Development and Validation of RP-HPLC Chromatographic Dissolution Method for the Simultaneous Estimation of Ramipril and Hydrochlorothiazide from Solid Dosage Formulation

The present study describes the dissolution method development and validation of Ramipril and Hydrochlorothiazide in tablet dosage form by HPLC Method. A simple, rapid, selective, reproducible and isocratic reversed-phase high performance liquid chromatographic (RP-HPLC) method has been developed and validated as per ICH guidelines. Analysis was performed on a Thermo, Sunniest C8 (150 mm x 4.6 mm, 5 μm) with the mobile phase consisting of mixing 500 mL of buffer solution and 500 mL of acetonitrile at a flow rate of 1.0mL/min. UV detection was performed at 210nm and the Run time for Ramipril and Hydrochlorothiazide were 10 minutes. The calibration curve was linear (correlation coefficient = 1.000) in the selected range for both analytes. Original Research Article Raut et al.; JPRI, 33(42B): 203-217, 2021; Article no.JPRI.73330 204 The optimized dissolution conditions include the USP Type 1 (Basket) rotation rate of 100 rpm and 750 mL of 0.1 N Hydrochloric acid as dissolution medium, at 37.0 ± 0.5°C. The method was validated for precision, linearity, specificity, accuracy, limit of quantitation and ruggedness. The system suitability parameters, such as theoretical plate, tailing factor and relative standard deviation (RSD) between six standard replicates were well within the limits. The stability result shows that the drug is stable in the prescribed dissolution medium.


INTRODUCTION
This medication is used to treat high blood pressure (hypertension). Lowering high blood pressure helps prevent strokes, heart attacks, and kidney problems [1][2][3].
Ramipril: Ramipril is a prodrug belonging to the angiotensin-converting enzyme (ACE) inhibitor class of medications. It is metabolized to ramiprilat in the liver and, to a lesser extent, kidneys. Ramiprilat is a potent, competitive inhibitor of ACE, the enzyme responsible for the conversion of angiotensin I (ATI) to angiotensin II (ATII). ATII regulates blood pressure and is a key component of the renin-angiotensin-aldosterone system (RAAS) [18][19][20][21]. Ramipril may be used in the treatment of hypertension, congestive heart failure, nephropathy, and to reduce the rate of death, myocardial infarction and stroke in individuals at high risk of cardiovascular events [22][23][24].
Hydrochlorothiazide: A thiazide diuretic always considered the prototypical member of this class. It reduces the reabsorption of electrolytes from the renal tubules [25,26]. 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 [27][28][29]. Hydrochlorothiazide is suddenly used for the treatment of hypertension, congestive heart failure, symptomatic edema, diabetes insipidus, renal tubular acidosis [30][31][32][33][34]. It is also used for the prevention of kidney stones in those who have high levels of calcium in their urine [35][36][37][38][39]

Specificity
Specificity is the ability to assess unequivocally the analyte in the presence of components which may be expected to be present.

Acceptance criteria:
 Relative standard deviation for five replicate injections of standard solution should be not more than 2.0%.  There should not be any interference of peak due to blank and placebo at the retention time of the Ramipril and Hydrochlorothiazide. Observations/Results: Standard Details: (Ramipril 10 mg & Hydrochlorothiazide 25 mg). Summary: No interference found due to blank and placebo at the retention time of the Hydrochlorothiazide and Ramipril.

Linearity And Range:
The linearity of an analytical procedure is its ability (within a given range) to obtain test results which are directly proportional to the concentration (amount) of analyte in the sample.   The range of an analytical procedure is the interval between the upper and lower concentration (amounts) of analyte in the sample (including these concentrations) for which it has been demonstrated that the analytical procedure has a suitable level of precision, accuracy and linearity.
Acceptance Criteria:  Relative standard deviation for five replicate injections of standard solution should be not more than 2.0%.  The value obtained for correlation coefficient (r2) should not be less than 0.98 Observations/Results:     The relative standard deviation for peak area of Ramipril and Hydrochlorothiazide of standard solution is within acceptance criteria.  The correlation coefficient obtained from the graph for Hydrochlorothiazide and Ramipril is 0.9999.

Precision
The precision of an analytical procedure expresses the closeness of agreement (degree of scatter) between a series of measurements obtained from multiple sampling of the same homogeneous sample under the prescribed conditions.

A) System Precision
Acceptance criteria: Relative standard deviation for five replicate injections of standard solution should be not more than 2.0%.
Observations/Results:  Intermediate precision of the method will be established by carrying out the analysis on different day, different column with different analyst and different instrument.       The relative standard deviation for peak area of Ramipril and Hydrochlorothiazide of standard solution is less than 2.0% % Release of all six units is more than The relative standard deviation for % s less than 5.0%. The difference in the mean value between the % releases at method precision and intermediate precision is less than 5.0%.
The accuracy of an analytical procedure expresses the closeness of agreement between the value which is accepted either as a conventional true value or an accepted reference value and the value found.