A combination tablet of ibuprofen and famotidine is indicated for the relief of signs and symptoms of rheumatoid arthritis and osteoarthritis and to decrease the risk of developing upper gastro-intestinal ulcer.
An RP-HPLC gradient elution method has been developed for the simultaneous estimation of ibuprofen and famotidine in presence of their degradation products of different polarity index. Separation was carried out on Qualisil BDS C8 column (250 Ã- 4.6 mm, 5 Âµm) using a mobile phase gradient consisting of methanol and water pH 3.0 at a flow rate of 1 mL/min. The detection and reference wavelengths were set at 263 nm (4 nm BW) and 360 nm (80 nm BW) respectively. Retention time of famotidine and ibuprofen was 6.34 Â± 1.53 and 21.76 Â± 0.38 min respectively. The method obeys Beer-Lambert's law in the range of 3-21 Âµg/mL for ibuprofen (r2 = 0.9998) and 0.1-0.7 Âµg/mL for famotidine (r2 = 0.9999). The proposed method was validated as per ICH Q2 (R1) guidelines. The mean percent recoveries were found to be 96.55 Â± 1.83 for ibuprofen and 102.83 Â± 0.85 for famotidine. The method was specific as it estimates the ibuprofen and famotidine in presence of their acidic, alkaline, oxidative, hydrolytic and photolytic degradation products.
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Keywords- RP-HPLC, ibuprofen, famotidine, gradient elution.
A combination tablet of ibuprofen (IBU) and famotidine (FAM) is indicated for the relief of signs and symptoms of rheumatoid arthritis and osteoarthritis and to decrease the risk of developing upper gastro-intestinal ulcer (1-2). Chemically, IBU is (RS)-2-(4-(2-methylpropyl) phenyl) propionic acid and FAM is 3-([2-(diaminomethyleneamino) thiazol- 4-yl] methylthio) -N'-sulfamoylpropanimidamide (3). Both the drugs are official in Indian Pharmacopoeia (3), British Pharmacopoeia (4), United States Pharmacopoeia (5) and European Pharmacopoeia (6).
For IBU estimation the reported method includes spectrophotometric (7-8), HPTLC (9), UPLC (10), HPLC (11-16), GC (17-18). And for FAM estimation spectrophotometric (19-24), HPTLC (9, 25-26), and HPLC (27-31) methods are available. But there is no single analytical method available for the simultaneous estimation of IBU and FAM in presence of their degradation products.
The HPLC is more accurate, specific and selective analytical method than other methods available for the estimation of drug in bulk and pharmaceutical formulations. Gradient elution has become an effective method for separation of mixture of compounds having different polarity index. Present work demonstrates a specific RP-HPLC gradient elution method for simultaneous estimation of IBU and FAM in presence of their degradation products.
Agilent technologies 1200 series HPLC instrument equipped with PDA detector, EZChrom Elite software, G 1311 A solvent delivery system (Quaternary pump), Rheodyne injector (20.0 ÂµL), and Qualisil BDS C8 column (250 Ã- 4.6 mm, 5 Âµm) was used.
The pH of solution was measured with MK IV pH meter.
Reagents and chemicals
IBU and FAM were obtained as gift samples from Centurion Laboratories, Baroda. Analytical grade chemicals and double distilled water were used during experimentation.
Separation and simultaneous determination IBU, FAM and their degradation products were performed on Qualisil BDS C8 column (250 Ã- 4.6 mm, 5 Âµm) using the gradient elution mode. A gradient programme with mobile phase consists of methanol (Solvent A) and water pH 3.0 adjusted with ortho-phosphoric acid (Solvent B) (Table 1). A mixture of methanol and water pH 3.0 (15:85 v/v) was used as diluent. The injection volume was 20 ÂµL. The mobile phase was pumped at flow rate of 1 mL/min. The detection and reference wavelengths were set at 263 nm (4 nm BW) and 360 nm (80 nm BW) respectively.
Preparation of mobile phase and standard solutions
The pH of water was adjusted to 3.0 with ortho-phosphoric acid. Methanol and water pH
3.0 were filtered through 0.45 Âµm Millipore filter and degassed by ultra-sonication for 3 min.
Standard stock solution
Standard stock solution of IBU (Solution A, 300 Âµg/ml)
An accurately weighed quantity of about 300.0 mg of IBU was dissolved in methanol and the volume was made up to 100.0 ml with the methanol. About 1.0 ml of above solution was diluted up to 10 ml with methanol.
Working standard solution of IBU (Solution B, 9 Âµg/ml)
About 0.3 ml of solution A was diluted up to 10.0 ml with diluent.
Standard stock solution of FAM (Solution C, 10 Âµg/ml)
An accurately weighed quantity of about 10.0 mg of FAM was dissolved in methanol and the volume was made up to 100.0 ml with methanol. About 1.0 ml of above solution was diluted up to 10 ml with methanol.
Working standard solution of FAM (Solution D, 0.3 Âµg/ml)
Always on Time
Marked to Standard
About 0.3 ml of solution C was diluted up to 10.0 ml with diluent.
Mix working standard solution (9 Âµg/ml of IBU + 0.3 Âµg/ml of FAM)
About 0.3 ml of solution A and solution C was diluted up to 10.0 ml with diluent.
All the solutions were degassed by ultra-sonication for 3 min.
Study of Beer - Lamberts Law
Aliquots of solution A and solution C (0.1 - 0.7 mL) were transferred separately in to a series of 10.0 mL volumetric flasks. The solutions were diluted up to a mark with diluent so as to obtain final concentrations in the range of 3-21 Âµg/mL for IBU and 0.1-0.7 Âµg/mL for FAM. A constant volume of 20.0 ÂµL of each solution was injected for HPLC analysis as per the optimized chromatographic conditions. All the measurements were repeated three times for each concentration and calibration curve was plotted between concentration Vs peak area.
Application of proposed method to laboratory mixture
The proposed method was applied on the laboratory mixture analysis.
A constant volume of mixed working standard solution 20.0 ÂµL was injected for HPLC analysis as per the optimized chromatographic conditions. The peak area was recorded for each analysis. Concentration and % drug content of both the drugs were determined using the formula mentioned below.
Cu- Concentration of sample solution
Cs- Concentration of standard solution
Au- Peak area of sample solution
As- Peak area of standard solution (24000 for FAM, 0.3 Âµg/ml) and (38701 for IBU, 9 Âµg/ml)
CEst = Estimated concentration (ppm)
CAct = Actual concentration (ppm)
The results of analysis of laboratory mixture are shown in Table 2.
Application of proposed method to synthetic mixture
Sample stock solution (Solution E)
An accurately weighed quantity of about 300 mg of IBU and 10 mg of FAM were transferred in to 100 mL volumetric flask. The contents were dissolved with methanol. Excipients used in the tablet formulation were added in this mixture (1), (Table 3) and sonicated for 20 minute. The final volume was made with same solvent. The solution was filtered through 0.45 Âµm filter paper. About 1.0 ml of above solution was diluted up to 10 ml with methanol.
Working sample solution (Solution F)
About 0.3 ml of solution E was diluted up to 10.0 ml with diluent.
A constant volume 20.0 ÂµL was injected for HPLC analysis as per the optimized chromatographic conditions. And peak area was recorded. Concentration and % drug content of both the drugs were determined using the formula mentioned in "Application of proposed method to laboratory mixture".
The results of analysis of synthetic mixture are shown in Table 2.
The proposed method was validated as per ICH Q2 (R1) guidelines (32) for different parameters like accuracy, precision, linearity, range, specificity, limit of detection, limit of quantitation, robustness etc.
System suitability test
System suitability tests were carried out on mixed working standard solution.
The accuracy of the proposed method was determined by recovery study. The known amount of pure drug was spiked in to pre-analyzed synthetic mixture. Accuracy study was carried out on solution F containing 9 Î¼g/mL IBU and 0.3 Î¼g/mL FAM. Analysis was carried out at three concentration levels such as 80%, 100% and 120% within the specified linearity range.
The content determination of IBU and FAM was done by using the formula mentioned in "Application of proposed method to laboratory mixture".
And the % recovery was calculated using the formula mentioned below.
E- Total amount of drug estimated
T- Amount of drug taken from pre-analyzed synthetic mixture
P- Amount of pure drug added.
The precision of the method was determined as inter-day and intra-day precision study. The repeatability study (inter-day precision) was performed by analyzing the homogeneous solution. The intermediate precision study was performed by variation in days of analysis. Six determinations of mix working standard solution were performed. The results were expressed as SD, % RSD.
Limit of detection and limit of quantitation
Limit of detection and limit of quantitation of developed analytical method was calculated using the formula mentioned below.
Ïƒ - Standard deviation of the response (57.63 for IBU and 162.74 for FAM)
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S - Slope of calibration curve (4197.2 for IBU and 78939 for FAM)
Specificity study/ Forced degradation study
Specificity is the ability to assess unequivocally the analyte in the presence of components which may be expected to be present. Typically these might include impurities, degradates, matrix, etc.
Stress studies were performed at initial concentration of 100 Âµg/mL of IBU and FAM to provide an indication of the stability indicating property. Intentional degradation was attempted to stress condition of hydrolytic (reflux for 1 h at 80oC), acid (5M HCl, reflux for 1 h at 80oC), base (5M NaOH, reflux for 1 h at 80oC), oxidation (15% H2O2, for 6 h at 30oC) and sunlight (4 h). From all forced degradation samples, about 1.0 mL solution was transferred to 10.0 mL volumetric flask and dilution was made with diluent. These samples were used for the HPLC analysis as per the optimized chromatographic conditions to evaluate the ability of the proposed method to separate IBU and FAM from their degradation products.
The robustness of an analytical procedure is a measure of its capacity to remain unaffected by small, but deliberate variations in method parameters and provides an indication of its reliability during normal usage. To determine the robustness of the method the experimental conditions were deliberately changed. The flow rate of the mobile phase was changed by Â± 0.1 mL.
Results and discussion
Structural, physical and chemical properties of analyzed substances are very important factors in establishing appropriate chromatographic conditions. According to that, non-polar HPLC columns were chosen for the analysis. Retention behavior of the IBU, FAM and their degradation products was analyzed using Qualisil BDS C8 (250 Ã- 4.6 mm, 5 Âµm) HPLC column.
It was noticed that the optimal retention of FAM requires mobile phase with low percent of organic solvent, i.e., less than 20%. and its degradation products requires mobile phase with low to high percent of organic solvent. However, on the other hand, IBU and its degradation products, as more lipophilic substances were retained for almost 70 min under the same experimental condition. Because of that, isocratic elution was not found to be economical to analyze the mixture, and hence efforts were aimed for settling an optimal gradient programme. Different trials were conducted to establish some initial gradient elution conditions.
From the literature survey it is revealed that, the mobile phases containing organic solvent and different buffer solutions were used in most of the HPLC methods reported for the analysis of IBU and FAM. Therefore, in order to establish an economical HPLC method, which could be applied in the routine analysis of pharmaceuticals containing IBU, FAM and their degradation products, it was decided to use mobile phase composition without buffer solution. As with the use of buffer solution, the column life was reduces.
Critical pairs in the present study were separation of IBU and its degradation products, as well as FAM and its degradation products. IBU contains benzene ring and carboxylic acid group; whereas, FAM contains sulphamide and primary amino group. So the key difference is in the polarity and acidity/alkalinity. For the analysis of such a mixture (compounds with high and low lipophilicity), demands a gradient programme starting with low percent of organic solvent and gradual increasing of organic solvent content so as to achieve an optimal separation and retention of all the components of the mixture.
Selection of detection wavelength and band width
From the overlain UV spectra of IBU and FAM of mixed working standard solution, the detection wavelength selected was 263 nm. The use of narrow band width has the advantage of increasing the signal selectivity of the detector. So, 4 nm band width was selected for analysis.
Selection of reference wavelength and bandwidth
In the Gradient analysis the absorbance value of sample is varying as the composition of the mobile phase is varying, refractive index also changes during the gradient. This change in sample absorbance is not because of the sample itself but because of change in composition of mobile phase. The use of a reference wavelength is highly recommended to reduce baseline drift induced by room temperature fluctuations or refractive index changes during a gradient. A reference wavelength of 350 nm with an 80 nm bandwidth is fine for a sample that doesn't have a visible absorption band.
Validation of developed method
System suitability test
The results of system suitability test were found to be within the acceptance criteria and thus the developed method passes the system suitability test. System suitability parameters are summarized in Table 4.
Linearity and range
The calibration graph was constructed between concentration Vs peak area for the proposed method under optimized HPLC conditions as described above. The proposed method shows linear relationship in the concentration range of 3 - 21 Î¼g/mL for IBU (r2 = 0.9999) and 0.1 - 0.7 Î¼g/mL for FAM (r2 = 0.9999) (Table 5, Fig. 3 and 4).
The mean % recoveries were found to be 96.55 Â± 1.83 and 102.83 Â± 0.85 for IBU and FAM respectively (Table 6).
The results of inter-day precision were expressed as % RSD and it was found to be 0.17 and 0.73 for IBU and FAM respectively. The results of intra-day precision were expressed as % RSD and it was found to be 0.14 and 0.57 for IBU and FAM respectively (Table 7). The % RSD value indicates the good precision of the method.
LOD and LOQ
The LOD was found to be 0.0453 Î¼g/mL and 0.0068 Î¼g/mL for IBU and FAM respectively. The LOQ was found to be 0.1373 Î¼g/mL and 0.02061 Î¼g/mL for IBU and FAM respectively (Table 5).
The results of specificity study indicate the degradation of IBU and FAM in different reaction condition. In the presence of degradation products IBU and FAM were specifically analyzed by proposed method. The chromatograms and results of specificity study are given in Fig. 6 and Table 8.
The proposed method was found to be robust. Results of robustness study were summarized in Table 9.
We would like to thanks to Dr. S. B. Bari Sir, Principal H. R. Patel Institute of Pharmaceutical Education and Research, Shirpur. Also we thankful to Centurion Laboratories, Baroda (INDIA) for providing gift sample.