IBUPROFEN

Ibuprofen is non steroidal anti inflammatory drug that displays potent anti inflammatory properties as well as strong analgesic and anti pyretic activity. It was introduced in 1969 in UK. It was widely used as a prescription only medicine. In 1983 it became available without prescription and currently it is used throughout world as an over-the-counter medicine for the treatment of pain and fever. The recommended single dose of ibuprofen for the treatment of mild to moderate pain is 200 to 400mg with a maxim daily dosage of 1200mg, compared with a daily dosage upto 3600mg for the treatment of rheumatic disease. IUPAC NAME: 2-[4-(2-methyl propyl)phenyl)] prop ionic acid Synonym: Hydratropic acid P-isobutyl; alpha-p-isobutyl phenyl propionic acid. CAS No: 15687-27-1. Product codes: 3751, 4036, 6280, 6333, 6454. Chemical formula: C13 H18 O2 Molecular weight: 206.30 Physical and chemical properties: Appearance: White to off-white crystalline solid. Odour: Characteristic fruity odour Solubility: Insoluble in water soluble in most organic solvents. Bulk density: 0.4gm/cc Melting point: 75-77c. MECHANISM OF ACTION: Ibuprofen is an NSAID which is to work through inhibition of cyclooxygenase (cox), thus inhibiting prostaglandin synthesis. There are at least 2 variants of cyclooxygenase (cox-1 and cox-2). Ibuprofen inhibits both cox-1 and cox-2. It appears that it acts as analgesic, antipyretic, and anti-inflammatory activity are achieved principally through cox-2 inhibition; where as cox-1 inhibition is responsible for its unwanted effects on platelet aggregation and GI mucosa. The role of individual cox isoforms in the analgesic and anti inflammatory and gastric damage effects of NSAIDS is uncertain and different compounds cause different degrees of analgesia and gastric damage. Adverse reactions and side effects: Ibuprofen appears to have lowest incidence of gastrointestinal adverse drug reactions of all the non-selective NSAIDS. However, this only holds true at lower doses of ibuprofen, so over the counter preparations of ibuprofen are generally labelled to advise a maxim dose of 1,200mg. Common adverse effects include: Nausea, dyspepsia, gastrointestinal ulceration/bleeding, raised liver enzymes, diarrhea, epistaxis, headache, dizziness, unexplained rash, salt and fluid retention, and hyper tension. Infrequent adverse effects include: Oesophageal ulceration, heart failure, hyperkalaemia, renal impairment, confusion, bronchospasm, and rash.

Dosage: Adults: Dosage of ibuprofen for the treatment of mild to moderate pain is 200 to 400mg with a maxim daily dosage of 1200mg, and a maximum up to 3,600mg per a day. Preparations containing Ibuprofen in the market are: Nurofen Brufen Ebufac Galprofen Ibuleve Ibuprofen is available in various dosage forms like: 1. Tablets 2. Caplets 3. Liquid gel capsules 4. Syrups and solutions 5. Ointments Before taking Ibuprofen the following conditions must be taken into consideration Use with caution in Elderly people History of disorders affecting the stomach or intestines. Inflammatory bowel disease such as crohns disease. Kidney disease Liver disease High blood pressure Heart failure People with blood clotting problems or taking anti coagulant medicines. History of asthma History of allergies Diseases affecting connective tissue. Not to be used in: People in whom asprin or other NSAIDS asthma attacks. Active peptic ulcer or history of this. Severe heart failure Severe liver failure Severe Kidney failure Ibuprofen should not be used during pregnancy and breast feeding.

METHOD VALIDATION
Method validation is the process used to confirm that the analytical procedure employed for a specific test is suitable for its intended use. Results from method validation can be used to judge the quality, reliability and consistency of analytical results; it is an integral part of any good analytical practice. Analytical methods need to be validated or revalidated:

Before their introduction in to their routine use. Whenever the conditions change for which the method has been validated (e.g. an instrument with different characteristics or samples with a different matrix); and Whenever method is changed and the change is outside the original scope of the method.

The USP has published specific guidelines for method validation compound evaluation. USP defines eight steps for method validation. Accuracy Precision Specificity Limit of detection Limit of quantification Linearity and range Ruggedness Robustness

Strategy for the validation of methods The validity of a specific method should be demonstrated in laboratory experiments using samples or standards that are similar to unknown samples analysed routinely. The preparation and execution should be follow a validation protocol, preferably written in a stepby-step instruction format. 1. Develop a validation protocol, an operating procedure or a validation master plan for the validation. 2. For a specific validation project define owners and responsibilities. 3. Develop a validation project plan. 4. Define the application, purpose and scope of the method. 5. Define the performance parameters and acceptance criteria. 6. Define validation experiments. 7. Verify relevant performance characteristics of equipment. 8. Quality materials, e.g. standards and reagents for purity, accurate amounts and sufficient stability. 9. Perform pre-validation experiments. 10. Adjust method parameters and/or acceptance criteria if necessary. 11. Perform full internal (and external) validation experiments. 12. Develop SOPs for executing the method in type routine. 13. Define criteria for revalidation. 14. Define type and frequency of system suitability tests and/or analytical quality control checks for the routine. 15. Document validation experiments and results in the validation report. STEPS IN METHOD VALIDATION Successful acceptance of the validation parameters and performance criteria, by all parties involved, requires the cooperative efforts of several departments, including analytical development, QC, regulatory affairs and the individuals requiring the analytical data.

The operating procedure should clearly define the roles and responsibilities of each department involved in the validation of analytical methods. These includes following questions. What analytes should be detected? What are the expected concentration levels? What are the samples matrices? Are there interesting substances expected, and if so, should they be detected and quantified? Are there any specific legislative or regulatory requirements? Should information be qualitative or quantitative? What is the expected concentration range? What are the required detection and quantitation limits? What precession and accuracy is expected? How robust should be the method be? Which type of equipment should be used? Is the method for one specific instrument, or should it be used by all instruments of the same type? Will the method be used in one specific laboratory or should it be applicable in all laboratories at one side or around the globe? What skills do the anticipated users of the method have?

1. Specificity/Selectivity The term selectivity and specificity are often used interchangeably. A detailed discussion of this term, as defined by different organisations, has been presented by Vessmann. He particularly pointed out the difference between the definitions of specificity given by IUPAC/WELAC and the ICH. The term specific generally refers to a method that produces a response for a single analyte only, while the term selective refers to a method that provides responses for a number of chemical entities that may or may not be distinguished from each other. If the response is distinguished from all other responses, the method is said to be selective. 2. Precession and Reproducibility The precision of a method is extent to which the individual test results of multiple injections of a series of standards agree. The measured standard deviation can be sub divided into three categories; repeatability, intermediate precision and reproducibility. Reproducibility is obtained when the analysis is carried out in a laboratory by an operator using a piece of equipment over a relatively short time span. At least 6 determinations of 3 different matrices at 2 or 3 different concentrations should be performed, and the rsd calculated. 3. Accuracy The accuracy of an analytical method is the extent to which test results generated by the method and the true value agree. Always can also be described as the closeness of agreement

between the value that is adopted, either as a conventional, true or accepted reference value, and the value found. The ICH document on validation methodology recommends accuracy to be assessed using a minimum of nine determinations over a minimum of three concentration levels covering specific range. Accuracy should be reported as percent recovery by the assay of known added amount of analyte in the sample or as the difference between the mean and the accepted true value, together with the confidence intervals. The range for the accuracy should be similar to the linearity range. The percentage recovery of 98-102% is accepted and the data beyond this level needs to be understood. 4. Linearity and calibration curve The linearity of an analytical method is its ability to elicit test results that are directly proportional to the concentration of analytes in samples within a given range or proportional by means of well-defined mathematical transformations. Linearity may be demonstrated directly on the test substance (by dilution of a standard stock solution) and/or by using separate weighings of synthetic mixtures of the test product components, using the proposed procedure. Linearity is determined by a series of 3 to 6 injections of 5 or more standards whose concentrations span 80-120 percent of the expected concentration range. The response should be directly proportional to the concentrations of the analytes or proportional by means of a well-defined mathematical calculations. A linear regression equation applied to the results should have an intercept not significantly different from 0. If a significant non-zero intercept is obtained, it should be demonstrated that this has no effect on the accuracy of the method. 5. Range The range of analytical method is the interval between the upper and lower levels that have been demonstrated to be determined with precision, accuracy and linearity using the method as written. The range is normally expressed in same units as the test results (e.g. percentage or parts per million) obtained by the analytical method. 6. Limit of detection Limit of detection is the lowest concentration level that can be determined so that is statistically different from blank. The LOD is generally determined in the region where the signal to noise ratio is greater than 5. Limit of detection can obtained using the standard deviation of response observed and the slope of the calibration curve. Signal to Noise Ratio: 12 This is a dimensionless ratio of the relative strength of an analytical signal (s) to the average strength of the background instrumental noise of a sample and are highly related to the detection level. The signal to noise ratio can be measured by dividing the arithmetic mean of series of replicates by standard deviation. 6. Limit of Quantification

Limit of quantification is the level above which the quantitative results can be obtained with a certain degree of confidence. Limit of quantification can also be mathematically defined as equal to 10 times the standard deviation of the results for a series of replicates which are used to determine limits of detection. 7. Robustness Robustness is the capacity or ability of the method to remain unchanged by the small variations in the process parameters. Robustness of a particular method can be determined by changing the method conditions such as temperature, ionic strength, pH etc and observing the effect on the final results of the method. 8. Ruggedness Ruggedness is the measure of the reproducibility of the data obtained by a method under specified conditions. Ruggedness of any method is expressed in terms of Relative Standard Deviation RSD%.

CHEMICALS AND REAGENTS USED

The different chemicals and reagents used for the HPLC validation of Ibuprofen are as follows: The mobile phase selected for the method was composed of Acetonitrile, water and ortho phosphoric acid. 1. Water: Water used for the mobile phase preparation is the de-ionised water from the laboratory. 2. Acetonitrile: Chemical compound with the molecular formula CH3 CN. This is one of the most widely used solvents for HPLC. IUPAC Name: Ethanenitrile. Synonyms: Cyanomethane. Ethylnitrile, Methyl Cyanide, Ethane nitrile. CAS Number: 75-05-8. Physical and Chemical properties: Density: 0.786g/ml. Boiling point: 81.6 C Melting point: -46C Odour: Acetonitirile smells like ether. Appearance: Acetonitirile is a clear and colourless liquid. Molecular weight: 41.05g/mol. Solubility: Soluble in organic solvents and miscibly soluble in water. Stability: Acetonitrile is usually stable under normal conditions of use. Batch Number is 0809411. Acetonitrile is taken from the university HPLC laboratory.

Class 3.

Phosphoric acid:

Chemical compound with the molecular formula H3PO4 Synonyms: Orthophosphoric acid. Molecular formula: H3PO4 CAS Number: 7664-38-2 Physical and Chemical properties: Appearance: Colourless odourless liquid. Molecular Weight: 98.0g/mol Melting point: 21C Boiling point: 158C Density: 1.685g/ml Stability: stable and is incompatible with metals. Phosphoric acid taken to prepare the mobile phase belongs to batch and code: Batch number: 0762682 Code: O/0500/PB08 UN number: 1805 Class: 8

4. Ibuprofen sodium salt that is taken to prepare the standard solutions from the HPLC laboratory in the University of the Batch number 027 k2 111 Manufacturer is SIGMA ALDRICH. 5. IBUPROFEN 200mg TABLETS: Ibuprofen tablets which contains 200mg of ibuprofen each are from Paydens Pharmacy which are manufactured by Galpharm Health care ltd., with the code number PL 16028/0013 and with the batch number 80097.

APPARATUS

Volumetric flask, measuring cylinders, Beakers, funnel. Auto pipette. Filter papers of Fisher brand QL100 were used. Analytical balance used was a Mettler AC88 Delta Range, Serial number 806430. Column: The column used was 150 4.6mm S No 81675 Type Chromosil.

EXPERIMENTAL CONDITIONS REQUIRED FOR THE METHOD The method selected for validation of Ibuprofen is Reverse phase HPLC and the system and the method requires some operating conditions which must be maintained through out the process. They include The UV detector was set to a wavelength of 220nm which should be the same through out the method. The flow rate should not be increased directly to the required rate it should be increased fro 0.5ml/min later to 2.0ml/min. The flow rate of the mobile phase should be adjusted to 2.0ml/min. The temperature was maintained at 40 C. The mobile phase composing of acetonitrile, water and phosphoric acid was degassed using the degasser each time before using it for the experiment. The waste bottle should be emptied before switching the system on every time. Once the mobile phase is kept and the column is adjusted the entire system that is the pumps, column and the detector must be turned on. The method which we are using should be selected before starting the runs so that there will not be any confusion with the others work. After starting the system the samples injection must be done only after the base line is stabilised and there are no interactions in the baseline. It must be ensured that the mobile phase is not below its required level that is 250ml.

PREPARATION OF MOBILE PHASE: In the first step the mobile phase of Acetonitrile, Deionised water and Phosphoric acid was prepared. The mobile phase composition selected was 600:400:2 of Acetonitrile, Deionised water and Phosphoric acid. So 600ml of Acetonitrile and 400ml of De-ionised water and 2ml of ortho phosphoric acid are taken in to a 1000ml bottle and mixed well to prepare the mobile phase. The mobile phase should be kept for degassing before using it all the times to prevent the bubbles. METHOD FOR STANDARD SOLUTIONS PREPARATION In the first step 0.2006g of Ibuprofen sodium salt is weighed and transferred in to 100ml volumetic flask and made up to mark with the solvent methanol. Volume calculations: the usual formula for calculating the volumes is C1V1=C2V2. Where C1 is the concentration of the standard stock solution

V1 is the volume of the standard stock solution needed to ;make the dilutions C2 is the concentration of standard solution required to be prepared. V2 is the volume to which the final solution is made up. 100ml of the solution contains 200mg of ibuprofen sodium salt so 1 ml of solution contains 2 mg of Ibuprofen salt a. To prepare solution containing 0.4mg/50ml of Ibuprofen sodium salt C1V1=C2V2 2 V1=0.4 50 V1= 1ml So 1 ml of standard solution contains 0.4 mg of Ibuprofen sodium salt which made up to 50 ml with mobile phase. b. To prepare solution containing 0.5mg/50ml of Ibuprofen sodium salt C1V1=C2V2 2 V1=0.5 50 V1= 1.25 ml of standard solution contains 0.5 mg of Ibuprofen sodium salt which made up to 50 ml with mobile phase. c. To prepare solution containing 0.6mg/50ml of Ibuprofen sodium salt C1V1=C2V2 2 V1=0.6 50 V1= 1.5ml of standard solution contains 0.6mg of Ibuprofen sodium salt which is made up to 50ml with mobile phase. d. To prepare solution containing 0.7mg/50ml of Ibuprofen sodium salt C1V1=C2V2 2 V1=0.7 50 V1= 1.75ml of standard solution contains 0.7mg of ibuprofen sodium salt which is made up to 50ml with mobile phase. e. To prepare solution containing 0.8mg/50ml Ibuprofen sodium salt C1V1=C2V2 2 V1=0.8 50 V1= 2ml of standard solution contains 0.8mg of Ibuprofen sodium salt which is made up to 50ml with mobile phase. 2. Hence from the standard stock solution, stock solutions of 0.4,0.5,0.6,0.7 and 0.8mg/50ml were prepared by taking 1ml.1.25ml,1.5ml,1.75ml and 2.0ml of standard stock solution and adding mobile phase up to 50ml mark in 50ml volumetric flasks. 3. All these solutions were injected into the HPLC column individually and the chromatograms are taken. PREPARATION OF SAMPLE SOLUTION 1. 20 tablets of Ibuprofen are taken and weight of each tablet is measured. 2. These tablelts are then crushed in a mortor and grinded to powder. 3. And the average weight of Ibuprofen 0.4732g of is taken in to a 100ml of volumetric flask and mixed well with the Deionised water. 4. This is then kept in an ultrasonic bath for 10 minutes so that the powdered Ibuprofen is dissolved completely in the solvent. 5. This is then made up to 100 ml with De ionised water 6. 1.5ml of above sample solution is taken in to a 50 ml of volumetric flask and then made up to 50 ml with the De ionised water.7.

7. The sample solution like the standard stock solutions is injected into the HPLC column and the chromatograms are obtained.

EXPERIMENTAL PROCEDURE The main objective of the project is to analyse the drug Ibuprofen and validate the analytical method. PROCEDURE: The Agilent Technologies 1200 series HPLC system was used for the reverse phase HPLC of the Ibuprofen. The sample and the standard solutions are prepared as said in the above section. The mobile phase prepared was kept for degassing using the degasser. Once the mobile phase is degassed the column is attached to it. The HPLC system is switched on by switching the pump and detector. A method was created for the selected drug in the system. The wavelength was adjusted to 220nm. The flow rate was adjusted to 1.0ml/min starting from 0.5ml/min. The base line was stabilised first before injecting the samples. The sample and the standard solutions are then taken into the syringe and injected in to the HPLC column. Parameters like Linearity, Limit of detection, Limit of quantification, Accuracy are determined and for each parameter the data is obtained separately. FOR LINEARITY: For linearity determination there should be at least five standard solutions. In this experiment there are five standard solutions. The standard and sample solutions are taken into the syringe and are injected in to the HPLC system. A sequence was created so that each of the solution is analysed five times each and the chromatograms are taken. From the chromatograms a graph was plotted of peak versus concentration of the Ibuprofen using regression analysis. From this graph the slope, Y-intercept and the correlation coefficient that is r2 are determined. LOQ and LOD are also calculated from the same data as used for linearity. FOR ACCURACY Accuracy was also reported using the same data.

LINEARITY
Linearity determination in HPLC is done as a part of validating the entire method. It is one of the important parameters of the method validation. Linearity in HPLC can be determined by injecting at least five concentrations of the standard solution in the range from above or below the sample concentration so that the sample concentration falls with in the standard solutions range. In this method five standard solutions of Ibuprofen are prepared from the standard stock solution of the concentrations of 0.4, 0.5, 0.6, 0.7 and 0.8mg/ml. Linearity for Ibuprofen was determined with in 0.4mg0.8mg/ml and the following data of results are obtained from the chromatograms. The standard deviation is calculated using the formula Standard deviation = [(x1-x)2+ (x2-x)2/n Where n is the number of variables X1= Variable number 1 X2= Variable number 2 X is the mean of variable value RSD that is relative standard deviation can be calculated using the formula RSD= SD/MEAN 100 The standard and the relative standard deviations for each solution were calculated using the above formula taking the peak areas. The sample and the standard solutions as said in the experimental procedure are injected into the HPLC and the chromatograms are taken eight times each for the determination of linearity. The same data is used for the determination of accuracy and limit of detection and limit of quantification.

DATA TABLES:

Number

Concentration mg/ml

Peak area 753.5662 742.3117 753.6782 747.4132 768.5170 953.437 922.776 928.500 921.924 915.974 1153.459 1156.105 1155.137 1159.488 1156.476 1331.167 1332.486 1333.511 1333.221 1339.170

Mean

SD

0.4

752.6

9.8135

0.5

927.8

14.7352

0.6

1155.8

2.21261

0.7

1333.6

3.0761

0.8

1558.394 1536.078 1521.190 1530.832 1530.009

1530.6

6.3179

The retention time for the Ibuprofen from the chromatograms obtained was found to be 2.98min. The linearity is determined by plotting the graph in excel. The calibration graph was plotted in excel taking the concentration of the standard solutions on the X-axis and peak areas obtained on the Y-axis. The graph is plotted using the regression analysis tool. Linearity is correctly determined by the regression coefficient R2 which should be 0.999. From the graph obtained the Regression coefficient was found to be 0.9994 which is very close to 1. This suggest that our data for linearity are with in the specified range and the linearity obtained is good. The chromatograms for linearity are attached at the end of the calculations page.

DETECTION LLIMITS
The limit of detection is the lowest quantity of the substance that can be differentiated from the absence of the substance that is the blank. Limit of detection is another performance parameter measure as a part of the method validation in HPLC. Limit of detection can be calculated using the formula LOD=3 SD/s where SD is the standard deviation and s is the slope. From the graph plotted the slope obtained was 1.916 10tothe power 5. From the peak areas obtained for each of the standard solutions the Standard deviations were calculated. Number 1 2 3 4 5 Concentration Mg/ml 0.4 0.5 0.6 0.7 0.8 SD 9.8315 14.7352 2.2161 3.0761 6.3179

Among the values obtained for standard deviation the highest value was selected to calculate the LOD and LOQ So LOD = 3 SD/s =3 14.7352/1.916 10 power 5 =23.07 10 power -5 =2.307 10 power -4mg/ml

Mg/L

=2.307 10 power -1 mg/l =0.23 mg/L -1 =0.00023mg/ml

Limit of quantification: The limit of quantification is the lowest amount of the analytical sample which can be determined quantitatively with precession and accuracy. Limit of quantification can be calculated using the formula 10 SD/s SD is standard deviation which is 14.7352 Slope is 1.916 10 power 5 LOQ = 10 14.7352/1.916 10 power 5 =76.91 10 power -5 =7.961 10 power -4mg/ml Mg/L =7.961 10 power -1 mg/l =0.77 mg/L -1 =0.00077mg/ml

ACCURACY
Accuracy is one among the several parameters measured during method validation. Accuracy determination is measuring how close the experimental value obtained to the true value. Accuracy is measured by analysing the sample of known concentration and by comparing the measured value with the true value. Accuracy is usually expressed in terms of percentage recovery which is defined by the formula Percentage recovery = Actual concentration/ Assumed concentration 100 Weight of Ibuprofen tablets Number 1 2 3 4 5 6 7 8 9 10 Weight (gm) 0.4571 0.4412 0.4247 0.4612 0.4820 0.4814 0.4380 0.4612 0.4689 0.4072

11 12 13 14 15 16 17 18 19 20

0.465 0.474 0.535 0.532 0.464 0.514 0.483 0.465 0.503 0.517

Total weight of 20 Ibuprofen tablets was9.474 gm. Average weight of Ibuprofen tablet is 473.4 mg. 473.4 mg of Ibuprofen tablet contain 200mg of Ibuprofen. So 473.4 mg of powdered drug is mixed in 100ml of deionised water. 100ml = 200mg of Ibuprofen

METHOD DEVELOPMENT
Method development is a critical step for any analytical process. Different compounds react in a different manner for various analytical methods. There are various analytical techniques available like Gas chromatography, Liquid chromatography, column chromatography and the final method selected for any compound is based up on its ability to get analysed by the method selected. Various parameters like mobile phase composition, stationary phase composition, column diameters and the type of detectors used are considered during method development. In this method of validation of HPLC of Ibuprofen the HPLC techniques used was reverse phase. As per the literature the wavelength selected was 220nm. The column which was selected for our method was a 150 4.6mm S.No 81675 and type is Chromosil. The manual HPLC of Agilent technologies was used with a variable wavelength detector. The standard solution is then injected through the column and peaks obtained for the standard solution are very good and the retention time was found to be 2.98min. The flow rate from the beginning was maintained at 2.0ml/min and it remained the same during the entire process. The chromatograms are attached at the end.