190

CHAPTER-VII

FAMOTIDINE AND IBUPROFEN

191

7.1 INTRODUCTION

7.1.1. Famotidine

Famotidine is a histamine H2-receptor antagonist used to treat ulcers

(sores on the lining of the stomach or small intestine); gastroesophageal

reflux disease (GERD, a condition in which backward flow of acid from the

stomach causes heartburn and injury of the esophagus [tube that connects

the mouth and stomach]); and conditions where the stomach produces too

much acid, such as Zollinger-Ellison syndrome (tumors in the pancreas or

small intestine that cause increased production of stomach acid). Over-the-

counter famotidine is used to prevent and treat heartburn due to acid

indigestion and sour stomach caused by eating or drinking certain foods or

drinks. Famotidine is in a class of medications called H2 blockers. It works by

decreasing the amount of acid made in the stomach. Unlike cimetidine, the

first H2 antagonist, famotidine has no effect on the cytochrome P450 enzyme

system, and does not appear to interact with other drugs.[1] Famotidine was

developed by Yamanouchi Pharmaceutical Co.[2] It was licensed in the mid-80s

by Merck & Co.[3] and is marketed by a joint venture between Merck and

Johnson & Johnson.

Figure: 7.A. Structure of Famotidine

Systematic (IUPAC) name : 3-([2-(diaminomethyleneamino)thiazol- 4- yl]methylthio)- N'- sulfamoylpropanimidamide Formula : C8H15N7O2S3

Mol. mass : 337.5

192

Routes : Oral, IV

Famotidine used in combination with antacids promotes

local delivery of these drugs to the receptor of the parietal cell wall.

Therefore, researchers are developing innovative formulations of tablets,

such as gastroretentive drug delivery systems. Such tablets are retained in the

stomach for a longer period of time and thereby improve the bioavailability of

drugs. Local delivery also increases bioavailability at the stomach wall

receptor site and increases the efficacy of drugs to reduce acid secretion[4].

Famotidine is given to surgery patients before operations to prevent

postoperative nausea and to reduce the risk of aspiration pneumonitis.

Famotidine is also given to some patients who take NSAIDs, to prevent peptic

ulcers.[5] It serves as an alternative to proton-pump inhibitors.[6] It is also given

to dogs with acid reflux. Famotidine has also been used in combination with

an H1 antagonist to treat and prevent urticaria caused by an acute allergic

reaction.[7] It has been found to decrease the debilitating effects of chronic

heart failure by blocking histamine[8].

Side effects are associated with famotidine use. In clinical trials, the

most common adverse effects were headache, dizziness, and constipation or

diarrhea[9]. Antacid preparations such as famotidine, by suppressing acid-

mediated breakdown of proteins, lead to an elevated risk of developing food

or drug allergies. This happens due to undigested proteins then passing into

the gastrointestinal tract where sensitization occurs. It is unclear whether this

risk occurs with only long-term use or with short-term use as well [10].

7.1.2. Ibuprofen

Ibuprofen is a non-steroidal analgestic, antipyretic and anti

inflammatory agent[11]. It was derived from propanoic acid by the research

193

arm of Boots Group during the 1960[12] and patented in 1961. Originally

marketed as brufen, ibuprofen is available under a variety of popular

trademarks, including motrin, nurofen, advil, and nuprin.[13] It reduces

inflammation ,fever and relieves pain . It is equivalent to aspirin in its anti

inflammatory effects. Ibuprofen is used primarily for fever, pain,

dysmenorrhea and inflammatory diseases such as rheumatoid arthritis[8]. It is

also used for pericarditis and patent ductus arteriosus[14].

Ibuprofen has an antiplatelet effect, though relatively mild and

somewhat short-lived compared with aspirin or prescription antiplatelet

drugs. In general, ibuprofen also acts as a vasoconstrictor. Ibuprofen is a 'core'

medicine in the World Health Organization's Model List of Essential Medicines

necessary to meet the minimum medical needs of a basic healthcare

system[15-18].

Fig : 7.B.Structure of Ibuprofen

IUPAC name : (RS)-2-(4-(2-methylpropyl)phenyl)propanoic acid

Molecular formula : C13H18O2

Molecular mass : 206.3

Route : Oral, rectal, topical, and intravenous

The exact mechanism of action of ibuprofen is unknown. Ibuprofen is a

non-selective inhibitor of cyclooxygenase, an enzyme invovled in

194

prostaglandin synthesis via the arachidonic acid pathway. Its pharmacological

effects are believed to be due to inhibition cylooxygenase-2 (COX-2) which

decreases the synthesis of prostaglandins involved in mediating inflammation,

pain, fever and swelling. Antipyretic effects may be due to action on the

hypothalamus, resulting in an increased peripheral blood flow, vasodilation,

and subsequent heat dissipation. Inhibition of COX-1 is thought to cause some

of the side effects of ibuprofen including GI ulceration. Ibuprofen is

administered as a racemic mixture. The R-enantiomer undergoes extensive

interconversion to the S-enantiomer in vivo. The S-enantiomer is believed to

be the more pharmacologically active enantiomer.

Side effects of Ibuprofen include peripheral edema and fluid retention.

Use caution in patients with congestive heart failure or severe uncontrolled

hypertension. It may cause dyspepsia, heartburn, nausea, vomiting, anorexia,

diarrhea, constipation, stomatitis, flatulence, bloating, epigastric pain, and

abdominal pain. Peptic ulcer and GI bleeding have been reported. It may also

cause dizziness, headache and nervousness. Acute renal failure accompanied

by acute tubular necrosis has been reported.

The list of available brand names, label claim details and manufacturer

company names of famotidine, ibuprofen are shown in the Table 7.1

Table: 7.1: Formulations of famotidine, ibuprofen

S.NO Brand

Name Available form Label claim Manufacturer

Famotidine formulations

1 Faltidin Tab 20mg Alkem

195

2 Famocid Tab 40mg Sun

3 Famodin Tab 20mg cadila

4 Famopril Tab 20mg Zydus

Ibuprofen formulations

I Ibugin Tab 200mg and

400mg Gsk

2 Ibugesic Fc Tab 200,400

and600mg Cipla

3 Brufen Fc Tab 200,400

and600mg Abbott

4 Ibuspan Tab 800 mg Emcure

5 Ibusynth Tab 200 mg Astra Zeneca

6 Inflapen Tab 300 mg Gsk

7.2. REVIEW OF LITERATURE

Traje stafilov et al [19] developed a rapid, specific and sensitive high-

performance liquid chromatographic method for the determination of

famotidine in human plasma. Famotidine and the internal standard were

chromatographically separated from plasma components using a Lichrocart

Lichrospher 60 RP selec B cartridge for solid-phase separation with a mobile

phase composed of 0.1 % (v/v) triethylamine in water (pH 3) and acetonitrile

(92:8, v/v). UV detection was set at 270 nm. The calibration curve was linear

in the concentration range of 10.0 – 350.0 ng mL -1. The method was

implemented to monitor the famotidine levels in patient samples.

N. Helali et al [20] developed a simple, sensitive, and rapid reversed-phase

high-performance liquid chromatographic method for determination of

196

famotidine (FMT) and its impurities in pharmaceutical formulations.

Separations were performed on a Supelcosil LC18 column with an isocratic

mobile phase—13:87 (v/v) acetonitrile–0.1 M dihydrogen phosphate buffer

containing 0.2% triethylamine (pH 3.0). The mobile phase flow rate was 1 mL

min−1 and the detection wavelength was 265 nm. Response was linearly

dependent on concentration between 1 and 80 μg mL−1 (regression

coefficient, R 2, from 0.9981 to 0.9999). RSD from determination of method

repeatability (intraday) and reproducibility (interday) were <2% (n=6). Lowest

detectable concentrations ranged from 0.08 to 0.14 μg mL−1. The proposed

liquid chromatographic method can be satisfactorily used for routine quality

control of famotidine in pharmaceutical formulations.

M. Vamsi Krishna et al [21] developed and validated an efficient reversed

phase high performance liquid chromatographic (RP-HPLC) method for

quantitation of famotidine and its process impurities which may coexist in

bulk drugs and in solid pharmaceutical dosage forms. The separation was

achieved on a C18 column (250 mm x 4.6 mm) using a mobile phase of

acetonitrile, methanol and 1-hexane sodium sulfonate. Flow rate was 1.5

ml/min. The photo diode array detector was operated at 266 nm. The method

was validated for specificity, linearity, precision, accuracy and limit of

quantification. The degree of linearity of the calibration curves, the percent

recoveries of famotidine and impurities, the limit of detection and

quantitation, for the HPLC method were determined. The method was found

to be simple, specific, precise, accurate and reproducible. The method was

applied for the quality control of commercial famotidine tablets to quantify

the drug and its related substances and to check the formulation content

uniformity.

197

C.Rambabu et al[22] developed a reverse phase liquid chromatographic

method (RP-HPLC) for the estimate the amount of famotidine in bulk and its

pharmaceutical formulations. Waters- Alliance HPLC system equipped with

auto sampler, ultra-violet detector and symmetry C8 (4.6mm ID x 150mm, 3.5

µm, Make: XTerra) column were used for the quantification of the drug.

Separation was carried out by using potassium dihydrogen phosphate buffer

of pH=7.0 and acetonitrile in the ratio 40:60 (v/v) as mobile phase at a flow

rate of 0.5mL/min. and the detection was carried out at a wavelength of 297

nm. The retention time, tailing factor and USP theoretical plates of famotidine

were found to be 3.338min., 1.3 and 2273.9 respectively. The area of the peak

was proportional to the concentration of the drug in the range 20-60 µg/mL

of famotidine. The values of LOD and LOQ for famotidine were found to be

0.019 and 0.06µg/ml respectively. The mean recovery of the substance was

found to be 99.8%. The developed method was found to be simple,

repeatable and reproducible and hence it can be used as an alternative

method in any pharmaceutical industries in the assay of famotidine.

Khoddam A et al [23] developed a rapid and sensitive HPLC method using a

monolithic column for quantification of famotidine in plasma. The assay

enables the measurement of famotidine for therapeutic drug monitoring with

a minimum detectable limit of 5 ng/ml. The method involves simple, one-step

extraction procedure and analytical recovery was complete. The separation

was carried out in reversed-phase conditions using a chromolith performance

(RP-18e, 100 mm x 4.6 mm) column with an isocratic mobile phase consisting

of 0.03 M disodium hydrogen phosphate buffer-acetonitrile (93:7, v/v)

adjusted to pH 6.5. The wavelength was set at 267 nm. The calibration curve

198

as linear over the concentration range 20-400 ng/ml. The coefficients of

variation for inter day and intra-day assay were found to be less than 8%.

Narendra Nyola et al [24] developed a new simple, accurate, precise and

reproducible RP-HPLC method for the simultaneous estimation of ibuprofen

and famotidine in tablet dosage forms using C18 column (Phenomenex, 250 x

4.6 mm, 5 μm) in isocratic mode. The mobile phase consisted of methanol:

water: phosphate buffer in the ratio of 70:20:10 (v/v/v). The flow rate was 1.0

ml/min and detection wavelength was carried out at 284 nm. The retention

times of ibuprofen and famotidine were 3.6 min and 7.8 min, respectively.

The method was linear over the concentration range for ibuprofen 2-10 μg/ml

and for and famotidine 2-10 μg/ml. The recoveries of ibuprofen and

famotidine were found to be in the range of 99.037-100.766% and 99.703-

100.433% respectively. The validation of method was carried out utilizing ICH

guidelines. The described HPLC method was successfully employed for the

analysis of pharmaceutical formulations containing combined dosage form.

Dimal A. Shah et al [25] developed an isocratic reversed phase liquid

chromatograpic assay method for the quantitative determination of ibuprofen

and famotidine in combined dosage form. A Brownlee C18 column with

mobile phase containing water: methanol: acetonitrile (30:60:10, v/v/v) was

used. The flow rate was 1.0 mL/min and effluents were monitored at 264 nm.

The retention times of ibuprofen and famotidine were 4.9 min and 6.8 min,

respectively. The linearity for ibuprofen and famotidine were in the range of 2

- 20 μg/ ml and 0.1 - 10 μg/ ml, respectively. The proposed method was

validated with respect to linearity, accuracy, precision, specificity and

robustness. The method was successfully applied to the estimation of

ibuprofen and famotidine in combined dosage form.

199

Mohit K. Joshi et al [26] described a simple reverse phase HPLC method for

the determination of Ibuprofen and famotidine in synthetic mixture. The

determination was carried out on a phenomenex C18 column (250 mm x 4.6

mm i.d., 5 µm particle size) column using a mobile phase consisting of

acetonitrile : buffer(KH2PO4) (75 : 25 , v/v) having pH 3.0 using 0.2%

triethylamine at a flow rate and run time of 1.0 ml/min and 7 min

respectively. The eluent was monitored at 222nm. The method was

reproducible, with good resolution between ibuprofen and famotidine. The

detector response was found to be linear in the concentration range of 120-

600 μg/ml and 4-20 μg/ml for ibuprofen and famotidine. The methods were

validated as per ICH guidelines (Q2R1).

Snezana s. Mitic et al [27] developed a new kinetic spectrophotometric

method for the determination of ibuprofen in pharmaceutical formulations.

Ibuprofen was determined in an acidic ethanolic medium by monitoring the

rate of appearance of 1-nitroso-2-naphthol, resulting from the displacement

by ibuprofen of Co(III) from the tris(1-nitroso-2-naptholato)cobalt(III)

complex. The optimum operating conditions regarding reagent concentrations

and temperature were established. The tangent method was adopted for

constructing the calibration curve, which was found to be linear over the

concentration range 0.21–1.44 and 1.44–2.06 μg/ml. The optimized

conditions yielded a theoretical detection limit of 0.03 μg /ml based on the

3.3 S0 criterion. The interference effects of the usual excipients of powdery

drugs, foreign ions and amino acids on the reaction rate were studied in order

to assess the selectivity of the method. The developed procedure was

successfully applied for the rapid determination of ibuprofen in commercial

pharmaceutical formulations and human control serum. The unique features

200

of this procedure are that the determination can be performed at room

temperature and the analysis time is short. The newly developed method is

simple, inexpensive and efficient for use in the analysis of a large number of

samples.

Prasanna Reddy Battu et al [28] developed and validated a simple, selective,

accurate High Performance Liquid Chromatographic (HPLC) method for the

analysis of paracetamol and ibuprofen. Chromatographic separation achieved

isocratically on a C18 column [Use Inertsil C18, 5m , 150 mm x 4.6 mm]

utilizing a mobile phase of acetonitrile/phosphate buffer (60:40, v/v, pH 7.0)

at a flow rate of 0.8 ml/min with UV detection at 260nm. Aceclofenac was

used as an internal standard. The retention time of ibuprofen, paracetamol

and aceclofenac was 2.48, 4.45 and 6.34 min respectively. The developed

method was validated in terms of accuracy, precision, linearity, limit of

detection, limit of quantitation.This study aimed at developing and validating

an HPLC method, being simple, accurate and selective, and the proposed

method can be used for the estimation of these drugs in combined dosage

forms.

7.3. EXPERIMENTAL

7.3.1. Instrumentation:

Chromatographic separation was performed on a PEAK

chromatographic system equipped with LC-P7000 isocratic pump; rheodyne

injector with 20μl fixed volume loop, variable wavelength programmable UV

detector UV7000 and the output signal was monitored and integrated by

PEAK Chromatographic Software version 1.06. Teccomp UV-2301 double

beam UV-Visible spectrophotometer was used to carry out spectral analysis

201

and the data was recorded by Hitachi software. Sonicator (1.5L) ultrasonicator

was used to sonicating the mobile phase and samples. Standard and sample

drugs were weighed by using Denver electronic analytical balance (SI-234) and

pH of the mobile phase was adjusted by using Systronics digital pH meter.

7.3.2. Chemicals and Solvents:

The drug samples, famotidine and ibuprofen working standards were

obtained as gift sample by Ranbaxy Pvt. Ltd, Hyderabad, AP, India. The

pharmaceutical formulation was procured from local market. methanol,

acetonitrile and water used were HPLC grade and were purchased from

Merck Specialties Private Limited, Mumbai, India. Orthophosphoric acid and

remaining buffer solutions used were AR Grade and purchased from Merck

Specialties Private Limited, Mumbai, India.

7.3.3. Preparation of standard stock solution:

Standard stock solution of famotidine and ibuprofen pure drug

(1mg/ml) was prepared by accurately weighing about 100 mg of each drug in

100 ml volumetric flasks separately. Then the drugs were dissolved with 25ml

of methanol, and sonicated to dissolve it completely and made up to the mark

with the same solvent. The contents were mixed well and filtered through

Ultipor N66 Nylon 6, 6 membrane sample filter paper. Appropriate volumes of

these solutions were further diluted with mobile phase to get at

concentrations of 50-100µg/ml separately. Equal quantities of the two drug

solutions were mixed and the resultant solution was used for simultaneous

analysis.

202

7.3.4. Preparation of sample solution:

Famotidine and ibuprofen (DUEXIS: 26.6 mg of famotidine and 8000mg of

ibuprofen) tablets were purchased from local pharmacy. Ten tablets were

weighed and average weighed calculated. Then they were ground to a fine,

uniform size powder. Tablet contains equal amount of the both drugs

famotidine and ibuprofen. An amount of drug equivalent to 10 mg of

famotidine and ibuprofen was accurately weighed and quantitatively

transferred into 100ml volumetric flask. Approximately 30 ml methanol was

added and the solution was sonicated for 15 min. The flask was made up to

volume with mobile phase, and mixed well. Then the solution was filtered

through 0.45µm nylon 6, 6 membrane filter paper. The solution results

100µg/ml of both famotidine and ibuprofen drugs. Then an amount of the

solution was diluted to a concentration of 70µg/ml of famotidine and

ibuprofen.

7.4. METHOD DEVELOPMENT

For developing the method, a systematic study of the effect of various

factors was under taken by varying one parameter at a time and keeping all

other conditions constant. Method development consists of selecting the

appropriate wave length and choice of stationary and mobile phases. The

following studies were conducted for this purpose.

7.4.1. Detection wavelength:

The spectrum of diluted solutions of the famotidine and ibuprofen in

methanol was recorded. The absorption spectrum of famotidine and

ibuprofen obtained by scanning the sample separately on spectrophotometer

in UV region (200-400nm) in spectrum mode showed that the drug has

203

maximum absorbance at 278nm. Analysis was carried out by adjusting the UV

detector of the HPLC system at 243nm.

7.4.2. Choice of stationary phase:

Preliminary development trials were performed with octadecyl

columns with different types, configurations and from different

manufacturers. Finally the expected separation and shapes of peak was

succeeded with analytical column Inertsil ODS C-18 column with 250 x 4.6mm

internal diameter and 5µm particle size.

7.4.3. Selection of the mobile phase:

Several systematic trials were performed to optimize the mobile phase.

Different solvents like methanol, water and acetonitrile in different ratios and

different PH values of the mobile phase ratios by using different buffer

solutions in order to get sharp peak and base line separation of the

components and without interference of the excipients. Satisfactory peak

symmetry, resolved and free from tailing was obtained in mobile phase water:

ACN: TEA 80:10:10 (v/v) in isocratic condition.

7.4.4. Selection of the mobile phase flow rate

Flow rates of the mobile phase were changed from 0.5 – 1.2 ml/min for

optimum separation. A minimum flow rate as well as minimum run time gives

the maximum saving on the usage of solvents. It was found from the

experiments that 1ml/min flow rate was ideal for the successful elution of the

analyte.

7.4.5. Optimized chromatographic conditions:

After completion of several systematic trials to optimize the

chromatographic conditions, a sensitive, precise and accurate RP-HPLC

204

method was developed for the analysis of famotidine and ibuprofen in

pharmaceutical dosage forms. The optimized chromatographic conditions

were shown in table 7.2. The chromatograms of blank, standard, single and

formulation were shown in figure 7.B, 7.C, 7.D, 7.E and 7.F

Table 7.2: optimized chromatographic conditions of famotidine and ibuprofen

Standard Concentration 70µg/ml

Pump mode Isocratic

Mobile phase Water: ACN: TEA 80:10:10 (v/v/v)

Mobile Phase PH 6.1

Wavelength 278nm

Column C18 column (250 X 4.6 mm, 5μ)

Column Temp Ambient

Diluent Methanol

Injector Rheodyne

Injection Volume 20μl

Flow rate 1 ml/min

Retention Time Famotidine 2.59

Ibuprofen 6.76

Run time 10min

Peak Area Famotidine 318644

Ibuprofen 346055

Theoretical plates Famotidine 7863

Ibuprofen 45105

Tailing Factor Famotidine 1.79

Ibuprofen 1.55

205

Pump Pressure 9.4psi

Figure7.B: Chromatogram of Blank

206

Figure 7.C: Chromatogram of Standard

Figure 7.D: Chromatogram of famotidine single

207

Figure 7.E: Chromatogram of ibuprofen single

Figure 7.F: Chromatogram of Formulation

208

7.5. VALIDATION OF THE PROPOSED METHOD

The proposed method was validated as per ICH guidelines. The

parameters studied for validation were specificity, linearity, precision,

accuracy, robustness, and system suitability, limit of detection and limit of

quantification.

7.5.1. Specificity:

The selectivity of an analytical method is its ability to measure

accurately and specifically the analyte of interest in the presence of

components that may be expected to be present in the sample matrix. If an

analytical procedure is able to separate and resolve the various components

of a mixture and detect the analyte qualitatively the method is called

selective. It was observed that there were no peaks of diluents and placebo at

main peaks. This confirms that the chromatographic system used for the

simultaneous estimation of famotidine and ibuprofen was selective and

specific. Specificity studies indicating that the excipients did not interfere with

the analysis. The standard solution showed symmetric peak with retention

times of 2.59 minutes for famotidine and 6.76minutes for ibuprofen. There

was no interference of excipients in the chromatogram. This indicates that the

proposed method is specific.

Table 7.3: Result of Specificity analysis

Name of the solution Retention Time in Min

Blank No peaks

Famotidin 2.59minutes

Ibuprofen 6.76minutes

209

7.5.2. System suitability:

System suitability tests were carried out on freshly prepared standard

stock solution of famotidine and ibuprofen. Equal volume of standard

concentration was mixed well. From the prepared solution 20μl of the sample

was injected into HPLC system and the results obtained were used to express

the system suitability of the developed method. System suitability results

were shown in Table 7.4.

Table 7.4: System suitability results

Retention Time Famotidine 2.59

Ibuprofen 6.76

Peak Area Famotidine 318644

Ibuprofen 346055

Theoretical plates Famotidine 7863

Ibuprofen 45105

Tailing Factor Famotidine 1.79

Ibuprofen 1.55

Resolution Factor Ezetimibe ……….

Simvastatin 18.52

7.5.3. Linearity

Linearity of the method was determined by mean of calibration graph

using an increasing amount of each analyst. Linearity was evaluated by visual

inspection of a calibration graph. Atleast three concentration levels were

tested in agreement to ICH. The slope, intercept was reported as required by

ICH. Under the experimental conditions, linear calibration curves for the two

drugs were obtained throughout the concentration ranges studied.

210

Regression analysis was done on the peak areas of the two drugs (Y) v/s

concentration (X). The linear ranges of famotidine and ibuprofen are 50-

100μg/ml with correlation coefficient (r2) 0.999 for both famotidine and

ibuprofen, regression equation of Y=5250X+1226 for famotidine, Y=

4987X+6341 for ibuprofen as shown in Fig 7.E. Results were shown in

Table7.5.

Table 7.5: Linearity results of famotidine and ibuprofen

S.no Conc µg/ml Area of

famotidine

Area of

ibuprofen

1 50 217240 228092

2 60 258879 294813

3 70 318644 346055

4 80 368791 389451

5 90 414797 442934

6 100 478024 498697

Concentration range

Slope (m)

Intercept (b)

Correlation coefficient

50-100µg/ml

1226

5250

0.999

50-100µg/ml

6341.4

4987.622

0.999

211

Figure 7.E: Linearity graph for famotidine and ibuprofen

7.5.4. Accuracy:

To ensure the accuracy of the analytical method, the recovery studies

were carried out. Known amount of famotidine and ibuprofen was added to a

pre quantified sample solution of its dosage form and the amounts of

famotidine and ibuprofen were estimated by measuring the peak area ratios

and by fitting these values to the straight line equation of calibration curve.

The recovery studies were carried out three times over the specified

concentration range of famotidine and ibuprofen. Accuracy was evaluated at

three different concentrations equivalent to 50%, 75% and 100% of the active

ingredient by calculating the recovery of famotidine and ibuprofen with

%RSD. The standard addition method was performed at 50%, 75% and 100%

levels of standard 70µg/ml. The solutions were analyzed in triplicate at each

level as per the proposed method. The percent recovery and % RSD was

calculated and results are presented in Table 7.6 Satisfactory recoveries

ranging from 99.2 to100.5%for ibuprofen and 99.4 to 101.2 for famotidine

-100000

0

100000

200000

300000

400000

500000

600000

0 20 40 60 80 100 120

Pe

ak a

rea

Concentration

212

were obtained by the proposed method. The values of recovery justify the

accuracy of the method. The percentage recovery values were obtained

within the standard limit which confirms that the method is accurate and free

from any positive or negative interference of the excipients. This indicates

that the proposed method was accurate.

Table 7.6: Recovery results for famotidine and ibuprofen

Recovery

Conc. of

sample

µg/ml

Famotidine

Recovery

Ibuprofen

Recovery

Famotidine

% of

recovery

Ibuprofen

% of

recovery

50 49.91 49.86 99.82 99.7

50% 50 50.03 49.73 100.6 99.46

50 49.9 50.12 99.8 100.2

75 75.09 74.95 100.12 99.9

75% 75 74.93 75.07 99.9 100.09

75 74.9 74.92 99.94 99.8

100 100.5 100.9 100.5 100.9

100 % 100 100.6 100.8 100.6 100.8

100 100.3 101.2 100.3 101.2

7.5.5. Precision:

Repeatability was studied by determination of intra-day and inter day

precision. Intra-day precision was determined by injecting five standard

solutions of three different concentrations on the same day and inter-day

precision was determined by injecting the same solutions for three

consecutive days. Relative standard deviation (RSD%) of the peak area was

then calculated to represent precision.

213

7.5.5.A. Intra-day precision

To study the intra-day precision, six replicate standard solutions

(70µg/ml) of famotidine and ibuprofen were injected. The percent relative

standard deviation (% RSD) was calculated and it was found to be 0.36 for

famotidine and 0.98 for ibuprofen, which are well within the acceptable

criteria of not more than 2.0. It was confirmed that the analytical technique

showed good repeatability. Results of system precision studies are shown in

Table 7.7.

7.5.5.B. Inter day precision:

To study the inter day precision, six replicate standard solutions

(70µg/ml) of famotidine and ibuprofen was injected on three consecutive

days. The percent relative standard deviation (% RSD) was calculated and it

was found to be 1.23 for famotidine and 0.4 for ibuprofen, which are well

within the acceptable criteria of not more than 2.0. It was confirmed that the

analytical technique showed good repeatability. Results of system precision

studies are shown in Table 7.8.

Table7.7: Intra-day precision results of famotidine and ibuprofen

Conc. Injection No. Famotidine peak

area response

Ibuprofen peak

area response

70µg/ml

1 318644 346055

2 317542 345223

3 318602 345647

4 316493 353973

5 316493 345891

6 316059 345547

RSD 0.36 0.98

214

Table7.8: Inter day precision results of famotidine and Ibuprofen

Conc. Injection No.

Famotidine

peak area

response

Ibuprofen peak area

response

70µg/ml

1 318858 345711

2 316421 346019

3 315686 347584

4 326115 348330

5 326577 347938

6 315424 346019

RSD 1.6 0.32

7.5.6. Robustness:

Premeditate variations were performed in the experimental conditions

of the proposed method to assess the method robustness. For this intention,

minor changes were made in mobile phase composition, flow rate and pH of

buffer solution. The effect of these changes on chromatographic parameters

such as retention time, tailing factor and number of theoretical plates was

then measured. The typical variations are given below: Variation in flow rate

by ±0.1ml/min. Variation in wavelength by ±2nm. Famotidine and ibuprofen

at 70µg/ml concentration were analyzed under these changed experimental

conditions. Percentage change in the results was calculated and was found to

be within the acceptance criteria of below 2. This indicates that the proposed

method is valid. Results were shown in Table 7.9

215

Table 7.9: Robustness results of famotidine and ibuprofen

7.5.7. Ruggedness:

Ruggedness of the method intermediate precision was estimated by

preparing six dilutions of the test sample as per the proposed method and

each dilution injected in duplicate using different columns and different

analyst on different days. 70µg/ml of the sample was injected. The percent

relative standard deviation (% RSD) was calculated and it was found to be 0.69

for famotidine and 0.73 for ibuprofen, which are well within the acceptable

criteria of not more than 2.0. It was concluded that the analytical technique

showed good repeatability. Results of system precision studies are shown in

Table 7.10.

Table7.10: Ruggedness results of famotidine and ibuprofen

Conc. Injection No.

Famotidine

peak area

response

Ibuprofen peak area

response

70µg/ml 1 316587 343259

2 314856 341447

Parameter Modification Famotidine

Area Ibuprofen

Area

Famotidine % of

change

Ibuprofen % of

change

Standard ………… 318644 346055 ……….. ………

MP Water:ACN:TEA

75:15:10 318946 347118 0.09 0.30

PH 5.7 319394 347316 0.23 0.36

Wavelength 284nm 320023 349194 0.43 0.91

216

3 316629 348561

4 312930 345590

5 319520 344009

6 316008 342669

RSD 0.69 0.73

7.5.8. Limit of Detection and Limit of Quantification:

Limit of quantitation and limit of detection values were determined by

the signal-to-noise (S/N) approach. To investigate the limit of quantitation and

limit of detection solutions of different concentrations were prepared by

spiking know amounts of famotidine and ibuprofen into excipients. Each

solution was prepared according to the procedure and analysed repeatedly to

determine the S/N ratio. The average S/N ratio from all the analyses at each

concentration level was used to calculate the limit of quantitation and limit of

detection. The concentration level that gives an S/N ratio of about 10:1 at

which analytes can be readily quantified with accuracy and precision was

reported as the limit of quantitation. The concentration level that gives an S/N

ratio of about 3:1 at which analytes can be readily detected was reported as

the limit of detection.

7.5.8.A. Limit of Detection:

Limit of detection is the concentration of the analyte that give signal-

to-noise (S/N) ratio of 3:1 at which analyte can be readily detected.

Determination of the signal-to-noise ratio is performed by comparing

measured signals from samples with known low concentrations of analyte

with those of blank samples and establishing the minimum concentration at

217

which the analyte can be reliably detected. A signal-to-noise ratio 2:1 is

generally considered acceptable for estimating the detection limit. LOD is

found to be 0.3µg/ml for famotidine and 0.5µg/ml for Ibuprofen.

7.5.8.B. Quantitation Limit:

Limit of quantitation is the concentration of the analyte that give a

signal-to-noise (S/N) ratio of 10:1 at which analyte can be readily quantified

with accuracy and precision. The quantitation limit is generally determined by

the analysis of samples with known concentrations of analyte and by

establishing the minimum level at which the analyte can be quantified with

acceptable accuracy and precision. LOQ is found to be 1µg/ml for famotidine

and 1.5µg/ml for ibuprofen.

7.5.9. Formulation:

From the prepared formulation solution, 20μl of sample solution was

injected into HPLC system and peak area of the detector response was used

to calculate % assay in the prepared solution. The % assay was found to be

99.83% for famotidine and 98.9% for ibuprofen. Results were shown in Table

7.11.

Table 7.11: Formulation results of famotidine and ibuprofen

S.NO

Drug

Formulations

Dosage

Sample

conc

Amount

found

% of Drug

Estimated

in Tablet

1

Famotidine Duexis

26.6mg

52µg/ml

51.86

µg/ml

99.73%

2 Ibuprofen 800mg

80µg/ml

79.12

µg/ml

98.9%

218

7.6. DISCUSSION ON THE RESULTS

A simple, specific, accurate, precise and sensitive reverse phase High

Performance Liquid Chromatographic method has been developed for the

quantification of famotidine and ibuprofen. Wavelength of the two drugs that

absorb maximum light was confirmed by using spectrophotometer. Selections

of stationary and mobile phases were selected by random change in the

different ratios of mobile phases and different stationary phases that separate

the drugs with high resolution and high theoretical plates and less tailing

factor. Finally it was succeeded at ODS C18 column with a mobile phase ratio

of water: acetonitrile : triethyl amine 80:10:10(v/v/v), detection wavelength

278 nm was found to be most suitable conditions for the simultaneous

analysis of famotidine and ibuprofen. The optimized chromatographic

conditions were shown in Table 7.2.

The linear regression response was obtained for a series of

concentration in the range of 50-100µg/ml and it was found to be linear. The

calibration curve equation was found to be Y= 5250X+ 1226 for famotidine, Y=

4987 X+6341 for ibuprofen with correlation coefficient (r2) value 0.999 for

both famotidine and ibuprofen. Both the drugs show best response on the

regression equation with less intercept and high correlation coefficient. The

data of regression analysis of the calibration curves are shown in Table 7.5.

Selectivity and specificity were studied for the examination of various

excipients generally present in the tablet dosage form of famotidine and

ibuprofen. The results indicated that they did not interfere in the assay.

A suitability test was applied to representative chromatograms for

various parameters. It was found that high number of theoretical plates for

both famotidine and ibuprofen was observed with less tailing factor and high

219

resolution. Both the compounds elute with in three min with high resolution

and short run time. The results obtained were within acceptable limits of

tailing factor ≤2.0 and theoretical plates >2000, resolution factor >2 (Table

7.3). Results indicate that the developed method having shortest run time

with high resolution. This confirms that the easy usage of the method, less

time for analysis. The proposed methods were validated as per the ICH

guidelines.

The precision was measured in terms of repeatability, which was

determined by sufficient number of aliquots of a homogenous sample with in

the day (intraday) and next consequent three days for inter day precision. For

each cases % RSD was calculated within the acceptable range of 2 in intraday

and interday precision for both famotidine and ibuprofen. This showed that

the precision of the methods were satisfactory.

To ensure the accuracy of the analytical method, the recovery studies

were carried out. Known amount of famotidine and ibuprofen was added to a

pre quantified sample solution of its dosage form and the amounts of

famotidine and ibuprofen were estimated by measuring the peak area ratios

and by fitting these values to the straight line equation of calibration curve.

The recovery studies were carried out three times over the specified

concentration range of famotidine and ibuprofen. Accuracy was evaluated at

three different concentrations equivalent to 50%, 75% and 100% of the active

ingredient by calculating the recovery of famotidine and ibuprofen with

%RSD. The percent recovery and % RSD was calculated and results are

presented in Table 7.6 Satisfactory recoveries ranging from 99.2 to100.5%for

ibuprofen and 99.4 to 101.2 for famotidine were obtained by the proposed

method. Recovery results were shown in Table 7.6.

220

The proposed method was validated by changing chromatographic

parameters and system suitability parameters were found to be with in

acceptable limits. The results indicate that the method was robust for all

variable. Hence the method sufficiently robust for normally expected

variations in chromatographic conditions. famotidine and ibuprofen at

70µg/ml concentration were analyzed under these changed experimental

conditions. Percentage change in the results was calculated and was found to

be within the acceptance criteria of below 2. Robustness results indicate that

there was no considerable change in the results with change in the developed

conditions. Hence the developed method is robust in nature. Robustness

results were shown in Table 7.9.

Ruggedness performed by using six replicate injections of standard

solution of concentrations which were prepared and analyzed by different

analyst on three different days over a period of one week. The percent

relative standard deviation (% RSD) was calculated and it was found to be 0.69

for famotidine and 0.73 for ibuprofen, which are well within the acceptable

criteria of not more than 2.0. It was concluded that the analytical technique

showed good repeatability. Ruggedness results were shown in Table 7.10.

A signal-to-noise ratio 2:1 is generally considered acceptable for

estimating the detection limit. LOD is found to be 0.3µg/ml for famotidine and

0.5µg/ml for ibuprofen and LOQ is found to be 1µg/ml for famotidine and

1.5µg/ml for ibuprofen.

The validated method was applied for the assay of commercial tablets

of famotidine and ibuprofen (duexis 26.6mg of famotidine and 800 mg of

ibuprofen). Peak area of the detector response was used to calculate % assay.

The % assay was found to be 99.73% for famotidine and 98.9% for ibuprofen.

221

Results were shown in Table 7.11. The results presented good agreement with

the labeled content.

Thus the method developed in the present investigation is simple,

sensitive, accurate, rugged, robust, rapid and precise. The absence of

additional peaks in the chromatogram indicated that there was no

interference of the common excipients used in the tablets. Hence, the above

said method can be successfully applied for the estimation of famotidine and

ibuprofen in tablet dosage forms.

7.6. REFERENCES

1. Humphries TJ, Merritt GJ (August 1999). "Review article: drug

interactions with agents used to treat acid-related diseases". Aliment.

Pharmacol. Ther. 13 Suppl 3: 18–26.

2. US patent 4283408, Hirata , et al., "Guanidinothiazole compounds,

process for preparation and gastric inhibiting compositions containing

them", issued 1981-08-11

3. "Sankyo Pharma". Skyscape Mediwire. 2002. Retrieved 2009-10-30.

4. "Formulation and Evaluation of Gastroretentive Floating Tablets of

Famotidine". Farmavita.Net. 2008. Retrieved 2009-01-30.

5. "Horizon Pharma, Inc. Announces FDA Approval of DUEXIS(R) for the

Relief of the Signs and Symptoms of Rheumatoid Arthritis and

Osteoarthritis is and to Decrease the Risk of Developing Upper

Gastrointestinal Ulcers" (Press release). Horizon Pharma. 25 April 2011.

Retrieved 26 April 2011.

6. http://www.medscape.com/viewarticle/705708 (membership

required)

222

7. TB Fogg; D Semple (29 November 2007). "Combination therapy with H2

and H1 antihistamines in acute, non compromising allergic reactions".

BestBets. Manchester, England: Manchester Royal Infirmary. Retrieved

26 April 2011.

8. http://www.rxpgnews.com/research/cardiology/congestive-heart-

failure/article_5010.shtml

9. "Pepcid Side Effects & Drug Interactions". RxList.com. 2008. Retrieved

2008-07-31.

10. Pali-Schöll I, Jensen-Jarolim E (April 2011). "Anti-acid medication as a

risk factor for food allergy". Allergy 66 (4): 469–77.

11. Van Esch, A; Van Steensel-Moll, HA; Steyerberg, EW; Offringa, M;

Habbema, JD; Derksen-Lubsen, G (1995). "Antipyretic efficacy of

ibuprofen and acetaminophen in children with febrile seizures".

Archives of pediatrics & adolescent medicine 149 (6): 632–7.

12. Adams, S. S. (1992). "The propionic acids: A personal perspective".

Journal of clinical pharmacology 32 (4): 317–323.

13. "PubMed Health - Ibuprofen". U.S. National Library of Medicine. 2010-

10-01. Retrieved 2011-01-20.

14. "Ibuprofen". The American Society of Health-System Pharmacists.

Retrieved 3 April 2011.

15. WHO Model List of Essential Medicines (16th ed.). World Health

Organization (WHO). March 2009. Retrieved 2011-03-30.

16. WHO Model List of Essential Medicines for Children (2nd ed.). World

Health Organization (WHO). March 2010. Retrieved 2011-03-30.

17. Stuart MC, Kouimtzi M, Hill SR, ed. (2009). WHO Model Formulary 2008

(2nd ed.). World Health Organization (WHO). Retrieved 2011-03-30.

223

18. WHO Model Formulary for Children 2010 (2nd ed.). World Health

Organization (WHO). 2010. Retrieved 2011-03-30.

19. Dragica zendelovska and traje stafilov, High-performance liquid

chromatographic determination of famotidine in human plasma using

solid-phase column extraction, J.Serb.Chem.Soc. (2003), 68(11)883–

892.

20. N. Helali, F. Darghouth, L. Monser, RP-HPLC Determination of

Famotidine and its Potential Impurities in Pharmaceuticals,

Chromatographia, October 2004, Volume 60, Issue 7-8, pp 455-460.

21. M.Vamsi Krishna, G. Madhavi, L. A. Rama Prasad and D. Gowri Sankar,

Impurity profiling of Famotidine in bulk drugs and pharmaceutical

formulations by RP-HPLC method using ion pairing agent, Der

Pharmacia Lettre, 2010, 2(3): 1-11.

22. T.Vijaya Bhaskara Reddy, G.Ramu , P.V.Lakshmana Rao and

C.Rambabu, development and validation of stability indicating reverse

phase liquid chromatographic method for the assay of famotidine in

bulk and formulations, 2012, Vol. 5, No.2, 250-255.

23. Zarghi A, Shafaati A, Foroutan SM, Khoddam A, Development of a rapid

HPLC method for determination of famotidine in human plasma using a

monolithic column, Journal of pharmaceutical and biomedical

analysis 39:3-4 2005 Sep 15 pg 677-80.

24. Narendra Nyola and Govinda Samy Jeyabalan, Simultaneous Estimation

of Ibuprofen and Famotidine in Pure and Combination Dosage Form by

RP-HPLC, Journal of Applied Pharmaceutical Science 02 (05); 2012: 79-

83.

224

25. Dimal A. Shah, Dixita J. Suthar, Sunil L. Baldania, Usman K. Chhalotiya,

Kashyap K. Bhatt, development and validation of liquid

chromatographic method for estimation of ibuprofen and famotidine

in combined dosage form.

26. Mohit K. Joshi, Lalit Lata Jha, Rajesh K.S, development and validation of

rp-hplc method for the simultaneous estimation of ibuprofen and

famotidine, International Journal of Institutional Pharmacy and Life

Sciences, September-October 2012, 2(5).

27. Snezana s. Mitic1, gordana z. Miletic1, aleksandra n. Pavlovic,

quantitative analysis of ibuprofen in pharmaceuticals and human

control serum using kinetic spectrophotometry, j. Serb. Chem. Soc. 73

(8–9) 879–890 (2008).

28. Prasanna Reddy Battu, RP-HPLC Method for Simultaneous Estimation

of Paracetamol and Ibuprofen in Tablets, Asian J. Research Chem. 2(1):

Jan.-March, 2009.