An RP-HPLC gradient elution method has been developed for the coincident appraisal of isobutylphenyl propionic acid and Pepcid in presence of their debasement merchandises. Separation was carried out on Qualisil BDS C8 column ( 250 A- 4.6 millimeter, 5 Aµm ) utilizing a nomadic stage gradient dwelling of methyl alcohol and H2O pH 3.0 at a flow rate of 1 mL/min. The sensing and mention wavelengths were set at 263 nanometers ( 4 nm bandwidth ) and 360 nanometer ( 80 nm bandwidth ) , severally. Retention clip of Pepcid and isobutylphenyl propionic acid was 6.34 A± 1.53 and 21.76 A± 0.38 min severally. The method obeys Beer – Lambert ‘s jurisprudence in the concentration scope of 3-21 Aµg/mL for isobutylphenyl propionic acid ( r2 = 0.9998 ) and 0.1-0.7 Aµg/mL for Pepcid ( r2 = 0.9999 ) . The assay consequence of man-made mixture was found to be 99.13 A± 0.14 and 100.73 A± 0.57 for isobutylphenyl propionic acid and Pepcid, severally. The proposed method was validated as per ICH Q2 ( R1 ) guidelines. The per centum recovery was found to be 96.55 A± 1.83 and 102.83 A± 0.85 for isobutylphenyl propionic acid and Pepcid, severally. The method was specific as it estimates ibuprofen and Pepcid in presence of their acidic, alkaline, oxidative, hydrolytic and photolytic debasement merchandises.
Ibuprofen ( IBU ) and Pepcid ( FAM ) in combination is indicated for the alleviation of marks and symptoms of arthritic arthritis and degenerative arthritis and to diminish the hazard 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 study revealed spectrophotometric ( 7, 8 ) , HPTLC ( 9 ) , UPLC ( 10 ) , HPLC ( 11-16 ) and GC methods ( 17-18 ) for appraisal of IBU entirely and in combination with other drug whereas spectrophotometric ( 19-24 ) , HPTLC ( 9, 25-26 ) , and HPLC methods ( 27-31 ) are reported for appraisal of FAM entirely and in combination with other drugs. But there is no individual analytical method available for the coincident appraisal of IBU with FAM in presence of their debasement merchandises.
The HPLC is more accurate, specific and selective analytical method than other methods available for the appraisal of drug in majority and pharmaceutical preparations ( 32 ) . Gradient chromatography is a powerful method to command keeping and declaration over a really wide analyte mutual opposition scope ( 33 ) . Present work demonstrates a specific RP-HPLC gradient elution method for coincident appraisal of IBU and FAM in presence of their debasement merchandises.
Agilent engineerings 1200 series HPLC instrument equipped with exposure rectifying tube array sensor, G 1311 A solvent bringing system ( Quaternary pump ) , Rheodyne injector ( 20.0 AµL ) Qualisil BDS C8 column ( 250 A- 4.6 millimeter, 5 Aµm ) and EZChrom Elite package was used.
Reagents and chemicals
IBU and FAM were obtained as gift samples from Centurion Laboratories, Baroda, India. Analytical class chemicals and dual distilled H2O were used during experimentation.
The separation and coincident finding of IBU, FAM with their debasement merchandises was performed on Qualisil BDS C8 column ( 250 A- 4.6 millimeter, 5 Aµm ) utilizing the gradient elution manner. A gradient programme consist of methyl alcohol ( Solvent A ) and H2O pH 3.0 adjusted with ortho-phosphoric acid ( Solvent B ) as shown in Table 1. A mixture of methyl alcohol and H2O pH 3.0 ( 15:85 v/v ) was used as dilutant. The injection volume was 20 AµL. The nomadic stage was pumped at flow rate of 1 mL/min. The sensing and mention wavelengths were set at 263 nanometers ( 4 nm bandwidth ) and 360 nanometer ( 80 nm bandwidth ) severally.
Preparation of nomadic stage and standard solutions
The methyl alcohol: H2O pH 3.0 was used as nomadic stage. The pH of H2O was adjusted to 3.0 with ortho-phosphoric acid. Methanol and H2O pH 3.0 were filtered through 0.45 Aµm Millipore filter and degassed by ultra-sonication.
Preparation of standard stock and working standard solution
Standard stock solution of IBU ( Solution A, 300 Aµg/ml )
An accurately weighed measure of about 300.0 milligrams of IBU was dissolved in 100 milliliters volumetric flask with methyl alcohol. Aliquot of approximately 1.0 milliliters of above solution was diluted to 10 milliliter with methyl alcohol.
Working standard solution of IBU ( Solution B, 9 Aµg/ml )
Aliquot of approximately 0.3 milliliters of solution A was diluted to 10.0 milliliter with dilutant.
Standard stock solution of FAM ( Solution C, 10 Aµg/ml )
An accurately weighed measure of about 10.0 milligrams of FAM was dissolved in 100 milliliters volumetric flask with methyl alcohol. Aliquot of approximately 1.0 milliliters of above solution was diluted to 10 milliliter with methyl alcohol.
Working standard solution of FAM ( Solution D, 0.3 Aµg/ml )
Aliquot of approximately 0.3 milliliters of solution C was diluted to 10.0 milliliter with dilutant.
Mixed working standard solution ( 9 Aµg/ml of IBU + 0.3 Aµg/ml of FAM )
About 0.3 milliliters of solution A and solution C was diluted to 10.0 milliliter with dilutant.
System suitableness trial
System suitability trial was carried out on assorted working standard solution.
Specificity is the ability to measure unambiguously the analyte in the presence of constituents which may be expected to be present. Typically these might include drosss, degradates, matrix, etc.
Stress surveies were performed at initial concentration of 100 Aµg/mL of IBU and FAM to supply an indicant of the stableness bespeaking belongings. Intentional debasement was attempted to emphasize status 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 ) , oxidization ( 15 % H2O2, for 6 H at 30oC ) and sunlight ( 4 H ) . From all forced debasement samples, approximately 1.0 mL solution was transferred to 10.0 milliliters volumetric flask and dilution was made with dilutant. These samples were analyzed by HPLC as per the optimized chromatographic conditions. The ability of the proposed method to divide IBU and FAM from their debasement merchandises was evaluated.
Study of Beer – Lambert ‘s Law
Aliquots of solution A and solution C ( 0.1 – 0.7 milliliter ) were transferred individually in to a series of 10.0 milliliters volumetric flasks. The solutions were diluted to a grade with dilutant to obtain concentration in the scope of 3-21 Aµg/mL for IBU and 0.1-0.7 Aµg/mL for FAM. A changeless volume of 20.0 AµL of each solution was injected for HPLC analysis as per the optimized chromatographic conditions. All the measurings were repeated three times for each concentration and standardization curve was plotted for drug concentration Vs peak country.
Application of proposed method to man-made mixture
Sample stock solution ( Solution E )
An accurately weighed measure of about 300 milligrams of IBU and 10 milligram of FAM were transferred in to 100 milliliters volumetric flask. The contents were dissolved with methyl alcohol. Excipients used in the tablet preparation were added in this drug mixture ( 1 ) , ( Table 5 ) and sonicated for 20 minute. The concluding volume was made with methyl alcohol. The solution was filtered through 0.45 Aµm filter paper. Aliquot of approximately 1.0 milliliter was diluted to 10 milliliter with methyl alcohol to obtain concentration 300 Aµg/mL of IBU and 10 Aµg/mL of FAM.
Working sample solution ( Solution F )
Aliquot of approximately 0.3 milliliters of solution E was diluted to 10.0 milliliter with dilutant to obtain concentration 9 Aµg/mL of IBU and 0.3 Aµg/mL of FAM. A changeless volume 20.0 AµL was injected for HPLC analysis as per the optimized chromatographic conditions. Peak country was recorded. Concentration and % drug content was determined utilizing the expression as
Cu- Concentration of sample solution ( Aµg/ml )
Cs- Concentration of standard solution ( Aµg/ml )
Au- Peak country of sample solution
As- Peak country of standard solution ( 24000 for FAM, 0.3 Aµg/ml ) and ( 38701 for IBU, 9 Aµg/ml )
CEst: Estimated concentration ( Aµg/ml )
CAct: Actual concentration ( Aµg/ml )
The truth of the proposed method was determined by recovery survey. The known sum of pure drug was spiked to pre-analyzed man-made mixture. Accuracy survey was carried out on solution F incorporating 9 I?g/mL IBU and 0.3 I?g/mL FAM. Analysis was carried out at three concentration degrees such as 80 % , 100 % and 120 % within the specified one-dimensionality and scope. The content finding of IBU and FAM was done by utilizing the expression mentioned in “ Application of proposed method to man-made mixture ” .
The % recovery was calculated utilizing the expression as
Tocopherol: Entire sum of drug estimated ( I?g/mL )
Thymine: Sum of drug taken from pre-analyzed man-made mixture ( I?g/mL )
Phosphorus: Sum of pure drug added ( I?g/mL )
The preciseness of the method was determined as inter-day and intra-day preciseness. The repeatability survey ( intra-day preciseness ) was performed by analysing the homogenous solution. The inter twenty-four hours preciseness survey was performed by fluctuation in yearss of analysis. Six findings of assorted working standard solution were performed. The consequences were expressed as SD, % RSD.
Limit of sensing and bound of quantitation
Limit of sensing and bound of quantitation of developed analytical method was calculated utilizing the expression mentioned below.
I? : Standard divergence of the response ( 57.63 for IBU and 162.74 for FAM )
Second: Slope of standardization curve ( 4197.2 for IBU and 78939 for FAM )
The hardiness of an analytical process is a step of its capacity to stay unaffected by little, but calculated fluctuations in method parametric quantities and provides an indicant of its dependability during normal use. To find the hardiness of the method the experimental conditions were intentionally changed. The flow rate and sensing wavelength was changed by A± 0.1 and 1 unit severally.
Consequences and treatment
Structural, physical and chemical belongingss of analyzed substances are really of import factors in set uping appropriate chromatographic conditions ( 34 ) . IBU and FAM were indissoluble in H2O. Harmonizing to that, non-polar HPLC columns were chosen for the analysis.
Retention behaviour of the IBU, FAM and their debasement merchandises was analyzed utilizing Qualisil BDS C8 ( 250 A- 4.6 millimeter, 5 Aµm ) HPLC column.
It was noticed that the optimum keeping of FAM ( log p value = -0.64 ) requires nomadic stage with low per centum of organic dissolver, i.e. , less than 20 % and its debasement merchandises requires nomadic stage with low to high per centum of organic dissolver. However, on the other manus, IBU ( log p value = 3.621 ) and its debasement merchandises are more lipotropic substances and they retained for about 70 min under the same experimental status. Because of this, isocratic elution was found to be wasteful, clip devouring to analyse the IBU and FAM mixture. Hence attempts were aimed for settling an optimum gradient programme. Different tests were conducted to set up some initial gradient elution conditions.
For IBU and FAM analysis, the usage of organic dissolvers with different buffer solutions was reported in literature ( 11-16, 27-31 ) . Therefore, in order to set up an economical HPLC method, the nomadic stage composing was decided to be used without buffer solution, as with the usage of buffer solution, the column life was reduces ( 35 ) .
Critical braces in the present survey were separation of IBU and its debasement merchandises, every bit good as FAM and its debasement merchandises. IBU contains benzene ring and carboxylic acid group ; whereas, FAM contains sulphamide and primary amino group. So the cardinal difference is in the mutual opposition and acidity/alkalinity. For the analysis of such a mixture ( compounds with high and low lipophilicity ) , demands a gradient programme get downing with low per centum of organic dissolver and gradual increasing of organic dissolver content so as to accomplish an optimum separation and keeping of all the constituents of the mixture.
Choice of sensing wavelength and set breadths: From the overlain UV spectra of IBU and FAM of assorted working standard solution, the sensing wavelength selected was 263 nanometer. The usage of narrow set breadth has the advantage of increasing the signal selectivity of the sensor ( 36 ) . So, 4 nm set breadth was selected for analysis.
Choice of mention wavelength and set breadth: In the gradient analysis the optical density value of sample was changing as the composing of the nomadic stage varies, refractile index besides changes during the gradient. This alteration in sample optical density is non because of the sample itself but because of alteration in composing of nomadic stage. The usage of a mention wavelength is extremely recommended to cut down baseline impetus induced by room temperature fluctuations or refractile index alterations during a gradient ( 37 ) . A mention wavelength of 360 nanometer with an 80 nanometer bandwidth is all right for a sample that does n’t hold a seeable soaking up set.
The representative chromatogram was shown in Fig. 3.
System suitability trial: The system suitableness trial was carried out utilizing an optimized chromatographic status with optimized gradient programme ( Table 1 ) and it was found to be within the credence bound. System suitableness parametric quantities are summarized in Table 2.
Peak pureness: A peak pureness value was determined for the chromatogram of isobutylphenyl propionic acid and Pepcid by utilizing the EZChrom Elite package. The entire peak pureness of IBU and FAM was found to be 1.000000 which is more than the threshold value as shown in Fig. 4.
Specificity survey: The consequences of specificity survey indicate the debasement of IBU and FAM in different reaction conditions except in oxidative and photolytic status debasement of IBU was non observed. In the presence of debasement merchandises, IBU and FAM were specifically analyzed by proposed method ( Fig. 5 ) . The consequences of specificity survey are summarized in Table 3.
One-dimensionality: The proposed HPLC method shows linear relationship in the concentration scope of 3 – 21 I?g/mL for IBU ( r2 = 0.9998 ) and 0.1 – 0.7 I?g/mL for FAM ( r2 = 0.9999 ) ( Table 4, Fig. 6 and 7 ) .
Analysis of man-made mixture: The assay consequence of man-made mixture was found to be 99.13 A± 0.14 and 100.73 A± 0.57 for IBU and FAM, severally ( Table 6 ) .
Accuracy: The % recovery was found to be 96.55 A± 1.83 and 102.83 A± 0.85 for IBU and FAM severally ( Table 7 ) .
Preciseness: The consequences of inter-day preciseness were expressed as % RSD and which was found to be 0.17 and 0.73 for IBU and FAM severally. The consequences of intra-day preciseness were found to be 0.14 and 0.57 for IBU and FAM severally ( Table 8 ) . The less % RSD value indicates the good preciseness of the method.
LOD and LOQ: The LOD was found to be 0.0453 I?g/mL and 0.0068 I?g/mL for IBU and FAM, severally. The LOQ was found to be 0.1373 I?g/mL and 0.0206 I?g/mL for IBU and FAM, severally.
Robustness: The method was found to be robust, as no important alteration was found in consequences after little but calculated fluctuations in chromatographic conditions. Consequences of hardiness survey are summarized in Table 9.
We thankful to Centurion Laboratories, Baroda ( India ) for supplying gift sample. Besides we would wish to state thanks to Dr. S. B. Bari Sir, Principal, H. R. Patel Institute of Pharmaceutical Education and Research, Shirpur for giving permission and supplying necessary installations.