Method Development and Validation of Famotidine Oral Suspension by RP-HPLC Method

For perseverance of Famotidine a simple, fast and selective procedure were developed in drug substance and its pharmaceutical preparations. In the proposed project, a successful attempt has been made to develop a simple, accurate, economic and rapid method for the estimation and to validate the method. As a result, a simple, economical, precise and accurate method was developed and validated by Reverse Phase High Performance Liquid Chromatography (RP-HPLC). The main objective for that is to improve the conditions and parameters, which should be followed in the development and validation. The developed Reverse phase HPLC technique was done utilizing iltered and degassed pH-6.0 Acetate buffer as a Mobile phase-A and pH-6.0 Acetate buffer andorganicmixture in the ratio of 30:70 as aMobile phase-B. By usingwaters X-Bridge C18 (150*4.6mm), 3.5μmcolumn chromatographic separation was achieved. The low rate and run time was 0.8mL/min and 45minutes. The detection wavelength was 265nm.The average percentage recovery for Famotidine related compound-Cwas found to be 94.3%, 95.9%, 96.0% represents the accuracy of themethod and for Famotidine related compoundD was found to be 95.8, 95.4 and 96.4. The %RSD for Famotidine related compound-C was found to be 5.576 and for Famotidine related compoundD was found to be 1.588 represents the precision of the method. The correlation coef icient square for Famotidne, Famotidine related compound-C and Famotidine related compound-D was found to be 0.999999, 0.9992 and 0.9991 respectively. Respective parameters met the acceptance criteria, from the results concluded that the developed method was precise and accurate.


INTRODUCTION
Famotidine is a competing suppressant or blocker of histamine H2-receptors.
Famotidine is a propanimidamide and H2receptor antagonist chemically called as 3-[[2-(diaminomethylideneamino)-1,3-thiazolyl-4]methylsulfanyl]-N'-sulfamoylpropanimidamide. It is white to pale yellow crystalline composite that is readily or amply solvable in glacial acetic acid, most moderately solvable in water, moderately solvable in methanol and almost insolvable in ethanol (Langtry et al., 1989). Famotidine is a competing suppressant or blocker of histamine H2-receptors, it hinder or prevent nocturnal gastric acid secretion and basal by competing blockage or prohibition of the activity of histamine at the histamine H2-receptors of the lateral cells and also hinder gastric acid secretion accelerated or excited by insulin, pentagastrin, food, caffeine, betazole and physiologic vagal re lex. Comparing to ranitidine famotidine is three fold high effective or dynamic and twenty times more effective when compared to cimetidine. Feeble inhibiting of hepatic cytochrome p450 mixed function oxidase system (Rockville and Convention, 1996), (Chicago, 1994). Famotidine is effective in boosting or facilitating the restoring of stomach and duodenal ulcers and additionally in diminishing ulcer agony (Kanayama, 1999) (Soga et al., 1999). High doses are utilized for healing circumstances in which there are characterized enhance or rise in acid excretion called Zollinger-Ellison syndrome, when provided in low doses for prolonged periods of time it has been ef icient in inhibiting or stopping repetition of ulcers (Borody et al., 1995) , (Hu et al., 2003). Famotidine additionally is utilized for healing heartburn and in treating or restoring ulceration and in lammation of the esophagus emerging from acid (Kirika et al., 2004) (Fujiwara et al., 2005). Prior or earlier operation famotidine provided to surgery patient (Escolano et al., 1992) to diminish the chance of aspiration pneumonitis (Vila et al., 1991) (Jahr et al., 1991) .

Method Development
Documentation or authentication and method development plays crucial part in development analysis and production of pharmaceuticals. Method development needs a lot of efforts and implies functioning on several concepts or thoughts concurrently and therefore eventually choosing one of those (Sethi, 2001) (Shethi and Hplc, 1996). Method development employed to make sure or secure the ef iciency of drug products, identi ication, potency and purity. There are several steps concerned in development process are:  (Sankar, 2006) (Breaux et al., 2003) 6. Documentation statement (Sankar, 2006) (Breaux et al., 2003)

Chemicals and Reagents
The utilized pharmaceutical preparation Famotidine Oral Suspension (Equivalent to 40mg) were formulated in-house. Famotidine API with a potency 99.68% were used. All reagents utilized were of an analytical grade. Methanol HPLC grade were procured from Finar Limited and Acetonitrile HPLC grade were procured from MerckLimited and water for HPLC ELGA puri ication system.

Instrumentation
Method development and validation was performed on HPLC instrument equipped with UV-detector using waters X-Bridge C 18 (150*4.6mm), 3.5µm column chromatographic separation was achieved. The injection volume was 20µL. The run time was set 45minutes and low rate 0.8mL/min and wavelength selected was 265nm. The Empower Software is used for processing data. Chromatographic parameters are shown in Table 1 and gradient program in Table 2.

Buffer Preparation
Acetate Buffer pH 6.0 The solution was prepared by dissolving 13.6 g of sodium acetate trihydrate in 1000 mL of water. Mixed well and then the solution adjusted to pH 6.0±0.05 with glacial acetic acid, then the solution iltered through 0.45 µm membrane ilter and sonicated the buffer solution to degas.

Phosphate Buffer pH 7.0
The solution was prepared by dissolving 13.6 g of sodium dihydrogen phosphate monohydrate in a suitable container containing 1000 mL of water. Mixed well and then the solution iltered through 0.45 µm membrane ilter and sonicated the buffer solution to degas.

Preparation of Organic Mixture
The organic mixture was prepared by mixing ACN: Methanol in the ratio of 80:20 and sonicated for 5 minutes to degas.

Preparation of Diluent
The diluents was prepared by mixing 900mL of pH 7.0 Phosphate buffer and 100mL of Organic mixture into suitable container and then sonicated to degas.

Mobile Phase -A
Used iltered and degassed pH 6.0 Acetate buffer as a Mobile Phase-A.    To verify the content of Famotidine related Compound-C and related compound-D 0.45µm Nylon/2mL discard 1 0.45µm Nylon/4mL discard 1 0.45µm Nylon/6mL discard 1 0.45µm Nylon/8mL discard 1

Figure 1: Optimized Chromatogram of Famotidine Standard Solution
The mobile phase was prepared by mixing 300 mL of pH 6.0 Acetate buffer and 700 mL of Organic mixture into a suitable container and thensonicated to degas.

Procedure Standard Stock Preparation
40.37mg of Famotidine RS was weighed and transferred into a 250mL volumetric lask. To that 3/4th volume of diluent was added. Sonicated to dissolve, diluted to volume with diluent and mixed well.

Standard Preparation
Pipetted out 2mL of Famotidine Standard Stock solution into 100mL volumetric lask. Diluted to volume with diluent and mixed well. An optimized chromatogram is shown in Figure 1

Preparation of Sample Solution
Transfer 5.0mL of sample into a 250-mL volumetric lask and noted down the weight of sample in mg. (Equivalent to about 40 mg of Famotidine). Added 150mL of diluent and then spiked the 10mL of Impurity-C and Impurity-D stock solution into the same sample solution.Further sonicated to 15 min-

Preparation of Placebo Solution
Transfer 5.0mL of sample into a 250-mL volumetric lask and noted down the weight of sample in mg.
(Equivalent to about 40 mg of Famotidine). Added 150mL of diluent and further sonicated to 15 minutes with frequent intermittent shake. After the sonication, diluted to volume with diluent and mixed well. Centrifuged the sample for about 5 minutes and collected the supernatant. Filtered the clear aliquot through 0.45-µm Nylon syringe ilter and collected the iltrate after discarded the irst 4mL of iltrate.

Initialization of the Instrument
Initially the column was positioned on the instrument and switch on the instrument and column

Validation of Developed Method
As stated by ICH guidelines the optimized technique was validated. In accordance with above developed technique, the mobile phase were prepared and organized all parameters.

Evaluation of System Precision
System precision was tested by injecting 6 replicates of Famotidine standard.The %RSD of peak area of

Acceptance criteria
The tailing factor for famotidine peak in standard preparation should not be more than 2.0. The theoretical plate count for famotidine peak in the stan-dard preparation should not be less than 5000. The relative standard deviation for the area of famotidine peak from six replicate injections of standard solution should not be more than 2.0%.

Linearity
Linearity was performed in the concentration of LOQ(10%), 30%, 50%, 80%, 100%, 120%, 150% of working concentration of respective Famotidine, Famotidine related compound C and Famotidine related compound D average area for each level was recorded and slope, y-intercept & correlation coef icient was calculated. Graph was plotted for respective analyte peak concentration on x-axis and area response on y-axis. Linearity graphs are shown in Figures 2, 3 and 4.

Standard Stock Preparation
40.37mg of Famotidine RS was weighed and transferred into a 250mL volumetric lask. To that 3/4th volume of diluent was added. Sonicated to dissolve, diluted to volume with diluents and mixed well.

Standard Preparation
Pipetted out 4mL of Famotidine Standard Stock solution into 200mL volumetric lask. Diluted to volume with diluent and mixed well.

Acceptance criteria
The correlation coef icient should not be less than 0.98 for famotidine.

Famotidine Related Compound-C Stock Preparation
2.12mg of Impurity-C was weighed and transferred into a 100mL volumetric lask added 75mL of diluent and sonicated to dissolve. After sonication diluted to volume with diluent and mixed well.

Famotidine Related Compound-C Preparation
Pipetted out 4mL of Impurity-C Stock solution into 100mL volumetric lask. Diluted to volume with diluent and mixed well.

Famotidine Related Compound-D Stock Preparation
2.21mg of Impurity-D was weighed and transferred into a 100mL volumetric lask added 75mL of diluent and sonicated to dissolve. After sonication diluted to volume with diluent and mixed well.

Famotidine Related Compound-D Preparation
Pipetted out 4mL of Impurity-D Stock solution into 100mL volumetric lask. Diluted to volume with diluent and mixed well.

Acceptance criteria
The correlation coef icient should not be less than 0.98 for famotidine related compound-C and related compound-D.

Method Precision
Method precision was evaluated by injecting a blank, standard, six sample injection and one bracketing standard injection.

Acceptance Criteria
The %RSD for %Impurity from six (6) sample preparations should be NMT 10.0.

Solution Stability
Stability of standard and sample solution was demonstrated by injecting standard and sample solution with different time interval from the time of preparation. A solution was injected once in initial, 12 hours, 24 hours, 48 hours, 72 hours and 96 hours. The stability of solution shall be decided based on the area obtained at different time interval. If the results are not meeting the acceptance criteria within the time interval speci ied, the test can be discontinued and reported the hours up-to which the solution is found to be stable.

Acceptance Criteria
1. The %difference in area between initial and time points should be NMT 25.0 for Standard.
2. The %difference in %Impurity between initial and time points should be NMT 25.0 for sample.

Acceptance Criteria
The %difference in Peak area for Impurity between the centrifuged sample and iltered sample should be NMT 25.0. Injection sequence for ilter study is shown in Table 3.

Speci icity
No interference should be observed from diluents, placebo and all known Impurities at the retention time of Famotidine peak.

Accuracy
Accuracy shall be assessed using 3 concentrations 50%, 100%, 150% by preparing triplicate sets of sample solutions. The active can be added to placebo at 50%, 100%, 150% concentrations. At each concentration, the average result shall be expressed as a percentage.
2. The %RSD for recovery of triplicate preparations at each level should be NMT 10%.

Inference
The system suitability parameters were within the acceptance criteria and the results are presented in     This method is to be employed on Famotidine oral suspensions for the purpose of determining the RS method.

Observation
The Correlation coef icient square (r 2 ) of Famotidine, Impurity-C and Impurity-D was found to be 0.999999, 0.9992 and 0.9991 respectively.

Report
The Correlation Coef icient Square (r 2 ) for Famotidine, Impurity -C and Impurity-D were met the acceptance criteria of not less than 0.998. The linear regression data shows that the method is linear over the entire concentration range (LOQ (10%)-150%) and it is adequate for its intended concentration range and results are shown in Table 5.

Observation
The S.D and %RSD of Impurity-C was found to be 0.027 and 5.576 then for Impurity-D 0.008 and 1.588 respectively.

Report
The %RSD for %Impurity from six (6)-sample preparations of Impurity-C and Impurity-D is less than 10 and the results are given in Table 6, hence the method is precise.

Report
1. Overall average recovery for Famotidine related compound-C is between 80.0-120.0%.
2. The %RSD for recovery of triplicate preparations at each level is NMT 10% and hence the method is accurate, results are presented in Table 7.

Report
1. Overall average recovery for Famotidine related compound-D is between 80.0-120.0%.
2. The %RSD for recovery of triplicate preparations at each level is NMT 10% and hence the method is accurate, results are presented in Table 8 Report 1. The %difference in area between initial and time points is NMT 25.0 for standard.
2. The %difference in %Impurity between initial and time points is NMT 25.0 for sample solution and results are reported in Tables 9 and 10.
All results met the acceptance criteria. Based on above results, it is concluded that standard and sample solutions were stable up to 96 hrs respectively when stored at Room temperature.

Speci icity
No interference was observed from diluents, placebo and all known Impurities at the retention time of Famotidine peak.

Report
The %difference in Peak area for Impurity between the centrifuged sample and iltered sample is NMT 25.0. Datas are reported in Table 11.

DISCUSSION
In the proposed project, a successful attempt has been made to develop a simple, accurate, economic and rapid RP-HPLC method for the determination of Famotidine Oral suspension, Famotidine related compound-C and related compound-D in pharmaceutical formulations. The method has been validated as per the guidelines given by ICH requirements to assure that the method consistently meets the predetermined speci ications and quality attributes. The average percentage recovery for Famotidine related compound-C was found to be 94.3, 95.9, 96.0 represents the accuracy of the method and for Famotidine related compound-D was found to be 95.8, 95.4 and 96.4. The %RSD for Famotidine related compound-C was found to be 5.576 and for Famotidine related compound-D was found to be 1.588 represents the precision of the method. The correlation coef icient square for Famotidne, Famotidine related compound-C and Famotidine related compound-D was found to be 0.999999, 0.9992 and 0.9991 respectively. Respective parameters met the acceptance criteria, from the obtained results concluded that the developed method was precise and accurate.

CONCLUSIONS
All respective validation parameters met the acceptance criteria and it was concluded that the Related substance determination of famotidine in oral suspension by using pH 6.0 Acetate buffer as mobile phase-A and pH 6.0 Acetate buffer: Organic mixture (30:70) as mobile phase-B. pH 7.0 Phosphate buffer: Organic mixture (90:10) is diluent. The separation is achieved by using column Waters, X-Bridge C18, (150*4.6mm), 3.5µm and low rate is 0.8mL/min. Detection wavelength is 265nm. Hence this method can be used for related substance determination of famotidine in oral suspension formulation by precise and accurate manner. The inal resultant of the established RS method for perseverance of Famotidine indicates that the technique or procedure was precise, simple, accurate and reproducible. The developed HPLC technique indicates satisfying outcome with precision, linearity, speci icity and accuracy. Hence the method is precise and accurate.