A Review on Development and Validation of Method for Determination of Ramipril and Hydrochlorothiazide in Bulk and Pharmaceutical Dosage Form

Abstract

Ramipril is an ACE inhibitor that serves primarily for the treatment of high blood pressure and heart failure, as well as for preventing cardiovascular events in patients at high risk. Generally well-tolerated, Ramipril's common side effects include dizziness, headaches, and dry cough, necessitating adherence to prescribed dosages and communication with healthcare providers regarding any adverse reactions. Notably, it is contraindicated in pregnancy due to potential harm to the fetus. Hydrochlorothiazide is a significant generic diuretic extensively prescribed for hypertension and edema associated with various medical conditions, including heart failure, liver disease, and kidney disease. It is listed among the World Health Organization's Essential Medicines, reflecting its critical role in healthcare. As a first-line therapy, HCTZ, classified as a thiazide diuretic, is recommended in major hypertension treatment guidelines. The review concludes that various analytical methods, primarily UV-Vis spectrophotometry and high-performance liquid chromatography (HPLC), have been developed and validated for the determination of ramipril and hydrochlorothiazide, either individually or in combination. These methods are proven to be specific, accurate, precise, and sensitive, allowing for reliable analysis of these drugs in pharmaceutical formulations and biological samples. Advanced techniques like Ultra-Performance Liquid Chromatography-Mass Spectrometry (UPLC-MS) offer even greater sensitivity and are suitable for detecting low concentrations, such as in cleaning validation.

Keywords

Introduction

Ramipril (Figure 1) is an ACE inhibitor that serves primarily for the treatment of high blood pressure and heart failure, as well as for preventing cardiovascular events in patients at high risk. Generally well-tolerated, Ramipril's common side effects include dizziness, headaches, and dry cough, necessitating adherence to prescribed dosages and communication with healthcare providers regarding any adverse reactions. Notably, it is contraindicated in pregnancy due to potential harm to the fetus (1, 2).

Figure 1: Ramipril

Mechanistically, Ramipril functions by inhibiting the angiotensin-converting enzyme, leading to a decrease in angiotensin II production. This effect elicits vasodilation, facilitating a reduction in blood pressure, while also mitigating the influence of hormones that elevate blood volume. While common side effects include a dry cough, dizziness, headaches, nausea, vomiting, and mild skin rash, there are serious adverse effects that require immediate medical attention. These include severe allergic reactions, hyperkalemia, and hyponatremia (3, 4). Additionally, long-term use may impact kidney function, warranting regular monitoring through blood tests. Critical considerations while using Ramipril include its contraindication during pregnancy, where cessation is mandatory upon pregnancy confirmation, as well as individualized dosage determination by a healthcare provider. Initial doses may be recommended at bedtime to mitigate the risk of dizziness, and regular monitoring of kidney function and potassium levels through blood tests is advised. Patients must also disclose all concurrent medications to their doctor to avoid potential interactions (5, 6).

Hydrochlorothiazide (Figure 2) is a significant generic diuretic extensively prescribed for hypertension and edema associated with various medical conditions, including heart failure, liver disease, and kidney disease. It is listed among the World Health Organization's Essential Medicines, reflecting its critical role in healthcare.  As a first-line therapy, HCTZ, classified as a thiazide diuretic, is recommended in major hypertension treatment guidelines (7, 8).

Figure 2: Hydrochlorothiazide

It has a long-standing history of over 60 years for effectively lowering blood pressure and mitigating the risk of serious cardiovascular events such as heart attacks and strokes. Moreover, HCTZ is commonly included in many fixed-dose combination medications with other antihypertensives like ACE inhibitors or ARBs, enhancing drug efficacy and patient compliance while managing potential side effects like potassium loss (9, 10).

In terms of managing edema, HCTZ is effective for treating fluid retention due to conditions such as congestive heart failure and hepatic cirrhosis. Particularly in cases of loop diuretic resistance in acute heart failure, the addition of thiazides like HCTZ may enhance diuretic response and decongestion. Besides its primary uses, HCTZ also has secondary applications, including the treatment of diabetes insipidus, prevention of calcium-containing kidney stones through calcium reabsorption promotion, and a potential role in improving bone mineral density. Clinical efficacy comparisons highlight that while HCTZ is crucial, its potency at lower doses (12.5-25 mg) may be less than that of other diuretic classes or longer-acting thiazide-like diuretics, such as chlorthalidone or indapamide (11).

Although some large clinical trials have shown substantial cardiovascular event reductions with chlorthalidone, newer studies indicate that both HCTZ and chlorthalidone could have similar efficacy, although chlorthalidone may present a higher risk of side effects like hypokalemia. Hence, HCTZ can be an appealing alternative for certain patients. As for side effects, they tend to be dose-dependent. Generally, at low doses, HCTZ is well-tolerated, but patients may experience electrolyte imbalances, elevated blood sugar and uric acid levels, and increased photosensitivity, which raises the risk of non-melanoma skin cancer with prolonged use (12).

2. REVIEW ON ANALYTICAL METHODS FOR THE DETERMINATION OF ACTIVE PHARMACEUTICAL INGREDIENTS (API)

Kondreddy VK et al., 2025 created and validated an analytical method for the RP-HPLC estimation of telmisartan and hydrochlorothiazide. The HPLC Binary Gradient System (HPLC 3000 series) was used to construct the analytical procedure. The column used was the Cosmosil C18, which had dimensions of 250mm x 4.ID and a particle size of 5µm. The mobile phase consisted of a 60:40 mixture of methanol and water, and the flow rate was 0.8 ml. The detected wavelength was 231nm. The half-life of hydrochlorothiazide was 5.5 minutes and that of telmisartan was 4.1 minutes. Two drugs, telmisartan and hydrochlorothiazide, were determined to have linearities of 0.998 and 0.999, respectively. The % recovery for Hydrochlorothiazide was 99.71% and the limit of detection for Telmisartan was 0.174, whereas for Hydrochlorothiazide it was 0.339. This method was created with accuracy, simplicity, precision and cost-effectiveness in mind. The approach provides precise estimations of hydrochlorothiazide and telmisartan (13).

Varungase AT et al., 2025 proposed LC-MS/MS method offers a rapid, sensitive, and reliable approach for the simultaneous determination of MPL and HCTZ in human plasma, suitable for clinical pharmacokinetic studies and therapeutic drug monitoring. A sensitive and selective liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) method was developed and validated for the simultaneous quantification of metoprolol succinate (MPL) and hydrochlorothiazide (HCTZ) in human plasma. Sample preparation involved a simple liquid-liquid extraction procedure using dichloromethane: tert-butyl ether (85:15% v/v). Chromatographic separation was achieved on an ACE column using a mobile phase consisting of methanol and water with 0.1% formic acid (70:30, v/v). The analytes were detected using electrospray ionization in negative ion mode and quantified using multiple reaction monitoring. MPL D4 and HCTZ 13C15N2 D2 were used as internal standards. The method was validated over concentration ranges of 10-5000 ng/mL for MPL and 1-500 ng/mL for HCTZ. The intra- and inter-day precision and accuracy were within acceptable limits. The method demonstrated excellent selectivity, recovery, and stability. Matrix effects were minimal, and no significant ion suppression or enhancement was observed. The validated method was successfully applied to a pharmacokinetic study in human subjects (14).

Wang N et al., 2025 conducted a systematic review and meta-analysis of randomised, double-blind, placebo-controlled trials involving adult participants randomly assigned to receive angiotensin-converting enzyme inhibitors, angiotensin II receptor blockers, β blockers, calcium channel blockers, or diuretics. The primary outcome was placebo-corrected reduction in systolic blood pressure. Blood pressure-lowering efficacy was estimated using fixed-effects meta-analyses standardised to mean baseline blood pressure across included trials. Drug regimens were categorised into low, moderate, and high intensity, corresponding to systolic blood pressure-lowering efficacy of <10 mm Hg, 10–19 mm Hg, and ≥20 mm Hg, respectively, from a baseline of 154 mm Hg. A model was developed to calculate efficacy for any combination of antihypertensives and validated on external trials of dual and triple combination antihypertensives (15).

Gewehr DM et al., 2025 analyzed several classes of antihypertensive medications, namely angiotensin-converting enzyme (ACE) inhibitors, angiotensin receptor blockers (ARBs), dihydropyridine calcium channel blockers (CCBs), β-blockers, mineralocorticoid antagonists (MRAs), and thiazide diuretics (TDs). A Bayesian random-effects network meta-analysis (NMA) was employed to calculate the risk ratios (RR) with a 95% credible interval (CrI), and the treatments were ranked based on the surface under the cumulative ranking (SUCRA). In total, 22 RCTs involving 66,156 patients with hypertension, diabetes, or existing AF were included in the study. The results indicated that combinations of ACE inhibitors with TD were associated with a significantly lower risk of new-onset or recurrent AF (RR 0.44; 95% CrI 0.23–0.82). Individual ACE inhibitors (RR 0.66; 95% CrI 0.44–0.95) and ARBs (RR 0.52; 95% CrI 0.38–0.70) also demonstrated reduced rates of AF compared to CCBs. The SUCRA analysis ranked ACE inhibitors combined with TDs as the most effective treatment (0.86), followed by ARBs (0.77), MRAs (0.75), and stand-alone ACE inhibitors (0.53), with these findings being consistent across secondary and sensitivity analyses (16).

Typlynska K et al., 2024 developed and validated analytical methods for the determination of ramipril and hydrochlorothiazide in wash waters by ultra-performance liquid chromatography–mass spectrometry method. In the study, standard samples of ramipril (USP RS) and hydrochlorothiazide (USP RS), as well as class A reagents, were used. Samples were analysed on a liquid chromatograph with an MS detector (Agilent 6420 and Waters Xevo TQD ACQUITY). Methods for the determination of ramipril and hydrochlorothiazide in wash waters by ultra-performance liquid chromatography–mass spectrometry have been developed. The developed methods have sufficient linearity, correctness and precision. The sensitivity of the techniques was confirmed at the level of 0.0026 μg/ml. The techniques can be used in the concentration range of 0.0026 – 0.0255 μg/ml (17).

Kale PR et al., 2024 uses shimadzhu700 and Elico UV spectrophotometers for simultaneous estimation utilising the first derivative approach and RP-HPLC. OLM has zero crossing point at 240nm in methanol and RAM has zero crossing point at 246 nm in methanol. Both these drugs obey Beer’s law in the concentration range employed for the present method. The result of analysis has been validated statistically by recovery studies. The slope and intercept for OLM were 0.0364x and 0.0078 and for RAM were 0.001 and -0.0001 respectively as determined by the method of least squares. Estimation of individual drug in the tablet formulation by Amplitude measurement method (peak to peak) was carried out at first derivative mode. The work is done within the UV range of 230nm to 280nm, where OLM has λmax at 256nm. showing the linearity range between 5-30 µg/ml. The slope and intercept for OLM were 0.4594x and 2.1693 respectively. The result of analysis has been validated statistically by recovery studies (18).

3. FUTURE SCOPE OF THE STUDY

The future scope of analytical methods for determining ramipril and hydrochlorothiazide is poised for significant advancements with an emphasis on sensitivity, speed, and environmental sustainability, particularly for trace analysis in both biological and environmental contexts. Key areas of focus include: There is an ongoing push towards the development of miniaturized analytical systems, such as microfluidics, alongside automated sample preparation techniques. These innovations aim to decrease analysis time, minimize solvent consumption, and reduce the need for manual intervention. A priority for future research will be the enhancement of sensitivity in bioanalytical techniques. This includes the adoption of Ultra-Performance Liquid Chromatography (UPLC) and Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS), which can accurately quantify the drugs and their metabolites, like ramiprilat, at extremely low concentrations amidst complex biological matrices, such as human plasma and urine. The field is increasingly leaning towards the principles of green chemistry, advocating for methods that utilize fewer hazardous solvents or alternative, environmentally-friendly mobile phases. This shift addresses both safety and environmental concerns. There is a continuous effort to develop robust stability-indicating methods capable of effectively separating the active pharmaceutical ingredients from potential degradation products and impurities. Such methods are essential to comply with regulatory standards set by organizations like the International Conference on Harmonisation (ICH). As new combination drug therapies involving ramipril, hydrochlorothiazide, and potentially other medications such as telmisartan arise, there will be a necessity for analytical methods tailored to facilitate their simultaneous quantification in single analytical runs. The integration of analytical methodologies directly into manufacturing processes is expected to enhance real-time monitoring and quality control. This approach aims to boost efficiency and ensure product consistency across production batches. In response to growing concerns over pharmaceutical contaminants in water sources, there is a clear future need for highly sensitive analytical methods capable of detecting and quantifying trace levels of these compounds in environmental samples, thereby contributing to public health and environmental safety.

4. CONCLUSION

The review concludes that various analytical methods, primarily UV-Vis spectrophotometry and high-performance liquid chromatography (HPLC), have been developed and validated for the determination of ramipril and hydrochlorothiazide, either individually or in combination. These methods are proven to be specific, accurate, precise, and sensitive, allowing for reliable analysis of these drugs in pharmaceutical formulations and biological samples. Advanced techniques like Ultra-Performance Liquid Chromatography-Mass Spectrometry (UPLC-MS) offer even greater sensitivity and are suitable for detecting low concentrations, such as in cleaning validation.

5. CONFLICT OF INTEREST

None

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