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1. RP-HPLC Analytical Method Development and Validation for Simultaneous Estimation of Enalapril Maleate and Hydrochlorothiazide in Tablet Dissertation Prepared By- Omraj R.Rathod Shri Wagheshwar Gramvikas Pratishthan’s LOKNETE SHRI DADAPATIL PHARATE COLLEGE OF PHARMACY Mandavgan Pharata , Tal- Shirur , Dist -Pune, 412211 2022-23

2. Introduction High Performance Liquid Chromatography/ High Pressure Liquid Chromatography is an analytical chromatographic method to separate ions/molecules from sample using mobile and stationery phases due to difference in adsorption, size, partitioning etc. It consists of 4 basic parts: 1. Pump system 2. The column 3. The injection system 4. The detector system

3. DRUG PROFILE Hydrochlorothiazide Name Hydrochlorothiazide A thiazide diuretic always considered the prototypical member of this class. It reduces the reabsorption of electrolytes from the renal tubules. This results in increased excretion of water and electrolytes, such as sodium, potassium, chloride, and magnesium. It has been used for the treatment of several disorders including edema, hypertension, diabetes insipidus, and hypoparathyroidism. Description is suddenly used for the treatment of hypertension, congestive heart failure, symptomatic edema, diabetes insipidus, renal tubular acidosis. It is also used for the prevention of kidney stones in those who have high levels of calcium in their urine

4. Structure Molecular Formula C 7 H 8 ClN 3 O 4 S 2 Synonyms hydrochlorothiazide 58-93-5 HCTZ Esidrix Hypothiazide Molecular Weight 297.7 g/mol Fig No.2.1 Structure of Hydrochlorothiazide

5. MATERIALS AND METHODS Instrument-HPLC Agilent 1120 series with degasser, binary gradient pump and UV detector Drug- Pure drug of Tapentadol hydrochloride was obtained from Emcure Pharmaceuticals Ltd., Pune. Stationery phase- Chromasil C18 (4.6 x 250 mm, 5 µm) column maintained at 27°C temperature Mobile phase- Methanol: water (60:40 v/v) and maintained at flow rate 1.0 ml/min. The mobile phase was kept in ultrasonicator for 30 min. and filtered through a 0.22 µm nylon membrane filter. Injection volume-_20 µl. Wavelength- 272.0 nm

6. Literature Survey The literature survey for the RP-HPLC Analytical Method Development and Validation for Simultaneous Estimation of Enalapril Maleate and Hydrochlorothiazide in Tablets focuses on the significance of accurate analysis for combined hypertension therapy. It reviews existing methods, highlighting challenges posed by distinct chemical properties. The chosen RP-HPLC approach is explained, covering factors like column selection, mobile phase optimization, and detector choice. Emphasis is placed on validation parameters' role in ensuring reliability. Comparative analysis underscores RP-HPLC's strengths, especially its sensitivity and relevance in pharmaceutical quality control. Recent innovations in RP-HPLC techniques are discussed, along with their contributions to efficiency.

7. Aim and Objective Aim: RP-HPLC Analytical Method Development and Validation for Simultaneous Estimation of Enalapril Maleate and Hydrochlorothiazide in Tablet . Objective: To developed sensitive, rapid, accurate, precise and desirable method for estimation of drug by RP-HPLC method. To develop RP-HPLC method for Enalapril Maleate and Hydrochlorothiazide. To validate Novel Qualitative and Quantitative Analytical method development for estimation of Enalapril Maleate and Hydrochlorothiazide in bulk and pharmaceutical formulation as per ICH guidelines. Statistical analysis of the recovery data obtained from different techniques for Enalapril Maleate and Hydrochlorothiazide.

8. Plan of Work 1. Selection of Drug: Online Journals, chemical and analytical abstracts were studied to find out drugs for which reported methods or the reported methods were observe, many methods were got costly and time consuming. Market survey was carried to check the availability of these drugs. The criteria for selection of drugs are explained in individual drug profile. 2. Enalapril Maleate and Hydrochlorothiazide 3. Selection of analytical techniques: HPLC method 4. Method development and validation:

9. HPLC Method: Selection of different mobile phase. Selection of range of pH. Selection of range of proportion of mobile phase with appropriate retention time. Design of experiment. Selection of suitable detection wavelength. Optimization by Using Software. To develop calibration curve of optimized result for Enalapril Maleate and Hydrochlorothiazide drug. Commercial formulation analysis. Method Validation as per ICH guidelines.

10. Materials and Methods

11. Method Validation

12. Summary The study revolves around the critical task of simultaneously estimating Enalapril Maleate and Hydrochlorothiazide in tablet formulations using RP-HPLC. This combination therapy is widely employed in hypertension treatment, making accurate analysis imperative. The literature survey reveals previous approaches' limitations due to the drugs' differing properties. The RP-HPLC method's development is carefully guided, encompassing aspects like stationary phase selection, mobile phase optimization, and detector choice. Notably, validation parameters are rigorously addressed to ensure the method's reliability. Comparative analysis demonstrates RP-HPLC's superiority, highlighting its sensitivity and applicability in pharmaceutical quality control. Recent advancements in RP-HPLC techniques are discussed, showcasing how they enhance analytical efficiency. The study underlines the clinical significance of precise estimation in achieving optimal therapeutic outcomes and patient safety.

13. RESULTS Mobile phase: Acetate buffer pH 5, Acetonitrile, Methanol (60:20:20v/v) Detection wavelength: 232 nm System suitability: satisfactory, chromatograms confirm the presence of Enalapril Maleate and Hydrochlorothiazide Concentration range: 10-30µg/ml for Enalapril Maleate and 10-30µg/ml for Hydrochlorothiazide Correlation coefficients: 0.999 and 0.999 for Enalapril Maleate and Hydrochlorothiazide respectively %RSD: 0.87 and 1.14 for Enalapril Maleate and Hydrochlorothiazide respectively

14. Conclusion In present research work separation of Enalapril Maleate and Hydrochlorothiazide was carried out in less than 8 min run time by using simple chromatographic conditions at ambient temperature using two component mobile phase (Methanol: Acetonitrile: Buffer pH 4.5 adjusted with OPA) in the ratio of 40: 30:30 at 230nm. System suitability parameters showed that the method is appropriate and found to be satisfactory the estimation of Enalapril Maleate and Hydrochlorothiazide in tablet dosage form. The HPLC method was validated as per ICH guidelines for specificity, linearity, precision, accuracy, robustness, ruggedness, LOD and LOQ. All the validation parameters were found to be well within the acceptable limit. The assessment of robustness and ruggedness of the method indicates that the method remains unaffected by slight changes in chromatographic conditions. Hence, method can be used for the routine analysis of Enalapril Maleate and Hydrochlorothiazide in bulk and dosage form.

15. REFERENCES Sharma, Y.R., (1991). Introduction of Organic Spectroscopy. 4th ed CBS Publishers and Distributors; New Delhi. pp.5-25. Beckett A.H., Stenlake J.B., (2004). Practical Pharmaceutical Chemistry. 4 th ed. Part 2. Publication of New Delhi, pp.275-300. Lampman, Pavia. Kriz , Vyvyan., Spectroscopy. 4th ed. Cengage Learning. pp. 379-406. Sethi P.D. (1997). Quantitative Analysis of drugs in Pharmaceutical Formulations. 3rd ed. CBS publishers and distributors, New Delhi, pp. 17-19. J. Bassett, R. C. Denney, G.H. Jeffery and J. Mendham. (1978). Vogel’s Text Book of Quantitative Inorganic Analysis. 4th ed. Longman Group Ltd: England, pp. 1-9.

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