BIOANALYTICAL METHOD DEVELOPMENT, VALIDATION AND QUANTIFICATION OF BOSENTAN BY HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY IN RAT PLASMA

: A Simple and rapid bioanalytical high performance liquid chromatographic (HPLC) method for the determination of Bosentan using Losertan as an internal standard was developed and validated as per regulatory requirements. Sample preparation was accomplished through liquid phase extraction and chromatographic separation on a reverse phase column. The mobile phase consists of mixture of methanol and water in the ratio of 50:50 at a flow rate of 1ml/min. The wavelength used for the detection of bosentan was 225nm with a total run time of 6minutes. The retention times of bosentan and losertan were found to be 2 and 4 respectively. The method was developed and tested for the linearity range of 250-750ng/ml. The method was validated for accuracy,precision,linearity, and recovery in compliance to international regulatory guidelines.


INTRODUCTION:
Bosentan, 4-t-butyl-N-(6-(2-hydroxyethoxy)-5-(2methoxyphenoxy)-2,2-bipyrimidin-4yl)benzenesulfonamide, is a endothelian receptor antagonist used in the treatment of pulmonary artery hypertension. Bosentan is available in the tablet dosage form. Hypertension also known as high blood pressure is a long term medical condition in which the blood pressure in the arteries is persistently elevated. High blood pressure usually does not cause symptoms. Endothelins are peptides that constrict blood vessels and raise blood pressure. They are normally kept in balance by other mechanisms, but when they are over-expressed, they contribute to high blood pressure (hypertension) and heart disease [1][2][3][4][5].
Endothelins are 21-amino acid vasoconstricting peptides produced primarily in the endothelium having a key role in vascular homeostasis. Endothelins are implicated in vascular diseases of several organ systems, including the heart, general circulation and brain [6][7][8][9][10]. Literature survey has revealed that there only few methods were reported for the determination of BOSENTAN in plasma by liquid chromatography. Methods reported in the literature for the estimation of bosentan in bulk and biological fluids include A rapid high-performance liquid chromatographic bioanalytical method development and validation for Bosantan in human plasma by taking 70% ammonium acetate & 30% acetonitrile as a mobile phase and Phenomenex luna C18 as column at 5.7-7.8mins as retention time. Development, estimation and validation of Bosentan in bulk and in its pharmaceutical formulation by UV-Vis Spectroscopic method using Methanol: Water as mobile phase. From the literature survey, various analytical method developments have been reported for the estimation of bosentan by RP-HPLC and UV-methods. Here an attempt was made to develop the bio-analytical method for the estimation of bosentan from plasma using losertan as internal standard and to validate as per international regulatory guidelines. Runtime was decreased while developing the method [11][12][13][14][15].

MATERIALS AND METHODS: Chemicals and reagents
Bosentan (Figure 1) of the highest quality has been purchased from sigma Aldrich (Mumbai, India) and Losertan (Internal Standard) (Figure 1)  Chromatographic condition LC (2010)AHT with the C18 column (4.6X250mm, 5µm), (X bridge C18 5µm) was used and the wave length used for the detection was 254nm. The other method conditions included were the column oven temperature of 23ºC, flow rate of 1ml/min, run time of 6min and injection volume of 5µl. Table 1: HPLCchromatographic condition.

Preparation of stock and standard solutions
The Bosentan standard was weighed twice separately and prepared separately using methanol and water (50:50) to yield two primary standard stock solutions (1 and 2) with a concentration of 50 µg/mL. Secondary and working stock solutions for the calibration curve were prepared from bosentan primary stock solution-1 by methanol and water (50:50). The quality controls working stock solution were prepared from primary stock solution-2 by methanol and water (50:50). These working stock solutions (1 and 2) were further diluted to obtain  Figure 2.

Bioanalytical method validation
Preparation of calibration curve:The linearity of the method was evaluated by a calibration curve in the range of 0.98 -998ng/ mL of metaxalone, including lower limit of Quantitation (LLOQ). The calibration curve was achieved by plotting the peak area ratios of metaxalone and internal standard versus the concentration of metaxalone by least-squares linear regression analysis. The calibration curve requires a correlation coeicient (R2) of >0.99. he acceptance criteria for each back-calculated standard concentration should be within 15% of the nominal concentration, except it should not exceed 20% for the LLOQ. Each validation run consisted of a double blank, system suitability sample, a zero standard, calibration curve consisting of ten non-zero samples covering the total range (0.98 -998 ng/mL) and QC samples at three concentrations (N = 6, at each concentration). Such validation runs were generated on six consecutive days. Accuracy and precision: Intra-day and Inter-day accuracy and precision were determined by duplicate analysis of six sets of samples spiked with four diferent concentrations of bosentan at low, medium, high quality control samples (200,500 and 750 ng/mL) including LLOQ (200 ng/mL) within a day or on 6 consecutive days. For acceptance criteria for intra and inter-day precision, accuracy should be within 85-115% of the nominal concentration and coeicient of variation (%CV) values should be <15% over the calibration range, except at the LLOQ, where accuracy should be between 80 -120% and %CV should not be more than 20% Selectivity: The selectivity of the assay methodology was established using a minimum of six independent sources of the same matrix. There were no interferences from the endogenous material at the retention time for both Bosentan and internal standard (Losertan). The representative chromatogram is shown in Figure 3. Recovery: Recovery of bosentan was evaluated by comparing the mean peak areas of three extracted low, medium and high quality control samples to mean peak areas of three neat reference solutions (un-extracted). Recovery of internal standard was evaluated at a concentration of 200 ng/mL and corresponding mean peak area of the extracted samples compared to the mean peak areas of neat reference solutions. Recovery of the analyte need not be 100%, but the extent of recovery for analyte (Bosentan) and internal standard (Losertan) should be consistent and reproducible. Stability: In order to ind out the stability of bosentan in rat plasma, bench top stability, freeze thaw stability, auto injector stability and long term stability studies were carried out by using six replicates of the low and high plasma quality control samples. For the bench top stability, frozen plasma samples were kept at room temperature for 24 hr before sample preparation. Freeze-thaw stability of the samples was obtained over three freeze-thaw cycles, by thawing at room temperature for 2-3 hr and refrozen for 12-24 hr for each cycle. Auto sampler stability of metaxalone was tested by processed and reconstituted low and high plasma QC samples, which were injected 24 hr ater reconstitution and were compared with freshly prepared QC samples. Long term stability of metaxalone in rat plasma was tested after storage at approximately −70°C for 30 days. For the acceptance criteria of stability, the deviation compared to the freshly prepared standard should be within ± 15% of the nominal concentration. Matrix efect: The matrix effect was performed in 6 different lots of rat plasma by taking 47.5 µl of rat plasma and 2.5 µl of methanol: water (50:50) solution. From the mixture, take 25 µl and add 375 µl of blank methanol containing. This blank mixture was vortexed for 5 min at 885 g and centrifuged at 19283 g for 8 min to prepare the extracted blank. The aqueous equivalent solution was prepared by taking 25 µl of water and adding 25 µl of internal standard along with 350 µl of methanol containing 0.1% trichloroacetic acid. The extracted blank supernatant and aqueous equivalent solution were mixed in a ratio of (1:1) solution. The blank aqueous solution was prepared by mixing 25 µl of water along with 375 µl of methanol and subjected to vortexing. The neat solution and post extracted solution. Were prepared by mixing aqueous equivalent solution to both extracted blank and blank aqueous solution in a ratio of (1:1) solution. Both solutions were vortexed and subjected to RP-HPLC for analysis. Matrix suppression or enhancement was calculated as follows: 100×mean peak area of post extracted sample/mean peak area of neat standard solution. The acceptance criteria for matrix efect implied that the %CV should be less than 15% of matrices tested and at least 80% of matrices should meet the above criteria. he results obtained were displayed in Table  2.

RESULTS: Selectivity and optimization of chromatographic conditions
Plasma matrices were obtained from six diferent sources and assayed to evaluate the selectivity of the method and the detection of interference. Metaxalone and Phenytoin (internal standard) were well separated from the co-extracted material under the described chromatographic conditions at retention times of 2 and 4.3 min respectively. No endogenous peak from plasma was found to interfere with the elution of either the drug or the internal standard. The LLOQ which could be measured with acceptable accuracy and precision for the analyte 200 ng/mL was established ( Figure 4). It indicates that the proposed method is highly selective and specific.

Calibration curve
Linear detector response for the peak-area ratios of the metaxalone to internal standard was observed in the concentration range between 50 -750 ng/mL with a mean correlation coeicient of 0.992.T he reason for choosing a wide calibration range for Bosentan. PK study is to analyze samples of higher and lower dose concentration and diferent route of administration like intravenous administration and per oral where the Cmax concentration will be higher for intravenous. he best it for the calibration curve could be achieved with the linear equation Y = MX + C. The mean linear regression equation of calibration curve for the analyte was Y = 0.14777x-510298, where Y was the peak area ratio of the analyte to the IS and X was the concentration of the analyte. he results were given in the Table 3.

Accuracy and precision
The intra-day accuracy and precision ranged between 96.2-104.11%, and 1.54-1.69%, respectively. The inter-day accuracy and precision ranged from 95.56 to 110.26% and 1.38 to 1.87%, respectively. The accuracy and precision for intra and interday at the LLOQ and at LQC, MQC, HQC control samples of metaxalone in plasma were within acceptable limits (N = 6). The results of the method validation studies presented in Table 4 and 5.

Recovery
The recovery of Bosentan in plasma was calculated at three QC levels.T he response (extracted) compared to that of unextracted samples of the reference solution. The percentage recovery of Bosentan and losertan (internal standard) were found to be above 80%. Results are displayed in the Table 6. Stability Stock solutions of Bosentan(5 mg/mL) and internal standard (5 mg/mL) were separately prepared. The solutions were stable for at least 1 month when stored under light-protected conditions at 4°C. The stability experiments were aimed at testing all possible conditions that the samples might experience after collecting and prior the analysis. All stability results were summarized in Table 7. The results of three freeze-thaw cycles and bench top stability testing (24 h) when the spiked samples were kept at room temperature indicated that Bosentan was stable in rat plasma under these conditions. Analyte spiked QC samples were stable for at least 30 days if stored in the freezer at −85°C. Testing of auto sampler stability of quality control samples indicated that Bosentan would be stable when kept in the auto sampler up to 24 h.

DISCUSSION:
A simple and selective LC method is described for the determination of BOSENTAN tablet dosage forms. Chromatographic separation was achieved on a c18 column using mobile phase consisting of a mixture of 50 volumes of METHANOL and 50 volumes of WATER (with detection of 225 nm. Linearity was observed in the range 50-150 µg /ml for BOSENTAN (r 2 =0.992) for the amount of drugs estimated by the proposed methods was in good agreement with the label claim. The proposed methods were validated. The accuracy of the methods was assessed by recovery studies at three different levels. Recovery experiments indicated the absence of interference from commonly encountered pharmaceutical additives. The method was found to be precise as indicated by the repeatability analysis, showing %RSD less than 2.
All statistical data proves validity of the methods and can be used for routine analysis of pharmaceutical dosage form.

CONCLUSION:
In conclusion a validated RP-HPLC method has been developed for determination of BOSENTAN in the bulk and combined tablet dosage forms. The results show that the method was found to be specific, simple, accurate, precise and sensitive. The method was successfully applied for the determination of both drugs in combined tablet dosage form. In the future, this method may be applied for routine analysis of both the drugs in API and in tablet formulation. Several analytical procedures have been proposed for the quantitative estimation of BOSENTAN separately and in combination with other drugs. To my knowledge simple, rapid analytical method for determination of BOSENTAN has not been reported so far. So attempt was taken to develop and validate a reversed-phase high performance liquid chromatographic method for the quality control of BOSENTAN in pharmaceutical preparations with lower solvent consumption along with the short analytical run time that leads to an environmentally friendly chromatographic procedure and will allow the analysis of a large number of samples in a short period of time.

ACKNOWLEDGEMENT
Authors are thankful to Principal Dr. Bhagavan Raju Sri Venkateshwara College Of Pharmacy for support, encouragement and providing facilities to carry out