Development and validation of a new stability indicating RP-UFLC method for the estimation of Bosentan

 

Kalyani Lingamaneni*, Mukthinuthalapati Mathrusri Annapurna

Department of Pharmaceutical Analysis & Quality Assurance, Gandhi Institute of Technology and Management (Deemed to be University), GITAM Institute of Pharmacy, Visakhapatnam, Andhra Pradesh-530045, India.

*Corresponding Author E-mail: kalyani.lingamaneni@gmail.com

 

ABSTRACT:

A new stability-indicating RP-UFLC method has been developed for the estimation of Bosentan in pharmaceutical dosage forms and the method was validated. Bosentan is used to lower the high blood pressure in lungs (pulmonary arterial hypertension). Bosentan acts by blocking the actions of endothelin -1and thereby lowers the blood pressure. Mobile phase mixture consisting of sodium acetate (pH 5.0) buffer solution and acetonitrile (50: 50 v/v) with flow rate 0.7 mL/min were the optimized chromatographic conditions (Detection wavelength 254 nm) for the present study. Linearity was observed in the concentration range of 0.1–100 μg/mL (R2 = 0.9998) with regression equation y = 126698 x – 392.49. The LOQ was found to be 0.08964 µg/mL and the LOD was found to be 0.02913 µg/mL. Stress degradation studies such as acidic, alkaline, oxidation, photolysis and thermal degradations were performed by exposing Bosentan and finally the proposed method was validated as per ICH guidelines. The assay of Bosentan was conducted by applying the proposed method to the marketed formulations. The proposed method is simple, specific, precise, and accurate and can be applied for the estimation of pharmaceutical formulations.

 

KEYWORDS: Bosentan, RP-UFLC, Stability-indicating, Validation, ICH.

 

 

 

INTRODUCTION:

Bosentan (Figure 1) is chemically, 4-tert-butyl-N-[6-(2-hydroxyethoxy) -5- (2-methoxyphenoxy) -2- (pyrimidin-2-yl) pyrimidin-4-yl] benzene-1 sulfonamide1. Bosentan is used to lower the pulmonary hypertension by blocking the action of endothelin-1 molecules responsible for narrowing the blood vessels and elevates high blood pressure2-3. Different analytical techniques such as Spectrophotometry4-8, HPTLC9, LC-MS10-14 and reverse phase liquid chromatography15-22 have been developed for the determination of Bosentan in biological fluids, dosage forms as well as its impurities and degradation products. In the present study a new simple stability indicating RP-UFLC method was proposed for the determination of Bosentan and in its tablet dosage forms and the method was validated as per ICH guidelines.

 

Figure 1: Structure of Bosentan

 

MATERIALS AND METHODS:

Acetonitrile (HPLC grade), sodium hydroxide (NaOH) and hydrochloric acid (HCl) and Hydrogen peroxide (H2O2) were obtained from Merck (India). Bosentan API was obtained from LUPIN Ltd (India), a pharmaceutical company as gift sample. Bosentan is available in pharmacy store with brand names BOSENTAS (62.5 mg and 125 mg) (CIPLA Ltd, India) and BOZETAN (62.5 mg and 125 mg) (LUPIN Ltd, India) and all other chemicals were of AR grade and used as received.

 

Chromatographic separation was achieved by using a Shimadzu Model CBM-20A/20 Alite HPLC system, equipped with PDA detector and Phenomenex C18 column (250 mm × 4.6 mm, 5 µm particle size) maintained at 25 ºC. Isocratic elution was performed using Sodium acetate buffer: Acetonitrile (50:50, v/v) with flow rate 0.7 mL/min. 20 µL of sample was injected into the UFLC system.

 

Preparation of Bosentan drug solution

Stock solution of Bosentan (1000 μg/mL) was prepared by weighing accurately 25 mg of Bosentan and dissolving in HPLC grade acetonitrile in a 25 mL volumetric flask. The working standard solutions were prepared from the stock solution on dilution with mobile phase [Sodium acetate buffer: Acetonitrile (50:50, v/v)] and the solutions were filtered filtered through 0.45 μm membrane prior to injection.

 

Method validation23

Linearity, Limit of quantification (LOQ) and limit of detection (LOD)

The method was validated for linearity, limit of quantitation (LOQ), limit of detection (LOD), precision, accuracy, robustness and selectivity. From the series of Bosentan drug solutions (0.1-100 µg/mL) prepared from the stock solution only 20 µL of each of the solutions were injected (n=3) in to the UFLC system and the area under the curve i.e. peak area observed at the retention time of the chromatogram was noted. The mean peak area was calculated and a calibration curve was drawn by plotting the concentration of the drug solutions on the x-axis and the corresponding mean peak area values on the y-axis.

 

The limit of quantification (LOQ) and limit of detection (LOD) were based on the standard deviation of the response and the slope of the constructed calibration curve (n=3), as described in International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use.

 

Precision, Accuracy and Robustness

The intra-day and inter-day precision studies were evaluated by carrying out independent assays of Bosentan at three concentration levels (10, 20 and 50 µg/mL) (n=3) on the same day and also on different days (Day 1, Day 2 and Day 3) respectively and the %RSD of the peak areas obtained was calculated.

 

The accuracy of the assay method was evaluated in triplicate by standard addition method (80, 100 and 120%) i.e. by the addition of API drug solution to the pre-analyzed formulation and thereby the percentage recovery was calculated. The robustness of the analytical method denotes the ability of the method to remain unaffected by the small and deliberate variations in the method optimized conditions.

 

The robustness of the method was evaluated by incorporating small changes in the chromatographic conditions such as mobile phase composition (± 2%; 48:52 v/v and 52:48 v/v), detection wavelength (± 2 nm; 252 and 256 nm), pH  (± 0.1 unit; 4.9 and 5.1) and flow rate (± 0.1 mL; 0.6 and 0.8 mL/min).

 

Forced degradation studies24

Forced degradation studies were performed by exposing Bosentan to stress conditions such as acidic (HCl), alkaline (NaOH), oxidation (H2O2) and thermal conditions to evaluate the stability indicating properties and the specificity of the proposed method. The solutions used for the degradation studies were prepared by refluxing Bosentan drug solutions (50 µg/mL) at        70 ºC for 30 min in a thermostat with the reagents as per the degradation study and were filtered before injection in to the UFLC system.

 

Acidic degradation was performed by treating the Bosentan solution (50 µg/mL) with 0.1 N hydrochloric acid for 30 min in a thermostat maintained at 70 ºC, cooled and then neutralized with sodium hydroxide followed by dilution with mobile phase before injecting in to the UFLC system.

 

Acidic degradation was performed by treating the Bosentan solution (50 µg/mL) with 0.1 N sodium hydroxide for 30 min in a thermostat maintained at 70 ºC, cooled and then neutralized with hydrochloric acid followed by dilution with mobile phase before injecting in to the UFLC system.

 

Oxidation degradation was performed by treating the Bosentan solution (50 µg/mL) with 0.1 ml of 30% H2O2 for 30 min in a thermostat maintained at 70 ºC, cooled and then diluted with mobile phase before injecting in to the UFLC system.

 

Photolytic degradation was performed by exposing the Bosentan solution (50 µg/mL) to UV light (365 nm) in a photo stability testing chamber for 4 hours and then diluted with mobile phase before injecting in to the UFLC system.

 

Thermal degradation was performed by heating Bosentan drug solution (50 µg/mL) in a thermostat maintained at 70 ºC for 30 mins. The stressed drug solution was cooled and then diluted with mobile phase and injected in to the UFLC system.

 

Analysis of Bosentan dosage forms (Tablets)

Twenty tablets of each brand were procured from the local market, weighed accurately and crushed in to fine powder. Powder equivalent to 25 mg Bosentan was weighed accurately and transferred into a 25 ml volumetric flask and made up to volume with acetonitrile, sonicated for 30 min, filtered. The filtrate was diluted as per the requirement with mobile phase and 20 µL of the solution was injected in to the UFLC system after filtering through 0.45 μm nylon filter before injection.

 

RESULTS AND DISCUSSION:

A new stability-indicating RP-UFLC method was proposed for the quantification of Bosentan in pharmaceutical formulations (Tablets). The previously reported liquid chromatographic methods in the literature were compared with the present proposed RP-UFLC method and summarized in Table 1.

 

HPLC method development and optimization

Initially many trial runs were made with mobile phase consisting of a mixture of sodium acetate (pH 5.0) buffer solution and acetonitrile solution with flow rate 1.0 mL/min. System suitability parameters such as tailing factor, capacity factor, theoretical plates etc were not within the  acceptable criteria and therefore the chromatographic conditions were changed and finally mobile phase mixture consisting of sodium acetate (pH 5.0) buffer solution and acetonitrile (50: 50, v/v) with flow rate 0.7 mL/min (Detection wavelength 254 nm) were the optimized chromatographic conditions for the present study. The chromatograms obtained in the trial runs (Figure 2A-2F) and the corresponding chromatographic conditions along with the system suitability parameters were given in Table 2.

 

Table 1: Literature survey

Method / Mobile phase (v/v)

Comment

Ref.

 

LC-MS-MS

Bio analytical method

10

LC-MS

Bosentan and its three major metabolites

11

LC-MS

Human plasma

12

ESI-MS(n) and LC-ESI-MS

5 mM Ammonium acetate buffer (pH 4.5): Methanol: Acetonitrile

Stability indicating

Characterization of degradants products

13

SPE-LC-MS/MS

Human Plasma Bosentan and its active metabolite Hydroxy Bosentan

14

HPLC

Methanol: Potassium dihydrogen orthophosphate buffer (pH 7.8) (60:40)

Not stability indicating

Retention time: 8.26 min

Linearity: 50-150

15

HPLC

10mM Phosphate Buffer (pH 6.0): Acetonitrile (50:50)

Not stability indicating

Retention time: 3.687 min

Linearity: 15-27

16

HPLC

Acetonitrile: 10 mM Ammonium acetate (pH 4.5) buffer (70:30)

Not stability indicating

Linearity: 5-70

17

UHPLC (Gradient mode)

0.1 % Acetic acid: Methanol

In process control of Bosentan monohydrate synthesis

Linearity: 0.3-200

18

HPLC (Gradient mode)

Process and degradation impurities

19

HPLC

Related substances

20

HPLC

Plasma

21

HPLC (Isocratic mode)

Tetra butyl ammonium hydrogen sulphate: Acetonitrile (35: 65)

Stability indicating

Linearity: 1-350

22

UFLC

Stability indicating

Linearity: 0.1-100

Present method

 

Table 2: Method optimization (Bosentan 10 µg/mL)

Trial

Flow rate (mL/min)

Mobile phase

(Aq: Org)

Rt (mins)

Theoretical

plates

Tailing

factor

Observation

Figure

1

1

70:30

1.303

2059

1.550

Retention time <2

2(A)

2

0.8

70:30

1.965

2680

1.494

Retention time <2

2(B)

3

0.8

70:30

1.970

2606

1.491

Retention time <2

2(C)

4

0.5

70:30

2.808

1427

1.325

Theoretical plates <2000

2(D)

5

0.6

60:40

2.698

1928

1.346

Theoretical plates <2000

2(E)

6

0.7

50:50

2.262

3092

1.481

Method Optimized

2(F)

 

Figure 2: Chromatograms (Trial runs) observed during method optimization

 

 

Method validation

Linearity

Beer-Lambert’s law was obeyed over the concentration range 0.1–100 μg/mL (Table 3) with linear regression equation y = 126698 x – 392.49 and correlation coefficient, R2 = 0.9998 (Figure 3). The chromatograms obtained for the placebo, Bosentan API were shown in Figure 4A-4B. The LOQ was found to be 0.08964 µg/mL and the LOD was found to be 0.02913 µg/mL.

 

Table 3: Linearity study of Bosentan

Conc. (µg/mL)

*Mean peak area ± SD

(n = 3)

*RSD (%)

0.1

12879 ± 32.1975

0.25

0.5

65183 ± 337.5424

0.26

1

129824 ± 402.4544

0.31

5

637825 ± 2870.2125

0.45

10

1274643 ± 5480. 9649

0.43

20

2516372 ± 12078.5856

0.48

40

5047896 ± 9591.0024

0.19

50

6410437 ± 45514.1027

0.71

60

7598263 ± 52428.0147

0.69

80

9987634 ± 53933.2236

0.54

100

12761852 ± 116132.8532

0.91

*Mean of three replicates

 

Figure 3: Calibration curve of Bosentan

 

Figure 4A:  Placebo

 

Figure 4B:  Typical chromatogram of Bosentan API (10 μg/mL)

(Rt 2.262 min; Theoretical plates 4192; Tailing factor 1.231)

 

Figure 4C:  Typical chromatogram of Bosentan tablet (10 μg/mL)   (Rt 2.180 min; Theoretical plates 2730; Tailing factor 1.441)

 

Precision, Accuracy and Robustness

The % RSD in precision studies was found to be 0.74-0.91 (Intra-day) and 1.02-1.38 (Inter-day) (<2.0 %) (Table 4) and 0.38-0.72 in accuracy studies (<2.0 %) (% Recovery 98.56-98.97) indicating that the proposed RP-UFLC method is precise and accurate. The % RSD value in the robustness study was found to be 0.39-0.1.21 which is less than 2.0% indicating that the method is robust (Table 5).

 

Table 4: Precision and accuracy studies of Bosentan

Conc.

(µg/mL)

Intra-day precision

Inter-day precision

*Mean peak area ± SD (% RSD)

*Mean peak area ± SD (% RSD)

10

1271987 ± (0.74)

1281895 ± 13075.33 (1.02)

20

2498361 ± (0.91)

2532478 ± 31149.48 (1.23)

50

6398521 ± (0.76)

6425136 ± 88666.88 (1.38)

Accuracy

Conc.

(µg/mL)

*Mean peak area ± SD

(% RSD)

*Drug found

(µg/mL)

%

Recovery

18

2247421 ± 16181.43 (0.72)

17.743

98.56

20

2507551 ± 9528.69 (0.38)

19.795

98.97

22

2747978 ± 16762.67 (0.61)

21.69

98.60

*Mean of three replicates

 

Table 5:  Robustness study of Bosentan (10 μg/mL)

S. No

Parameter

Condition

*Mean peak area

Statistical analysis

Mean peak area ± SD (% RSD)

1

Flow rate (mL/min)

0.6

1275598

1269760.67 ± 15364.10 (1.21)

0.7

1264731

0.8

1268953

2

Detection wavelength

(± 2 nm)

252

1274602

1274528.67 ± 9304.06  (0.73)

254

1273999

256

1274985

3

Mobile phase

(Sodium acetate buffer: Acetonitrile)

52:48

1272879

1273043 ± 4964.87 (0.39)

50:50

1273102

48:52

1272984

4

pH (± 0.1unit)

4.9

1272936

1273684 ± 13883.16 (1.09)

5.0

1274219

5.1

1273897

*Mean of three replicates

 

Assay of Bosentan tablets

The proposed method was applied to the marketed formulations i.e. tablets (Figure 4C) for carrying out the assay of Bosentan and the % recovery was found to be 98.29- 99.13 (Table 6).

 

Table 6: Assay of Bosentan tablets

Sample No.

Formulation

Labeled claim (mg)

*Amount found   (mg)

*Recovery (%)

1

Brand I

125

122.86

98.29

2

Brand II

125

123.91

99.13

*Mean of three replicates

 

Forced degradation studies

The representative chromatograms obtained during the forced degradation studies were shown in Figure 5. A very slight decomposition i.e. less than 12 % was observed during the acidic, alkaline, oxidation, thermal and photolytic degradation conditions indicating that the drug is very much resistant (Table 7). The system suitability parameters such as tailing factor (<1.5-2.0), capacity factor and theoretical plates (>2000) were within the acceptable criteria.

 

Table 7: Forced degradation studies of Bosentan (50 μg/mL)

Stress

Conditions

*Mean peak area

Retention time (min)

*Drug

recovered (%)

*Drug

decomposed (%)

Theoretical

plates

Tailing factor

Standard Drug

6410698

2.226

100

0

3894

1.123

Acidic degradation

5649874

2.234

88.13

11.87

3256

1.386

Alkaline degradation

5712896

2.219

89.11

10.89

2356

1.319

Oxidation degradation

6103172

2.240

95.20

4.80

3129

1.123

Thermal degradation

6409773

2.226

99.99

0.01

3746

1.328

Photolytic degradation

6371051

2.224

99.38

0.62

2257

1.417

*Mean of three replicates

 

Figure 5:  Typical chromatograms of Bosentan (50 μg/mL) during stress degradation studies

 

 

CONCLUSIONS:

The proposed stability-indicating RP-UFLC method for the determination of Bosentan was sensitive, selective and specific and no excipients interfere with the Bosentan drug peak during the assay. The proposed stability-indicating RP-UFLC method is precise, accurate and robust and can be applied successfully to perform stability studies and pharmacokinetic studies.

 

ACKNOWLEDGEMENTS:

The authors are grateful to Lupin Ltd (India) for providing the gift samples of Bosentan.

 

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Received on 10.07.2021           Modified on 23.07.2021

Accepted on 31.07.2021         © RJPT All right reserved

Research J. Pharm. and Tech. 2021; 14(8):4445-4451.

DOI: 10.52711/0974-360X.2021.00772