Development and Validation of a UV Spectroscopic Method to Estimate Eltrombopag Olamine along with Bulk and In-house Formulation
Mohammed Aabid1*, Shakeel Memon1, Dilnawaz Pathan2, Tabbussum Hangad1
1Department of Quality Assurance, M. C. E. Society’s Allana College of Pharmacy, Azam Campus, Camp, Pune.
2HOD, Department of Pharmaceutics, Trinity College of Pharmacy, Pune.
*Corresponding Author E-mail: memon.shakeel@gmail.com
ABSTRACT:
The current UV Spectroscopic method developed involving ethanol as a solvent is simple, fast, specific, precise, and sensitive for the estimation of Eltrombopag Olamine in bulk in day-to-day analysis. As per the ICH Q2 (R1) guideline, the method was validated. Eltrombopag Olamine is a drug used to treat thrombocytopenia (a low blood platelet count) in adults and youngsters with chronic immune idiopathic thrombocytopenic purpura that didn't get well with different treatments. Eltrombopag Olamine is additionally accustomed to treating severe aplastic anemia. It’s conjointly being studied within the treatment of different conditions and kinds of cancer. Eltrombopag Olamine binds to the thrombopoietin receptor, which causes the bone marrow to create more platelets. It’s class of thrombopoietin receptor agonists, also known as Promacta. Eltrombopag is also recently approved (2012) for the treatment of thrombocytopenia in a patient with chronic hepatitis C to start and sustain interferon-based therapy. The solvent used in the entire method development and validation was ethanol. The maximum wavelength of absorption was found to be 423nm. Beer’s law was obeyed in the concentration range of 5 to 30ug/ml with a correlation coefficient of 0.9966. The method was precise with an RSD of less than 2%, the LOD, and LOQ were found to be 6.18ug/ml and 18.7ug/ml respectively, % recovery of the drug is 98 to 100%. The method was validated for linearity, precision, accuracy, and robustness and all parameters were found to be satisfactory which proves that this method can be used for routine analysis of Eltrombopag Olamine.
KEYWORDS: Eltrombopag, UV Spectrophotometer, Method development, Validation, Ethanol.
INTRODUCTION:
Eltrombopag (ELT) is a new therapeutic medicinal agent accepted for the management of chronic immune thrombocytopenia. Eltrombopag Olamine is an orally bioavailable, small-molecule TPO-receptor agonist that interacts with the transmembrane domain of the human TPO-receptor. Eltrombopag Olamine is used to treat low blood platelet counts in adults with chronic immune thrombocytopenia (idiopathic thrombocytopenia i.e., ITP), when certain other medicines or surgery to remove the spleen, have not worked well enough ITP is a disorder that may cause unusual swelling or bleeding due to an abnormally low number of platelets in the blood.1
The IUPAC name of Eltrombopag Olamine 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.2,3
A double beam UV-visible spectrophotometer (Jasco Companies, Tokyo, Japan), a high precisionelectronic weighing balance (Mettler Toledo, Switzerland) are used for weighing the reagents. Ultrasonication was used for the solubilization of the drug.
Pharmaceutical grade Eltrombopag Olamine was supplied by Hetero Drugs Ltd, Hyderabad, India. Analytical grade ethanol is used as a solvent.
Solubility test for the drug EltrombopagOlamine was performed by using various solvents, which include distilled water, sodium acetate (pH 6.2), according to the literature review, and also with ammonium acetate (6M), sodium citrate (1.25M), sodium glycinate (1M), sodium chloride (1M). However, ethanol was chosen as the solvent for developing the method.
In a volumetric flask of 100ml, Eltrombopag Olamine 100mg was collected. Dissolved with 100ml of ethanol, make it up to label (1000μg/ml), and sonicated.
From the stock solution, 10ml was further diluted to 100ml with ethanol to get the solution having a concentration of 100μg/ml.
As the pharmaceutical formulation of Eltrombopag Olamine is not obtainable in the local Indian market, hence in-house tablets were prepared with 250mg of ELT and frequent excipients. The sample solution was prepared from in-house formulated ELT tablets. Accurately weighed powder drug equivalent to 250mg, ELT was quantitatively transferred into 100ml volumetric flask dissolved and diluted volume with ethanol. The solution was sonicated for 15minutes. From that, an appropriate volume of solution was diluted to get a final concentration of 100μg/ml using the solvent (Ethanol).
From the above working standard solution, 1, 2, 3, 4, 5ml was pipette out into a 10ml volumetric flask and the volume was made up to the mark with ethanol to prepare a concentration of 10, 20, 30, 40 and 50μg/ml. Then the sample was scanned in UV-VIS Spectrophotometer in the range of 300-600nm using ethanol as blank. The derived result gives the maximum wavelength.
Figure 1: UV spectra of Eltrombopag Olamine expressing maximum absorbance (λmax) at 423nm
From the working standard solution, pipette out 0.5ml, 1ml, 1.5ml, 2ml, 2.5ml, and 3ml and diluted to 10ml using ethanol to produce 5μg/ml, 10μg/ml, 15μg/ml, 20µg/ml, 25ug/ml and 30μg/ml solutions respectively. The solutions were scanned at 423nm using solvent (Ethanol) as blank. Then, the calibration curve was plotted by taking concentration on X-axis and absorbance on Y-axis (Figure). The curve shows linearity in the concentration range of 5-30μg/ml.
Validation is a process of building documented evidence, which provides a high degree of assurance that a particular activity will consistently produce a desired result or product meeting its pre-determined specifications and quality characteristics. ICH has set down different method performance attributes for an analytical method in ICH guidelines Q2 (R1). Thus, for a spectrophotometric analytical method, it's appropriate to validate the method in keeping with ICH guidelines Q2 (R1) on the chosen critical parameters.4-5
Hence by using selected critical parameters, the developed method is further validated as per ICH Q2 (R1). The validation parameters studied using the developed method were system suitableness, linearity, accuracy, precision, detection limit (LOD), quantification limit (LOQ), and robustness.6
System suitableness is administrated to illustrate the suitableness of the UV spectrophotometric system which is utilized for the analysis. Six replicates of standard solution (10ug/ml) of Eltrombopag Olamine were prepared from a working standard solution within the selected solvent (Ethanol) and absorbance was determined at 423nm of every replicate employing a UV spectrophotometer. Further, the percentage relative standard deviation (%RSD) was calculated for the absorbance.7-8
As per the ICH Q2 (R1) guidelines, the linearity of an analytical procedure verifies that the test results have a direct relationship with the concentration (amount) of the analyte within the sample. For the linearity study, six solutions of various concentrations (5, 10,15, 20, 25, and 30ug/ml) were ready in ethanol from a working standard solution of ELT, and therefore the absorbance of each solution was noted at 423nm. The graph of the calibration curve was plotted between absorbance and concentration and percentage relative standard deviation and the coefficient of correlation was determined by regression analysis.9
According to the ICH guidelines, the precision of an analytical procedure indicates the closeness of results obtained by multiple measurements of the identical homogenized sample. Repeatability (intra-day precision) and intermediate precision (inter-day precision) were done to signify the precision of the method. To demonstrate the repeatability (intra-day precision) of the test methodology, six replicates of the 10g/ml concentration (n=6) were examined on a similar day. % relative standard deviation of assay results of six replicates was calculated. Similarly, for intermediate precision (inter-day precision), six replicates of the 10 g/ml concentrations were analyzed for assay on 3 consecutive days, and the percentage relative standard was calculated.10-11
The accuracy of a commenced investigation was appraised by standard addition methods, where a known amount of the standard was added in three different levels, i.e., 50, 100, and 150% to the in-house tablet formulation of ELT and analyzed by the commenced method in a set of three. The %recovery studies for ELT were carried out by spiking three different amounts of ELT standard (50, 100, and 150%) to the in-house tablet formulation. The %recovery of ELT was estimated for each level.12-14
The limit of detection is the lowest amount of analyte in a sample that may be detected, but not necessarily quantified as an actual value and also the limit of quantification is the lowest concentration of an analyte within the sample will be determined with accuracy and precision. The limit of detection and limit of quantification concentrations for ELT were determined based on the residual standard deviation of response and slope method as per ICH guidelines. A calibration curve prepared in the linearity study was used for this purpose. For LOD calculation equation (3.3 x σ )/S and LOQ equation (10 x σ)/S were used. Where σ indicates the standard deviation of the response and S is the slope of the calibration curve.15-16
Robustness is often interpreted as the capability to reproduce the (analytical) method in diverse laboratories or under different conditions without the occurrence of unexpected differences within the obtained results, and a robustness test as an experimental set-up to assess the robustness of a method. to check the capability of the proposed method, different UV spectrophotometers were used present within the different laboratories for the determination of absorbance.17-20
The absorbance of six replicates of the standard solution (10 g/ml) of ELT is outlined in Table 1. For system suitability percentage and RSD of absorbance of replicate solutionsshouldn't be more than 2. The results obtained was meets the system’s suitability needs, which means that the system was appropriate for the analysis.
Table 1: Result of system suitability
|
Concentration (ug/ml) |
Absorbance at 423nm |
|
10 |
0.3520 |
|
10 |
0.3532 |
|
10 |
0.3608 |
|
10 |
0.3506 |
|
10 |
0.3509 |
|
10 |
0.3518 |
|
Mean |
0.353 |
|
SD |
0.004 |
|
%RSD |
1.084 |
Figure 2: Calibration curve plot of Eltrombopag Olamine at 423nm
Fig 3 Overly spectrum of Eltrombopag Olamine (5 to 30 µg/ml) at 423nm
Table 2: Result of linearity study of Eltrombopag Olamine
|
Concentration (µg/ml) |
Absorbance (nm) |
|
5 |
0.185 |
|
10 |
0.340 |
|
15 |
0.543 |
|
20 |
0.755 |
|
25 |
0.996 |
|
30 |
1.182 |
|
R2 |
0.9966 |
|
%RSD |
0.003 |
|
Linear regression equation |
y= 0.0399x -0.0266 |
The calculated RSD of intra-day and inter-day preciseness tests are outlined in Tables 3 and 4. For a precise analytical technique, the % RSD of assay results of six replicates mustn't be more than 2. The RSD of the intra-day assay of six replicates was 1.032%, 0.546%, and 0.855% severally for 3 completely different times in a day, and for the interday assay, RSD was 1.067%, 1.263%%, and 1.144% severally for 3 consecutive days. a low % RSD value shows that the method was precise.
Table 4: Result of Intr-day precision (Intermediate precision)
|
No. |
Absorbance at 423 nm |
|||
|
10 µg/ml |
10 am |
1 pm |
4 pm |
|
|
1 |
0.3608 |
0.3622 |
0.3585 |
|
|
2 |
0.3614 |
0.3625 |
0.3518 |
|
|
3 |
0.3626 |
0.3609 |
0.3511 |
|
|
4 |
0.3604 |
0.3601 |
0.3515 |
|
|
5 |
0.3617 |
0.3635 |
0.3501 |
|
|
6 |
0.3704 |
0.3657 |
0.3522 |
|
|
Average |
0.3629 |
0.3625 |
0.3525 |
|
|
SD |
0.004 |
0.002 |
0.003 |
|
|
%RSD |
1.032 |
0.546 |
0.855 |
|
Table 3: Result of Intra-day precision (Repeatability)
|
Concentration |
No. |
Absorbance at 423 nm |
||
|
10 µg/ml |
Day 1 |
Day 2 |
Day 3 |
|
|
1 |
0.3338 |
0.3324 |
0.3301 |
|
|
2 |
0.3319 |
0.3308 |
0.3283 |
|
|
3 |
0.3329 |
0.3411 |
0.3271 |
|
|
4 |
0.3405 |
0.3303 |
0.3291 |
|
|
5 |
0.3319 |
0.3315 |
0.3231 |
|
|
6 |
0.3305 |
0.3301 |
0.3206 |
|
|
Average |
0.3336 |
0.3327 |
0.3264 |
|
|
SD |
0.004 |
0.004 |
0.004 |
|
|
%RSD |
1.067 |
1.263 |
1.144 |
|
Table 5: Result of accuracy
|
Concentration taken (15pm) 50% |
Concentration taken (20ppm) 100% |
Concentration taken (25ppm) 150% |
|||
|
Absorbance |
Conc. found |
Absorbance |
Conc. found |
Absorbance |
Conc. found |
|
0.5713 |
14.99 |
0.7699 |
19.97 |
0.9187 |
24.25 |
|
0.5877 |
14.74 |
0.7750 |
19.43 |
0.9243 |
24.42 |
|
0.5873 |
14.73 |
0.7719 |
19.89 |
0.8934 |
24.89 |
|
Mean of conc. |
14.82 |
|
19.77 |
|
24.52 |
|
SD of conc. |
0.15 |
|
0.29 |
|
0.33 |
|
RSD |
1.02 |
|
1.47 |
|
1.35 |
|
%Recovery |
98.79 |
|
98.83 |
|
98.08 |
The limit of detection is the lowest concentration of the analyte in a sample that can be detected with a satisfactory level of precision and accuracy under the expressed working condition of the method. LOD can fluctuate with the type of method utilized and the nature of the sample.
The limit of detection of ELT was found 6.19µg/ml and, The limit of quantification was found 18.75µg/ml.
The LOD and LOQ were determined by the equations from the calibration diagram and reported.
Table 6: Result of LOD & LOQ
|
Concentration (ppm) |
Absorbance at 423 |
|
1 |
0.0323 |
|
2 |
0.0866 |
|
3 |
0.1210 |
|
4 |
0.1697 |
|
5 |
0.2170 |
|
6 |
0.2478 |
|
Std. deviation |
0.08 |
|
Slope |
0.04 |
|
LOD |
6.19 |
|
LOQ |
18.75 |
The change in system and laboratories with the same concentration of 10µg/ml gave reproducible results. Hence the parameters were found to be validated.
Table 7: Result of Robustness
|
Conc. ug/ml |
Absorbance on system 1 |
Absorbance on system 2 |
|
10 |
0.3849 |
0.3740 |
|
10 |
0.3859 |
0.3787 |
|
10 |
0.3853 |
0.3719 |
|
10 |
0.3788 |
0.3811 |
|
10 |
0.3791 |
0.3767 |
|
10 |
0.3819 |
0.3745 |
|
Mean |
0.3827 |
0.3762 |
|
Std. deviation |
0.0032 |
0.0034 |
|
%RSD |
0.8316 |
0.8954 |
The proposed UV Spectroscopic method was used to estimate Eltrombopag Olamine in prepared in-house tablet formulation. The % amount of drug found in triplicate analysis for Eltrombopag olamine was found to be 98.85 ± 0.13%, respectively. In the future, the developed method can be frequently utilized for the investigation of Eltrombopag Olamine.
A precise, accurate, cost-effective, and robust UV Spectroscopic method has been produced for the assessment of Eltrombopag Olamine in bulk and in-house tablet formulation. The developed UV Spectroscopic method for the estimation of Eltrombopag Olamine is simple and rapid. Data obtained from precision shows result in terms of RSD less than 2, which conclude that the method is reproducible and precise. The accuracy range is between 98-99% recovery ensures good accuracy and specificity. The excellent % recovery of a drug shows that the excipients present in the tablet formulation have no obstruction in the determination of Eltrombopag Olamine indicating that the method is specific with a good response for the estimation ofEltrombopag Olamine.Therefore, the developed method can be used for the regular analysis of Eltrombopag Olamine in bulk and a pharmaceutical formulation.
The authors would like to thank Mr. Shakeel Memon professor of Allana College of Pharmacy, Pune for their kind support during method development and all other lab studies.
All the authors contributed equally
The authors state that there is no conflict of interest in publishing this paper.
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Received on 01.06.2022 Modified on 04.12.2022
Accepted on 15.04.2023 © RJPT All right reserved
Research J. Pharm. and Tech 2023; 16(11):5010-5014.
DOI: 10.52711/0974-360X.2023.00811