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An RP-HPLC gradient elution method has been developed for the simultaneous estimation of ibuprofen and famotidine in presence of their degradation products. Separation was carried out on Qualisil BDS C8 column (250 Ã- 4.6 mm, 5 µm) 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 bandwidth) and 360 nm (80 nm bandwidth), respectively. Retention time of famotidine and ibuprofen was 6.34 ± 1.53 and 21.76 ± 0.38 min respectively. The method obeys Beer - Lambert's law in the concentration range of 3-21 µg/mL for ibuprofen (r2 = 0.9998) and 0.1-0.7 µg/mL for famotidine (r2 = 0.9999). The assay result of synthetic mixture was found to be 99.13 ± 0.14 and 100.73 ± 0.57 for ibuprofen and famotidine, respectively. The proposed method was validated as per ICH Q2 (R1) guidelines. The percent recovery was found to be 96.55 ± 1.83 and 102.83 ± 0.85 for ibuprofen and famotidine, respectively. The method was specific as it estimates ibuprofen and famotidine in presence of their acidic, alkaline, oxidative, hydrolytic and photolytic degradation products.
Ibuprofen (IBU) and famotidine (FAM) in combination 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 (Fig. 1) whereas FAM is 3-([2-(diamino- methylene amino) thiazol-4-yl] methylthio)-N'-sulfamoylpropanimidamide (Fig. 2) (3). Both the drugs are official in Indian Pharmacopoeia, British Pharmacopoeia, United States Pharmacopoeia and European Pharmacopoeia (3-6).
Literature survey revealed spectrophotometric (7, 8), HPTLC (9), UPLC (10), HPLC (11-16) and GC methods (17-18) for estimation of IBU alone and in combination with other drug whereas spectrophotometric (19-24), HPTLC (9, 25-26), and HPLC methods (27-31) are reported for estimation of FAM alone and in combination with other drugs. But there is no single analytical method available for the simultaneous estimation of IBU with 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 (32). Gradient chromatography is a powerful method to control retention and resolution over a very broad analyte polarity range (33). 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 photo diode array detector, G 1311 A solvent delivery system (Quaternary pump), Rheodyne injector (20.0 µL) Qualisil BDS C8 column (250 Ã- 4.6 mm, 5 µm) and EZChrom Elite software was used.
Reagents and chemicals
IBU and FAM were obtained as gift samples from Centurion Laboratories, Baroda, India. Analytical grade chemicals and double distilled water were used during experimentation.
The separation and simultaneous determination of IBU, FAM with their degradation products was performed on Qualisil BDS C8 column (250 Ã- 4.6 mm, 5 µm) using the gradient elution mode. A gradient programme consist of methanol (Solvent A) and water pH 3.0 adjusted with ortho-phosphoric acid (Solvent B) as shown in Table 1. A mixture of methanol and water pH 3.0 (15:85 v/v) was used as diluent. The injection volume was 20 µL. The mobile phase was pumped at flow rate of 1 mL/min. The detection and reference wavelengths were set at 263 nm (4 nm bandwidth) and 360 nm (80 nm bandwidth) respectively.
Preparation of mobile phase and standard solutions
The methanol: water pH 3.0 was used as mobile phase. The pH of water was adjusted to 3.0 with ortho-phosphoric acid. Methanol and water pH 3.0 were filtered through 0.45 µm Millipore filter and degassed by ultra-sonication.
Preparation of standard stock and working standard solution
Standard stock solution of IBU (Solution A, 300 µg/ml)
An accurately weighed quantity of about 300.0 mg of IBU was dissolved in 100 mL volumetric flask with methanol. Aliquot of about 1.0 ml of above solution was diluted to 10 ml with methanol.
Working standard solution of IBU (Solution B, 9 µg/ml)
Aliquot of about 0.3 ml of solution A was diluted to 10.0 ml with diluent.
Standard stock solution of FAM (Solution C, 10 µg/ml)
An accurately weighed quantity of about 10.0 mg of FAM was dissolved in 100 mL volumetric flask with methanol. Aliquot of about 1.0 ml of above solution was diluted to 10 ml with methanol.
Working standard solution of FAM (Solution D, 0.3 µg/ml)
Aliquot of about 0.3 ml of solution C was diluted to 10.0 ml with diluent.
Mixed working standard solution (9 µg/ml of IBU + 0.3 µg/ml of FAM)
About 0.3 ml of solution A and solution C was diluted to 10.0 ml with diluent.
System suitability test
System suitability test was carried out on mixed working standard solution.
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 µg/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 analyzed by HPLC as per the optimized chromatographic conditions. The ability of the proposed method to separate IBU and FAM from their degradation products was evaluated.
Study of Beer - Lambert's 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 to a mark with diluent to obtain concentration in the range of 3-21 µg/mL for IBU and 0.1-0.7 µg/mL for FAM. A constant volume of 20.0 µL 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 for drug concentration Vs peak area.
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 drug mixture (1), (Table 5) and sonicated for 20 minute. The final volume was made with methanol. The solution was filtered through 0.45 µm filter paper. Aliquot of about 1.0 ml was diluted to 10 ml with methanol to obtain concentration 300 µg/mL of IBU and 10 µg/mL of FAM.
Working sample solution (Solution F)
Aliquot of about 0.3 ml of solution E was diluted to 10.0 ml with diluent to obtain concentration 9 µg/mL of IBU and 0.3 µg/mL of FAM. A constant volume 20.0 µL was injected for HPLC analysis as per the optimized chromatographic conditions. Peak area was recorded. Concentration and % drug content was determined using the formula as
Cu- Concentration of sample solution (µg/ml)
Cs- Concentration of standard solution (µg/ml)
Au- Peak area of sample solution
As- Peak area of standard solution (24000 for FAM, 0.3 µg/ml) and (38701 for IBU, 9 µg/ml)
CEst: Estimated concentration (µg/ml)
CAct: Actual concentration (µg/ml)
The accuracy of the proposed method was determined by recovery study. The known amount of pure drug was spiked 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 and range. The content determination of IBU and FAM was done by using the formula mentioned in "Application of proposed method to synthetic mixture".
The % recovery was calculated using the formula as
E: Total amount of drug estimated (Î¼g/mL)
T: Amount of drug taken from pre-analyzed synthetic mixture (Î¼g/mL)
P: Amount of pure drug added (Î¼g/mL)
The precision of the method was determined as inter-day and intra-day precision. The repeatability study (intra-day precision) was performed by analyzing the homogeneous solution. The inter day precision study was performed by variation in days of analysis. Six determinations of mixed 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)
S: Slope of calibration curve (4197.2 for IBU and 78939 for FAM)
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 and detection wavelength was changed by ± 0.1 and 1 unit respectively.
Results and discussion
Structural, physical and chemical properties of analyzed substances are very important factors in establishing appropriate chromatographic conditions (34). IBU and FAM were insoluble in water. 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 µm) HPLC column.
It was noticed that the optimal retention of FAM (log p value = -0.64) 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 (log p value = 3.621) and its degradation products are more lipophilic substances and they retained for almost 70 min under the same experimental condition. Because of this, isocratic elution was found to be uneconomical, time consuming to analyze the IBU and FAM mixture. Hence efforts were aimed for settling an optimal gradient programme. Different trials were conducted to establish some initial gradient elution conditions.
For IBU and FAM analysis, the use of organic solvents with different buffer solutions was reported in literature (11-16, 27-31). Therefore, in order to establish an economical HPLC method, the mobile phase composition was decided to be used without buffer solution, as with the use of buffer solution, the column life was reduces (35).
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 (36). So, 4 nm band width was selected for analysis.
Selection of reference wavelength and band width: In the gradient analysis the absorbance value of sample was varying as the composition of the mobile phase varies, 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 (37). A reference wavelength of 360 nm with an 80 nm bandwidth is fine for a sample that doesn't have a visible absorption band.
The representative chromatogram was shown in Fig. 3.
System suitability test: The system suitability test was carried out using an optimized chromatographic condition with optimized gradient programme (Table 1) and it was found to be within the acceptance limit. System suitability parameters are summarized in Table 2.
Peak purity: A peak purity value was determined for the chromatogram of ibuprofen and famotidine by using the EZChrom Elite software. The total peak purity of IBU and FAM was found to be 1.000000 which is more than the threshold value as shown in Fig. 4.
Specificity study: The results of specificity study indicate the degradation of IBU and FAM in different reaction conditions except in oxidative and photolytic condition degradation of IBU was not observed. In the presence of degradation products, IBU and FAM were specifically analyzed by proposed method (Fig. 5). The results of specificity study are summarized in Table 3.
Linearity: The proposed HPLC method shows linear relationship in the concentration range of 3 - 21 Î¼g/mL for IBU (r2 = 0.9998) and 0.1 - 0.7 Î¼g/mL for FAM (r2 = 0.9999) (Table 4, Fig. 6 and 7).
Analysis of synthetic mixture: The assay result of synthetic mixture was found to be 99.13 ± 0.14 and 100.73 ± 0.57 for IBU and FAM, respectively (Table 6).
Accuracy: The % recovery was found to be 96.55 ± 1.83 and 102.83 ± 0.85 for IBU and FAM respectively (Table 7).
Precision: The results of inter-day precision were expressed as % RSD and which was found to be 0.17 and 0.73 for IBU and FAM respectively. The results of intra-day precision were found to be 0.14 and 0.57 for IBU and FAM respectively (Table 8). The less % 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 0.0206 Î¼g/mL for IBU and FAM, respectively.
Robustness: The method was found to be robust, as no significant change was found in results after small but deliberate variations in chromatographic conditions. Results of robustness study are summarized in Table 9.
We thankful to Centurion Laboratories, Baroda (India) for providing gift sample. Also we would like to say thanks to Dr. S. B. Bari Sir, Principal, H. R. Patel Institute of Pharmaceutical Education and Research, Shirpur for giving permission and providing necessary facilities.