A review on validated analytical methods for Ramipril

 Corresponding author: P. Sai Teja. Copyright © 2025 Author(s) retain the copyright of this article. This article is published under the terms of the Creative Commons Attribution Liscense 4.0. A review on validated analytical methods for Ramipril P. Sai Teja *, CH. Yedukondalu, P. Bhargavi and B. Thangabalan SIMS College of Pharmacy, Mangaldas Nagar, Vijayawada Road, Guntur-522001. A.P., India. World Journal of Biology Pharmacy and Health Sciences, 2025, 22(01), 200-212 Publication history: Received on 25 February 2025; revised on 03 April 2025; accepted on 05 April 2025 Article DOI: https://doi.org/10.30574/wjbphs.2025.22.1.0357 Abstract Ramipril is the drug is official in British Pharmacopoeia used to treat high blood pressure (hypertension) and heart failure. The official method of analysis of Ramipril is potentiometric method. Several UV spectrophotometric, HPLC methods in pure form, pharmaceutical formulation and HPTLC methods have been reported to determine. This review provides an overview of various analytical techniques used for Ramipril determination both in a single preparation and in combination. Keywords: Ramipril; spectrophotometric; LC-MS; Flourimetry; UV; HPLC; HPTLC 1. Introduction Transforming ramipril into ramipril takes place on the periphery as well where it is partly carried out in the kidneys. Both congestive heart failure and nephropathy and is used for treatment of hypertension. Angiotensin converting enzyme (ACE) is responsible for the conversion of angiotensin-I into angiotensin-II. This angiotensin II is called ACE-II, and it’s the most important part of the RAAS system (Renin angiotensin aldosterone system). It is in control of blood circulation. Ramipril is the most efficient ester of Angiotensin II and Ace inhibitor, and he inhibits the conversation of angiotensin-I to angiotensin-II. Ramipril is relatively long acting and is rapidly metabolized, becoming a potent inhibitor of angiotensin converting enzyme. It has the same action as that of Captopril and Enalapril. Its active form or metabolite, ramiprilat have a long elimination half-life that makes it easy to take, as only one dose a day is needed. The daily dose of ramipril in a hypertensive patient is 2.5 to 20mg which is usually effective in lowering high blood pressure and is satisfactorily controlled during long term treatment. Those who do not respond to, respond well with the addition of diuretics. ramipril monotherapy Patients who have diabetes mellitus continue to take advantage in of the antihypertensive action of ramipril and the initial data suggest that the drug has the additional value of decreasing urinary albumin excretion in diabetic patients with nephropathy. Ramipril also have a beneficial effect in the treatment of patients with more established heart failures World Journal of Biology Pharmacy and Health Sciences, 2025, 22(01), 200-212 201 Figure 1 Skeletal Structure IUPAC Nomenclature: [2S,3aS,6aS)-1- [(2S)-2- [ [ [(2S)-1-Ethoxy-1-oxo-4-phenylbutan-2-yl] amino] propanoyl]]- 3,3a,4,5,6,6a. Its melting point: 109 OC. Its molecular weight: 416.518 g. mol-1. Indications for use: Ramipril 1. Hypertension 2. Prevention of the heart failure after myocardial infarction from getting worse. 3. Heart Failure with a reduced ejection fraction. 4. Reduction of MI and stroke risks. 1.1. Pharmacokinetic Data of Ramipril A ramipril drug is a professional who becomes an active metabolite known as ramiprilat. Ramipril undergoes a phase of depressurized oral bioavailability of around 28% with ultra-rapid distribution to various tissues. Concentration of the drug is higher in the blood cortex than in the liver, kidneys, and lungs. The drug has a protein binding capacity of 73% while its active metabolite ramiprilat is relatively low at 56%. Ramipril is liver metabolized adrenally and focally excreted with 60% and 40% elimination ratio. The drug has an elimination half-life of 60% and 40% elimination ratio. The drug has an elimination half-life of 13-17 hours. 1.1.1. Adverse effects of Ramipril • Cough • Orthostatic hypotension • Rise in creatinine levels in blood • Hyperkaliemia • Swelling of deeper layers of skin • In the initial stages, feeling tired and dry mouth Initiated studies for the calculation. Along with various environmental models, there are many different approaches for determination of ramipril in pharmaceutical formulations and human blood serum samples. Some of these include UV, HPLC, HPTLC, UPLC, LC/MS, and others. This current review is an attempt made to compile all the analytical methods which have been used for the analysis of ramipril. 1.2. UV-spectroscopic methods A new, simple, rapid and novel spectrophotometric method has been developed for estimation of Ramipril (RAM). For this Absorption maximum Method (method A) and Area under Curve Method (Method B) is used. The method involved measurement of absorbance at wavelengths 210 nm for method A and method B involved measurement of area under curve in the wavelength range 202 to 237.5 nm for RAM. Beer’s law obeyed in concentration range of 0.1 to 3.5 µg/ mL by both the methods. The proposed methods are recommended for routine analysis since they are rapid, simple, accurate and also sensitive and specific. The results obtained are reproducible with a coefficient of variation less than 2%. These methods were validated for precision, reproducibility, linearity and accuracy as per ICH guidelines. [1] A new, simple, rapid and novel spectrophotometric method has been developed for estimation of Ramipril (RAM) and Hydrochlorothiazide (HCT) in bulk and combined pharmaceutical formulations using absorbance correction method (ACM). λmax of RAM was found at 209nm and HCT was at 270nm respectively. This method involved measurement of absorbance at two wavelengths of RAM & HCT i.e., 209 nm and 270 nm for RAM and 270nm & 209nm for HCT. The combination is also estimated by ACM. It showed linearity of both the drugs at two wave lengths. Calibration curve was constructed at 209nm & 270nm for of 0.1‐ 0.5 μg/ mL for RAM and 0.25‐ 1.25 μg/ mL for HCT respectively by the World Journal of Biology Pharmacy and Health Sciences, 2025, 22(01), 200-212 202 method. These methods were validated for precision, reproducibility, linearity and accuracy as per ICH guidelines. The proposed methods are recommended for routine analysis since they are rapid, simple, accurate, cost effective and also sensitive and specific. It involves neither heating nor use of any organic solvent for separation of the combination. This study thus which may be required in pre-clinical and clinical study in near future.exploits the possibility for determining Pharmacokinetic data of the combined formulation [2] A new, simple, rapid and novel spectrophotometric method has been developed for estimation of Ramipril (RAM). For this Absorption maximum Method (method A) and Area under Curve Method (Method B) is used. The method involved measurement of absorbance at wavelengths 210 nm for method A and method B involved measurement of area under curve in the wavelength range 202 to 237.5 nm for RAM. Beer’s law obeyed in concentration range of 0.1 to 3.5 μg/ mL by both the methods. The proposed methods are recommended for routine analysis since they are rapid, simple, accurate and also sensitive and specific. The results obtained are reproducible with a coefficient of variation less than 2%. These methods were validated for precision, reproducibility, linearity and accuracy as per ICH guidelines [3]. The current study focuses on the development and analytical validation of a unique, precise, and accurate UV-Visible Spectrophotometric method for estimating Ramipril and Olmesartan medoxomil simultaneously. The regression strength of Ramipril and Olmesartan medoxomil over its absorbances were obtained as y=0.0378x-0.05347 and y=0.0397x-0.0042 respectively with a correlation coefficient (r2) of 0.9917 for Ramipril and 0.9999 for Olmesartan medoxomil. The intra-day precision in addition inter-day precision for Ramipril and its % RSD were obtained as 1.46% and 1.40% respectively. The intra-day precision in addition inter-day precision for Olmesartan medoxomil and % RSD were obtained as 2.28% and 1.46% respectively [4]. Two newly introduced pharmaceutical mixtures of amlodipine/celecoxib and amlodipine/ramipril were developed to manage hypertension and the associated osteo arthritis. The current work presents three newly developed UV spectrophotometric methods depending on minimal mathematical manipulations on the zero-order spectrum namely: absorption correction, induced dual-wavelength, and Fourier self-deconvoluted method; for the simultaneous determination of celecoxib and ramipril in their pharmaceutical combined dosage forms with amlodipine. In absorption correction and induced dual-wavelength method, celecoxib and ramipril were determined at 253 and 222 nm for absorption correction and (251–270 nm) and (222–230 nm) for induced dual-wavelength method, respectively from the zero-order spectrum after calculating the absorption correction and equality factors foramlodipine. Amlodipine itself was determined at 361 nm from the zero-order spectrum in both methods. In Fourier self-deconvoluted method, celecoxib and amlodipine zero-order spectra were deconvoluted, using the spectrophotometer software built-in Fourier wavelet function, and then was determined at 360 and 269 nm, respectively. The proposed methods were simple, accurate, and sensitive requiring minimal mathematical manipulations saving the time needed for analysis. The methods were linear over the range of (5–60 μg/ml), (5– 30 μg/ml), and (5–110 μg/ml) for each of amlodipine, celecoxib, and ramipril, respectively. The limit of detection was in the range of (0.5781–0.7132 μg/ml) for amlodipine, (0.6497– 1.0450 μg/ml) for celecoxib, and (0.0001–0.0003 μg/ml) for ramipril that indicated the sensitivity of these suggested methods. All methods were validated as per ICH recommendations regarding linearity, range, accuracy, precision, and selectivity. A statistical comparative study executed for the proposed methods with each other and with the reported methods showed no significant difference between the proposed methods and the reported methods. [5] A new, simple, rapid and novel spectrophotometric method has been developed for simultaneous estimation of Ramipril and Amlodipine. For this, simultaneous equation method is used. The method involved measurement of absorbance at two wavelengths, 210 nm and 238 nm, λ max of Ramipril and Amlodipine respectively. Beer’s law obeyed in concentration range of 1535 µg/ mL and 525 µg/ mL for Ramipril and Amlodipine respectively. The proposed method is recommended for routine analysis since it is rapid, simple, accurate and also sensitive and specific by no heating and no organic solvent extraction. This paper describes the development and validation of UV spectroscopic method for Simultaneous estimation of Ramipril and Amlodipine in combined solid dosage form. [6] A new, simple, rapid and novel spectrophotometric method has been developed for estimation of Ramipril (RAM). For this Absorption maximum Method (method A) and Area under Curve Method (Method B) is used. The method involved measurement of absorbance at wavelengths 210 nm for method A and method B involved measurement of area under curve in the wavelength range 202 to 237.5 nm for RAM. Beer’s law obeyed in concentration range of 0.1 to 3.5 μg/ mL by both the methods. The proposed methods are recommended for routine analysis since they are rapid, simple, accurate and also sensitive and specific. The results obtained are reproducible with a coefficient of variation less than 2%. These methods were validated for precision, reproducibility, linearity and accuracy as per ICH guidelines [7]. World Journal of Biology Pharmacy and Health Sciences, 2025, 22(01), 200-212 203 2. HPLC method The aim of the present study was to develop a procedure based on high-performance liquid chromatography (HPLC) for determination of ramipril in pharmaceutical preparations. Separation of ramipril was achieved on a Ace C18 column (5 µm, 250×4.6 mm i.d.) using UV detection with λ=208 nm. The mobile phase consisted of 20 mM phosphate buffer (pH 2.5) containing 0.1% trifluoroacetic acid (TFA)- acetonitrile (50:50, v/v). The analysis was performed in less than 5 min with a flow rate of 1.0 mL min-1. Calibration curve was linear over the concentration range of 0.25-7.5 µg mL-1. Intraand inter-day precision values for ramipril were less than 4.95, and accuracy (relative error) was better than 4.00%. The mean recovery of ramipril were 99.7% for pharmaceutical preparations. The limits of detection (LOD) and quantification (LOQ) were 0.10 and 0.25 µg mL-1, respectively. Also, the method was successfully applied for the quality control of commercial ramipril dosage forms to quantify the drug and to check the formulation content uniformity [8]. An RP-HPLC method for the simultaneous determination ofRamipril(RP) andAmlodipine(AL) in tablets was developed and validated by Chinese Pharmacopoeia 2010. The linearity of the proposed method was investigated in the range of 0.01–0.25 mg/mL (r2=0.9998) for RP and 0.014–0.36 mg/mL (r2=0.9997) for AL. The limits of detection (LOD) were 0.06 μg/mL and 0.02 μg/mL for RP and AL, and the limits of quantitation (LOQ) were 0.2 μg/mL and 0.07 μg/mL, respectively. Some major impurities and degradation products did not disturb the detection of RP and AL and the assay can thus be considered stability-indicating [9]. The primary objective of this study is to develop and optimize a simple, novel, reproducible, and efficient RP-HPLC method using quality by design (QbD) approach for the routine analysis of ramipril. Thechromatographic separation was carried out by C18 column with an isocratic elution of a mobile phase of 65:35(%v/v) acetonitrile: water at a flow rate of 0.9 mL/min. The detection was done at a wavelength of 210 nm using aphoto-diode array plus (PDA+) detector. A 32 full-factorial design was employed for the development of analyticalmethod using Design Expert® software in which the mobile phase composition and flow rate were taken as independent variables and the retention time (RT), tailing factor (TF) and theoretical plate count (TP) were chosen asresponses of the study. Statistically significant models were obtained for the development of the method (p<0.05). The empirical responses perfectly fitted to that of predicted, with an error within the tolerance of ± 2%. This indicatesthat the model efficiently identified the optimum levels of independent variables, i.e. the composition of mobile phaseand its flow rate, to get the desirable responses. The validation of the developed method followed the ICH Q2 (R1) guidelines, demonstrating that the method is robust and well-suited for the routine analysis of ramipril in activepharmaceutical ingredients and in drug products. [10] A simple, sensitive and validated HPLC method has been developed to determine valsartan and ramipril simultaneously in synthetic mixture. Chromatographic separation was achieved on a C-18 column using a mixture of acetonitrile and water in the ratio 55:45 (v/v), pH adjusted to 3.6 with 88% orthophosphoric acid at a wavelength of 215 nm. Linearity of the method was found to be in the concentration range of 50-250 µg/ml for valsartan andl00-500 µg/ml for ramipril with correlation coefficient greater than 0.999. The total eluting time for the two components is less than five minutes. The method can be used for simultaneous determination of valsartan and ramipril [11]. 3D printing has shown its usefulness as a drug manufacturing technology over the past decade. However, the lack of regulated methods for quality control of finished printed drugs imposes a limitation on the widespread use of 3D printing methods in pharmaceutical practice. Thus, the development of methods for the analysis of printed dosage forms is of interest in pharmaceutical development. To develop a specific method for the determination of ramipril in filaments and printlets by HPLC. Materials and methods. Substance: ramipril. Excipients: Kollidon® VA 64, Kollidon® CL-F, PEG1500, sodium carbonate anhydrous, Poloxamer-188, sodium stearyl fumarate. Reagents: hydrochloric acid, acetonitrile for ultra-HPLC, sodium octanesulfonate for HPLC, orthophosphoric acid 85 %, sodium perchlorate analytical grade, triethylamine. Standard: ramipril USP (No 1598303). A special HPLC method in accordance with an ion-pair reagent (sodium octanesulfonate) for the determination of ramipril in the composition of filaments and printets was proposed.Conclusion. The developed chromatographic method should be adapted for ramipril release determination. This method can be used to quantify ramipril in further studies. [12] The present work explains the development and validation of a simple and reliable RP-HPLC method for the quantitative determination of Ramipril (RMP). Chromatography was carried out by reversed phase technique on a Fortis C18 (100 mm × 4.6 mm; 2.5 μm particle size). The optimized mobile phase was consisted of methanol and citric acid sodium citrate buffer solution (50:50 v/v) having pH 3.0. The retention times were 3.645 min for RMP. The detection was carried out at 270 nm and a column temperature of 25°C. The method was evaluated for the various validation parameters, such as linearity, accuracy, precision, LOD, LOQ, specificity, selectivity, and sample stability. The proposed World Journal of Biology Pharmacy and Health Sciences, 2025, 22(01), 200-212 204 method was validated and successfully applied for the analysis of pharmaceutical formulations and laboratory prepared mixture containing Ramipril respectively. Keywords: Ramapril; LOD; LOQ; RP-HPLC; Column temperature [13] A rapid and sensitive High Performance Liquid Chromatography (HPLC) method has been developed and validated as per ICH guideline for simultaneous determination of ramipril and felodipine binary mixture. Chromatographic separation was achieved on a Hyperchom C18 column (250 × 4.6 mm i.d., 5 μm) using an isocratic mobile phase of potassium di-hydrogen phosphate (pH = 3.4): methanol: acetonitrile in the ratio 15:15:70 (v:v:v). The flow rate was 1.5 mL/min, temperature of the column was maintained at 30 °C and detection was made at 210 nm. Linearity studies indicated that the drugs obey Beer’s law over the range of 10-80 μg/mL for ramipril and 5-80 μg/mL for felodipine. The proposed method is precise, accurate, linear and robust. The short retention time allows the analysis of a large number of samples in a short period of time and, therefore, considered to be cost-effective that can be used for routine analysis of both drugs in the pharmaceutical industry. [14] A Simple and precise HPLC method was developed for the simultaneous estimation of Ramipril and Amlodipine in pure drug and pharmaceutical dosage forms. The separation was carried out using C18 Column (250 × 4.6 mm i.d. 5 μm particle size), with mobile phase compressing of Acetonitrile, Sodium phosphate buffer and Methanol in the ratio of 50: 20:25 v/v/v, pH= 6.8 (pH adjusted with OPA). The flow rate was 0.8 ml/min and the detection was carried out using PDA detector at 210 nm. The retention times were 2.64 and 7.45 mins for Ramipril and Amlodipine respectively. Calibration curves were linear with correlation coefficient 0.998 and 0.996 over concentration range of 1 - 16 µg/ml for Ramipril and 0.2 – 3.2 µg/ml for Amlodipine respectively. Recovery was found in between 100.21% and 100.82% for Ramipril and Amlodipine respectively. Method was found to be reproducible with relative standard deviation (R.S.D) for intra and inter day precision less than 2%. The method was validated by evaluation of different parameters such as accuracy, linearity, precision, LOD and LOQ. [15] The development and validation of a reversed-phase liquid chromatographic method for the determination of the related substances of 2- [N- [(S)-1-Ethoxycarbonyl-3-phenylpropyl]-l-alanyl] -(1S, 3S, 5S) -2-azabicyclo [3.3.0] octane3carboxylic acid (ramipril) in Altace capsules is described. The method utilizes an ion-pairing agent and a simple twostep gradient for the separation of ramipriland ten related substances from each other in a 40-min run time. Four of the related substances are ramipril diastereomers. To the best of our knowledge, no method described previously in the literature has demonstrated resolution of ramipril from this set of related substances. No method for the determination of the related substances of ramipril is currently described in the United States Pharmacopoeia or the European Pharmacopoeia. The proposed method was validated with respect to accuracy, precision, linearity, and specificity. Also, the method was determined to be robust with regards to the following parameters: mobile phase apparent pH; mobile phase organic content; mobile phaseperchloratecon centration; detection wavelength and time dependence of sample and standard stability [16]. A simple and precise stability indicating RP-HPLC method was developed and validated for simultaneous determination of Metoprolol succinate and Ramipril in bulk and Pharmaceutical marketed formulation. Chromatography was carried out on Altima C18 (150 x 4.6 mm, 5µ particle size) column in an isocratic mode with mobile phase containing phosphate buffer (adjusted to pH 4.8 with dilute othophosphoric acid, acetonitrile and methanol in the ratio of 35:10:55% v/v/v at a flow rate of 1ml/min. The analyte was monitored using PDA detector at 210 nm. The retention time was found to be 2.203 min and 3.283 min for Metoprolol succinate and Ramipril respectively. The proposed method was found to be having linearity in the concentration range of 5-30 µg/ml for Metoprolol succinate and 0.53.0 µg/ml for Ramipril with correlation coefficient value of 0.999 respectively. The mean % recoveries obtained were found to be 99.87-100.24 % for Metoprolol succinate and 99.64-100.08 % for Ramipril respectively. Stress testing which covered acid, base, peroxide, UV light, neutral and thermal degradation was performed on under test to prove the specificity of the method and the degradation was achieved. The developed method has been statistically validated according to ICH guide lines. Thus the proposed method can be successfully applied for the stability indicating simultaneous determination of Metoprolol succinate and Ramipril in bulk and combined tablet dosage form and in routine quality control analysis. [17] 3. HPTLC This paper describes validated high-performance thin-layer chromatography (HPTLC) methods for simultaneous estimation of ramipril (RAM) and losartan potassium (LOS) in pure powder and formulation. The HPTLC separation was achieved on an aluminum-backed layer of silica gel 60F254 using methanol: ethyl acetate: toluene: glacial acetic acid (1:9:1:0.2 v/v/v/v) as mobile phase. Quantification in HPTLC method was achieved with UV detection at 210 nm over the concentration range of 300 – 1300 ng/spot for RAM and 3000 – 13000 for LOS, respectively, with recovery of 98.93 - 99.73 and 98.96 - 100.11 % for RAM and LOS, respectively. These methods are simple, specific, precise, sensitive World Journal of Biology Pharmacy and Health Sciences, 2025, 22(01), 200-212 205 and robust; they are applicable for the simultaneous determination of RAM and LOS in pure powder and formulation. [18] A simple, precise, accurate and rapid high-performance thin-layer chromatographic method has been developed and validated for the estimation of ramipril and valsartan simultaneously in combined dosage form. The stationary phase used was precoated silica gel 60F254. The mobile phase used was a mixture of chloroform: ethyl acetate: methanol: glacial acetic acid (5.0:5.0:1.0:02 v/v/v/v). The detection of spots was carried out at 210 nm. The method was validated in terms of linearity, accuracy, and precision. The calibration curve was found to be linear between 0.4 to 2.0 µg/spot for ramipril and 0.2 to 1.0 µg/spot for valsartan. The limit of detection and the limit of quantification for ramipril were found to be 200 ng/spot and 640 ng/spot for ramipril and 100 ng/spot and 330 ng/spot respectively. The proposed method can successfully be used to determine the drug content of marketed formulations. [19] Telmisartan, an angiotensin II antagonist and Ramipril, a long-acting ACE inhibitor are found in combination in tablet dosage form used for the treatment of high blood pressure. The present study deals with development of validated stability indicating method for simultaneous estimation of Telmisartan and Ramipril using TLC plate precoated with Silica gel 60 F254 and the mobile phase consisting of Methanol: Chloroform in the ratio of 1:6v/v. Telmisartan and Ramipril were well resolved with Rf 0.68 ± 0.03 and 0.38±0.03 respectively Wavelength selected for quantization was 210nm. At this wavelength, Telmisartan and Ramipril show high absorbance. Inherent stability of these drugs was studied by exposing both drugs to various stress conditions as per ICH guidelines viz. Dry heat, oxidative, photolysis (UV and cool white fluorescent light), and hydrolytic conditions under different pH values. Both drugs were not degraded under acidic condition. Both drugs show degradation under alkaline, dry heat, oxidative condition and photolytic condition. The developed method is found to be simple, specific, precise and stability indicating. The specificity of the method was confirmed by peak purity profile of the resolved peaks. [20]. A simple, sensitive, precise and accurate high performance thin layer chromatographic (HPTLC) method has been developed for the simultaneous estimation of atorvastatin (AT) calcium, ramipril (RA) and aspirin (AS) in combined dosage form. The method was developed using precoated silica gel 60F254 as stationary phase. The mobile phase used was a mixture of benzene: ethyl acetate: toluene: methanol: glacial acetic acid (4.0:4.5:1.0:0.5:0.1 v/v/v/v/v). Detection was carried out with ultra-violet detection at 220 nm. The Rf values were about 0.45±0.02, 0.28±0.01 and 0.72±0.02 for AT, RA and AS, respectively. The developed method was validated for linearity, accuracy, precision, limit of Detection (LOD), limit of quantification (LOQ) and robustness. The linearity ranges were 0.5-2.5 µg/spot for AT, 0.5-2.5 µg/spot for RA and 0.75-3.75 µg/spot for AS with mean recoveries of 100.29±0.94, 99.06±1.79 and 98.85±0.61 for AT, RA and AS, respectively. The proposed method can be used for the estimation of these drugs in combined dosage forms. [21] This paper describes a new, simple, precise, and accurate HPTLC method for simultaneous estimation of Ramipril and Metolazone as the bulk drug and in tablet dosage forms. Chromatographic separation of the drugs was performed on aluminum plates precoated with silica gel 60 F254as the stationary phase and the solvent system consisted of toluene : ethyl acetate : methanol : glacial acetic acid (4 : 4 : 1 : 0.2 v/v/v/v). Densitometric evaluation of the separated zones was performed at 223 nm. The two drugs were satisfactorily resolved with RF values 0.33 ± 0.02 and 0.59 ±0.02 for Ramipril and Metolazone respectively. The accuracy and reliability of the method was assessed by evaluation of linearity (6002100 ng/spot for Ramipril and 100-350 ng/spot for Metolazone, precision (intra-day % RSD was 1.28 – 1.58 and interday % RSD was 1.14 – 1.83 for Ramipril and intraday % RSD was 0.67 – 1.03 and inter-day % RSD was 0.49 – 1.18 for Metolazone), accuracy 99.44 ± 0.15 for Ramipril and 99.85 ± 0.39 for Metolazone), and specificity in accordance with ICH guidelines. [22] A simple, selective, rapid, precise and stability‐indicating HPTLC method has been developed for the quantitative simultaneous estimation of Telmisartan and Ramipril in combined pharmaceutical dosage form and validation was done. The proposed HPTLC method involves the use of HPTLC plates (Merck) precoated with silica gel 60F254 on aluminium sheets and a mobile phase comprising of toluene: acetonitrile: formic acid: water (5:5:0.3:1). Densiometric analysis of both the drugs was carried out in the a bsorbance mode at 212 nm. This method has been successfully applied for estimation of Telmisartan and Ramipril in combined tablets formulation. Both the drugs were subjected to acid‐alkali hydrolysis, oxidation and photolytic degradation and both of them were found to be susceptible to acid‐alkali hydrolysis, oxidation and photolytic degradation. Linearity of Telmisartan was found to be within the range of 500‐2500 ng/spot and f or Ramipril the range was found to be 250‐1250 ng/spot, with significant high values of correlation coefficient for both the drugs. The method was validated for the precision, robustness and recovery. As method. could effectively separate the drug from its degradation products, it can be employe d as a stability indicating one. [23] World Journal of Biology Pharmacy and Health Sciences, 2025, 22(01), 200-212 206 Hypertension is a serious medical condition that significantly increases the risk of heart, Brain, kidneys and other diseases. Blood pressure is a force exerted by circulating body against the walls of the body’s arteries, the major blood vessels in the body. It is the elevation of systolic blood pressure, Diastolic blood pressure or both above the normal levels. It effects above 1-Billion people worldwide and it is estimated that by 2025 up to 1.58 billion adults worldwide will suffer from complications of hypertension. It is the major public health problem in India. It accounts for 57% of all stroke deaths and 24% of all coronary heart diseases, deaths in India. Adequate management of hypertension can eventually reduce the risk of stroke, myocardial infection, chronic kidney disease and heart failure. The initial antihypertensive agent should be generally selected from one of the drug classes like Thiazide diuretics, Angiotensin converting enzyme (ACE) Inhibitors, Angiotensin receptor blockers(ARB’S) & Calcium channel blockers shown to reduce cardiovascular events. One of the angiotensin-converting enzyme (ACE) inhibitor class of medication is Ramipril. It is used to treat high blood pressure and lowering of high bold pressure which in turn helps to prevent strokes, heart attacks, & kidney problems. Ramipril is also used to improve survival after heart attacks and also used to treat heart failure in patents who have had a recent heart attack. Various analytical methods such as UV-Spectroscopy, High performance thin layer chromatography (HPTLC), High performance liquid chromatography (HPLC), Ultraperformance liquid chromatography (UPLC), Mass spectroscopy, Liquid chromatography-Mass spectroscopy (LC-MS), and UVSpectrophotometric methods for the determination of Ramipril as single and in combination with other drugs have been reported. The present review covers the analytical methods which have been used for analysis of Ramipril. [24] 4. Calorimetry Hypertension is a serious medical condition that significantly increases the risk of heart, Brain, kidneys and other diseases. Blood pressure is a force exerted by circulating body against the walls of the body’s arteries, the major blood vessels in the body. It is the elevation of systolic blood pressure, Diastolic blood pressure or both above the normal levels. It effects above 1-Billion people worldwide and it is estimated that by 2025 up to 1.58 billion adults worldwide will suffer from complications of hypertension. It is the major public health problem in India. It accounts for 57% of all stroke deaths and 24% of all coronary heart diseases, deaths in India. Adequate management of hypertension can eventually reduce the risk of stroke, myocardial infection, chronic kidney disease and heart failure. The initial antihypertensive agent should be generally selected from one of the drug classes like Thiazide diuretics, Angiotensin converting enzyme (ACE) Inhibitors, Angiotensin receptor blockers (ARB’S) & Calcium channel blockers shown to reduce cardiovascular events. One of the angiotensin-converting enzyme (ACE) inhibitor class of medication is Ramipril. It is used to treat high blood pressure and lowering of high bold pressure which in turn helps to prevent strokes, heart attacks, & kidney problems. Ramipril is also used to improve survival after heart attacks and also used to treat heart failure in patents who have had a recent heart attack. Various analytical methods such as UVSpectroscopy, High performance thin layer chromatography (HPTLC), High performance liquid chromatography (HPLC), Ultraperformance liquid chromatography (UPLC), Mass spectroscopy, Liquid chromatography-Mass spectroscopy (LC-MS), and UV-Spectrophotometric methods for the determination of Ramipril as single and in combination with other drugs have been reported. The present review covers the analytical methods which have been used for analysis of Ramipril. [25] Ramipril is an angiotensin-converting enzyme (ACE) inhibitor, used to treat high blood pressure and congestive heart failure. In the present study, urea, a wellknown adductor for linear compounds was successfully employed for inclusion of Ramipril—a substituted cyclic organic compound through a modified technique using oleic acid as rapidly adductibleendocyte (RAE). The proportion of oleic acid and urea in the inclusion compounds was optimized for maximum dissolution of ramipril using 22 factorial designs. Formation of urea inclusion compounds was confirmed by FTIR, DSC and XRD. Urea–Ramipril–RAE inclusion compounds containing varying proportions of guests were prepared and their thermal behavior studied by DSC. The inclusion compounds were also found to exhibit high content uniformity and markedly improved dissolution profile as demonstrated by increased dissolution efficiency in acidic, neutral and alkaline medium. Stability studies revealed that lesser amount of Ramipril in urea inclusion compounds degraded at 4oC, 25oC and 40oC than pure drug kept under same conditions. Studies reveal the possibility of exploiting co-inclusion of the drug in urea host lattice for simultaneous improvement of dissolution and stability. [26] A good Transdermal patch containing can be formulated by solvent casting technique using as film former and propylene glycol as plasticizer such Transdermal patches are advantages in providing effective treatment for Hypertension with enhanced patient compliance. From the invitro release results observed that the films prepared by using different ratios of HPMCK 15M, PVPK30 and EC Transdermal Ramipril patches were formulated using DBP as a plasticizer and DMSO as a penetration enhancer proved to exhibit better release characteristics. It can be reasonably concluded that Ramipril can be formulated into Transdermal patches to prolong its release characteristics. Thus, the formulation HPMCK15M and Ethyl Cellulose was found to be the best for controlled release. The Cumulative drug World Journal of Biology Pharmacy and Health Sciences, 2025, 22(01), 200-212 207 release from Formulation R10 was found to be 99.64% after 24 hrs. So, the formulation R10 is emerged as ideal formulation for Ramipril because it showed better release with sustained effect as compared to other formulations. [27] Transdermal drug delivery system is being extensively investigated as a viable alternative to drug delivery with improved bioavailability. The aim of the present investigation is to develop membrane type Transdermal drug delivery patches of antihypertensive agent Ramipril. Transdermal patches of Ramipril were prepared by using polymers, like HPMCK15M, Ethyl Cellulose and PVPK30. The patches were transparent, smooth and flexible. The results of weight variation, thickness, moisture content, moisture uptake, Folding Endurance, Tensile strength, drug content with the range. The patches were evaluated by DSC and SEM to ensure compatibility of drug with polymer and. The optimized formulation R5 is 96.42% drug release after 24 hours uniform distribution of the drug. [28] 5. LCMS A selective, rapid and sensitive liquid chromatography tandem mass spectrometry method has been developed for the simultaneous determination of ramipril and ramiprilat in human plasma using enalapril as the internal standard via one-step extraction with ethyl acetate under acidic condition. The analysis was carried out on a Diamonsil C18 column (150 mm × 4.6 mm i.d., 5 μm) with a mobile phase consisting of 1% formic acid-acetonitrile (25:75, v/v) at a constant flow rate of 0.5 mL min−1. The detection was performed on a triple-quadruple tandem mass spectrometer by selective reaction monitoring mode via electrospray ionization. Linear calibration curves of ramipril and ramiprilat were obtained in the concentration range of 0.107–107.0 and 0.262–105.0 ng mL−1, respectively. The intraand inter-day precision (RSD) values were below 8.2 and 4.8% for ramipril, 10.4 and 12.3% for ramiprilat, and accuracy (RE) were within ±5.5 and ±3.2%, respectively at all QC levels. The method was utilized to support clinical pharmacokinetic studies in healthy volunteers following oral administration of ramipril tablets. [29]. A rapid, simple, sensitive and specific LC-MS/MS method has been developed and validated for the simultaneous estimation of atorvastatin (ATO), amlodipine (AML), ramipril (RAM) and benazepril (BEN) using nevirapine as an internal standard (IS). The API-4000 LC-MS/MS was operated under the multiple-reaction monitoring mode using electrospray ionization. Analytes and IS were extracted from plasma by simple liquid–liquid extraction technique using ethyl acetate. The reconstituted samples were chromatographed on C18 column by pumping 0.1% formic acid– acetonitrile (15:85, v/v) at a flow rate of 1 mL/min. A detailed validation of the method was performed as per the FDA guidelines and the standard curves were found to be linear in the range of 0.26–210 ng/mL for ATO; 0.05–20.5 ng/mL for AML; 0.25–208 ng/mL for RAM and 0.74–607 ng/mL for BEN with mean correlation coefficient of ≥0.99 for each analyte. The intra-day and inter-day precision and accuracy results were well within the acceptable limits. A run time of 2.5 min for each sample made it possible to analyse more than 400 human plasma samples per day. The developed assay method was successfully applied to a pharmacokinetic study in human male volunteers.Copyright © 2010 John Wiley & Sons, Ltd. [30] A new method development and validation approach is proposed to develop a reliable method for the simultaneous quantitation of ramipril and ramiprilat in the presence of numerous labile metabolites. This new approach involves the usage of a synthesized labile acyl glucuronide of ramipril as well as individual and pooled incurred (study) samples in the development and validation process. Following the method validation and prior to its application to a large clinical study, a mini pilot study was performed to evaluate the performance of the method. When the samples from the mini pilot study were analyzed by two different scientists, 100% of the results from incurred sample reanalysis (ISR) matched within 8% of difference and the mean differences were 0.21% and 1.40% for ramipril and ramiprilat, respectively. The validated concentration range reported in this article is 0.2–80 ng/mL for both analytes. Various stabilities, such as bench-top, autosampler, freeze/thaw, and long-term, were also successfully evaluated. The key to the success were low sample processing temperature (4 °C), proper choice of sample extraction procedure, and adequate chromatographic conditions to obtain good peak shape without the need of derivatization and baseline separation between the analytes and their glucuronide metabolites. [31] The monitoring of the plasmatic concentrations of cardiovascular drugs is crucial for understanding their pharmacokinetics and pharmacodynamics. A simple, sensitive, specific, and high-throughput liquid chromatography/tandem mass spectrometry (LC–MS/MS) method was developed and validated for the simultaneous estimation and pharmacokinetic study of losartan (LOS), losartan carboxylic acid (LCA), ramipril (RAM), ramiprilat (RPT), and hydrochlorothiazide (HCZ) in rat plasma using irbesartan (IBS) and metolazone (MET) as internal standards (ISs). After solid phase extraction (SPE), analytes and ISs were separated on an Agilent Poroshell 120, EC-C18 (50 mm × 4.6 mm, i.d., 2.7 μm) column with a mobile phase consisting of methanol/water (85:15, v/v) containing 5 mmol/L ammonium formate and 0.1% formic acid at a flow rate of 0.4 mL/min. The precursor → product ion transitions for the analytes and ISs were monitored on a triple quadrupole mass spectrometer, operating in the multiple reaction World Journal of Biology Pharmacy and Health Sciences, 2025, 22(01), 200-212 208 monitoring (MRM) mode and switching the electrospray ionization (ESI) mode during chromatography from positive (to detect LOS, LCA, RAM, RPT, and IBS) to negative (to detect HCZ and MET). The method was validated as per the FDA guidelines, and it exhibited sufficient specificity, accuracy, and precision. The method was found to be linear in the range of 3–3000 ng/mL for LOS and LCA, 0.1–200 ng/mL for RAM and RPT, and 1–1500 ng/mL for HCZ. The described method was successfully applied to the preclinical pharmacokinetic study of analytes after oral administration of a mixture of LOS (10 mg/kg), RAM (1 mg/kg), and HCZ (2.5 mg/kg) in rats. [32] The present study describes a novel liquid chromatographic-tandem mass spectrometric (LC-MS/MS) method for the simultaneous estimation of ramipril (RAM) and hydrochlorothiazide (HCTZ) in human plasma using liquid–liquid extraction technique. This method made use of electrospray ionization in positive mode for RAM and in negative mode for HCTZ using triple quadrupole mass spectrometry where carbamazepine was used as an internal standard (IS). Analytes were recovered by methyl tertiary butyl ether: dichloromethane (85:15) subsequently separated on an Enable C18 G column (150 mm × 4.6 mm, 5 μm) using methanol:0.1% formic acid in water (85:15) as a mobile phase, at a flow rate of 0.5 mL/min. Quantification of RAM, HCTZ and IS was performed using multi-reaction monitoring mode (MRM) where transition of m/z 417.2 → 234.1 (RAM) and 237.0 → 194.0 (IS) in positive mode and 296.1 → 205.0 for HCTZ in negative mode. The calibration curve was linear (r2 > 0.99) over the concentration range of 2–170 ng/mL for RAM and 8–680 ng/mL for HCTZ. The intra-day and inter-day precisions were <15% and the accuracy was all within ±15% (at LLOQ level ±20%). Additionally, the LC-MS/MS method was fully validated for all the other parameters such as selectivity, matrix effect, recovery and stability as well. In conclusion, the findings of the present study revealed the selectivity and sensitivity of this method for the simultaneous estimation of RAM and HCTZ in human plasma. [33] A fast and robust liquid chromatography–mass spectrometry (LC–MS–MS) method has been developed for simultaneous quantitation of the angiotensin-converting enzyme (ACE) inhibitor, ramipril and its metabolite ramiprilat in human plasma. The method involves a solid-phase extraction from plasma, simple isocratic chromatography conditions and mass spectrometric detection that enables a detection limit at sub-nanogram levels. The proposed method has been validated with a linear range of 0.5–250 ng/ml for both ramipril and ramiprilat. The overall recoveries for ramipril and ramiprilat were 88.7 and 101.8%, respectively. [34] 6. Flourimetry Sensitive and selective analytical method is required for the estimation of ramipril in human plasma as ramipril has been reported to have high intra-subject variability and phase II inactive metabolite (ramipril acyl glucuronide) back converted to ramipril in an in vitro system. If this back conversion is not restricted, it could lead to pseudo estimation of ramipril in human plasma. [35] A novel, simple and specific spectrofluorimetric method was developed and validated for the determination of perindopril erbumine (PDE). The method is based on the fluorescence quenching of Rhodamine B upon adding perindopril erbumine. The quenched fluorescence was monitored at 578 nm after excitation at 500 nm. The optimization of the reaction conditions such as the solvent, reagent concentration, and reaction time were investigated. Under the optimum conditions, the fluorescence quenching was linear over a concentration range of 1.0–6.0 μg/mL. The proposed method was fully validated and successfully applied to the analysis of perindopril erbumine in pure form and tablets. Statistical comparison of the results obtained by the developed and reference methods revealed no significant differences between the methods compared in terms of accuracy and precision. The method was shown to be highly specific in the presence of indapamide, a diuretic that is commonly combined with perindopril erbumine. The mechanism of rhodamine B quenching was also discussed. [36] Ramipril, as a secondary amine compound, reacts with 7-fluoro-4-nitrobenzo-2-oxo-1,3-diazole (NBD-F) producing the corresponding fluorescent NBD-ramipril. According to this fact, spectrophotometric and fluorimetric methods for the determination of ramipril were developed. The effect of these parameters on the reaction product were carefully studied to optimize reaction conditions. The relationship between the absorbance at 465 nm and the concentration was found to be linear over the range 1–10 μg/ml. Moreover, the fluorescence intensity was also found to be directly proportional at the concentration over the range of 20–100 ng/ml at 530 nm after excitation at 465 nm. The proposed procedure was successfully applied to the determination of ramipril in both tablet dosage form and in plasma. Spectrophotometric determination of ramipril tablets yielded a percentage recovery of 98.66±0.38, while the percentage recovery of spectrofluorimetric determination of ramipril in spiked human plasma was 99.08±1.11%. The results obtained are in good agreement with those obtained by the reference method. No interference could be observed from the co-administered drug (hydrochlorothiazines). [37]