QUANTITATIVE DERTERMINATION IN COMPARISION OF RAMIPRIL IN PURE FORM WITH OTHER PHARMACEUTICAL DOSAGE FORM BY USING RP – HPLC

About Authors:
Rajkumar Bolledula*, Priyanka.Mare¹, Sunanda.Mare²
*Department of pharmaceutical analysis, Assistant professor in MITS college of pharmacy, H.No.41-64-s, saibabanagar, Kurnool-518004, A.P, India.
1.Department of pharmaceutics, Assistant professor, Mits college of pharmacy, Block .no.mc2/2, singareni colony, tekulapally,yellandu,khammam-507123,A.P,India
2.Department of pharmacology, J.K.K.Natarajan college of pharmacy, Salem, Tamil Nadu

ABSTRACT
This article describes the quantitative determination of percentage of drug concentration in pure and in formulated pharmaceutical dosage forms by using RP-HPLC. Procedure does not require prior separation of components from the sample.The mobile phase consists of methanol-water (80:20 v/v) for RP-HPLC with injection volume of 20μl.The RP-HPLC method was developed on a C-8 (150x4.6mm) column with detection carried out by variable wavelength detector at 209nm and 220 nm for ramipril, cardace, odopril respectively…Thus the Rt values obtained for different dosage forms are 1.59,1.44,1.34,1.35…..respectively with the concentration of 100% for pure drug and 97.54%,91.03%,65.76%.......respectively.

Reference Id: PHARMATUTOR-ART-1150

INTRODUCTION
Ramipril is an angiotensin-converting enzyme (ACE) inhibitor, used to treat hypertension and failurewith molecular weight 416.51 g/mol Systematic (IUPAC) (2S,3aS,6aS)-1-[(2S)-2-{[(2S)-1-ethoxy-1-oxo-4-phenylbutan-2yl]amino}propanoyl] octahydrocyclopenta[b]pyrrole-2-carboxylicacid.ACE inhibitors lower the production of angiotensin II, therefore relaxing arterial muscles while at the same time enlarging the arteries, allowing the heart to pump blood more easily, and increasing blood flow due to more blood being pumped into and through larger passageways.Ramipril is a prodrug and is converted to the active metabolite ramiprilat by liver esterase enzymes.Ramiprilat is mostly excreted by the kidneys. The half-life of ramiprilat is variable (3-16 hours), and is prolonged by heart and liver failure, as well as kidney failure. Ramipril is marketed in India under the brand names of Cardace, .Cardiopril, Odipril, Ramistar, Ramicor, Zigpril and Zorem.Ramipril is used to treat high blood pressure (hypertension), heart failure, and to improve survival after a heart attack. Ramiprilat, the diacid metabolite of Ramipril, is a non-sulfhydryl angiotensin converting enzyme inhibitor.Ramipril is converted to Ramiprilat by hepatic cleavage of the ester group.Ramipril is supplied as hard shell capsules for oral administration containing 1.25 mg, 2.5 mg, 5 mg, and 10 mg of Ramipril.

Mechanism of Action:
Ramipril and Ramiprilat inhibit angiotensin-converting enzyme (ACE) in human subjects and animals. ACE is a peptidyl dipeptidase that catalyzes the conversion of angiotensin I to the vasoconstrictor substance, angiotensin II. Angiotensin II also stimulates aldosterone secretion by the adrenal cortex. Inhibition of ACE results in decreased plasma angiotensin II, which leads to decreased vasopressor activity and to decreased aldosterone secretion. The latter decrease may result in a small increase of serum potassium. While the mechanism through which Ramipril lowers blood pressure is believed to be primarily suppression of the renin-angiotensin-aldosterone system, Ramipril has an antihypertensive effect even in patients with low-renin hypertension.

Pharmacokinetics:
Following oral administration of Ramipril, peak plasma concentrations of Ramipril are reached within one hour. The extent of absorption is at least 50–60% and is not significantly influenced by the presence of food in the GI tract, although the rate of absorption is reduced. Cleavage of the ester group (primarily in the liver) converts Ramipril to its active diacid metabolite, Ramiprilat. Peak plasma concentrations of Ramiprilat are reached 2–4 hours after drug intake. The serum protein binding of Ramipril is about 73% and that of Ramiprilat about 56%; in vitro, these percentages are independent of concentration over the range of 0.01 to 10μg/ml.Ramipril is almost completely metabolized to Ramiprilat, which has about 6 times the ACE inhibitory activity of Ramipril, and to the diketopiperazine ester, the diketopiperazine acid, and the glucuronides of Ramipril and Ramiprilat, all of which are inactive. After oral administration of Ramipril, about 60% of the parent drug and its metabolites is eliminated in the urine, and about 40% is found in the feces. Drug recovered in the feces may represent both biliary excretions of metabolites and/or unabsorbed drug, however the proportion of a dose eliminated by the bile has not been determined. Less than 2% of the administered dose is recovered in urine as unchanged Ramipril.Plasma concentrations of Ramiprilat decline in a triphasic manner (initial rapid decline, apparent elimination phase, terminal elimination phase). The initial rapid decline, which represents distribution of the drug into a large peripheral compartment and subsequent binding to both plasma and tissue ACE, has a half-life of 2–4 hours. Because of its potent binding to ACE and slow dissociation from the enzyme, Ramiprilat shows two elimination phases. The apparent elimination phase corresponds to the clearance of free Ramiprilat and has a half-life of 9–18 hours. The terminal elimination phase has a prolonged half-life (>50 hours) and probably represents the binding/dissociation kinetics of the Ramiprilat/ACE complex. It does not contribute to the accumulation of the drug. After multiple daily doses of Ramipril 5–10 mg, the half-life of Ramiprilat concentrations within the therapeutic range was 13–17 hours.In patients with impaired liver function, the metabolism of Ramipril to Ramiprilat appears to be slowed, possibly because of diminished activity of hepatic esterases, and plasma Ramipril levels in these patients are increased about 3-fold. Peak concentrations of Ramiprilat in these patients, however, are not different from those seen in subjects with normal hepatic function, and the effect of a given dose on plasma ACE activity does not vary with hepatic function.

Pharmacodynamics:
Single doses of Ramipril of 2.5–20 mg produce approximately 60–80% inhibition of ACE activity 4 hours after dosing with approximately 40–60% inhibition after 24 hours. Multiple oral doses of Ramipril of 2.0 mg or more cause plasma ACE activity to fall by more than 90% 4 hours after dosing, with over 80% inhibition of ACE activity remaining 24 hours after dosing. The more prolonged effect of even small multiple doses presumably reflect saturation of ACE binding sites by Ramiprilat and relatively slow release from those sites.

Uses:
This medication belongs to a group of drugs called ACE inhibitors. ACE inhibitors prevent certain enzymes in the body from constricting blood vessels. This helps to lower blood pressure and makes the heart beat stronger. This medication is used to treat hypertension (high blood pressure) and heart failure.

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Contraindications:
Ramipril is contraindicated in patients who are hypersensitive to this product or any other angiotensin converting enzyme inhibitor (e.g., a patient who has experienced angioedema during therapy with any other ACE inhibitor).Renovascular disease, severe renal impairment (especially in patients with one kidney or with bilateral renal artery stenosis), volume-depleted patients, history of angioedema while on an ACE inhibitor, pregnancy, hypotension.

Reversed phase HPLC:
In this case, the column size is the same, but the silica is modified to make it non-polar by attaching long hydrocarbon chains to its surface - typically with either 8 or 18 carbon atoms in them. A polar solvent is used - for example, a mixture of water and an alcohol such as methanol. In this case, there will be a strong attraction between the polar solvent and polar molecules in the mixture being passed through the column. There won't be as much attraction between the hydrocarbon chains attached to the silica (the stationary phase) and the polar molecules in the solution. Polar molecules in the mixture will therefore spend most of their time moving with the solvent. Non-polar compounds in the mixture will tend to form attractions with the hydrocarbon groups because of van der Waals dispersion forces. They will also be less soluble in the solvent because of the need to break hydrogen bonds as they squeeze in between the water or methanol molecules, for example. They therefore spend less time in solution in the solvent and this will slow them down on their way through the column. That means that now it is the polar molecules that will travel through the column more quickly. Reversed phase HPLC is the most commonly used form of HPLC.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.

MATERIALS AND METHODS
Chemicals Required: Methanol and Water from Make-Merck

Instruments Used:
Electronic balance-ELB300, DIGISUN P^H meter.
PEAK7000 isocratic HPLC with following configurations.
1. PEAK 7000 delivery system.
2. Rheodyne manual sample injector with switch (77251)
3. Analytical column C8.   (150×4.6mm).

PROCEDURE
Trial 1: The Mobile Phase 100% Methanol was passed through Stationary Phase C8Column at a flow rate 2ml/min at a wave length of 210nm. The drug concentration of 1mg/ml Ramipril dissolved in the solvent is used and HPLC process is carried out. The RT Value noted is 1.05 min.The Absorbance was found to be 217mAU.

Trail 2: The Mobile Phase Methanol and Water (80:20) was passed through Stationary Phase  C₈ Column at a flow rate 1ml/min at a wave length of 210nm. The drug concentration of 1mg/ml Ramipril dissolved in the solvent is used and HPLC process is carried out. The RT Value noted is 2.16min.The Absorbance was found to be 591mAU.

Trail 3: The Mobile Phase Methanol and Water (80:20) was passed through Stationary Phase  C₈ Column at a flow rate 1ml/min at a wave length of 209nm. The drug concentration of 1mg/ml Ramipril dissolved in the solvent is used and HPLC process is carried out. The RT Value noted is 1.63min.The Absorbance was found to be 1528mAU

Trail 4: The Mobile Phase Methanol and Water (80:20) was passed through Stationary Phase  C₈ Column at a flow rate 1ml/min at a wave length of 208nm. The drug concentration of 1mg/ml Ramipril dissolved in the solvent is used and HPLC process is carried out. The RT Value noted is 1.65min.The Absorbance was found to be 1447mAU.

Trail 5: The Mobile Phase Methanol and Water (50:50) was passed through Stationary Phase  C₈ Column at a flow rate 1ml/min at a wave length of 209nm. The drug concentration of 1mg/ml Ramipril dissolved in the solvent is used and HPLC process is carried out. The RT Value noted is 1.64min. The Absorbance was found to be 1418mAU.

Trail 6: The Mobile Phase Methanol and Water (50:50) was passed through Stationary Phase  C₈ Column at a flow rate 1ml/min at a wave length of 208nm. The drug concentration of 1mg/ml Ramipril dissolved in the solvent is used and HPLC process is carried out. The RT Value noted is 1.60min. The Absorbance was found to be 1318mAU.

Based on the above trails the satisfactory results are attained by using the mobile phase methanol and water (80:20) passing through the column C₈ at a wave length of 209nm with a flow rate of 1ml/min.By using the above parameters the analysis of various brands of Ramipril are performed.

Initial Chromatographic Conditions:
Column C₈, 150×4.6mm.Mobile phase: The mobile phase composed of methanol and water (80:20) was filtered through 0.45 µ nylon membrane filters before use and P^H was find that is 6.9. Prepare a filtered and degassed mixture of methanol and water (80:20).
Detector wave length 209nm, Operating Pressure-3000psi.Temperature –ambient. Injection volume-20µl.Run time: 4 min.Flow rate: 1ml per min.

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METHODOLOGY
Preparation of Standard Solution:About 5mg of   Ramipril was accurately weighed and transferred in to 5ml volumetric flask and dissolved and make up with sufficient mobile phase. Volumetric flask contains standard solution kept for sonication for10minutes.The standards solution was filtered with 0.45µm membrane filter paper with sample filter.

Procedure for Analysis:About, 5mg of given Commercial sample was accurately weighed add 5 ml mobile phase to prepare 1 mg/ml sample solutions.
The sample Solutions were filtered with 0.45 µm membrane sample filter.
With the optimized chromatographic conditions for the Ramipril was set at the conditions mentioned earlier a study baseline was recorded.
After the Stabilization of base line for about 30min successive aliquots of the sample Solution were injected separately and the chromatograms were recorded, until the reproducibility of the peak areas was satisfactory.
This procedure was repeated for every commercial sample into the column at flow rate of 1ml/min.The samples used were Cardace, Cardiopril, Odiopril, Ramistar and Ramicor.

RESULTS AND DISCUSSIONS

Table 1: Different samples containing ramipril and standard with Rt and conc %

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Thus the standard ramipril with sample their Rt values and Conc % obtained to be 1.59, 1.44, 1.34, 1.35, 1.35, 1.45 and 100%, 97.5%, 91.03%,65.76%, 48.52%,34.77% respectively and the obtained Rt values with their peak areas are shown in fig:1,2,3,4,5 and 6 respectively. The detection methods are used for the analysis of drugs. These methods are highly automated and extremely sensitive. Using RP HPLC method the method development for Ramipril is found out and various brands of Ramipril are analysed for the drug content. The Standard drugs have the Retention Time 1.59 min and the Drug Concentration is 100%. The  Various brands of Drugs have the Retention Time ranging from 1.34 -1.44 min and the Drug Concentration is from 34 – 97.8

CONCLUSION
The concentration of Ramipril is maximum in Odiopril when compared to the other samples where as the concentration of Ramipril in Ramicor.H is minimum because it is in combination with Hydrochlorthiazide. Cardace is having the maximum no. of impurities when compare to the other samples.

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