190 CHAPTER-VII FAMOTIDINE AND IBUPROFEN
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
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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
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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
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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
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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.
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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
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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.
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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.
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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
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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
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Pump Pressure 9.4psi
Figure7.B: Chromatogram of Blank
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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
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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
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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.
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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
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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
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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.
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