INTRODUCTION:

High-performance liquid chromatography (HPLC) is the fastest growing analytical technique for analysis of drugs. Its simplicity, high specificity, and wide range of sensitivity make it ideal for the analysis of many drugs in both dosage forms and biological fluids¹.

High-performance liquid chromatography (HPLC) is the term used to describe liquid chromatography in which the liquid mobile phase is mechanically pumped through a column that contains the stationary phase. An HPLC instrument, therefore, consists of an injector, a pump, a column, and a detector².

Spironolactone is chemically (17-hydroxy-7α-mercapto-3-oxo-17α-pregn-4-ene-21-carboxylic acid γ-lactone acetate). Spironolactone is most commonly used anti-diuretic agent in clinical practices. Spironolactone site of action is intracellular aldosterone receptors in the distal tubule cells. This increases the excretion of water and sodium and decreases the excretion of potassium. Spironolactone has ant androgen activity by binding to the androgen receptor and preventing it from interacting with dihydrotestosterone ³. High Performance Liquid Chromatographic (HPLC) methods for determination of SPR in human plasma in single and in combination with other drugs ⁴.

Fig 1: Chemical Structure of Spironolactone⁵

Hydrochlorothiazide: Chemically its is 6-chloro-3,4-dihydro-2H-1, 2, 4-benzothiadiazine-7-sulfonamide 1,1-dioxide Molecular formula: C7H8ClN3O4S2 Soluble in water, methanol, ethanol, acetone, Dimethyl formamide ⁶. It exerts its effect by reducing the reabsorption of electrolytes from the renal tubules, thereby increasing the excretion of sodium and chloride ions, and consequently of water ⁷. Hydrochlorothiazide is Diuretic and Antihypertensive used to treat excessive fluid accumulation and swelling (edema) of the body caused by heart failure, cirrhosis, chronic kidney failure, corticosteroid medications, and nephrotic syndrome ⁸. Hydrochlorothiazide is not metabolized but is eliminated rapidly by the kidney. Hydrochlorothiazide crosses the placental but not the blood-brain barrier and is excreted in breast milk⁹.

Fig 2: Chemical Structure of Hydrochlorothiazide ¹⁰

Hydrochlorothiazide is the most usually recommended thiazide diuretic. It is shown to treat edema and hypertension. Hydrochlorothiazide use is normal yet declining for angiotensin changing over catalyst inhibitors. Numerous mix items are accessible containing hydrochlorothiazide and angiotensin changing over protein inhibitors or angiotensin II receptor blockers¹¹. The major site of action in the nephron appears on an electroneutral NaCl co-transporter by competing for the chloride site on the transporter. By impairing Na+ transport in the distal convoluted tubule, hydrochlorothiazide induces a natriuresis and concomitant water loss.¹²

Hypertension is sustained elevation of blood pressure. Once when the time it develops, a patient should have his blood pressure checked frequently because hypertension is a life time condition. The status of hypertension control has improved considerably over the past twenty years.¹³ Sympathetic nervous system reactions from stress cause vasoconstriction, which increases arterial blood pressure (BP). In addition, stress increases the production of neurotransmitters, such as epinephrine, which place physiologic stress on the heart and increase oxygen demand.¹⁴

High blood pressure is a contributing factor in the development of many cases of heart disease, stroke, and kidney failure. However, since hypertension has no symptoms itself, it is difficult to convince patients of the importance of taking antihypertensive medication according to the physician’s prescription.¹⁵

MATERIALS AND METHODS:

Spironolactone from Sura labs, Hydrochlorothiazide from Sura labs, Water for HPLC from LICHROSOLV (MERCK), Methanol for HPLC from LICHROSOLV (MERCK), Acetonitrile for HPLC from Merck.

Validation methods procedures followed as per ICH guidelines^16-19.

RESULTS AND DISCUSSION:

Optimized Chromatographic Conditions:

Mobile phase: Phosphate Buffer (pH-4.8): Methanol (55:45% v/v)

Column: Inertsil C18 (4.6mm ×250mm, 5µm particle size)

Flow rate: 1ml/min

Diluent Phosphate Buffer (pH-4.8): Methanol (55:45% v/v)

Injection Volume : 20µl

Wavelength: 282nm

Column temp: 35ºC

Run mode: Isocratic

Runtime : 6 minutes

Optimized Chromatogram:

System Suitability Parameters:

Table 2: Observation of system suitability parameters

S. No	Parameter	Spironolactone	Hydrochlorothiazide
1.	Retention Time (min)	1.688	3.282
2.	Theoretical Plates	7586	6235
3.	Tailing factor	1.69	1.58
4.	Area	1658768	426589
5.	Resolution	10.89

Accuracy:

Spironolactone

Table 3: Accuracy Observation of Spironolactone

% Concentration (at specification Level)	Average Area	Amount Added (ppm)	Amount Found (ppm)	% Recovery	Mean Recovery
50%	879537	150	150.048	100.032	100.112%
100%	1743252	300	300.521	100.172
150%	2609693	450	450.598	100.132

Hydrochlorothiazide

Table 4: Accuracy Observation of Hydrochlorothiazide

%Concentration (at specification Level)	Average Area	Amount Added (ppm)	Amount Found (ppm)	% Recovery	Mean Recovery
50%	224271	25	25.114	100.456%	100.16%
100%	445748.3	50	49.952	99.904%
150%	670006.3	75	75.101	100.134%

Precision:

System Precision:

Table 5: Observation of System Precision

S. No	Sample Area 1	Sample Area 2
1	1658254	426598
2	1658952	426589
3	1654857	426985
4	1659854	426587
5	1653298	426515
Mean	1657043	426654.8
Std.dev	2820.29	187.5692
%RSD	0.1702	0.043963

Linearity

Fig 4: Calibration Curve for Spironolactone

Table 6: Linearity Observation of Spironolactone

S. No	Concentration Level (%)	Concentration mg/ml	Average Peak Area
1.	I	100	585985
2.	II	200	1182468
3.	III	300	1768785
4.	IV	400	2326852
5.	V	500	2856874
Correlation coefficient			0.999

Fig 5: Calibration Curve for Hydrochlorothiazide

Table 7: Linearity Observation of Hydrochlorothiazide

S. No.	Concentration Level (%)	Concentration mg/ml	Average Peak Area
1	I	30	268764
2	II	40	356958
3	III	50	445631
4	IV	60	535186
5	V	70	624698
Correlation coefficient			0.999

Limit of Detection (LOD)

The detection limit of an individual analytical procedure is the lowest amount of analyte in a sample which can be detected but not necessarily quantitated as an exact value.

LOD= 3.3 × S.D / Slope

Table 8: LOD results of the method

Drug	Amount(µg/ml )
Spironolactone	2.1
Hydrochlorothiazide	1.28

From the above, the LOD values of Spironolactone and Hydrochlorothiazide were found to be 2.1 and 1.28µg/ml respectively.

Limit of Quantitation (LOQ):

The quantitation limit of an individual analytical procedure is the lowest amount of analyte in a sample which can be quantitatively determined.

LOQ=10× S.D / Slope

Table 9: LOQ results of the method

Drug	Amount(µg/ml )
Spironolactone	6.3
Hydrochlorothiazide	3.84

Robustness

Table 10: Flow rate Observation of Spironolactone

System suitability Results for Spironolactone

Flow Rate (ml/min)		System suitability Results
Flow Rate (ml/min)		USP Plate Count	USP Tailing	Retention Time (min)
Less Flow rate	0.8	7365	1.62	1.868
Actual Flow rate	1	7586	1.69	1.688
More Flow rate	1.2	7254	1.61	1.544

Results for actual flow rate have been considered from assay standard.

Table 11: Flow rate Observation of Hydrochlorothiazide System suitability Results for Hydrochlorothiazide

Flow Rate (ml/min)		System suitability Results
Flow Rate (ml/min)		USP Plate Count	USP Tailing	Retention Time (min)
Less Flow rate	0.8	6284	1.51	3.621
Actual Flow rate	1	6235	1.58	3.282
More Flow rate	1.2	6168	1.56	2.998

Organic Composition:

Table 12: System suitability results Spironolactone

Organic phase		System suitability Results
Organic phase		USP Plate Count	USP Tailing	Retention Time (min)
Less organic phase	50:50	7269	1.61	1.868
Actual organic phase	55:45	7586	1.69	1.688
More organic phase	60:40	7496	1.64	1.675

Table 13: System suitability result Hydrochlorothiazide

Organic phase		System suitability Results
Organic phase		USP Plate Count	USP Tailing	Retention Time (min)
Less organic phase	50:50	6182	1.54	3.621
Actual organic phase	55:45	6235	1.58	3.282
More organic phase	60:40	6322	1.56	2.302

CONCLUSION:

In the present investigation, a simple, sensitive, precise and accurate RP-HPLC method was developed for the quantitative estimation of Spironolactone and Hydrochlorothiazidein bulk drug and pharmaceutical dosage forms.

This method was simple, since diluted samples are directly used without any preliminary chemical derivatisation or purification steps.

Spironolactone was found to be practically insoluble in water; soluble in chloroform, ethanol. Hydrochlorothiazide was found to be is slightly soluble in water, freely soluble in sodium hydroxide solution, in n-butyl amine, and in dimethyl formamide; sparingly soluble in methanol; insoluble in ether, in chloroform, and in dilute mineral acids.

Phosphate Buffer (pH-4.8): Methanol (55:45% v/v) was chosen as the mobile phase. The solvent system used in this method was economical.

The %RSD values were within 2 and the method was found to be precise.

The results expressed inTablesfor RP-HPLC method was promising. The RP-HPLC method is more sensitive, accurate and precise compared to the Spectrophotometric methods.

This method can be used for the routine determination of Spironolactone and Hydrochlorothiazidein bulk drug and in Pharmaceutical dosage forms.

ACKNOWLEDGEMENT:

Thе Authors arе thankful to the Management and Principal, Department of Pharmacy, Samskruti College of Pharmacy, Hyderabad, for extending support to carry out the research work. Finally, the authors express their gratitude to the Sura Pharma Labs, Dilsukhnagar, Hyderabad, for providing research equipment and facilities.

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Received on 22.10.2022 Modified on 17.11.2022

Asian J. Pharm. Tech. 2023; 13(3):166-170.

DOI: 10.52711/2231-5713.2023.00029

S. No	Peak Name	Retention Time	Area	Height	USP Tailing	USP Plate Count	USP Resolution
1	Spironolactone	1.688	1658785	385669	1.69	7586	10.85
2	Hydrochlorothiazide	3.282	425631	65245	1.58	6235	10.85