect of bosentan was investigated by comparing the amount of bosentan solutions with processed blank samples reconstituted with bosentan solutions. The blank plasmas used in this study were from six different batches of rat blank plasma. If the ratio 115%, a matrix effect was implied. 2.6.5. Stability experiments The stock solution stability studies indicated that bosentan was stable when kept refrigerated at −20 ◦ C for six months. The stability of bosentan in rat plasma was evaluated by analyzing six replicates of low and high QC samples. The samples were exposed to different time and temperature conditions. The freeze–thaw stability was determined after three freeze–thaw cycles. Post-preparation stability was estimated by analyzing QC samples in the autosampler at 4 ◦ C for 12 h. QC samples were stored at −20 ◦ C for 20 days and at ambient temperature for 4 h to determine long- and shortterm stability, respectively. All stability testing QC samples were determined using calibration curves of freshly prepared standards. The concentrations obtained were compared with the nominal values. 2.7. Animals 10 male Albino Wistar rats weighing 200–230 g were used in the study. The experiments were conducted according to the ethical norms approved by the Ethics Committee of the Experimental Animal Teaching and Research Centre (No. 93722986.03-678). Rats were obtained from Medicinal and Experimental Application and Research Centre, Erzurum, Turkey (ATADEM). They were kept in standard laboratory conditions under a natural cycle of light and dark. The animals were given water and fed a normal diet. The 10 rats were divided into two groups (n = 5 in each group; Group 1: 50 mg/kg bosentan group; Group 2: 100 mg/kg bosentan group). Each dose was administered to the rats (via oral gavages). The rats received bosentan with an oral gavage suspended in saline to a final concentration of 2 mL. 3. Results and discussion 3.1. Optimization of chromatography Chromatographic conditions, especially the type of column and composition of the mobile phase, were optimized to achieve good peak shape, high response and short retention time. The best peak shape and highest peak area for bosentan and IS were obtained using 0.1% acetic acid with 5 mM ammonium acetate in water for solvent A and 5 mM ammonium acetate in acetonitrile–methanol (50:50, v/v) for solvent B. In addition, formic acid and ammonium acetate were added in the mobile phase separately to improve the response. The effect of ammonium acetate at different concentrations (5, 10 and 15 mmol/L) on the response and peak shape of
35
analytes was investigated and 5 mmol/L ammonium acetate was found to be the best. The gradient during each run ensures the column was clean for the next analytical run. Compared with other literatures 24 min [6], 16 min [7] and 7 min [11], the method is advantageous because of suitable retention time which is 4.6 min for bosentan. However to make sure that the colon is completely cleaned between two injections we allow the mobile phase to flow until the 10th minute. If one needs to perform a faster analysis it is also possible to perform second injection after the 5th minute (after observing bosentan and IS peaks completely). 3.2. Optimization of mass spectrometry In order to optimize ESI conditions for bosentan and IS, the MS full scans were carried out in positive/negative ion detection modes. The MS spectra of bosentan and IS were recorded in positive ion mode. The negative ion mode was also tested, but the intensity obtained was very low for this analyte. For optimizing the MS parameters, standard solution (1.0 g/mL) of bosentan and the IS were directly infused given with the mobile phase into the mass spectrometer. The MS/MS parameters were optimized to maximize the response for the precursor/product ion combination of m/z 551.90 → 201.90 for bosentan and 288.20 → 172.00 for IS (Fig. 2). 3.3. Selection of the extraction method Liquid–liquid extraction and protein precipitation have been widely used in the preparation of biological samples for accurate and reliable assays [16]. In the earlier stage of the method development, several extraction methods were investigated. Methanol was chosen as the protein precipitant for its satisfactory efficiency in the precipitating process. During the optimization of the extraction procedure, diethyl ether, chloroform, dichloromethane and ethyl acetate were tested as extraction solvents in different volume ratios. The extraction efficiency of bosentan was found to be very high, and a significant increase was observed in the extraction efficiency using diethyl ether–chloroform (4:1, v/v). These results indicated that this technique was likely to efficiently extract bosentan from rat plasma. Compared with other sample preparation procedures, this extraction method was cheaper than procedure based on solid phase extraction on Oasis HLB cartridges [9]. Also, this extraction method does not take as much time as reported in other papers [9–11]. 3.4. Method validation 3.4.1. Selectivity and specificity The assay condition had sufficient specificity for analytes as no endogenous interference peaks were observed at the retention time of bosentan and IS. As shown in Fig. 3, the peak response with two different transitions had the same retention time for bosentan and IS, but varied in response. Typical MRM chromatograms for blank rat plasma, blank rat plasma spiked with IS, blank rat plasma spiked with bosentan, and response for rat samples (50 mg/kg and 100 mg/kg; 5 h after oral administration) are presented in Fig. 3. 3.4.2. Linearity and LLOQ The calibration curve for bosentan was linear well within the range of 0.5–2000 ng/mL. The mean value of the regression equation was y = 0.011x + 1502, with a correlation coefficient >0.997, where y is the peak-area ratio of bosentan to IS and x is the plasma concentration of bosentan. The LLOQ for bosentan was determined as 0.5 ng/mL. For this value, the accuracy and precision were less
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Fig. 2. Product ion mass spectra in the positive ionization mode for (A) bosentan (m/z 551.90 → 201.90), (B) IS (m/z 288.20 → 172.00).
Fig. 3. Typical MRM chromatograms of (C) blank rat plasma and plasma spiked with bosentan (25 ng/ml); (D) blank rat plasma and IS (100 ng/ml); (E) rat plasma after 5.0 h oral dose of 50 mg/kg and 100 mg/kg bosentan.
A. Atila et al. / Journal of Pharmaceutical and Biomedical Analysis 97 (2014) 33–38
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Table 2 Precision and accuracy for the determination of bosentan in rat plasma (mean ± SD, in three consecutive days, six replicates for each concentration). Analyte
Bosentan
Intra-day (n = 6)
Nominal conc. (ng/mL) 1 600 1800
Inter-day (n = 6)
Found (ng/mL)
RSD (%)
RE (%)
Found (ng/mL)
RSD (%)
RE (%)
1.03 ± 0.05 621.49 ± 35.88 1715.19 ± 64.51
4.85 5.77 3.76
3.00 3.58 −4.71
0.98 ± 0.04 561.65 ± 21.11 1742.92 ± 75.24
4.08 3.76 4.32
−2.00 −6.39 −3.17
Table 3 Stability of bosentan in rat plasma under various conditions (mean ± SD, six replicates for each concentration). Stability
3 ng/mL Found (ng/mL)
Short-term Long-term Post-preparative Freeze–thaw stability
3.05 2.91 2.87 3.10
± ± ± ±
0.06 0.14 0.12 0.17
1800 ng/mL RSD (%)
RE (%)
Found (ng/mL)
1.97 4.81 4.18 5.48
1.67 −3.00 −4.33 3.33
1751.19 1897.44 1865.21 1708.25
± ± ± ±
71.42 105.41 67.15 87.24
RSD (%)
RE (%)
4.08 5.56 3.60 5.11
−2.71 5.41 3.62 −5.09
than 6.95% (RE%) and 7.59% (RSD%), respectively. Although there are few reports about quantitative analysis of bosentan in plasma, some of them sensitivity are limited for bioanalysis studies [6,7]. With the present LLOQ, bosentan was determined in rat plasma, which was more sensitive than the previously reported methods [6–8,11–13].
guidelines. The results of the validation studies demonstrated that the developed method had high sensitivity, recovery, precision, accuracy and reproducibility. Finally, the method was successfully used for real rat plasma samples.
3.4.3. Precision and accuracy Accuracy and precision data for intra- and inter-day plasma samples are presented in Table 2. The assay values on both the occasions (intra- and inter-day) were found to be within the accepted variable limits [15].
The author(s) declares that they have no conflicts of interest to disclose.
3.4.4. Extraction recovery and matrix effect A simple liquid–liquid extraction proved to be robust and provided the cleanest samples. The extraction recoveries from rat plasma were estimated for bosentan at 3, 600 and 1800 ng/mL and the mean recovery was found to be 85.15 ± 8.52, 87.58 ± 6.54 and 83.02 ± 3.86%, respectively. At the same concentration levels, the matrix effects on bosentan were 94.31 ± 11.72, 88.52 ± 6.36 and 91.55 ± 9.74%, respectively. The matrix effect of IS was 103.91 ± 4.93%. The results demonstrated that no co-eluting substance interfered during the ionization of bosentan and IS. Additionally, the small sample volume (250 L) is an advantage over other method [10]. 3.4.5. Stability of samples According to all the stability tests shown in Table 3, bosentan was stable under routine laboratory conditions. For this reason, this method is proved to be applicable for routine analysis. 3.4.6. Application of the method to real rat plasma samples The rats were killed 5 h after administration of drugs with an overdose of a general anaesthetic (thiopental sodium, 50 mg/kg). The blood samples that were collected from the hearts of rats were transferred in 2 mL EDTA vacuum glass tubes to determine the concentrations of bosentan. The plasma was immediately separated by centrifugation at 4000 rpm for 10 min. Plasma samples were prepared as described in Section 3.3. The method was successfully applied to determine of bosentan in rat plasma. 4. Conclusion In summary, a new, simple, fast and high throughput UFLC–MS/MS method was developed for the analysis of bosentan in rat plasma. The method was fully validated in accordance to FDA
Conflict of interest
References [1] M. Clozel, V. Breu, G.A. Gray, B. Kalina, K. Burri, J.M. Cassal, G. Hirth, M. Müller, W. Neidhart, Pharmacological characterization of bosentan, a new potent orally active nonpeptide endothelin receptor antagonist, J. Pharmacol. Exp. Ther. 270 (1994) 228–235. [2] S. Motte, K. McEntee, R. Naeije, Endothelin receptor antagonists, Pharmacol. Ther. 110 (2006) 386–414. [3] M.L. Spangler, S. Saxena, Warfarin and bosentan interaction in a patient with pulmonary hypertension secondary to bilateral pulmonary emboli, Clin. Ther. 32 (2010) 53–56. [4] TracleerTM (bosentan) Product Information, Actelion Pharmaceuticals. Available from , 2011. [5] C. Weber, R. Gasser, G. Hopfgartner, Absorption, excretion, and metabolism of the endothelin receptor antagonist bosentan in healthy male subjects, Drug Metab. Dispos. 27 (1999) 810–815. [6] M. Taguchi, F. Ichida, K. Hirono, T. Mivawaki, N. Yoshimura, T. Nakumura, C. Akita, T. Nakavama, T. Saji, Y. Kato, I. Horiuchi, Y. Hashimoto, Pharmacokinetics of bosentan in routinely treated Japanese pediatric patients with pulmonary arterial hypertension, Drug Metab. Pharmacokinet. 26 (2011) 280–287. [7] I. Horiuchi, Y. Mori, M. Taguchi, F. Ichida, T. Miyawaki, Y. Hashimoto, Mechanisms responsible for the altered pharmacokinetics of Bosentan: analysis utilizing rats with bile duct ligation-induced liver dysfunction, Biopharm. Drug Dispos. 30 (2009) 326–333. [8] G. Hopfgartner, D. Tonoli, E. Varesio, High-resolution mass spectrometry for integrated qualitative and quantitative analysis of pharmaceuticals in biological matrices, Anal. Bioanal. Chem. 402 (2012) 2587–2596. [9] J.M. Parekha, D.K. Shahb, M. Sanyalc, M. Yadavd, P.S. Shrivastav, Development of an SPE-LC-MS/MS method for simultaneous quantification of bosentan and its active metabolite hydroxybosentan in human plasma to support a bioequivalence study, J. Pharm. Biomed. Anal. 70 (2012) 462–470. [10] B. Lausecker, G. Hopfgartner, Determination of an endothelin receptor antagonist in human plasma by narrow-bore liquid chromatography and ionspray tandem mass spectrometry, J. Chromatogr. A 712 (1995) 75–83. [11] B. Lausecker, B. Hess, G. Fischer, M. Mueller, G. Hopfgartner, Simultaneous determination of bosentan and its three major metabolites in various biological matrices and species using narrow bore liquid chromatography with ion spray tandem mass spectrometric detection, J. Chromatogr. B 749 (2000) 67–83. [12] N. Ganz, M. Singrasa, L. Nicolasb, M. Gutierrezb, J. Dingemanseb, W. Döbelinc, M. Glinskia, Development and validation of a fully automated online human dried blood spot analysis of bosentan and its metabolites using the Sample Card And Prep DBS System, J. Chromatogr. B 885–886 (2012) 50–60. [13] P.L.M. van Giersbergen, A. Halabi, J. Dingemanse, Single- and multiple-dose pharmacokinetics of bosentan and its interaction with ketoconazole, Br. J. Clin. Pharmacol. 53 (2002) 589–595.
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[14] G. Hopfgartnert, W. Vetter, W. Meister, H. Ramuz, Fragmentation of Bosentan® (Ro 47-0203) in ionspray mass spectrometry after collision-induced dissociation at low energy: a case of radical fragmentation of an even-electron ion, J. Mass Spectrom. 31 (1996) 69–76. [15] U.S. Food And Drug Administration, Guidance for Industry Bioanalytical Method Validation. Available from: , 2001. [16] Y. Kadioglu, A. Atila, Development and validation of gas chromatography–mass spectroscopy method for determination of prilocaine HCl in human plasma using internal standard methodology, Biomed. Chromatogr. 21 (2007) 1077–1082.
Contents lists available at ScienceDirect
Journal of Pharmaceutical and Biomedical Analysis journal homepage: www.elsevier.com/locate/jpba
Short communication
Development and validation of UFLC–MS/MS method for determination of bosentan in rat plasma Alptug Atila a,∗ , Murat Ozturk b , Yucel Kadioglu a , Zekai Halici c , Didar Turkan a , Muhammed Yayla c , Harun Un d a
Ataturk University, Department of Analytical Chemistry, Faculty of Pharmacy, Erzurum 25240, Turkey Turkish Public Health Association, Directorate of Erzurum, Erzurum 25100, Turkey c Ataturk University, Department of Pharmacology, Faculty of Medicine, Erzurum 25240, Turkey d Ataturk University, Department of Biochemistry, Faculty of Pharmacy, Erzurum 25240, Turkey b
a r t i c l e
i n f o
Article history: Received 29 October 2013 Received in revised form 20 March 2014 Accepted 24 March 2014 Available online 4 April 2014 Keywords: Bosentan UFLC–MS/MS Rat plasma Validation Liquid-liquid extraction
a b s t r a c t A rapid, simple and sensitive UFLC–MS/MS method was developed and validated for the determination of bosentan in rat plasma using etodolac as an internal standard (IS) after liquid–liquid extraction with diethyl ether–chloroform (4:1, v/v). Bosentan and IS were detected using electrospray ionization in positive ion multiple reaction monitoring mode by monitoring the transitions m/z 551.90 → 201.90 and 288.20 → 172.00, respectively. Chromatographic separation was performed on the inertsil ODS-4 column with a gradient mobile phase, which consisted of 0.1% acetic acid with 5 mM ammonium acetate in water for solvent A and 5 mM ammonium acetate in acetonitrile–methanol (50:50, v/v) for solvent B at a flow rate of 0.3 mL/min. The method was sensitive with 0.5 ng/mL as the lower limit of quantitation (LLOQ) and the standard calibration curve for bosentan was linear (r > 0.997) over the studied concentration range (0.5–2000 ng/mL). The proposed method was fully validated by determining specificity, linearity, LLOQ, precision and accuracy, recovery, matrix effect and stability. The validated method was successfully applied to plasma samples obtained from rats. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Bosentan, [(4-tert-butyl-N-[6-(2-hydroxyethoxy)-5-(2methoxyphenoxy)-2-(pyrimidin-2-yl) pyrimidin-4-yl], is a competitive oral dual endothelin receptor antagonist which is non-selective for endothelin A and B receptors (Fig. 1). Bosentan is used as an oral drug for the treatment of pulmonary arterial hypertension [1,2]. It has a high protein binding (98%), especially to albumin and is rapidly absorbed after oral administration. Its bioavailability is 45–50%. The peak plasma concentration occurs within 3–5 h [3,4]. Bosentan is eliminated mainly by hepatic metabolism; renal elimination occurs for only 0.9% of the administered dose [5]. Few studies of high performance liquid chromatography with UV detection (HPLC-UV) [6,7] and liquid chromatography-tandem mass spectrometry (LC–MS/MS) methods have been presented for the determination of bosentan in biological matrices [8–14]. HPLC methods were long chromatographic run time, low sensitivity and, usually had large volumes of biological samples. The analytical time of each run in these reports were 24 min [6] and 16 min [7]. ∗ Corresponding author. Tel.: +90 442 2315216; fax: +90 442 2360962. E-mail address: [email protected] (A. Atila). http://dx.doi.org/10.1016/j.jpba.2014.03.039 0731-7085/© 2014 Elsevier B.V. All rights reserved.
The relatively long chromatographic run time could not satisfy the requirement of high throughput determination of bosentan in plasma. Therefore, mass spectrometric detection coupled to an ultra fast liquid chromatography (UFLC) method has been considered as very important to performing bioanalytical analysis with speed, selectivity and sensitivity. Therefore, we developed UFLC–MS/MS method for determination of bosentan in rat plasma. Then, the developed method was validated by using linearity, stability, precision, accuracy and sensitivity parameters according to Food and Drug Administration (FDA) guidelines [15]. Finally, plasma samples obtained from rats after oral administration of bosentan were analyzed in order to demonstrate the applicability of the method. 2. Experimental 2.1. Chemicals and reagents The reference standard of bosentan (purity > 99%) was obtained from Actelion Pharmaceuticals (Allschwil, Switzerland). Etodolac (IS, purity > 99%) was generously supplied by Novagenix Company (Ankara – Turkey). High-purity grade acetonitrile, methanol, acetic
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A. Atila et al. / Journal of Pharmaceutical and Biomedical Analysis 97 (2014) 33–38
phase. All the solutions were stored at −20 ◦ C until analysis and were brought to room temperature before use. The standard and QC samples were extracted on each analysis run along with the procedure described below. 2.4. Plasma sample preparation
acid, ammonium acetate and all other reagents were purchased from Merck (Darmstadt, Germany) and were used without further purification.
An aliquot of 250 L plasma sample was transferred to a polypropylene tube; 20 L of IS solution (100 ng/mL) were added and vortex was mixed for 30 s. 100 L of methanol and 500 L of 1 M HCl were added to the mixture. The mixture was vortexed for 30 s and extracted with 5 mL of diethyl ether–chloroform (4:1, v/v) by vortexing for another 60 s. After centrifugation at 3500 rpm for 8 min, the upper organic layer (4.9 mL) was then transferred into another clean polypropylene tube and evaporated to dryness at 40 ◦ C under a gentle stream of nitrogen. The residue was reconstituted with mobile phase solvent A–solvent B (20:80, v/v), and transferred into an autosampler vial. An aliquot of 1 L was injected into the UFLC–MS/MS system for analysis.
2.2. Instrumentation and operation conditions
2.5. Selection of internal standard
The Shimadzu UFLC-XR-MS/MS system consisted of an 8030 LC-MS/MS system, a CBM-20ALite system controller, two LC20ADXR pumps with a micro gradient mixer and DGU-20A3R degasser, one SIL-20ACXR autosampler with a cooling function and, one CTO-20AC column oven (Shimadzu Co., Kyoto/Japan). All of the operations and analysis of data obtained were controlled by lab solutions Main software. Inertsil ODS-4 column (3 m, 2.1 mm × 50 mm; GL Science, Japan) was employed for the separation at 40 ◦ C. The mobile phase was composed of 0.1% acetic acid with 5 mM ammonium acetate in water for solvent A and 5 mM ammonium acetate in acetonitrile–methanol (50:50, v/v) for solvent B. The gradient was 30% B (0–3.50 min), 95% B (3.51–6.5 min) and 30% B (6.51–10 min). Efficient and symmetrical peaks were obtained at a flow rate of 0.3 mL/min and injection volume of 10 L. Mass spectrometric detection was performed using ESI ion source in the positive ionization mode; the nebulizing gas, and drying gas flow rates and the ESI voltage were 3 L/min, 15 L/min and 4500 V, respectively. The collision gas was argon and had a pressure of 230 kPa. The gas used for nebulizing and drying was high pure nitrogen. MS data acquisition was conducted with the MRM mode in order to quantify and identify the investigated analytes. Detailed information for MS-parameters is represented in Table 1.
To meet the internal standard, short analysis time, good extraction recovery, chromatographic and mass spectrometric behaviour similar to the analytes is required. Also, analytes should be readily available. We evaluated different analytes such as etodolac, nimesulide, meloxicam, indomethacin, lidocaine, prilocaine and amlodipine as internal standards. Etodolac was selected as the internal standard for its similarity in retention time, mass conditions and extraction efficiency to those of the analytes.
Fig. 1. Chemical structure of bosentan.
2.3. Preparation of standards and quality control samples Stock solutions of bosentan and IS were prepared separately in the mobile phase solvent A–solvent B (20:80, v/v) at concentrations of 100 g/mL. Working solutions of bosentan were prepared by serial dilution of the stock solution in the mobile phase. Calibration standards were prepared by spiking 20 L of the appropriate standard solutions to 250 L of blank plasma giving concentrations of 0.5, 5, 25, 100, 500, 1000 and 2000 ng/mL. Quality control (QC) samples were prepared in the same way as the calibration standards, to achieve low, medium and high concentrations of 3, 600 and 1800 ng/mL. IS working solution (1 g/mL) for routine use was freshly prepared by diluting IS stock solution in the mobile
2.6. Method validation This method was fully validated for selectivity, linearity, precision and accuracy, recovery, matrix effect and stability according to FDA guidance for validation of bioanalytical methods [15]. 2.6.1. Selectivity and specificity The samples were prepared to determine whether endogenous matrix constituents interfere with the mass transitions chosen for bosentan and IS. From six different batches of blank rat plasma, blank (spiked with IS) and the LLOQ (at concentration of 0.5 ng/mL) samples were tested for interferences using the proposed liquid phase extraction procedure and UFLC–MS/MS conditions. 2.6.2. Linearity and sensitivity The seven-point calibration curve over the concentration range of 0.5–2000 ng/mL was constructed by plotting the peak area ratio of BOS/IS against the nominal concentration of calibration standards in blank rat plasma. Calibration curves of peak area ratio as a function of nominal concentration were linear using weighted (1/×2) linear regression. The LLOQ was defined as the lowest concentration on the calibration curve. It has an acceptable accuracy (relative error, RE) within ±15% and a precision (relative standard deviation, RSD) below 15% can be obtained. 2.6.3. Precision and accuracy The intra- and inter-day precision and accuracy in rat plasma were evaluated using three QC samples on the same day and on
Table 1 Detailed information of MS parameters for bosentan and IS. Analyte
Precursor
Product
Dwell time
q1 pre-bias (V)
Collision energy (V)
q3 pre-bias (V)
Bosentan IS
551.90 288.20
201.90 172.00
100.00 100.00
−26.00 −15.00
−38.00 −14.00
−13.00 −18.00
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three consecutive days. Each run consisted with six replicates of each concentration level (low, medium and high) sample. The precision was expressed by relative standard deviation (RSD) and accuracy by relative error (RE). The precision determined at each concentration level should not exceed 15% of the RSD and accuracy must be within 15% relative error of the actual values. 2.6.4. Extraction recovery and matrix effect Extraction recovery was obtained by comparing the peak areas six replicates of samples at three QC concentration levels after extraction with those of the processed blank plasma spiked with standard solutions at corresponding concentrations. Matrix effect was defined as the direct or indirect alteration or interference in response due to the presence of unintended analytes or other interfering substances in the sample [15]. The matrix effect of bosentan was investigated by comparing the amount of bosentan solutions with processed blank samples reconstituted with bosentan solutions. The blank plasmas used in this study were from six different batches of rat blank plasma. If the ratio 115%, a matrix effect was implied. 2.6.5. Stability experiments The stock solution stability studies indicated that bosentan was stable when kept refrigerated at −20 ◦ C for six months. The stability of bosentan in rat plasma was evaluated by analyzing six replicates of low and high QC samples. The samples were exposed to different time and temperature conditions. The freeze–thaw stability was determined after three freeze–thaw cycles. Post-preparation stability was estimated by analyzing QC samples in the autosampler at 4 ◦ C for 12 h. QC samples were stored at −20 ◦ C for 20 days and at ambient temperature for 4 h to determine long- and shortterm stability, respectively. All stability testing QC samples were determined using calibration curves of freshly prepared standards. The concentrations obtained were compared with the nominal values. 2.7. Animals 10 male Albino Wistar rats weighing 200–230 g were used in the study. The experiments were conducted according to the ethical norms approved by the Ethics Committee of the Experimental Animal Teaching and Research Centre (No. 93722986.03-678). Rats were obtained from Medicinal and Experimental Application and Research Centre, Erzurum, Turkey (ATADEM). They were kept in standard laboratory conditions under a natural cycle of light and dark. The animals were given water and fed a normal diet. The 10 rats were divided into two groups (n = 5 in each group; Group 1: 50 mg/kg bosentan group; Group 2: 100 mg/kg bosentan group). Each dose was administered to the rats (via oral gavages). The rats received bosentan with an oral gavage suspended in saline to a final concentration of 2 mL. 3. Results and discussion 3.1. Optimization of chromatography Chromatographic conditions, especially the type of column and composition of the mobile phase, were optimized to achieve good peak shape, high response and short retention time. The best peak shape and highest peak area for bosentan and IS were obtained using 0.1% acetic acid with 5 mM ammonium acetate in water for solvent A and 5 mM ammonium acetate in acetonitrile–methanol (50:50, v/v) for solvent B. In addition, formic acid and ammonium acetate were added in the mobile phase separately to improve the response. The effect of ammonium acetate at different concentrations (5, 10 and 15 mmol/L) on the response and peak shape of
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analytes was investigated and 5 mmol/L ammonium acetate was found to be the best. The gradient during each run ensures the column was clean for the next analytical run. Compared with other literatures 24 min [6], 16 min [7] and 7 min [11], the method is advantageous because of suitable retention time which is 4.6 min for bosentan. However to make sure that the colon is completely cleaned between two injections we allow the mobile phase to flow until the 10th minute. If one needs to perform a faster analysis it is also possible to perform second injection after the 5th minute (after observing bosentan and IS peaks completely). 3.2. Optimization of mass spectrometry In order to optimize ESI conditions for bosentan and IS, the MS full scans were carried out in positive/negative ion detection modes. The MS spectra of bosentan and IS were recorded in positive ion mode. The negative ion mode was also tested, but the intensity obtained was very low for this analyte. For optimizing the MS parameters, standard solution (1.0 g/mL) of bosentan and the IS were directly infused given with the mobile phase into the mass spectrometer. The MS/MS parameters were optimized to maximize the response for the precursor/product ion combination of m/z 551.90 → 201.90 for bosentan and 288.20 → 172.00 for IS (Fig. 2). 3.3. Selection of the extraction method Liquid–liquid extraction and protein precipitation have been widely used in the preparation of biological samples for accurate and reliable assays [16]. In the earlier stage of the method development, several extraction methods were investigated. Methanol was chosen as the protein precipitant for its satisfactory efficiency in the precipitating process. During the optimization of the extraction procedure, diethyl ether, chloroform, dichloromethane and ethyl acetate were tested as extraction solvents in different volume ratios. The extraction efficiency of bosentan was found to be very high, and a significant increase was observed in the extraction efficiency using diethyl ether–chloroform (4:1, v/v). These results indicated that this technique was likely to efficiently extract bosentan from rat plasma. Compared with other sample preparation procedures, this extraction method was cheaper than procedure based on solid phase extraction on Oasis HLB cartridges [9]. Also, this extraction method does not take as much time as reported in other papers [9–11]. 3.4. Method validation 3.4.1. Selectivity and specificity The assay condition had sufficient specificity for analytes as no endogenous interference peaks were observed at the retention time of bosentan and IS. As shown in Fig. 3, the peak response with two different transitions had the same retention time for bosentan and IS, but varied in response. Typical MRM chromatograms for blank rat plasma, blank rat plasma spiked with IS, blank rat plasma spiked with bosentan, and response for rat samples (50 mg/kg and 100 mg/kg; 5 h after oral administration) are presented in Fig. 3. 3.4.2. Linearity and LLOQ The calibration curve for bosentan was linear well within the range of 0.5–2000 ng/mL. The mean value of the regression equation was y = 0.011x + 1502, with a correlation coefficient >0.997, where y is the peak-area ratio of bosentan to IS and x is the plasma concentration of bosentan. The LLOQ for bosentan was determined as 0.5 ng/mL. For this value, the accuracy and precision were less
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A. Atila et al. / Journal of Pharmaceutical and Biomedical Analysis 97 (2014) 33–38
Fig. 2. Product ion mass spectra in the positive ionization mode for (A) bosentan (m/z 551.90 → 201.90), (B) IS (m/z 288.20 → 172.00).
Fig. 3. Typical MRM chromatograms of (C) blank rat plasma and plasma spiked with bosentan (25 ng/ml); (D) blank rat plasma and IS (100 ng/ml); (E) rat plasma after 5.0 h oral dose of 50 mg/kg and 100 mg/kg bosentan.
A. Atila et al. / Journal of Pharmaceutical and Biomedical Analysis 97 (2014) 33–38
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Table 2 Precision and accuracy for the determination of bosentan in rat plasma (mean ± SD, in three consecutive days, six replicates for each concentration). Analyte
Bosentan
Intra-day (n = 6)
Nominal conc. (ng/mL) 1 600 1800
Inter-day (n = 6)
Found (ng/mL)
RSD (%)
RE (%)
Found (ng/mL)
RSD (%)
RE (%)
1.03 ± 0.05 621.49 ± 35.88 1715.19 ± 64.51
4.85 5.77 3.76
3.00 3.58 −4.71
0.98 ± 0.04 561.65 ± 21.11 1742.92 ± 75.24
4.08 3.76 4.32
−2.00 −6.39 −3.17
Table 3 Stability of bosentan in rat plasma under various conditions (mean ± SD, six replicates for each concentration). Stability
3 ng/mL Found (ng/mL)
Short-term Long-term Post-preparative Freeze–thaw stability
3.05 2.91 2.87 3.10
± ± ± ±
0.06 0.14 0.12 0.17
1800 ng/mL RSD (%)
RE (%)
Found (ng/mL)
1.97 4.81 4.18 5.48
1.67 −3.00 −4.33 3.33
1751.19 1897.44 1865.21 1708.25
± ± ± ±
71.42 105.41 67.15 87.24
RSD (%)
RE (%)
4.08 5.56 3.60 5.11
−2.71 5.41 3.62 −5.09
than 6.95% (RE%) and 7.59% (RSD%), respectively. Although there are few reports about quantitative analysis of bosentan in plasma, some of them sensitivity are limited for bioanalysis studies [6,7]. With the present LLOQ, bosentan was determined in rat plasma, which was more sensitive than the previously reported methods [6–8,11–13].
guidelines. The results of the validation studies demonstrated that the developed method had high sensitivity, recovery, precision, accuracy and reproducibility. Finally, the method was successfully used for real rat plasma samples.
3.4.3. Precision and accuracy Accuracy and precision data for intra- and inter-day plasma samples are presented in Table 2. The assay values on both the occasions (intra- and inter-day) were found to be within the accepted variable limits [15].
The author(s) declares that they have no conflicts of interest to disclose.
3.4.4. Extraction recovery and matrix effect A simple liquid–liquid extraction proved to be robust and provided the cleanest samples. The extraction recoveries from rat plasma were estimated for bosentan at 3, 600 and 1800 ng/mL and the mean recovery was found to be 85.15 ± 8.52, 87.58 ± 6.54 and 83.02 ± 3.86%, respectively. At the same concentration levels, the matrix effects on bosentan were 94.31 ± 11.72, 88.52 ± 6.36 and 91.55 ± 9.74%, respectively. The matrix effect of IS was 103.91 ± 4.93%. The results demonstrated that no co-eluting substance interfered during the ionization of bosentan and IS. Additionally, the small sample volume (250 L) is an advantage over other method [10]. 3.4.5. Stability of samples According to all the stability tests shown in Table 3, bosentan was stable under routine laboratory conditions. For this reason, this method is proved to be applicable for routine analysis. 3.4.6. Application of the method to real rat plasma samples The rats were killed 5 h after administration of drugs with an overdose of a general anaesthetic (thiopental sodium, 50 mg/kg). The blood samples that were collected from the hearts of rats were transferred in 2 mL EDTA vacuum glass tubes to determine the concentrations of bosentan. The plasma was immediately separated by centrifugation at 4000 rpm for 10 min. Plasma samples were prepared as described in Section 3.3. The method was successfully applied to determine of bosentan in rat plasma. 4. Conclusion In summary, a new, simple, fast and high throughput UFLC–MS/MS method was developed for the analysis of bosentan in rat plasma. The method was fully validated in accordance to FDA
Conflict of interest
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