INTRODUCTION:

Chemically 5-(4-Bromophenyl)-4-[2-(5-bromopyrimidin-2-yl) oxyethoxy]-6-(sulfamoylamino) pyrimidine. It is also be spelled as "Tryvio”. ¹^-⁷It is available in the Indian market as tablet dosage form in the brand names Tryvio.⁸

Aprocitentan is a medicine used to treat Hypertension. Aprocitentan belongs to a class of drugs called Hypertension. It is Highly soluble in Dimethylsulfoxide, Water. ⁹Mode of action Aprocitentan is an endothelin receptor antagonist that inhibits the protein endothelin-1 from binding to endothelin A and endothelin B receptors. Endothelin-1 mediates various adverse effects via its receptors, such as inflammation, cell proliferation, fibrosis, and vasoconstriction. Literature survey reveals that only RP-HPLC method has been developed for Aprocitentan so there is no UV spectroscopic method available to developed either from bulk drug or from the pharmaceutical dosage forms. The present investigation was proposed to developed and validated Aprocitentan in bulk and pharmaceutical dosage form by UV spectroscopic method which is rather simple, sensitive, specific, precise, accurate method. The present work was to develop and validate as per ICH guidelines so the method was developed validated according to ICH guidelines for accuracy, precision, reproductivity, repeatability and robustness.¹⁰

MATERIALS AND METHODS:

Materials and Reagents:

A gift sample of Aprocitentan Idorsia Pharmaceuticals, Idorsia is a Swiss pharmaceutical research company in Allschwil, near Basel, Switzerland. (Tryvio 12.5mg) film coated tablet dosage form bought from the local market. Other chemicals like Dimethylsulfoxide were bought from SD fine chemicals, Mumbai, India.

Apparatus and Eqiupments Required:

1. UV-Vis double beam spectrophotometer (Model; SHIMADZU: 1700S, Japan), Electric Sonicator, Volumetric flasks (10ml, 50ml, 100ml), Calibrated analytical pipettes, Electronic digital balance (Techno, Mumbai)

Preparation of Concentration Range (Beer’s limit):

Determination of concentration range which obeys the Lambert and Beer’s law is necessary for accuracy and reproducibility in Spectrophotometric analysis for quantitative determination of any drug. For this; Aprocitentan stock solution (100µg/ml) was prepared using pure drug in Dimethylsulfoxide. Further dilutions were made using 0.2ml, 0.4ml, 0.6ml, 0.8ml, 1.0ml, 1.2ml, 1.4ml, 1.6ml and 1.8ml of above solution was transferred to a series of 10ml volumetric flasks this gave a series of concentrations ranging from 2 to 18 µg/ml of Dimethylsulfoxide.The final volume was made up to 10ml mark using Dimethylsulfoxide, sonicated for 5 minutes. The resultant solutions were measured using a double beam uv-vis-spectrophotometer at wave length of 226.2nm against a reagent blank. A calibration curve was plotted with concentration against absorbance. From the graph it was clear that Beer’s law was obeyed in concentration range of 2-14µg/ml and deviation was observed above these concentrations.

Preparation of Standard Calibration Curve:

100 mg of pure Aprocitentan drug was dissolved in little quantity of Dimethylsulfoxide as in a 100 ml volumetric flask, the volume was made up to the mark using Dimethylsulfoxide. The solution was sonicated for 10 minutes. This gave a Aprocitentan solution with concentration of 1mg/ml (1000µg/ml). 10ml of this solution was further diluted 100ml in volumetric flask using Dimethylsulfoxide as to obtain a concentration of 100µg/ml. Further dilutions were made using 0.2, 0.4, 0.6, 0.8, 1.0, 1.2, 1.4, 1.6 and 1.8ml solution was transferred to a series of 10ml volumetric flasks (to Obtain a series of concentrations ranging from 2-14 µg/ml of Aprocitentan). The final volume was made up to 10ml mark using Dimethylsulfoxide as, sonicated for 5 minutes, the absorbances were measured at of 226.2nm against a reagent blank. A calibration curve was plotted with concentration against absorbance.

Estimation of Aprocitentan in Tablet Dosage Forms:

Twenty tablets were weighed accurately and powdered. The tablet powder equivalent to 100mg of Aprocitentan was transferred into a 100ml volumetric flask and dissolved in little quantity of Dimethylsulfoxide. Then the solution was sonicated for 30 minutes and filtered using whatman filter paper No#41. The filtrate so obtained was diluted with Dimethylsulfoxide as to produce 100ml. Further dilutions were made with Dimethylsulfoxide as to get required concentrations within Beer’s - Lambert limits. The resultant solutions were measured at wave length of 226.2nm against a reagent blank. The concentration of drug was calculated with the help of standard calibration curve.

Analytical Method Validation:

Validation of an analytical procedure is the process by which it is established, by laboratory studies, that the performance characteristics of the procedure meet the requirements for its intended use. All analytical methods that are intended to be used for analyzing any clinical samples will need to be validated. Validation of analytical methods is an essential but time-consuming activity for most analytical development laboratories. It is therefore important to wave length. Understand the requirements of method validation in more detail and the options that are available to allow for optimal utilization of analytical resources in a development laboratory.

RESULTS AND DISCUSSION:

Results of Determination of Beer's Limit:

Table:1 showing Beer’s range for Aprocitentan:

Sl. No.	Concentration in µg/ml	**Absorbance at λmax 226.2nm
1.	0.0	0.000
2.	2	0.085
3.	4	0.180
4.	6	0.270
5.	8	0.375
6.	10	0.470
7.	12	0.550
8.	14	0.640
9.	16	0.820
10.	18	1.000

(** Average of three determinations)

Figure: 1 showing Beer’s limit for Aprocitentan pure drug:

(Figure:1 showing Beer's limit for Aprocitentan at 226.2nm)

Table 3 showing absorption of drug from tablet dosage form

Volume	Amount of drug	Absorbance	Amount of drug	Percentage purity
of stock	(Label claim)	at	found (µg/ml)	found ±S. D**
Solution	(µg/ml)	226.2nm		(%w/w)
Used

0.2ml	2	0.1093	2.019	100.75±0.16
0.6ml	6	0.317	6.043	100.68±1.47
1.0ml	10	0.514	9.985	99.85±0.66
1.4ml	14	0.698	13.984	99.90±0.51

(** Average of three determinations)

Determination of Accuracy:

Table:4 Accuracy results for Aprocitentan:

	Initial amount	Amount of pure drug	Amount	% Recovery
Brands	(µg/ml)	added (µg/ml)	recovered (µg/ml)	±S. D**
	10	8(80%)	8.031	100.37± 0.147
TRYVIO	10	10(100%)	9.901	98.01± 0.231
	10	12(120%)	12.030	100.25± 0.421

(**Average of six determinations, n=6)

Determination of Precision:

Table: 5 Precision results for Aprocitentan

Sl.	Conc. in (µg/ ml)	Inter-day	% C. V	Intra-day absorbance	% C. V
		absorbance		Mean±S.D**
		Mean±S.D**

1.	8	0.417±0.009	0.42	0.416±0.021	0.69
2.	10	0.516±0.030	0.41	0.516±0.025	0.30
3.	12	0.609±0.023	0.67	0.612±0.031	0.31

(**Average of three determinations, n=3)

Table: 9 Showing calibration data for Aprocitentan at 226.2nm

Parameters	Calibration data at 226.2nm
λmax	226.2nm
Beer’s law limit (µg/ ml)	2 -14µg/ml
Molar Absorptivity	1.1161Lmol^-1cm^-1
Regression Equation(Y=a+bc)	Y= 0.055X+0.016
Slope (b)	0.01426 to 0.01472
Intercept(a)	- 0.004520 to 0.0016757
Correlation Coefficient (R²)	0.998
Limit of detection (LOD)	1.226µg/ml
Limit of quantitation (LOQ)	5.226µg/ml

Determination of Beer’s limit:

The Beer’s limit felled in the range of 2-14µg/ml under given experimental conditions.

Determination of absorption maxima:

100µg/ml stock solution of Aprocitentan was prepared and absorbances were measured from 200nm to 340nm. The optimum wave length was found to be 226.2nm.

Assay:

Marketed tablets contained Aprocitentan were used for the assay. After extraction, proper dilution, measurement, the concentration was determined using standard calibration curve. The amount of drug found in the range of 99.80 – 100.70%w/w.

Method validation:

The proposed method was validated in accordance to ICH guidelines.

a) Accuracy: Percentage of recoveries of Aprocitentan in tablets was found in the range of 99.01 – 100.37% w/w.

b) Precision: The percent coefficient of variations (% C.V) was between 0.34-0.60 for intra-day and 0.42-0.67 for inter-day absorbances.

c) Repeatability: Repeatability was determined by analyzing the sample at the given concentration wavelength for at least six times and it was found that the variability in the results was not more than 0.5%.

d) Reproducibility: The standard solution of Aprocitentan by analyst-I and analyst-II separately. The values obtained were evaluated using F-test and t-test to verify their reproducibility. Calculated value for t-test was found to be less than the tabulated (standard) value it can calculated that no significant difference was observed in the result of analysis.

e) Ruggedness: Ruggedness of the developed method was determined by changing the analytical tools such as laboratory, instruments, analyst and chemicals. The result (in terms of %RSD) of six determinations indicated that there were no significant variations in the data.

f) Robustness: Robustness of the method was established by slightly changing the temperature and PH of the reaction mixture. The data so obtained showed no significant variation in the absorption pattern.

g) Limit of detection and limit of quantitation: were determined from the standard deviation of y – intercepts of six calibration curves and average slope of six calibration curves. LOD and LOQ of Finerenone was found to be 1.226µg/ml and 5.226µg/ml respectively.

CONCLUSION:

A new spectrophotometric method was developed and validate Aprocitentan in pure and tablet dosage forms. Dimethylsulfoxide as solutions of Aprocitentan was developed by using UV-spectrophotometer (Shimadzu 1700S, Japan) with matched 1cm quartz cell. It showed maximum absorption at the range of 2-14µg/ml at 226.2nm with coefficient of correlation (R2) of 0.998. The method so developed was validated according to ICH guide lines for accuracy, precision (inter and intra-day precisions), repeatability, reproducibility, ruggedness, robustness etc. The proposed method was found to be simple, accurate, sensitive, precise, reproducible and rapid. This method can be successfully employed for routine quantitative analysis of Aprocitentan in bulk and tablet dosage form.

ACKNOWLEDGMENT:

The authors are thankful to Head of the Department of Chemistry, Department of Quality Assurance.

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Received on 13.11.2024 Revised on 18.02.2025

Accepted on 31.03.2025 Published on 06.05.2025

Available online from May 10, 2025

Asian Journal of Pharmaceutical Analysis. 2025; 15(2):80-84.

DOI: 10.52711/2231-5675.2025.00013

This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License. Creative Commons License.

Parameters	Laboratory	Name of the instrument	Manufacturer of the chemicals used
Lab. 1 with analyst I	M.M.U. College of Pharmacy, Ramanagara	Shimadzu- (model: 1700S, Japan) double beam uv-vis spectrophotometer	S.D Fine Chemicals, Mumbai.
Lab. 2 with analyst II	Dr.H.L.T. College of Pharmacy, Kengal, Channapatna	SystronicUv-Vis Double beam spectrophotometer	Loba Chemicals, Mumbai

Sl. No	Brand	Label claim(mg)	*Lab. 1 with analyst I**		*Lab. 2 with analyst II**
			Amount found(mg)	% Recovery±S.D**	Amount found (mg)	% Recovery±S.D**
1.	TRYVIO	10	10.09	100.9±0.749	9.97	99.7±0.349

Type	Sl. No.	Conc. In (µg/ml)	Change in temperature		Change in P^H
Type	Sl. No.	Conc. In (µg/ml)	+5⁰C	-5⁰C	2drops of 0.1N NaOH	2drops of 0.1N HCl
Pure	1	8	0.411±0.032	0.412±0.022	0.410±0.017	0.412±0.017
Drug	2	10	0.521±0.028	0.519±0.023	0.520±0.016	0.521±0.033
	3	12	0.615±0.028	0.616±0.032	0.615±0.055	0.614±0.054

Sl. No.	Concentration in µg/ml	Absorbance at λmax 226.2nm
1.	0.0	0.000
2.	2	0.085
3.	4	0.180
4.	6	0.270
5.	8	0.375
6.	10	0.470
7.	12	0.550
8.	14	0.640