Method Development and Validation of Fludrocortisone Acetate Tablets by Reverse Phase HPLC Method

The purpose or intent of this current studywas to establish a fast and sensitive HPLC technique for the perseverance of Fludrocortisone acetate and utilizing best frequently used HPLC technique. This method had been validated as per the ICH requirements to assure that themethod consistentlymeets the predetermined speci ications and quality attributes.Utilizing iltered and degassed pH 3.0 Phosphate buffer and Acetonitrile in the ratio 90:10 as a Mobile phaseA and pH 3.0 Phosphate buffer and Acetonitrile in the ratio 65:35 as a Mobile phase-B the established RP-HPLC technique was done. The separation was achieved by using Waters, X-Bridge Shield RP18, (150 X 4.6-mm), 3.5-μm column. Run time and Flow rate was set 45minutes and 1.2mL/min. Injection volume100μL andwavelengthwas set 240nm.The correlation coef icient square for ludrocortisones acetate and Fludrocortisone Impurity was found tobe0.9991and0.99997. TheSDand%RSD forFludrocortisone Impuritywas found to be 0.02 and 1.48 represents method precision. Following validated parameters lies within the limit. Hence, the developed method was precise, simple, fast and accurate.


INTRODUCTION
Disorder and then postural hypotension. It is usually consumed along with hydrocortisone in case of adrenal inadequacy and medication is utilized utmost in its acetate form (Elks, 2014;Index Nominum , 2000;Fludrocortisone Acetate, 2016;Day and and, 2010). Fludrocortisone acetate is an adrenal corticosteroid and has controlled level of glucocorticoid action and has extremely maximum level of mineralocorticoid action. Though it is utilized just for its mineralocorticoid issues. Mineralocorticoid functions in the distal tubules to enhance hydrogen ion evacuation, sodium resorption, to enhance potassium evacuation and later water retention. In further secretory cells cation transfer is equally in luenced, evacuation of water by sweat glands and salivary glands is also modiied, though to a lower degree. At the cellular levels corticosteroids spread throughout or over cell membranes and intricate with particular or individual cytoplasmic recipient, such complexes afterwards join the cell nucleus attach to DNA and encourage arrangement of mRNA and later protein combination of different enzymes thought to be inally liable for the physiologic issues of such hormones (Lemke and Williams, 2008;Polito et al., 2016). Fludrocortisone is utilized mainly to substitute the lacking hormone aldosterone in diverse forms of adrenal inad-equacy for instance classic salt squandering form of congenital adrenal hypertrophy and Addison's illness. It has been utilized in the therapy of cerebral salt squandering disorder. Because of its issues on enhancing sodium levels and hence blood volume and then used to heal postural hypotension and to heal low blood pressure (Taplin et al., 2006;Freitas et al., 2000).

Method Development
Method development is the procedure or method of choosing a precise testing process to establish the constitution of a preparation. Analytical process must be utilized under GLP and GMP conditions and utilizing the customs or properties ixed out in the ICH guidelines the analytical procedure should be established. It is the method of evidence that an analytical procedure is tolerable for employ in laboratory to assess the compactness of consequent specimens. Suitable and quanti ied or measured instruments, Certi ied procedure, Change control, Dependable reference standard, Competent analysts, Integrity and Specimen choice (ICH, 2000;European Commission, 2001;Mcdowall, 2005).

Chemicals and reagents
Fludrocortisone Acetate tablets (Equivalent to about 2.5mg) the used pharmaceutical preparation were formulated in-house and used Fludrocortisone Acetate API with a potency 100%. HPLC grade Acetonitrile were purchased from Merck limited and HPLC grade Methanol were purchased from Finar Limited and HPLC grade water. Analytical grade reagents were used.

Instrumentation
Automated Reverse Phase HPLC equipped with UVdetection chromatographic separation was achieved utilizing Waters, X-Bridge Shield RP18, (150 X 4.6mm), 3.5-µm column. Run time and Flow rate was set 45minutes and 1.2mL/min. Injection volume 100µL and wavelength was set 240nm. For processing data Empower software were used.
The chromatographic Parameters and gradient progam are listed in Tables 1 and 2. From Table 3, developed technique were met the acceptance criteria. Hence the developed method was found to be accurateTables 4, 5, 6, 7, 8 and 9 .
The correlation coef icient square (r 2 ) for Fludrocortisone Acetate and Fludrocortisone Impurity were met the acceptance criteria. The linear regression data shows that the method is linear over the entire concentration range (LOQ (10%)-150%) and the developed method was found to be precise and accurateFigures 1, 2 and 3.
From Figure 4, developed method were met the acceptance criteria. Hence the developed method was found to be accurate and precise.
Overall average recovery for Fludrocortisone Impurity is between 80.0-120.0%.The %RSD for recovery of triplicate preparations at each level is NMT 10% and hence the method is accurate No interference was observed from diluents, placebo and all known Impurities at the retention time of Fludrocortisone Acetate peak.
The %difference in area between initial and time points should be NMT 25.0 for Standard. The %difference in %Impurity between initial and time points should be NMT 25.0 for sample.
All results met the acceptance criteria. Based on above results, it is concluded that standard and sample solutions were stable up to 96 hours respectively when stored at Room temperature.
The %difference in Peak area for Impurity between the centrifuged sample and iltered sample should be NMT 25.0.

Preparation of Buffer
Phosphate Buffer pH 3.0 The buffer was prepared by dissolving 2.07 g of sodium di-hydrogen phosphate monohydrate in 1000 mL of water. Mixed well and then the buffer adjusted to pH 3.0±0.05 with diluted phosphoric acid, then the solution iltered through 0.45 µm membrane ilter and sonicated the buffer solution to degas.

Mobile Phase -A
900mL of pH 3.0 Phosphate buffer and 100mL of Acetonitrile into a suitable container and then sonicated to degas.

Mobile Phase-B
650 mL of pH 3.0 Phosphate buffer and 350 mL of Acetonitrile into a suitable container and then sonicated to degas.

Preparation of Diluent
Used Mobile Phase-B as a diluent.

Procedure Standard Stock Preparation
50.23mg of Fludrocortisone Acetate RS was weighed and transferred into a 100mL volumetric lask. To that 3/4th volume of diluent was added. Sonicated to dissolve, diluted to volume with diluent and mixed well.

Intermediate Stock Preparation
Pipetted out 6mL of Fludrocortisone Acetate Standard Stock solution into 100mL volumetric lask. Diluted to volume with diluent and mixed well.

Standard Preparation
Pipetted out 2.5mL of Fludrocortisone Acetate Intermediate Stock solution into 50mL volumetric lask. Diluted to volume with diluent and mixed well.

Preparation of Sample Solution
Randomly selected the twenty-ive (25) tablets and recorded the weight of tablets. Transferred the tablets into a 25mLof volumetric lask. (Equivalent to about 2.5 mg of Fludrocortisone Acetate) added about 15mL of diluent and sonicated the lask until the tablets dispersed. Then spiked the 0.75mL of Fludrocortisone Impurity stock preparation into the same sample solution. Further sonicated to 15 minutes with frequent intermittent shake. After the sonication, diluted to volume with diluent and mixed well. Kept the lask on bench top for about 10 min-

Intermediate Stock Preparation
Pipetted out 5mL of Fludrocortisone Acetate Standard Stock solution into 250mL volumetric lask. Diluted to volume with diluent and mixed well.

Standard Preparation
Pipetted out 5mL of Fludrocortisone Acetate Intermediate Stock solution into 100mL volumetric lask. Diluted to volume with diluent and mixed well.

Fludrocortisone Impurity Stock Preparation
2.52mg of Fludrocortisone Impurity was weighed and transferred into a 50mL volumetric lask added 35mL of diluent and sonicated to dissolve. After sonication diluted to volume with diluent and mixed well.

Fludrocortisone Impurity Preparation
Pipetted out 3mL of Fludrocortisone Impurity stock

Initialization of Instrument
By running mobile phase continuously stabilization of chromatographic system and saturation of column is done and baseline graph developed.

RESULTS AND DISCUSSION
The %difference in peak area for Impurity between the centrifuged sample and iltered sample was calculated and found less than 25.0. Based on ilter study data, it is concluded that samples should be iltered through 0.45µm Nylon ilter after discarding irst 4mL of iltrate.
The purpose or intent of current study was to establish a fast and sensitive HPLC technique for the perseverance of Fludrocortisone acetate and Fludrocortisone Impurity utilizing best frequently used HPLC technique. 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 system suitability parameter i.e., correlation coef icient square for ludrocortisones acetate and ludrocortisones impurity was found to be 0.9991 and 0.99997. The SD and %RSD for Fludrocortisone Impurity was found to be 0.02 and 1.48 represents method precision. Following validated parameters lies within the limit. Hence the developed method was precise,

CONCLUSIONS
The developed method was validated as per the ICH guidelines instructions. From the determined values it is concluded that the established technique was simple, fast, precise and accurate.