Chromatographic Analysis of Famotidine, Paracetamol and Ibuprofen from Tablet Formulation

 

Wrushali A. Panchale*, Rajnandini Suroshe, Minakshi S. Rathod, Yuvraj L. Pandhare

MUP’s College of Pharmacy (B.Pharm), Degaon 444506, Dist. Washim, M.S., India

 

ABSTRACT:

Aim of the present work was to develop Reversed phase HPLC method for simultaneous estimation of famotidine (FAM), paracetamol (PCT) and ibuprofen (IBU)in bulk drug and tablet formulation. Separation was carried out on Nucleosil C-18 (15cm, 4.6 mm I.D × 250 mm) using a mobile phase methanol and water (o-phosphoric acid, 0.05%) (83:17, v/v) at a flow rate of 0.7 ml/min. The detection wavelength was set at 270 nm. Retention time of FAM, PCT and IBU were found to be 1.81, 2.58 and 8.58 min respectively. Linear relationship was found in the concentration range of 0.5-3 μg/ml for FAM (r² = 0.999), 5-25 μg/ml for PCT (r² = 0.999) and 5–30 μg/ml for IBU (r² = 0.999). The % purity was found to be 98.40±1.49, 99.86±0.27 and 100.31±0.76, for FAM, PCT and IBU, respectively. The proposed method was validated as per ICH Q2 (R1) guidelines. The mean % recoveries were found be 100.90±1.99, 99.96±0.97 and 100.51±1.72 for FAM, PCT and IBU, respectively. 

 

 

 

1. INTRODUCTION:

Famotidine (FAM), chemically N2- (aminosulfonyl)-3-[[[2[(diaminomethylene) amino] thiazol-4 yl] methyl] thio] propanamidine is a potent histamine H₂-receptor antagonist (Fig. 1).¹ It is commonly used in the treatment of gastric and duodenal ulcers.² It is official in IP, BP, and USP.^3-5 It works by decreasing the amount of acid produced by the stomach.

 

 

Fig. 1: Structure of famotidine

 

Ibuprofen (IBU), chemically α-Methyl–4-(2–methylpropyl) benzene acetic acid, is a non-steroidal anti-inflammatory drugs (Fig. 2).⁶

 

 

 

 

 

It is nonselective COX inhibitor, which converts arachidonic acid to prostaglandin H₂.⁷ Ibuprofen is a nonsteroidal anti-inflammatory drug (NSAID) that is used to treat pain, inflammation, swelling, stiffness, and joint pain.⁸

 

Fig. 2: Structure of ibuprofen

 

Paracetamol (PCT), chemically N-(4-hydroxyphenyl) acetamide is antipyretic drugs (Fig. 3). It is used to relieve mild to moderate pain. It is also useful for lowering a raised temperature (fever), such as during a cold or after childhood immunisations.⁹ It is also official in various pharmacopoeias such as IP, BP, and USP.^3-5

 

 

Fig. 3: Structure of paracetamol

 

Combination of IBU, FAM and PCT is available in the market, which is prescribed for relieving pain and reduces inflammation and fever. It is indicated for the relief of headache from Musculo-skeletal origin, Feverishness, Muscular, Menstrual and Dental pain.  

 

Literature survey revealed various methods have been reported for estimation of these drugs alone and in combination with other drugs.^9-13 In the literature, one HPTLC and one HPLC method was reported for simultaneous estimation of famotidine, paracetamol and ibuprofen.^14-15 Reported HPLC method validation studies which is essential to probe the chemical behavior of the molecule. Validation shows that the test performs according to specifications when executed for the first time using the personnel, equipment, and reagents available.

 

There are number of analytical methods for the determination of drugs from bulk and various formulations like tablets, capsules, injections, etc. These methods include Uv-spectrophotometry, HPLC, UPLC, Gas chromatography, etc.^16-27  In the present paper, attempts have been made to develop a simple, rapid and reproducible reversed phase HPLC method for the simultaneous estimation of these drugs from bulk drug and pharmaceutical formulation.

 

2. MATERIALS AND METHODS:

2.1. Reagents and Chemicals:

Standard drugs of famotidine, ibuprofen and paracetamol were kindly supplied as a gift sample by Winston Pharmaceuticals Ltd., Agra, India. All the solvents used in the study were of HPLC grade. Commercial tablet formulations containing all the drugs (IBU C®, Winston Pharma) were purchased from local market.

 

2.2. Instrumentation:

The apparatus used was an Agilent 1220 Infinity LC system coupled with a gradient mixer and degasser. The temperature of the column could be kept at any desired point between 15 and 50◦C using an oven. A Rheodyne injector (manual loading) with a 20 l external loop was used. The analyte was chromatographed on a Nucleosil C-18 (15cm, 4.6 mm I.D × 250 mm) column, and detection was performed with a UV detector.

 

2.3 Selection of solvent for preparing solution:

These studies were carried out with a view to find an ideal solvent in which the standard drug is completely soluble and stable for a period of 6 hrs. Various solvents were tried for checking solubility of each drug. It was found that all the drugs were freely soluble in methanol. Therefore after examining these parameters, methanol was selected as the suitable solvent for further studies.

 

2.4. Preparation of standard solutions:

2.4.1. Standard stock solution of IBU (1000 µg/ml) (Sol. A):

An accurately weighed quantity of about 50.0 mg of IBU was dissolved in methanol and the volume was made up to 50.0 ml with methanol. Working standard solution of IBU was prepared by diluting 0.1 ml stock solution with 10 ml mobile phase.

 

2.4.2. Standard stock solution of FAM (200 µg/ml) (Sol. B):

An accurately weighed quantity of about 10.0 mg of FAM was dissolved in methanol and the volume was made up to 50.0 ml with methanol. Working standard solution of FAM was prepared by diluting 0.025 ml stock solution with 10 ml mobile phase.

 

 2.4.3. Standard stock solution of PCT (200 µg/ml) (Sol. C):

An accurately weighed quantity of about 10.0 mg of PCT was dissolved in methanol and the volume was made up to 50.0 ml with methanol. Working standard solution of PCT was prepared by diluting 0.5 ml stock solution with mobile phase.

 

2.4.4. Mixed working standard solution:

An accurately weighed quantity of about 40.0 mg IBU, 2 mg FAM and 33 mg PCT was dissolved in methanol and the volume was made up to 100.0 ml with methanol. From the above solution, about 0.25 ml solution was diluted up to 10 mL with mobile phase.

 

2.5. Selection of wavelength:

Each solution of drug was scanned separately by using UV absorption spectrophotometer for spectrum in the range of 200-400 nm. The overlay spectrum of these drugs was taken using computer. After overlapping, wavelength 270 nm was selected for further analysis (Fig. 4).

 

2.6. METHODOLOGY:

Chromatographic conditions were selected on a trial and error basis by considering the physicochemical properties of drugs such as its pKa value, solubility in solvents, and nature (acidic or basic). As pKa value of IBU, FAM and PCT were 4, 7.06 and 7.0  and 9.5, respectively, the pH of water was made to 7.0 by adding 0.05% o-phosphoric acid to suppress the ionization of the acidic drug, resulting in a higher retention. This low pH also prevents the ionization of the column packing material silanols.

 

Chromatographic separations were achieved using a Nucleosil C-18 (15 cm, 4.6 mm I.D × 250 mm) column. The mobile phase consisting of methanol and water pH 7.0 (0.05% o-phosphate acid (OPA)) in the ratio 83:17, v/v  was passed through 0.45μ membrane filter and degassed by ultrasonication. The flow rate was maintained at 0.7 mL/min and the measurements were made at 270 nm. The column and HPLC system was kept at ambient temperature. A column equilibration time of 30 min between each run was maintained. The mixed working standard solution was analyzed using the optimized chromatographic conditions as mentioned in above table.

 

2.7. Assay of tablet formulation:

Twenty tablets were weighed and powdered. An accurately weighed quantity of tablet powder equivalent to about 400.0 mg of IBU, 20.0 mg of FAM and 325 mg PCT were transferred in to 100.0 ml volumetric flask. The contents were dissolved in 0.1N NaOH solution and sonicated for 20 minute. The final volume was made with same solvent. About 1.0 ml of above solution was transferred to 10.0 ml volumetric flask and final dilution was made with mobile phase. From this about 0.25 ml solution diluted up to 10 ml with mobile phase. The solution was filtered through whatman filter paper (0.45μ) and was analyzed for drug content. The resulting solution was analyzed for drug content. The drug content of each drug in sample solution was calculated from the regression equations of standard calibration graph (Fig. 5).

 

2.8. Validation of Method:

The method was validated according to the ICH guidelines.²⁸ Parameters studied for validation were accuracy, precision, linearity, LOD, LOQ and specificity for the analyte.

 

2.8.1. Accuracy of method:

Accuracy of the method was determined by performing recovery studies using standard addition method. Recovery study was performed by applying the method to preanalysed drug sample to which known amount of standard drug corresponding to 80%, 100% and 120% of label claim was added. At each level of the amount, six determinations were performed and the results obtained were compared with expected results.

 

2.8.2. Precision of method:

Precision of method was determined with respect to both repeatability and reproducibility. An amount of the pre-analyzed tablet powder equivalent to 100% of the label claim of each drug was accurately weighed and assayed. The repeatability study (inter day precision) was performed by analyzing homogeneous tablet sample of formulation. The intermediate precision study was performed by variation in days of analysis.

 

The repeatability of sample application and measurement of peak area for active compound were expressed in terms of % RSD (relative standard deviation). Method repeatability was obtained from RSD value by repeating the assay three times in same day for intra-day precision. Inter-day precision was assessed by the assay of three sample sets on different days (inter-day precision).

 

2.8.3. Linearity and range:

Aliquot of solution A (0.05 – 0.5 ml) was transferred separately in to six 10.0 ml volumetric flasks and diluted up to a mark with mobile phase to obtain final concentrations in the range of 5 - 50 µg/ml for IBU.

 

Aliquot of solution B (0.025 – 0.15 ml) was transferred separately in to six 10.0 ml volumetric flasks and diluted up to a mark with mobile phase to obtain final concentrations in the range of 0.5 - 3 µg/ml for FAM.

 

Aliquot of solution C (0.25 – 1.25 ml) was transferred separately in to five 10.0 ml volumetric flasks and diluted up to a mark with mobile phase to obtain final concentrations in the range of 5 - 25 µg/ml for PCT. All the solutions were analysed using optimized chromatographic conditions.

 

2.8.4. Limit of detection (LOD) and limit of quantitation (LOQ):

A signal-to-noise ratio between 3:1 and 10:1 is generally considered acceptable for estimating the limit of detection and limit of quantitation, respectively.²⁶ LOD and LOQ were experimentally verified by diluting known concentrations of drugs until the average responses were approximately 3 or 10 times the standard deviation of the responses for six replicate determinations.

 

3. RESULTS AND DISCUSSION:

In this work, attempts were made to develop a simple, rapid and reproducible reversed phase HPLC method for the simultaneous estimation of these drugs from bulk drug and pharmaceutical formulation. Solvent methanol was used to prepare solutions of standard drugs. From the overlay of UV spectrum of FAM, PCT and IBU the analytical wavelength 270 nm was selected (Fig. 4).

 

Fig. 4: Overlain UV spectra of IBU, FAM and PCT

 

The mobile phase selection for HPLC method development was initially started with trial and error method using different solvents. Initially methanol, water, and water in different ratios were tried. Combination of methanol and water had given better resolution but it lacks good peak symmetry. Hence, peak symmetry asymmetry was achived with the help of rendering pH of water to 7.0 with 0.05% o-phosphate acid and by varying flow rate of mobile phase. The presence of water in mobile phase acetonitrile resulted in excellent overall chromatography with appropriate peak symmetry and complete base line resolution. Based on results of trial and error method, methanol and water (0.05% OPA) (83:17, v/v) combination was selected for separation of drugs study, as all the drugs was eluted within a period of 10 minutes with sharp peak.

 

 

 

 

 

 

The following Chromatographic conditions were maintained for analysis of drug throughout the experimental work.

 

System	: The Agilent 1220 Infinity LC Gradient System VL
Column	: Thermo (4.6 mm I.D x 250 mm) C-18
Detector	: Variable wavelength detector
Pump	: Gradient with degasser
Mobile phase	: Methanol : Water (0.05% OPA) (83  : 17, v/v)
Detection wavelength	: 270 nm
Mode	: Isocratic
Sample size	: 20 µl
Flow rate	: 0.7 ml/min.
Type of Injector	: Loop injector
Temperature	: 25°C
Column eq’m time	: 30 min (between each run)
Software	: Open LAB CDS, Chemostation Workstation

At the above-mentioned chromatographic conditions FAM , PCT and IBU were separated with the retention time 1.81, 2.58 and 8.53, respectively. The HPLC chromatogram obtained for IBU, FAM and PCT is shown in Fig. 5.

 

Fig. 5: Chromatograms of FAM, paracetamol and IBU

 

Table 1. Assay of tablets formulation and recovery study (n=6)

Parameters	Tablet assay			Recovery study
	Label claim*	% Drug estimated	% RSD	% Level			% Recovery	% RSD
				80	100	120
Famotidine	10	100.31	0.76	0.4	0.5	0.6	100.51	1.72
Paracetamol	10	99.86	0.27	6.6	8.25	9.9	99.96	0.97
Ibuprofen	0.5	98.40	1.49	8	10	12	100.90	1.99

*mg/tablet

 

The proposed method was applied to the determination of drugs in tablet formulation. The results of the assay indicate that the method is selective for the assay of drug without interference from the excipients used in these tablets. The recovery from the marketed tablet formulation was found to be 99.96-100.90%  with % RSD of 0.97-1.99 (Table 1).

 

The famotidine, paracetamol and ibuprofen were found to be linear over the concentration range 0.5-3 µg/ml, 5-25 µg/ml and 5-30 µg/ml, respectively. The co-efficient of correlation for all the drugs was found to be 0.999. The precision of the method was determined as repeatability and intermediate precision study. The results indicate that the selected factors were unaffected by small variations.

 

The results of intra-day precision were expressed as % RSD and it was found to be 0.63, 0.62 and 0.99 for IBU, FAM and PCT respectively. The results of inter-day precision were expressed as % RSD and it was found to be 0.83, 1.82 and 1.91 for IBU, FAM and PCT respectively. The % RSD value indicates the good precision of the method. The results of precision by determining system suitability parameters are shown in Table 2.

 

 

Table 2: Results of precision study

Parameter	Peak area
	Intra-day			Inter day
	IBU	FAM	PCT	IBU	FAM	PCT
Mean	14091	2357	15181	14135	2319	14990
SD	88.23	14.72	150.38	117.77	42.24	286.44
% RSD	0.63	0.62	0.99	0.83	1.82	1.91
LOD (µg/ml)	0.20	0.01	0.35	-	-	-
LOQ (µg/ml)	0.61	0.03	1.05	-	-	-

 

 

4. CONCLUSION:

The proposed HPLC methods provide simple, accurate, reproducible and stability indicating for quantitative analysis for simultaneous estimation of famotidine, paracetamol and ibuprofen from bulk drug and tablet formulation, without any interference from the excipients. Statistical tests showed that the proposed HPLC methods reduce the duration of analysis and appear to be equally suitable for routine determination of drug in pharmaceutical formulation. The method can be used to determine the purity of the drug available from various sources by detecting the related impurities and in stability studies.

 

5. CONFLICT OF INTEREST:

The authors declare no conflict of interest.

 

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Accepted on 28.07.2018        © RJPT All right reserved

Research J. Pharm. and Tech 2019; 12(1): 231-236.