A Study On Ranitidine Hydrochloride Biology Essay

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By John Bradshaw in the Ware research laboratories of Allen and Hanburys Limited, which became a part of Glaxo organization, in 1976 Ranitidine was first prepared in the name of AH19065. In November 1976 in the United Kingdom by Sir James Black at Kline and French, Smith launched that Ranitidine belongs to the classification of histamine H2-receptor antagonist. Eventually both companies merged as GlaxoSmithKline by following the sequences and the acquisitions of Allen and Hanbury's Limited, Glaxo started to form a Glaxo Group Research in 1979 and eventually with the union of Glaxo Wellcome and SmithKline Beecham in 2000. Ranitidine was the outcome of a rational drug-design process and it became a refined model of quantitative structure-activity relationships (QSAR) and Histamine H2-receptor.Later Glaxo changed the model; the imidazole-ring of the cimetidine was replaced with furan-ring substituent, later on Ranitidine was developed. Ranitidine has fewer adverse reactions. It has long-lasting action. In activity it is ten times greater than the cimetidine. In 1981 Ranitidine was first introduced; by 1988 it was the world's biggest selling prescription drug. Later it was superseded by Omeprazole because it has more effective proton pump inhibitors.


Ranitidine Hydrochloride is "histamine-2 or H2 blockers or H2 receptor antagonist".The properties of ranitidine hydrochloride are that it is a pale-yellow or white crystalline powder. It has a sulfur-like odor and slightly bitter taste. Ranitidine hydrochloride is an amino-alkyl substituted furan. Structurally it is different from cimetidine by lacking the cyanogaunidine group and imidazole ring. Ranitidine HCl is freely soluble in water and methanol and sparingly soluble in ethanol.It is used in the treatment of gastric ulcer, duodenal ulcer (which is associated with non-steroidal anti-inflammatory agents), Zollinger Ellison syndrome, erosive disease, and erosive esophagitis. Ranitidine Hydrochloride also indicated for the treatment of "Post-operative ulcer". Ranitidine Hydrochloride is also used for the treatment of where reduced acid output is desirable and reduction of gastric secretion. It is used for the treatment of "chronic episodic dyspepsia" which is related to food and disturbs the sleep. Ranitidine Hydrochloride is a drug which reduces the acid production by the cells and blocks the behavior of histamine on the parietal cells in the stomach. The H2 antagonists are competitive inhibitors of histamine. It will suppress the acid secreted by the food and the secretion of acid by parietal cells.


Figure1. Structure of Ranitidine hydrochloride.

Ranitidine hydrochloride is non-imidazole blockers of H2-receptors. It is amino-alky substituent of furan. The imidazole ring of cimetidine was replaced with furan. It comes under the class of H2-blockers. Zantac is brand name of ranitidine hydrochloride. The chemical formula of ranitidine hydrochloride is C13H22N4O3S, as shown in the above figure 1. The IUPAC name of ranitidine hydrochloride is"{dimethyl [(5-{[(2-{[(E)-1-(methyl amino)-2-nitro-ethenyl]amino} ethyl)sulfanyl] methyl}-furan-2-yl) methyl] amine".


It is used in the treatment of peptic ulcers, stress ulcer prophylaxis, dyspepsia, and GRED [Gastro-esophageal reflux disease]. The mechanism of action of ranitidine hydrochloride is at the parietal cell H2-receptor, the H2- antagonists are competitive inhibitors of histamines. It will suppress the acid secreted by the parietal cells which is stimulated by the food intake. It has two mechanisms of action: ECL cells release the histamines in the stomach, where they are blocked from binding on the parietal cells H2-receptors which stimulate acid secretion. The other mechanism is the H2-receptors are blocked which have a reduced effect on parietal cells which stimulates the acid secretion. The bioavailability of ranitidine hydrochloride is 50% approximately.

Ranitidine hydrochloride is Histamine of H2-blockers which is similar to cimetidine and Famotidine. The H2-receptor antagonist is reduced to H2-antagonist, it is a drug used to block the histamines on parietal cell which secretes the acid and decrease the acid production by the cells. It has 15% protein binding. The metabolism of ranitidine hydrochloride is a Hepatic metabolism. Ranitidine was metabolized to S-oxide, N-oxide and N-des-methyl metabolites. The route of elimination of ranitidine hydrochloride is through urine.


Ranitidine hydrochloride can be administered in many pharmaceutical forms like tablets, injectable solutions, and oral liquids. The tablets are preferable when compared to oral liquids. Oral liquids are needed in the presence of anti-microbial agents like parabens. Liquid form is preferred for infants or children or geriatric patients rather than the solid dosage form because it is easier and safer to swallow. Liquids formulations will favor a most rapid absorption of the active substances rather than a solid form. Ranitidine hydrochloride is an H2-anti-histamine drug which is mainly used for the treatment of peptic ulcers and 50% orally absorbed. To increase its oral bioavailability, the drug undergoes hepatic metabolism so as attempt has been made to fast dissolution of tablet. By using the sublimation method using ammonium bicarbonate as sublimating agent the tablets were prepared and these tablets were evaluated for wetting time, dispersion time, hardness, disintegrating time. By using the sodium starch glycolate and cross carmellose sodium as super dis-integrant,the tablets were prepared. By comparing to sublimation method and super dis-integrant method, the super dis-integrant methods have better dis-integrant properties when compared to sublimation method.


The excipients used in the preparation of ranitidine hydrochloride are microcrystalline cellulose, magnesium stearate, Hypromellose (E 464), titanium dioxide (E 171), red iron oxide (E 172) and triacetin. The microcrystalline cellulose is used as a binding agent in the preparation of ranitidine hydrochloride. Hyprocelluse (E 464) is a brand of Hydroxy-propyl cellulose, from fibrous plants; it is used as binder, diluent and lubricant. Titanium-dioxide (E 171) is used as a white pigment but chemically it is not obtained from any starch source. The iron oxide (E 172) used as a coloring agent. Tri -acetin is a derivative of glycerin, it is used as plasticizer. The magnesium stearate is used as a lubricant in the preparation of ranitidine HCl.

1.6 Procaine hydrochloride:

Procaine Hydrochloride used as an internal standard in my project. The chemical formula of Procaine hydrochloride is C13H20N2O2. Molecular weight of Procaine hydrochloride is 272.8 g/mol. Procaine was replaced in the place of cocaine as an anesthetic. Because cocaine was addictive and toxic and destructive to the central nervous system due to these reason it was outlawed. The procaine can be divided into two words, pro means" in the place of "and "caine" means "cocaine" now procaine means "in the place of cocaine". Procaine acts as anesthetic when injected into the muscles. Procaine hydrochloride solutions are also used as anesthetics. The trade name of Procaine Hydrochloride is NOVOCAINE. Chemically it is the P-amino benzoic acid {PABA} ester of the amino acid alcohol n,ndiethylaminoethanol{DEAE}. PABA is a vitamin B and DEAE is the precursor of the vitamin B choline. Procaine was usually used in the form of hydrochloride salt because it is highly water soluble. The problem with procaine hydrochloride is the blood enzymes will destroy it rapidly and its half-life is only 36seconds (0.6 minutes). If small amount of benzoic acid added to the procaine solution it becomes stabilized and some of the procaine can be protected from blood enzyme. This protected procaine named as Gerovital H3 or GH3 is very helpful in supplying of DEAE and PABA to the damaged cells and later it is named as Procaine vitamin H3. Procaine hydrochloride when combined with biological active acids like biotin, folic acid, pantothenic acid, ascorbic acid, and citric acids and other amino acids, they act as a food supplement or as vitamins. When procaine hydrochloride complexes with biologically active acids and vitamin acids it will protect the procaine from the blood enzymes and allows the procaine to enter the cells. Vitamin complex is useful for cell assimilation and utilization of nutrients more efficiently. Hence these procaine compounds were named as procaine based nutrients [PBNs]. When procaine HCl is mixed with biologically active acids it will protects from the diseased enzymes and tissues. It helps the cells for rebuilding, repair and detoxification.


The present invention is involved with hydrochloride salt of ranitidine. For the production of ranitidine hydrochloride there is a novel process. According to the present invention, the Form 1 ranitidine hydrochloride is obtained by dissolving ranitidine base in a "suitable solvent" and refluxing it maintains the temperature at 100C. Here suitable solvents are lower alkanols containing hydrochloride, stir the solution and collect the product.

There are many advantages if the above condition has been followed. They are

The obtained product is easily filtered and dried.

The suitable solvents which are used in the preparation are easily recoverable.

It is an economical and convenient process on plant scale.

The product has >99.5% degree of purity (high).

The obtained product is stable.

Suitable solvents: The term "suitable solvent" means any lower alkanol, which has primary, secondary and tertiary alcohols which containing 1to 6 carbon atoms. The suitable solvents that are "lower alkanols" include ethanol, isopropanol, n-propanol, isobutanol, n-butanol, t-butanol, and amyl alcohol. N-propanol, isopropanol or n-butanol are "suitable solvents" in the preparation of form 1 ranitidine hydrochloride. Isopropanol is the most preferable solvent. Mixture of two or more suitable solvents are used which have elements of carbon and hydrogen like hydrocarbons such as hexane, toluene, benzene, and xylene, or ester, or ketone, and having 1 to 10 carbons such as methyl ethyl ketone, acetone, 4-methyl pentan-2-one, 2-butanone, n-butyl acetate, acetonitrile or ethyl acetate are contemplated.

1.7.1. Process for the manufacture of form1 Ranitidine hydrochloride: The process for the preparation of ranitidine hydrochloride is as follows: firstly it was dissolved in a suitable solvent and stir the solution so that we can obtain the hydrochloric acid to form a reaction mixture. From the reaction we obtain the crystallized form of the Form 1 ranitidine hydrochloride. For example the ranitidine was mixed with methylene chloride; to this solution add hydrochloride with or without cooling and mix it to form reaction mixture, heat this reaction mixture under reflux and strip off the water under azeotropic conditions there by the form 1 ranitidine hydrochloride crystals are produced. These crystals are washed with ethanol and dried.

1.7.2 Process for the manufacture of Form 2 ranitidine hydrochloride: Form 2 ranitidine hydrochloride is the stable form of form 1 ranitidine hydrochloride. By warming the solution the lower alkanol or suitable solvents are added. Stir and cool the solution until the crystals are formed. To this solution add miscible solvent like ethyl acetate solution to complete the crystallization process. Generally the product can be collected by using any standard filtration method like filtration under vacuum or decantation and drying.


In this project, HPLC and NMR instruments are used.


HPLC method was performed at Sheffield Hallam University. It is a Perkin Elmer model 2000.

HPLC is one method of chromatography. It is one of the widely used analytical techniques. Chromatographic procedure can be explained as a separation technique, it involves mass-transfer between stationary and mobile phase. In HPLC liquid mobile phase is used to separate the components in the mixture. Here stationary phase can be either liquid or a solid phase. The components are dissolved in a solvent and then flow through the column under high pressure. The mixture separates into it components in the column. Here resolution is more important; it depends on the interaction between the solute components and the stationary phase. The immobile packing material in the column is defined as the stationary phase. Through different selections of both solvents and stationary phase the interactions of the solute with stationary and mobile phases can be manipulated. The HPLC requires a high degree of flexibility which is not found in other chromatographic systems. It can easily separate a wide diversity of chemical mixtures.

HPLC is an active adsorption process. When analyte molecules move through the porous packing beads it will interact with the surface adsorption sites. In the retention process the different types of the adsorption forces are included depending on the HPLC mode: Non-specific (Hydrophobic) interactions are the important ones in reversed-phase separations. Polar (Dipole-dipole) interactions are the main in normal phase (NP). In ion exchange chromatography the ionic interactions are responsible for the retention. Analyte molecules will compete with the eluent molecules for adsorption sites. The stronger analyte will interact with the surface. The analyte will be retained on the surface for a prolonged time when there is a weaker eluent interaction.


In this classification there are three modes based on the stationary and separation process.

Adsorption chromatography: Adsorbent (like silica gel) is stationary phase and the separation phase is based on the recurrent adsorption-desorption steps.

Ion-exchange chromatography: the stationary bed is ionically charged surface which is opposite charge to the sample ions. It is extensively used technique with ionic or ionizable samples. If there is strong charge on the sample, there will be stronger attraction to the ionic surface due to which it takes longer time to elute. To control elution time both pH and ionic strength aqueous buffer is used.

The size exclusion chromatography: it has controlled pore sizes where the columns are filled with material, and according to the solvated molecular size the sample is simply screened. Bigger molecules are swiftly washed through column where as small molecules will penetrate through the pores of the packing particles and eluted later. This technique is also known as gel permeation or gel filtration chromatography.

Taking the first type, two modes are explained contingent on the relative polarity of 2 phases: the normal and reverse phase chromatography. The stationary bed is strongly polar in nature in normal phase chromatography, and the mobile phase is non-polar. In reversed-phase chromatography it is inverse to the normal phase here the stationary phase is non-polar and mobile phase is polar in nature. Example of non-polar is n-hexane and polar liquids such as a mixtures of water and acetonitrile or methanol. In 90% of all chromatographic applications the reverse-phase chromatography is used. In all types of HPLC the eluent polarity plays the major role. There are two types of elution they are isocratic and gradient type. In isocratic the constant eluent composition was pumped through the column during the experiment analysis. In gradient type the eluent composition was steadily changed during the process.


HPLC separations are based on the types of adsorbents used and on the surface interactions. The small porous particles with high surface area are the modern HPLC adsorbents. The parameters of the adsorbents are:

The particle size should be 3 to 10 micrometer.

The particle size distribution is possible if it is narrow within 10% of the mean.

The final parameter in the catalogue is adsorbent surface chemistry. It depend on the type of ligand attached to the surface, the adsorbent may be reverse phase (C5, C8, C18, CN, NH2) or normal phase (-OH, -NH2) and even cation (R-SO3-H+) or anion (CH2NR3+OH-) exchangers.

HPLC instrumentation:

HPLC instrument embraces a pump, injector, column UV detector and a data system. The heart of the HPLC instrument is the column where separation takes place. The stationary phase consists of micrometer sized porous particles due to this high pressure is needed to move the mobile phase across the column. By injecting the sample at the top of the column the chromatographic process will begin. When the analyte and the mobile phase is pumped across the column the separation will takes place. Finally, each component will elute from the column as a peak on the data system or recorder. The main important component of detection is elution and it can be either universal or selective based on the detector used. Each component will display on the computer screen or recorded it is the response given by the detector hence it is known as chromatogram. It is shown in fig1 and fig 2


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MOBILE PHASE: In HPLC large variety of solvents are used they have several common properties like: Detector compatibility, Purity, Low viscosity, Solubility of sample and Chemical inertness.

The solvents are mainly non-polar for normal phase mode, for reverse-phase it is a mixture of water with some polar organic solvents like acetonitrile or methanol.


Mobile phase reservoir




Detector and

Data system


Glass bottle is the most common type of the solvent reservoir. These bottles supplied by the manufacturers with special caps. Teflon tubing and filters are connected to the pump inlet and the helium gas (purge gas) is used to remove dissolved air.


To force the solvents through the packed stationary phase beds, high pressure pumps are needed. High pressure is needed for smaller bed particles. By using smaller particles there are many advantages, but it may not be crucial for all separations.

The main advantages are:

Faster analysis

Higher resolution

Increased sample load capacity

By using the larger particle packing which requires less pressure many problems can be solved. The important pump feature that distinguishes the pumps is "flow rate stability". For analytical chromatography very stable flow rates are not important. If the user plans to operate a system in size exclusion mode, then pump is needed which provides an extremely stable flow rate. When we are using isocratic method it is an undesirable feature. The parameters of the modern pumps are:

Flow rate range will be 0.01 to 5mL/min

The stability of flow rate is not more than one percent

For SEC the flow rate stability must be less than 0.2%

The maximum pressure is up to 300 hPa.

It is necessary to have an integrated degassing system that is either membrane filtering or helium purging.

Fig 3: the pump of HPLC


The sample introduction can be done in many ways. The simplest method is to use an injection valve. The automatic sampling devices are integrated where the sample is introduced with the help of microprocessor and auto-samplers in more hi-tech LC systems. The liquid samples can be injected directly in liquid chromatography. Solid samples are dissolved in an appropriate solvent. To remove particles or impurities from the sample it should be filtered over a 5micrometer filter or centrifuged. Due to continuous injections of sample it may cause blockages in the in the injection devices or columns. The sizes of sample may vary.


There are 5, 10, 15 and 25 cm long of typical HPLC columns and they are filled with small diameter such as 3, 5 or 10 micrometer particles. Usually 4.6mm is the internal diameter of the column, it is considered as the best for mobile phase consumption, sample capacity, resolution and speed. For pure substances the lager diameter columns are required.


Now-a-days in liquid chromatographic system the optical detectors are used. These detectors will pass a beam of light through the column effluent which flows as it passes through low volume [~10 microliters] flow cell. UV absorption, change in refractive index or fluorescence emission will cause the variations in the light intensity, the sample components will pass through the cell, where they are monitored as changes in the output voltage. On a strip chart recorder the changes in voltage are recorded and to provide retention time and peak area data recently they are fed into a computer. The UV absorption detector is the most commonly used detector in LC. For the detection of the majority samples there is a wavelength detector which is capable of monitoring 190 to 400nm. The other detectors commonly used are: Refractive index, Photodiode array ultra violet detector [PAD], electrochemical [EC] and fluorescence [FLU] detector. The universal detector is refractive index but it is less sensitive. EC and FLU detectors are quite selective but they are quite sensitive [up to 10-15 pmole].

Data system:

By using the modern data collection we can promote the signal analysis because the detector signal is electronic. Computer analysis can be done later by storing a data in some systems in retrieving format. The important goal of using this electronic system is to increase the analysis of precision and accuracy. The specific software is used to reduce the accidental misuse of the computer system.

The process of HPLC has five major steps

Pump ----> Injector ----> Column -----> Detector -------> Computer.


To perform the experiment analysis, Perkin Elmer HPLC model 2000 was used. To retrieve the data Total Chromatogram Navigator software was used. The instrument consists of Mobile phase reservoir, column and UV detector. The column used for the analysis was ODS-2 5-micrometer column (150mm x 4.6mmID). To inject the sample using a syringe the instrument consists of injection valve. UV detector used was PERKIN-ELMER UV-Vis spectrometer. The volume to inject the sample through the injection valve was 20 microliters, flow rate is 0.5 mL to 1mL was maintained, the detector wavelength was 230nm.


Apart from HPLC, NMR method is used. It is explained briefly as follows.


NMR at Sheffield Hallam University:

NMR (company): Bruker

Detector: Avance 3(400MHz) [Bruker]

Software: Topsin 2.1.

Signals from nuclei is measured in PPM [parts per million] of the static field strength. DMSO-d6 is a heavy hydrogen which is used as NMR solvent. F19, P131, C13 are not used because it has a magnetic moment. DMSO-d6 is used a reference. NMR has a strong combination of magnetic and radio field, for number of nucleus. Heavy hydrogen is not magnetic hence it won't detect DMSO but it observes the signal. In olden days TMS was used as internal standard, it gives peaks from zero. New solvent is used as internal standard, it has low radio frequency to detect solvents (sample). The liquids used for NMR are acetone, DMSO, CDCl3, it will give the signals at different radio frequencies.

The process of sample detection by using NMR:

Lift up --------> Lock -------> Tuning -----> Shimming -----> Running.

Tuning: The nucleus which we use has to be tuned. It contains different parameters. Before setting the equipment nucleus tuning should be done or else good results are not obtained.

Shimming: Shim coils are present around the magnetic field to isolate the tuning.

Thermal flask: In this, liquid gases are used for cooling down the instrument. Super conducting magnets are used for good magnetic fields. In this NMR, He and Ni are used as liquid gases. He has temperature of (-296 0C), (-452 0F), (42K). Ni has temperature of (72K), (-321 0F), (-196 0C). Helium is present at the center of the container flask because helium will blow off. Nitrogen will be present at the corners of the container flask because it stops the blowing off of the helium. Usually magnets are made up of Nickel or Rhodium. Due the presence of electric field the magnetic field will become stronger. Insulator is used to avoid evaporation. Radio frequency enters through the pore head. It is based "BOLTZMANN EXCESS". Higher energy was applied [against the magnetic field] and then radio frequency was pulled and enters through the sample. Lower energy was applied only when it was not against the magnetic field. It is a free induction decay where sample will absorbs radio frequency.


HPLC instrument was used to separate the mixture of chemical compounds. The well separated and resolved peaks are obtained. In the validation method the linearity/range, system precision, intermediate precision, accuracy/ recovery, specificity and selectivity, stability of analytical solutions, robustness are known. NMR instrument was used to find the compounds in the solution. It is used for the structure elucidation and identification of biological, organometallics and organic molecules. In this project by using the HPLC the well separated and resolved peaks of ranitidine hydrochloride are obtained. HPLC instrument was used for the development of the Ranitidine hydrochloride. By using the NMR instrument all the compounds present in the sample are separated and the compounds present in that are known.


Azad et al (2008) have developed a sensitive, simple and rapid spectroscopic method for the determination of ranitidine HCl in Pharmaceutical dosages and pure forms. To give a colored complex, there is a formation of charge-transfer complex between the ranitidine based as n-electron with chloranil as acceptor at 550 nm. In the concentration ranges from 2-40 microgram/mL with the molar absorptivity of 2.5104 L/mol-1/cm-1 the Beer's laws is obeyed. For accuracy and precision the statistical comparison of the results was performed by using the student's F-ratio and t-test at 95% confidence level.

Lin et al (2006) have developed the High-performance liquid chromatography (HPLC) method for determination of ranitidine HCl capsules. In this method Hypersil ods C18 column was used. Acetonitrile (0.05 mol/L-1) citric acid; where here the pH was adjusted to 3.5 with TEA (tri-ethylamine) which is used as the mobile phase. The detection of wavelength was 314nm and flow rate was 1mL/min-1 and the linear range was 5-50.0 microgram/mL-1 or r = 0.9999. The average recovery was 100% and the relative standard deviation was 0.95% that is n=6.

Lau-cam et al (1994) have developed the rapid assay of samples from tablet dissolution testing and rapid assay of ranitidine in dosage form. They have enrooted the HPLC method with spectrophotometric detection. On Micros orb- MV C18 column the analysis were carried out. Mixture of methyl-hydroxide (Me OH) 0.01M, disodium hydrogen phosphate (Na2HPO4) (pH 7.0) was used as mobile phase. The wavelength detection was at 320 nm. Flow rate was 1.0mL/min. The typical retention time for S-oxide and ranitidine were 1.95 in and 3.50 min appropriately. The concentrations of ranitidine and ranitidine S-oxide is in the range of 0.035-9.00 microgram and 0.005-0.320 microgram, appropriately and detector responses were linearly related to column concentrations. The recoveries of ranitidine from spiked formulations simulating injection, syrups and tablets ranged from 99.70% - 100.5% of the added amount.

Rafiqul et al (1990) have developed an accurate, sensitive and reproducible method for the determination of ranitidine HCl in pharmaceuticals. It was an isocratic HPLC elution method which requires about 10 minutes to be performed. The concentration of ranitidine was 98.19% in Zantac and 98.33% in Apo-ranitidine tablets, appropriately.

Das Gupta V enrooted the reverse-phase high-performance liquid chromatography to quantify the ranitidine in pharmaceutical dosage form. This method was accurate and precise with relative standard deviation percent of 1.5% based on five injections. There was no interference from the excipients and the extraction procedure for form 1 from tablets is very simple. Form 1 appears to be very susceptible to decomposition on the basic side and stable to heat on the acidic- side. It losses potency of 84.4% on 20 minutes boiling with Sodium hydroxide and a new peak was observed in the chromatogram. It lost 37.8% of potency on 20 minutes boiling with Hydrogen-peroxide at room temperature and two new peaks were observed in the chromatogram.

Mahulikar et al (2006) have enrooted a very simple, rapid and reproducible reverse-phase isocratic HPLC assay method for the determination of Ranitidine in solid dosage form. YMC C18, ODS-AM stainless steel column [150mm x 4.6 mm, particle size 5 microns] was used for the chromatographic separation. 50mM ammonium acetate in water maintaining pH at 5 and methyl-hydroxide mixture was used as the mobile phase. By using the PDA detector the detection was performed. The percentage recovery of ranitidine in the given tablets was in the range of 100.9% - 102%. The linearity was in the range of 100 to 500 microgram/mL with correlation coefficient greater than 0.99.


For HPLC method:

Ranitidine Hydrochloride, Procaine hydrochloride, TEA (Tri Ethyl Amine), Acetonitrile, sodium di-hydrogen phosphate and Methanol. All solid materials are obtained from Sigma-Aldrich Company. All the solvents are obtained from Fischer-Scientific Company.

For NMR method:

Ranitidine Hydrochloride (obtained from Sigma-Aldrich company) 2-chloroacetophenone (obtained from Sigma-Aldrich company) and DMSO-d6 (obtained from Fischer-scientific company).

2.6 Preparation of solutions:

2.6.1 Preparation of mobile phase: To prepare a mobile phase solution of reservoir "A"; 800mL of water was taken and in that Potassium di-hydrogen phosphate (5.6gms) was mixed gently until it totally dissolved in water, keep this in degasser. The powder was mixed well and solution will be clear without any particles. To this add Methanol (87mL), acetonitrile (50mL). The Tri-ethylamine (TEA) [1.7mL] was added which acts as buffer to maintain the pH 7.0. The TEA was added to the Acetonitrile instead of adding directly to the water solution. Reservoir "B" contains only methanol.

2.6.2 Preparation of standard solution of Ranitidine Hydrochloride:

The 0.1gm of Ranitidine hydrochloride was dissolved in methanol and make up to the volume of 100mL using methanol, to prepare a stock solution. Using these stock solutions the dilutions are made up to 50, 75, 100, 125, 150, 175 and 200 microgram/mL for the calibration.

2.6.3 Preparation of Internal Standard solution of Procaine Hydrochloride:

The500mg of Procaine Hydrochloride was dissolved in Methanol up to the volume of 100mL. Dilutions are made up to 100mL, 150mL and 200mL with mobile phase. The amount of solution was mixed with standard solution of ranitidine hydrochloride to get calibration graph. The dilutions were prepared to obtain good resolution peaks which were well separated. The 20microL of the above solution was injected into the injection valve using syringe to obtain the results.

2.6.4 Preparation of Sample Solution:

6mg of ranitidine hydrochloride [each tablet contains 75 mg of the ranitidine hydrochloride] tablets was triturated and made in to a fine powder with the help of mortar and pestle and then it was weighed and taken into the volumetric flask. To this suitable amount of internal standard stock solution and methanol were added and the mixture was sonicated with occasional stirring. Make up the volume with same solvent. The solution was filtered through the 0.45micrometer membrane filter.

2.6.5 Preparation of NMR solution:

Ranitidine hydrochloride tablets were weighed, powdered and mixed. A portion of 90-110mg of it is weighed and transferred to a glass-stoppered tube. 30-50mg of 2-chloroacetophenone was weighed and 1.5-3ml of DMSO- d6 was added to the glass- stoppered tube. This solution was well mixed by means of vortex mixer and it is centrifuged. About 0.5ml of the supernatant solution was transferred to a PMR tube. In the NMR spectrometer the PMR tube was placed and the spectrum was recorded.

2.7 Aims of this project:

The main aim of this project is the development of ranitidine hydrochloride by using HPLC and NMR instruments. In this analysis by using HPLC instrument, tried to obtain well separated and resolved peaks by changing the flow rate of the mobile phase and making dilutions of the stock solution of ranitidine hydrochloride. By using NMR the resonance signals of the ranitidine hydrochloride are known.