INTRODUCTION:

Bosentan is chemically, 4-tetrabutyl-N-[6-(2-hydroxyethoxy)-5-(2-methoxyphenoxy)-2-(pyrimidin-2-yl) pyrimidin-4-yl] benzene-1-sulfonamide^2. (Fig.1).It is a white crystalline powder .chemical formula C₂₇H₂₉N₅O₆S, and Molecular weight of551.6. It is soluble in methanol and slightly soluble in ethanol and insoluble in water. It is an Antihypertensive Agents drug, which is used in the treatment of pulmonary hypertension. Bosentan is metabolized in the liver by the cytochrome P450 enzymes CYP2C9 and CYP3A4 (and possibly CYP2C19), producing three metabolites, one of which, Ro 48-5033, is pharmacologically active and may contribute 10 to 20% to the total activity of the parent compound.[1-4]

Received on 05.03.2015 Modified on 01.04.2015

Research J. Pharm. and Tech. 8(5): May, 2015; Page 549-553

DOI: 10.5958/0974-360X.2015.00091.8

Fig: 1 structure of Bosentan (BST)

Bosentan is eliminated by biliary excretion following metabolism in the liver. Literature survey reveals that few HPLC and UV Spectrophotometry methods for developed for estimation of bosentan in bulk and biological fluids. There is no simple accurate precise and economical RP-HPLC methods. Hence the authors were developed a validated simple and economical RP-HPLC method for the estimation of bosentan tablets in routine quality control analysis[5,6].

MATERIALS AND METHODS:

Apparatus:

Reverse phase HPLC column Teflon coated C18 (250 mm x 4.6 mm I.D; particle size 5μm), Agilent 1200HPLC with on EZ Chrome Elite 2 software, Diode array detector, Elico pH meter ,LC-GC Weighing balance, Ultra sonicator-DSK Industries

Chemicals and solvents:

Methanol HPLC Grade (Qualigens), 0.2%TEA (HPLC Grade; Qualigens), Triple distilled water, Ortho phosphoric acid (AR-GRADE; Qualigens)

Drug sample:

Working standard TT (99.75%) was procured from Aurabindo Labs, Hyderabad, India. Tracler (Ranbaxy) was taken for study which contains Bosentan 125mg were purchased from local pharmacy.

Method development

Preparation of mobile phase

The mobile phase was prepared by mixing Methanol: water (80:20) and 0.2%TEA was added and the pH was adjusted with Orthophosphoric acid. Then mobile phase filtered through 0.45µm membrane filter and degassed before use

Preparation of stock and working standard solutions:

Accurately weighed quantity of Bosentan (10mg) was transferred to 10mL of volumetric flask. Then small amount of methanol was added and diluted up to the mark with methanol (Concentration: 1000µg/mL). It was further diluted until to get concentration of 100 μg/ml with mobile phase. It was injected into the c₁₈ column using syringe and the peak response was observed. After getting optimised condition, the working standards of 20-120μg/ml were made and the calibration curve was plotted.

Optimized chromatographic conditions

Chromatographic separation was achieved on Agilent TC C₁₈ (250 x 4.6 mm, 5 μ) column using mobile phase composition of water: Methanol (20:80 v/v) (0.2% Tri Ethyl Amine) and pH adjusted to 3 with Ortho Phosphoric acid. Flow rate was maintained at 1mL/min with 268 nm UV detection. The retention time obtained for Bosentan was at 5.3min with injection volume.

Calibration curve of proposed method:

The series of aliquots 20-120μg/ml were made and the average peak area were observed at 268 nm. He calibration curve was plotted against Concentration vs Peak area and the regression equation was calculated. The drug content in tablet dosage form was calculated using above regression equation. The results were shown in fig no: 3.

Estimation of Bosentan in tablet dosage form:

20 tablets of traclear (containing 125mg of bosentan) were weighed and the powder equivalent to 10mg of Bosetan was transferred to 10mL standard flask and small amount methanol was added. The solution was sonicated for 5min, and the final volume was made with same to obtain solution of Bosentan (1000µg/mL). The mixture was then filtered through a nylon 0.45mm membrane filter. The above solution was suitably diluted with mobile phase to obtain final dilution of Bosentan (50µg/mL). The results were shown in table no 2

Method validation

The method was validated for its linearity range, accuracy, precision, sensitivity and specificity. Method validation is carried out as per ICH guidelines

System Suitability Studies:

Injected 20µL of the diluted standard solution in to the chromatographic system (n=6) and recorded the chromatogram. The column efficiency as determined from Bosentan peak is not less than 2000 USP plate count and the tailing factor for Bosentan peak is in between 1 to 2. The Standard and Sample chromatograms for optimized method are shown in fig. no. 4,5.

The system suitability studies were evaluated from standard chromatogram and obtaining the parameters includes Retention time, Column efficiency, Resolution and Tailing factor. The results were shown in table no 3

Linearity

In order to find out the linearity range of the proposed HPLC method, studies were carried out by plotting peak areas of analyte against concentrations of the analyte. A good linear relationship (r=0.999) was observed between the concentrations of Bosentan and the corresponding peak areas. The regression of Bosentan concentration over its peak area was found to be Y=79183x+185459 (where y is the peak area and x is the concentration of Bosentan). The slope, intercept and the correlation coefficient of the drug were shown in table no.4

Accuracy

The accuracy of the methods was determined by calculating recoveries of Bosentan by the standard addition methods. The accuracy of the method was determined by preparing solutions of different concentrations that is 80%, 100% and 120% in which the amount of marketed formulation (Traclear 125 mg) was kept constant (50µg/mL) and the amount of pure drug was varied that is 40µg/mL, 50µg/mL and 60µg/mL for 80%, 100% and 120% respectively. The solutions were prepared in triplicates and the accuracy was indicated by % recovery was shown in table no 5

Precision

The precision of the instruments was checked by repeatedly injecting (n=6) solutions of BST (50µg/mL) . The results were shown in table no 6

Intermediate Precision (Reproducibility)

The intra-day and inter-day precision of the proposed methods were determined by the corresponding responses three times on the same day and on three different days over a period of one week for concentration of 50µg/mL

Robustness:

Robustness of the method was determined by carrying out the analysis at two different mobile phase ratio (12:82, 22:78) and two different flow rates (i.e. 0.9, 1.1 mL/min), at two different wavelengths (2,213) . The results were shown in table no 7

Limit of detection and Limit of quantification :The limit of detection (LOD) limit of quantification (LOQ) of the drug carry was calculated using the following equation as per international conference harmonization (ICH) guidelines. The results were shown in table no 8

LOD = 3.3 X SD/S

LOQ = 10 X SD/S

Specificity

Specificity is the degree to which the procedure applies to a single analyte and is checked in each analysis by examining blank matrix samples for any interfering peaks. The specificity of the method was evaluated with regard to interference due to presence of any other excipients. Two different samples were injected and studied with respective excipients. HPLC chromatograms recorded for the drug-matrix (mixture of the drug and excipients) showed almost no interfering peaks with in retention time ranges. Fig4 and 5 show the respective chromatograms for Bosentan blank and standard. The figures shows that the selected drugs were cleanly separated. Thus, the HPLC method proposed in this study was selective.

RESULTS AND DISCUSSIONS:

The proposed method discussed in the present work provide a convenient and accurate way for the analysis of Bosentan in bulk and in pharmaceutical dosage form. The absorbance Maxima of Bosentan was found to be 268nm was selected for the analysis. Linearity for detector response was observed in the concentration range of 20-120 µg/ml for HPLC method. The validation of proposed methods were further confirmed by recovery studies, the %recovery values vary from 98- 102%. Based on results obtained it was found that the proposed methods were accurate, precise, reproducible and can be employed for routine quality control of Bosentan tablet dosage form.

Table no: 1: Optimised Method

PARAMETER	OBSERVATION
Elution	Isocratic
Temperature	Ambient
Mobile phase	Methanol:Water(80:20) with 0.2%TEA
P^H	3
Column	Agilent Teflon Coated C₁₈ (250 x 4.6 mm, 5 μ)
Wave length	268nm
Flow	1ml/min
Retention time	5.3 min
Run time	7mins

Table no 2: Results of tablet analysis

Formulation

Labeled claim

(mg/

tablet)

Test conc.

(μg/mL)

Mean Amount found (μg/mL)

% of mean amount found

%RSD

Bosentan

(Tracler)

125mg

50μg/mL

49.6

98.4%

0.66

Table no: 3 System Suitability Parameters

S.No	Parameter	Value
1	Retention time	5.3 min
2	Theoritical plates(N)	6423
3	Assymetric factor(As)	1.19
4	Tailing factor	1.32
5	LOD	0.55
6	LOQ	1.71

Table no: 4 Linearity of Bosentan

Parameter	Result
Λmax	268nm
Linearity range	20-120µg//mL
Regression equation	79183(x)+185457
Slope	59715
Intercept	82272
Correlation coefficient	0.999

Table no: 5. Recovery studies of Bosentan

Amount of sample taken (µg/mL)	Amount of standard added (µg/mL)	% of std added	Amount recovered (µg/mL)^*	% Amount recovered^*	% RSD
50	40	80	88.89	99.72	0.911
50	50	100	100.12	100.24
50	60	120	109.88	98.80

*Average of three determinations (n=3

Table no: 6: Intra-day And Inter-day Precision studies of Bosentan

Amount of std taken (µg/mL)	Intra-day precision *Mean^±% RSD**	Inter-day precision *Mean^±% RSD**
50	3736766±2467.78	3736678±4498.23
50	3736766±2467.78	3736766±2467.78
50	3736766±2467.78	3736766±2467.78

*Average of three determinations (n=3)

Table no: 7 Robustness of Bosentan

Parameters	Change in parameters	Peakarea	%assay	Results
Flow rate (1mL/min)	0.9mL/ min	4208951	101	Robust
	1.1mL/ min	3538110	91	Not robust
Mobilephase ratio(20:80)	22:78	3591619	98	Robust
	18:82	3762246	99	Robust
Wavelength(268nm)	270nm	3716439	121	Not robust
	266nm	3760675	76	Not robust

Table no: 8 LOD and LOQ

STD	LOD (µg/mL)	LOQ (µg/mL)
Bosentan	0.55	1.71

Fig no: 2 Chromatogram of bosentan with Methanol: Water(80:20) with 0.2%TEA P^H3

Fig no: 3 Calibration curve of Bosentan

Fig no.4: Chromatogram of Standard

Fig no.5: Chromatogram of sample