Quantitative Estimation of Eltrombopag in Bulk and Tablet Dosage Form by UV- Spectroscopy

 

Saroj Kanta Bisoyi*1,2, Debasish Pradhan1, Sudhir Kumar Sahoo2

1University Department of Pharmaceutical Sciences, Utkal University,

Vani Vihar, Bhubaneswar, 751004, Odisha, India.

2Royal College of Pharmacy and Health Sciences, Berhampur - 760002, Odisha, India.

*Corresponding Author E-mail: sarojbisoyi2010@gmail.com

 

ABSTRACT:

The present work aimed to develop a straightforward, simple and cost-effective UV Spectroscopic technique employing methanol and water (1:1) as a solvents. The method focused on a simple, rapid, specific, accurate, precise, and selective approach in the estimation of Eltrombopag in bulk and tablet dosage form for routine analysis. The ICH Q2 (R1) guideline was followed during method development and validation. Eltrombopag is the drug of choice in the treatment of thrombocytopenia and severe aplastic anemia. Eltrombopag is a BCS Class II medication that is sparingly soluble in water and has a pKa of about 3.5. Its solubility varies with pH, becoming more soluble at lower pH levels and more soluble in the organic solvent methanol. The method utilizes water and methanol (1:1), with a maximum absorption wavelength detected at 245nm. The method is specific; no interference with blank and placebo. Linearity in the concentration range of 1.00 to 40.00 µg/mL, exhibiting a high correlation coefficient of 0.999. Precision was demonstrated with an RSD below 2%, while LOD and LOQ were determined as 1.5µg/mL and 4.51µg/mL respectively. The drug recovery rate falls within the range of 99.94 to 100.65%. The solution was stable for 24hours at room temperature condition. The parameters validated were precision, linearity, accuracy, robustness and specificity as per ICH guidelines were found to be satisfactory for the routine analysis of Eltrombopag by UV spectroscopy.

 

KEYWORDS: Eltrombopag, UV Spectroscopy, Method Development, Validation, ICH.

 

 


1. INTRODUCTION: 

Eltrombopag (ELP) is a USA-approved therapeutic drug used for the treatment of chronic immune thrombocytopenia in children1-3. It belongs to the class of small-molecule TPO-receptor agonists and is administered orally,4-5 by interacting with the transmembrane domain of the human TPO-receptor, ELP effectively increases platelet production6.

 

 

 

This medication is indicated for adults with chronic immune thrombocytopenia (ITP) who has not responded adequately to other treatments or spleen removal  surgery 7. ITP is characterized by low platelet counts, leading to unusual bleeding or swelling8. The structure and IUPAC name is as follows.

 

Figure 1: Structure of Eltrombopag.

 

The IUPAC name of ELP is Bis (2-aminoethan-1-ol); 3-[(5E)-5-{2-[2(3, 4dimethylphenyl)-5-methyl-3-oxo-2, 3-dihydro-1H-pyrazol-4-yl] hydrazin-1-ylidene} 6oxocyclohexa-1, 3-dien-1-yl] benzoic acid 9.   

The ELP UV-spectroscopic method was performed by selecting wavelengths in the UV-visible region of 400–800nm detection. Thus, an attempt was made to develop and validate a UV spectrophotometric method for the estimation of ELP in bulk and tablet dosage form in UV region 200-400nm.

 

2. MATERIALS AND METHODS:

Instruments used:

The reagents were weighed using a high-precision electronic weighing balance from Mettler Toledo, Switzerland, while a double-beam UV-visible spectrophotometer (UV-1700,UV-1800) from Shimadzu Companies, Tokyo, Japan, and methanol form  Merck make and distill water was employed for analysis. Ultrasonication facilitated the drug's solubilization process.

 

METHOD DEVELOPMENT:

Solubility:

The solubility of ELP was determined using different solvents such as distilled water, ethanol, methanol, 0.1N hydrochloric acid, 0.1N sodium hydroxide and pH-4.5 acetate buffers was selected as the solvent for method development10

 

Preparation of Standard Stock Solution:

Accurately weighed 100mg of ELP and transferred in a 100mL volumetric flask and dissolve with diluents water and methanol (1:1) and final volume was made up with diluents to achieve 1000μg/mL11-12

 

Preparation of working standard solution:

Working standard solution was prepared by transferring 10mL of standard solution in to a100mL volumetric flask and final volume made up with diluents to get a concentration of 100μg/mL concentration.

 

Preparation of Sample solution:

Due to the unavailability of a commercial pharmaceutical formulation of ELP in the local market, tablets were prepared in-house containing 50mg of ELP along with common excipients. The sample solution was prepared from these in-house formulated ELP tablets. An accurately weighted amount of powder equivalent to 25mg of ELP was quantitatively transferred into a 100mL volumetric Flask, add diluents and sonicated for 15minutes finally volume made up with diluents. Filter through 0.45μm nylon filter and further diluted 1 mL to 25mL with diluents (10μg/mL)13-14.

 

λ max Determination:

Using the working standard solution, aliquots of 5mL were pipette into separate 50mL volumetric flasks and diluted with diluents to achieve concentrations of 10 μg/mL respectively. Subsequently, these solutions were scanned in a UV-Vis Spectrophotometer with the wavelength range of 200- 800nm, with methanol and water (1:1) as the blank15-17.

 

Validation:

It is a structural process aimed to generating documented evidence to ensure that a particular procedure consistently produces outcomes or products that meet predetermined specifications and quality standards. In accordance with the guidelines outlined by the international council for harmonization (ICH) in Q2 (R1), the developed spectrophotometric method further validated using the following critical parameters. This parameter includes system suitability, linearity, accuracy, and precision, limit of detection, limit of quantification, solution stability and robustness18-20.

 

System suitability:

System suitability tests were conducted to demonstrate the appropriateness of the UV spectrophotometric system employed for the analysis. Six replicates of a standard (10μg/mL) of ELP were prepared from a working standard solution in diluents, and the absorbance at 245nm was measured for each replicate using a UV spectrophotometer. The percentage relative standard deviation (% RSD) of the absorbance was then calculated21.

 

Specificity:

Blank as diluents, Placebo, Standard, sample was prepared and absorbance was taken at 245nm and checked for any interference22.

 

Precision:

Precision indicates the closeness of results obtained by multiple measurements of the same homogenized sample. Repeatability (intraday precision and intermediate precision (inter-day precision) were evaluated to demonstrate the precision of the method. Repeatability involved analyzing six replicates of the 10 μg/mL concentration on the same day, while intermediate precision involved analyzing six replicates of the same concentration on the consecutive days. The percentage relative standard deviation of assay results was calculated for both scenarios23,24.

 

Linearity:

As per ICH Q2 (R1) guidelines, the linearity of the analytical procedure verifies the direct relationship between test results and analyte concentration within the sample. Seven solutions of varying concentrations (1, 5, 10, 15, 20, 25, 30, 35 and 40μg/mL) were prepared in ethanol from a working standard solution of ELP. The absorbance of each solution at 245nm was recorded, and a calibration curve for absorbance and concentration was plotted. The percentage relative standard deviation and coefficient of correlation were determined through regression analysis25-27.

 

Accuracy:

Accuracy was assessed through standard addition methods where known amounts of the standard were added at three different levels (80,100 and 120%) of the in-house tablet formulation of ELP and analyzed using the method. %Recovery studies were conducted by spiking ELP standard at these levels and estimating the % recovery for each level28.

 

Solution stability:

Standard and sample solutions were prepared and absorbance was taken at 245nm for initial time and 24 hour interval and reported the stability of the solution29.

 

Robustness:

It assess the ability of the method to be reported in slightly change in method parameters like change in wavelength (247nm and 243nm), change in solvent composition (55:45 and 45:55) in different laboratories or under different condition without unexpected differences in results, thus evaluating the robustness of the proposed method30-31.

 

Limit of detection and limit of quantification:

The limit of detection is the minimum concentration of a substance in a sample that can be detected but is not necessarily precisely quantified. Conversely, the limit of quantification is the lowest concentration of a substance that can be accurately and precisely determined within the sample. These thresholds for the ELP were established according to the ICH guidelines, using the residual standard deviation of response and slope method. This involved utilizing a calibration curve from the linearity study. The LOD was calculated using the formula (3.3* σ)/S, where σ represents the standard deviation of the response, and S is the slope of the calibration curve. Similarly, the LOQ was determined using equation (10* σ)/S 32-35.

 

Analysis of in-house tablet formulation:

The UV Spectroscopic method proposed was employed to quantify ELP in the prepared in-house tablet formulation. Triplicate analyze revealed that the % amount of the drug found was 95.47%. This indicates the reliability and consistency of the method. It is anticipated that the developed method will be routinely employed for the assessment of ELP in the future investigations

 

3. RESULTS AND DISCUSSION:

Table 1: Solubility study was carried out in different solvents and results are given in table No-1.

Solvent

Solubility

Dist. Water

Slightly soluble

Ethanol

Soluble

Methanol

Soluble

0.1(N)  NaOH

Soluble

0.1(N) HCL

Insoluble

pH 4.5 acetate buffer

Insoluble

 

Determination of Working Wave Length (λ max):

Standard ELP 10µg/mL was prepared in diluent and scan by using UV spectrophotometer in the range of 200-400nm. The maximum absorbance found at 245nm and UV spectra is given in figure No-2.

 

 

Figure 2: UV spectra of ELP

 

Specificity:

Checked the absorbance of the Blank, Placebo, Standard and test sample solution of tablets by UV-VIS spectrophotometer for the determination of the interference. Table No- 2 shows the results.

 

Table 2: Specificity

Sample

Absorbance

% Interference

Blank

0.000

NA

Placebo

0.000

NA

Standard

0.741

NA

Test 25 mg

0.685

NA

 

System Precision:

Prepared the standard solution as per the method and checked the absorbance for six times into UV system. The mean, SD and % RSD for absorbance of ELP were calculated. The results are listed in Table No- 3.

 

Table: 3 System precision data:

Sr. No.

Absorbance of ELP

1

0.764

2

0.765

3

0.768

4

0.761

5

0.765

6

0.763

Mean

 0.764

SD

0.003 

%RSD

0.34

Method Precision:

Performed six sample, analyzed as per the test method and Calculated the % RSD for assay (%) of six units and tabulated in Table No - 4.

 

 

Table: 4 Method precision data

Sr. No

Absorbance

% Release of ELP

1

0.681

95.89

2

0.686

96.45

3

0.688

95.83

4

0.676

95.45

5

0.685

95.98

6

0.677

95.24

Mean

95.81

SD

0.424

% RSD

0.44

 

 

Intermediate Precision:

Performed on six units of sample by different analyst, by using different system on different day. Calculated the %RSD for %assay of six preparations. Calculated the overall %RSD for %release of above experiment results along with method precision results. RSD of %assay for twelve results of method precision and intermediate precision. Table No - 5 given the results.

 

Table: 5 Intermediate precision data

% Assay of ELP

Sr. No.

Absorbance

Intermediate precision

Method Precision

1

0.685

95.29

95.89

2

0.693

95.04

96.45

3

0.691

95.09

95.83

4

0.696

95.60

95.45

5

0.688

95.65

95.98

6

0.689

96.12

95.24

Mean

95.47

95.81

SD

0.408

0.424

%RSD

0.43

0.44

Over all Mean

95.64

Over  all SD

0.44

Over all %RSD

0.46

Analyst

Analyst II

Analyst I

UV-Spectrophotometer ID

UV-1800

UV-1700

 

Linearity and Range:

The linearity of ELP was determined in the range of 1.00µg/ml to 40.00µg/ml. The results are tabulated in Table –6, calibration spectra and graphically represented in Figure No-3 and 4 respectively.

 

Table 6: Linearity and Range data

Sr.No

Concentration of ELP µg/Ml in µg/mL

Absorbance

1

1.00

0.246

2

5.00

0.483

3

10.00

0.741

4

15.00

0.999

5

20.00

1.229

6

25.00

1.501

7

30.00

1.806

8

35.00

2.025

9

40.00

2.249

Slope

0.051

Intercept

 0.216

Correlation Coefficient

0.999

 

Fig No: 3 Calibration curve of ELP     

 

 

Fig No: 4 Linearity UV spectra of ELP

 

Accuracy:

Known amount of ELP spiked at 80%, 100% and 120% of test concentration in triplicate. The amount of ELP was quantified as per the test method. The % recovery was calculated from the amount recovered and actual amount added. The results are given in Table No – 7.


Table No: 7 Accuracy as recovery data

Level

Amount Added in (mg)

Abs

Amount Recovered (mg)

% Recovery

Mean

% Recovery

SD

% RSD

Level 1

 (80%)

8.12

0.612

8.17

100.62

100.65

0.07

0.07

8.16

0.615

8.21

100.61

8.21

0.619

8.27

100.73

Level 2 (100%)

10.32

0.769

10.27

99.52

99.94

0.5

0.5

10.56

0.789

10.54

99.81

10.25

0.771

10.30

100.49

Level 3 (120%)

12.56

0.939

12.54

99.84

100.11

0.54

0.54

12.21

0.921

12.30

100.74

12.45

0.93

12.42

99.76

Over all Mean

100.24

Over all SD

0.49

Over all % RSD

0.49

 


Solution stability:

Prepared one standard and sample solution as per test method and take absorbance by using UV Spectrophotometer at Initial and 24hours.  Determine percentage assay at different time intervals. The results are tabulated in Table No – 8.

 

Table No-8  solution stability data

Time

(In hours)

ELP Standard

ELP Sample

Absorbance

% Difference

% Assay

 

Initial

0.741

-

95.8

-

24

0.751

1.3

97.2

1.45

 

Robustness:

Little deviations were made in the method parameters, and the effects of these changes on the outcomes were investigated, in order to evaluate the robustness of the proposed approaches. The results of change in wavelength and change in solvent composition given in table No-10.

 

Table No-10 Robustness data

Parameter

% Assay

 

% RSD

 

Over All% RSD

of Assay

Plus Wavelength (247 nm)

95.71

0.43

0.88

Minus Wavelength (243 nm)

96.49

0.35

0.99

Plus Organic Methanol: Water (55: 45)

96.75

0.89

1.14

Minus Organic  Methanol: Water (45: 55)

96.10

0.32

0.92

 

Limit of detection and Limit of Quantification:

The ELP limit of detection was determined to be 1.5µg/mL, while its limit of quantification was measured at 4.51µg/mL.

 

4. CONCLUSION:

An accurate, precise, cost-effective and robust UV-Spectroscopic method has been developed for the quantitative determination of ELP in both bulk and in-house tablet formulations. The method is characterized by its simplicity and rapidity, precision data with %RSD value less than 2. Demonstrate the method’s reproducibility and precision. The recovery range of 98-99% indicates the method is accurate and specific. The high % recovery of the drug suggests that the presence of excipients in the tablet formulation does not interfere with the determination of ELP confirming the method's specificity and reliability. Therefore, the developed method is suitable for routine analysis of ELP in bulk and pharmaceutical formulations.

 

5. CONFLICTS OF INTEREST:

In relation to the publishing of this paper, the authors declare that they have no conflicts of interest.

 

6. ACKNOWLEDGMENTS:

The authors extend their gratitude to the Royal College of Pharmacy and Health Sciences Berhampur, Odisha for providing facilities for the successful completion of this research.

 

7. CONTRIBUTION:

Each author contributed a similar contribution to this work.

 

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Received on 17.03.2024      Revised on 13.07.2024

Accepted on 30.09.2024      Published on 02.05.2025

Available online from May 07, 2025

Research J. Pharmacy and Technology. 2025;18(5):2101-2106.

DOI: 10.52711/0974-360X.2025.00301

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