INTRODUCTION:

Eprosartan mesylate is a member of angiotensin-II receptor (AT1) antagonists that is a non-biphenyl or a tetrazole. 4-(2-butyl-5-[(1E)-2-carboxy-2-(thiophene-2-ylmethyl) eth-1-en-1-yl] is a selective non-peptide compound. benzoic acid (1H-imidazol-1-ylmethyl) (Fig 1). Eprosartan mesylate has the chemical formula C24H28N2O7S2 and white/off white colour powder, crystalline in nature and is freely flowable which is not soluble in water practically but soluble very freely in ethanol. Eprosartan mesylate is used to treat high blood pressure and high cholesterol. It has two effects on the renin-angiotensin system. By blocking angiotensin II from attaching to AT1, the vascular smooth muscle relaxes, and vasodilation occurs.

Blood pressure is decreased further by blocking norepinephrine production^1-2. HCT is a thiazide diuretic that is 6-chloro-3, 4-dihydro-2 H 1, 2, 4-benzothiadiazine-7-sulfonamide 1, 1-dioxide. The empirical formula of the compound is C₇H₈ClN₃O₄S₂. It is an almost white andit is a crystalline powder that is somewhat soluble in water. The Food and Drug Administration (FDA) recently gave approval of stable dose mixture of Eprosartan and Hydrochlorothiazide (600mg/25mg) administration in combination with an angiotensin II receptor (AT1subtype). Eprosartan eases blood flow by relaxing blood vessels. It is a member of the angiotensin-receptor blocker family of medications (ARBs). Hydrochlorothiazide increases the amount of urine your body produces to cleanse itself of excess salt and water. These drugs are known collectively as diuretics or water pills^3-6.

MATERIALS AND METHODS:

The analytical grade pure samples of Hydrochlorothiazide (99.28%) and Eprosartan obtained from hetero labs, Andrapredesh, India supplied mesylate (99.55%). Methanol HPLC grade solvent and all solvents were purchased from Merck Ltd, Mumbai, India. Potassium dihydrogen phosphate bought from Qualigens. Fine Chemicals, Mumbai, India. The HPLC standard grade water was obtained from a MilliQ RO water purification system which is sonicated, and used. Distilled water HPLC grade, HPLC water Analytical Reagent grade, Acetonitrile AR Grade, Phosphate buffer AR Grade, Methanol AR Grade Sodium dihydrogen phosphate AR Grade, Ortho-phosphoric acid AR Grade manufactured by Merck, Mumbai were purchased. Aimex Pharma, Hyderabad gifted Eprosartan pure drug, and Hydrochlorothiazide pure drug. Free expired Human plasma: K2 EDTA control purchased from Red Cross Society, Warangal. A water HPLC 2695 System was used. Centrifuge by Thermo Fisher make, Electronic balance by Sartorius make, pH meter by elico make was used.

Fig.1: Chemical structure of Eprosartan and Hydrochlorothiazide

DEVELOPMENT OF METHOD:

Diluent selection:on the basis of solubility of drugs, diluents were selected, and various trails performed with different solvent systems like Methanol: Acetonitrile (50:50); 0.01N Potassium dihydrogen phosphate pH (3.0): Acetonitrile (50:50); 0.1% of Orthophosphoric acid (pH-2.2): Acetonitrile (50:50); 0.1% of Ortho phosphoric acid (pH-2.2): Acetonitrile (60:40).

Preparation of Eprosartan Stock solution (160 µg/ml):

16mg of Eprosartan in a 100ml volumetric flask with diluent made to give 160µg/ml solution.

Preparation of Eprosartan Spiking Solutions:

0.05ml, 0.1ml, 0.15ml, 0.6ml, 1.0ml, 1.2ml, 1.6ml, and 2.0ml of Eprosartan stock solution were pipetted into 8 individual 10ml volumetric flasks and made up to the mark with diluent to give 0.8µg/ml, 1.6µg/ml, 2.4µg/ml, 9.6µg/ml, 16µg/ml, 19.2µg/ml, 25.6µg/ml and 32.0µg/ml. To create the analytes, blank plasma was spiked with working stock dilutions of the substances 80ng/ml, 160ng/ml, 240ng/ml, 960ng/ml, 1600ng/ml, 1920ng/ml, 2560ng/ml, and 3200ng/ml calibrating the standards.

Preparation of Hydrochlorothiazide Stock solution (17 µg/ml):

1.7mg of Hydrochlorothiazide in 100ml diluent to produce 17µg/ml in a volumetric flask.

Preparation of Hydrochlorothiazide Spiking Solutions:

0.05ml, 0.1ml, 0.15ml, 0.6ml, 1.0ml, 1.2ml, 1.6ml, and 2.0ml of the hydrochlorothiazide stock solution was pipette and transferred to eight separate 10ml volumetric flasks, which were then brought up to the required concentration using diluentto give 0.085µg/ml, 0.17µg/ml, 0.255µg/ml, 1.02µg/ml, 1.7µg/ml, 2.04 µg/ml, 2.72µg/ml and 3.4µg/ml. Blank plasma was spiked with working stock dilutions of analytes to create 8.5ng/ml, 17ng/ml, 25.5ng/ml, 102ng/ml, 170ng/ml, 204 ng/ml, 272ng/ml, and 340ng/ml calibration standards and quality control (QC) samples.

Preparing internal standard (Valsartan) solution:

Stock 1:

Valsartan was placed in a 100mL volumetric flask and the volume was filled up with diluent to get the desired result of 500µg/ml.

Stock 2:

Using the aforesaid solution, 1mL of the solution is put into a 10mL volumetric flask and the volume is filled up with diluent to make 50µg/mL solutions (see illustration).

Final solution:

10g/ml ISD concentration from the aforementioned solution by mixing 0.5ml of solution with blank plasma and working stock dilutions of the analyte were prepared.

Extraction procedure:

Centrifuge tube filled with 750µl of plasma and 500µl of internal standard, 250µl of Eprosartan and 250µl of Hydrochlorothiazide from the spiking solutions of each, and 1ml of Acetonitrile, and cyclomix for 15sec. Then vertex for 2 minutes before centrifuging for 5 minutes at 3200rpm. The samplewas collected after centrifugation and immediately injected 20µl into HPLC after filtering.

Optimized method: Chromatographic conditions:

Mobile phase: 0.1% Ortho phosphoric acid pH (2.2): Acetonitrile (60:40); Flow rate: 1.0ml/min; Column: Azilent C18 (150mm x 4.6 mm, 5m); Detector wavelength: 240nm; Column temperature: 30⁰C; Injection volume: 20µL.

Validation of Method:

The analytical method was validated on the basis of the International Conference on Harmonization (ICH) guidelinesand also based on the literature survey^7-21. The validation of method with parameters checked were linearity, specificity, and accuracy, and precision, the limits of detection andquantitation, and robustness. Resulted are depicted in table 1-4 and figures 2-4.

1. Selectivity/Specificity:

High selectivity of both drugs was displayed during the detection of extracted blank in plasma and internal standards as compared to other methods. Neither analyte nor internal standard retention times showed interferences.

2. Linearity:

Calibration was linear for Eprosartan (80-3200ng/ml) and Hydrochlorothiazide (8.5-340ng/ml). The value of r² was more than 0.999 consistently. This demonstrates good linearity in the data as well as the ratios of the peak areas against concentration of each analyte was tested in this study. The figure 3 and 4 shows a representative calibration curve from the third precision and accuracy batch. Three calibration curves had their concentrations recalculated.

3. Precision and Accuracy:

6 replicates at 5 QC levels (LQC, LLOQ, MQC, and HQC) were used to examine the accuracy and precision in intraday and interday. 6 replicate analyses were performed for Eprosartan at four concentration levels: 80ng/ml (LLOQ), 640ng/ml (LQC), 1600ng/ml (MQC), and 2560g/ml (HQC), and Hydrochlorothiazide at 8.5ng/ml (LLOQ), 25.5ng/ml (LQC), 170ng/ml (MQC), and 272ng/ml (HQC) (HQC), After examining the accuracy of plasma samples over time, it was discovered that the mean percent accuracy ranged from 100.13 to 100.66 percent intra-day and 99.16 to 100.87 percent inter-day, resulting in an exceptional mean percent accuracy. Calculating the accuracy (% CV) of the analytes and plasma samples revealed that they ranged between 0.91 and 1.76 % for intraday and 0.98 to 1.58 % for interday.

4. Recovery:

Eprosartan and Hydrochlorothiazide had 98.85% and 98.67% mean recovery. Valsartan had a 98.11 %overall mean recovery.

5. Stabilities:

Long-term stock solution stability for Eprosartan: Six duplicates of LQC and HQC samples (240 and 2560 ng/ml) were tested for 9 hours at room temperature. The percent mean stability was found to be 99.96% for LQC and 99.19% for HQC.

Long term Hydrochlorothiazide stock solution stability: Six LQC and HQC samples (25.5 and 272 ng/ml) were evaluated for bench-top stability for 9 hours at normal temperature. The percent mean stability was found to be 99.69% for LQC and 99.89% for HQC.

Matrix stability of samples at 28±5 ⁰C and 80±5 ⁰C for 1 month: It was determined that the stability of eposartan stock solution at a concentration of LQC-HQC after 37 days of storage at -28°C and -80°C in the refrigerator was satisfactory. At 28°C, the % mean stability of Eprosartan was established to be 99.90 %, while at 80°C, it was determined to be 100.16 %.The LQC-HQC concentration of Hydrochlorothiazide was evaluated after 37 days of storage at -28°C and -80°C in the refrigerator. The percent mean stability of Hydrochlorothiazide was determined to be 100.94% at 28°C and 101.11% at 80°C.

RESULTS AND DISCUSSIONS:

The estimation of Eprosartan, Hydrochlorothiazide, using current ICH recommendations. The first trial failed to elute peaks, therefore another was run. That trial was needed because the study didn't get to valsartan. The Valsartan USP plate count was low in trial 3. Trail 4 was optimized for peak form and retention time.

Fig. 2: Chromatogram of the optimized method

After the trials, orthophosphoric acid pH (2.2): Acetonitrile (60:40) becomesan optimized chromatogram.

Table 1: System Suitability of Eprosartan and Hydrochlorothiazide

	System Suitability of Eprosartan			System Suitability of Hydrochlorothiazide
S.No	Analyte Rt (min)	IS Rt (min)	Area Ratio	Analyte Rt (min)	IS Rt (min)	Area Ratio
MEAN	2.839	2.329	0.57789	3.379	2.329	0.17960
SD	0.0268	0.0152	0.003891	0.0087	0.0152	0.001588
%CV	0.94	0.65	0.67	0.26	0.65	0.88

Note: Mean ± SD of Six observations

All system suitability criteria were within ICH recommendations. The percent CV for system compatibility test was 0.94 for Eprosartan retention time (RT), 0.26 for Hydrochlorothiazide retention time (RT), and 0.88 percent for Valsartan area ratio (analyte area/IS area).

Table 2: Linearity of Eprosartan

Mean	79.3663	159.6980			238.0607	641.9563			1585.1803		1912.7910	2562.4600		3230.6987
SD	4.49484	5.1161			5.9980	3.2165			11.2299		12.7384	17.52342		5.3339
%CV	5.66	3.20			2.52	0.50			0.71		0.67	0.68		0.17
% Mean Accuracy	99.21	99.81			99.19	100.31			99.07		99.62	100.10		100.96
Linearity of Hydrochlorothiazide
Mean	8.5160		17.0420	25.3470			68.2743	170.8073		205.2137			270.5627		342.6617
SD	0.05543		0.12679	0.20001			0.73270	1.52081		0.36950			3.18791		3.05800
%CV	0.65		0.74	0.79			1.07	0.89		0.18			1.18		0.89
% Mean Accuracy	100.19		100.25	99.40			100.40	100.47		100.59			99.47		100.78

Note: Mean ± SD of three observations

Table 3: Precision and Accuracy

	intra-day runs of Eprosartan				inter-day runs of Eprosartan
	HQC	MQC1	LQC	LLOQ QC	HQC	MQC1	LQC	LLOQ QC
Mean	2541.1675	1590.7003	645.2742	80.1798	2552.7912	1597.7666	644.9650	80.0362
SD	38.14939	30.52685	9.36619	1.46237	40.29934	19.23303	6.32403	3.64750
%CV	1.50	1.92	1.45	1.82	1.58	1.20	0.98	4.56
% Mean Accuracy	99.26	99.42	100.82	100.22	99.72	99.86	100.78	100.05

Note:Mean ± SD of six observations

Table 4: Precision and Accuracy

	intra-day runs of Hydrochlorothiazide				inter-day runs of Hydrochlorothiazide
	HQC	MQC1	LQC	LLOQ QC	HQC	MQC1	LQC	LLOQ QC
Mean	271.2353	171.6132	25.4173	8.4220	271.0886	171.3558	25.3175	8.4956
SD	2.69068	1.94441	0.26702	0.18782	3.03792	1.59060	0.29169	0.16072
%CV	0.99	1.13	1.05	2.23	1.12	0.93	1.15	1.89
% Mean Accuracy	99.72	100.95	99.68	99.08	99.66	100.80	99.28	99.95

Note: Mean ± SD of six observations

Fig.3: Calibration curve of Eprosartan

Fig.4:Calibration curve of Hydrochlorothiazide

CONCLUSION:

This study's findings support the conclusion that the current validated approach may be used to accurately estimate Eprosartan and Hydrochlorothiazide concentrations in human plasma throughout a wide dose response range (80 to 3200 ng/ml for Eprosartan and 8.5 to 340ng/ml for Hydrochlorothiazide). The technique for measuring Eprosartan and Hydrochlorothiazide in human plasma using high-performance liquid chromatography (HPLC) detection passed all of the acceptance criteria in terms of selectivity, precision, accuracy, linearity, and recovery. Testing for stability in human plasma, stock solutions, and stock dilutions in k2EDTA fulfilled the approval requirements, with Eprosartan and Hydrochlorothiazide indicating minimal deterioration during the specified storage periods and circumstances, respectively. An affordable, easy, sensitive, precise, and accurate technique for determining Hydrochlorothiazide and Eprosartan in the solid dosage form has been devised for the simultaneous determination of both drugs. The simplicity in the constitution of the mobile phase and relatively cheap cost of the components of the mobile phase coupled with its accuracy make this method the best choice in routine analysis of HCTZ and EPRO in the pharmaceutical industry.

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Received on 02.11.2021 Modified on 18.03.2022

Research J. Pharm. and Tech 2023; 16(3):1095-1099.

DOI: 10.52711/0974-360X.2023.00182