INTRODUCTION:

Ibandronic acid¹ is a substance that acts on the central nervous system, e.g. narcotic, hallucinogen, barbiturate, or psychotropic drug² a substance that kills or inactivates disease causing infectious organisms³. A substance that affects the activity of a specific bodily organ or function⁴. According to the Food and Drug Administration (FDA), a drug is a substance intended for use in the diagnosis, cure, mitigation, treatment or prevention of disease or to affect the structure or function of the body⁷. Ibandronic acid is a potent bisphosphonate drug used in the prevention and treatment of osteoporosis. It may also be used to treat hypercalcemia (elevated blood calcium levels). Ibandronic acid is more effective than pamidronate in lowering Serum calcium in patients with severe Hypercalcemia of Malignancy (HCM), and has at least equal efficacy to Pamidronate in HCM Patients with Lower Baseline Calcium Levels¹⁰.

A literature survey reveals that only a few methods have been developed for the quantification of individual drug Ibandronic acid that too only by potentiometric analytical techniques and no easy, accurate and fast techniques by HPLC methods were till date available, hence the method was thought to be developed by HPLC.

There were no simple and reproducible methods so far reported for determination of Ibandronic Acid by RP- HPLC in solid dosage form⁷.

It was essential to develop simple, precise, accurate HPLC method for determination of this drug in solid dosage form. Therefore, in this study we developed reproducible method which can be used in laboratory. HPLC method can be applied in future for estimation of same drugs even from biological samples. The validation of this method carried out as per ICH guidelines.

MATERIAL AND METHODS:

Reagents and Materials

Pharmaceutical grade Ibandronic acid was used⁵. All chemicals and solvents of HPLC grade and were purchased from Qualigens fine Chemicals, Mumbai, India⁸. Water HPLC grade was obtained from a Milli-Q RO water purification system.

Instrumentation of HPLC⁸

Agilent 1100 series HPLC system was used, LC system used consisted of

VWD - Detector - G1314A Serial # JP92111193

Autosampler - ALS G1313A Serial # DE91611467

BIN Pump – G1312A Serial # DE70301025

Degasser – G1322A Serial# JP05026555

OVEN Colcomp – G1316A Serial# DE64302313

The column used was C₁₈ (25 cm Χ 4.6 mm i.d., 5µm particle size) Qualisil, BDS, at ambient temperature. Different mobile phases were tested in order to find the best conditions⁶.

The optimal composition of the mobile phase was consisted of EDTA and Hexane sulphonic acid buffer by adjusting pH (pH 2.5) and acetonitrile in ratio of (95:5 % v/v) was delivered at the flow rate of 1.0 mL/min and UV detection was carried out at 195 nm. due to better absorbance of the component spectrum observed.

IN SOLID DOSAGE FORM BY RP-HPLC⁴

Preparation of working solutions

Stock solution was prepared by dissolving 25.0 mg of Ibandronate Sodium (WS) in 25 mL volumetric flask and added 10ml of water and mixed and final dilution made to 25 ml with water to get a concentration of 1.0 mg/ml, and a volume of 20 µL is injected. The injection were done five times and calibration curve was constructed by plotting the peak area ratios of analyte versus the corresponding drug concentration ⁶.

All solutions were stored at + 20⁰C, these solutions were shown to be stable during the period of study. From the above stock solutions, Final concentration of 1.0mg/ml was prepared and a volume of 20 µL of the working standard was injected into column to check system suitability, for results refer Table 1.

System Suitability: Table 1

System suitability testing is performed to ensure system performance before and during analysis, which demonstrates that system is operating properly and is ready to deliver results with acceptable precision.

Standard Solution

Weigh accurately and transfer 25mg of Ibandronate sodium WS into 25ml volumetric flask. Dissolve and dilute to volume with water. (1mg/ml of std. Ibandronate Sodium)

Sr. No	Peak area Obtained of Ibandronate Sodium
1	751.84882
2	751.17963
3	756.72455
4	751.22809
5	757.12677
Avg	753.62157
SD	3.03106
% RSD	0.402

Remark :

· Relative standard deviation of Peak Area Obtained is found to be within the acceptable limits.

· Hence complies for the System suitability study for Ibandronate Sodium.

From the above Stock Solution dilutions were made, of working standard to get the concentration range of Ibandronic acid 0.5 - 1.5 µg/ml and a volume of 20 µL of each concentration of working standard was injected into column and Linearity was done refer Table 2.

Linearity: Table 2

The linearity of analytical procedure is its ability to obtain test results, which are directly proportional to concentration of analyte in sample.

Accurately weigh and transfer following quantity of Ibandronic Sodium WS into 25ml volumetric flask, respectively as follows.

a. 50% 12.5mg of Ibandronate Sodium -- 25ml with water. (0.5mg/ml of Standard Ibandronate Sodium)

b. 75% 18.75mg of Ibandronate Sodium -- 25ml with water (0.75mg/ml of Standard Ibandronate Sodium)

c. 100% 25.0mg of Ibandronate Sodium -- 25ml with water. (1.0mg/ml of Standard Ibandronate Sodium)

d. 125% 31.25mg of Ibandronate Sodium -- 25ml with water (1.25mg/ml of Standard Ibandronate Sodium)

e. 150% 37.5mg of Ibandronate Sodium -- 25ml with water (1.5mg/ml of Standard Ibandronate Sodium)

Sample	Conc. in mg/ml	Peak Area obtained of Standard Ibandronate Sodium			Average Peak Area
A.(50%)	0.5	375.4476	372.3168	371.95444	373.23961
B. (75%)	0.75	562.36884	561.1759	563.69843	562.41439
C. (100%)	1	753.34802	756.57776	754.3385	754.75476
D. (125%)	1.25	940.24512	941.57501	940.45654	940.75889
E. (150%)	1.5	1133.31128	1132.3385	1135.43994	1133.69657

Plot graph between Concentration of standard Ibandronate Sodium obtained on X axis and peak area obtained on the Y axis.

Linear Regression: Y = (Slope) X + Intercept

Y = 759.7X – 6.7305

Correlation Coefficient(r) : = 1.0

Remark:

· Graph plotted between Area Vs Concentration was found to be linear.

Coefficient of correlation was within the acceptable range.

Accuracy: Table 3

RESULTS FOR ACCURACY STUDY

Observations and Readings :
Standard Weight taken :	25.0mg
Standard Peak Area Obtained :	833.11074
Standard Purity :	99.24%

No.	Added mg	Peak Area obtained of IBANDRONATE SODIUM			Average Peak area	Found mg	Recovery %	RSD %
1	80	722.74933	717.74469	721.15527	720.54976	80.320	100.40	0.05
2	90	812.33167	810.11035	808.47382	810.30528	90.325	100.36
3	100	903.48834	898.59558	899.76239	900.61544	100.392	100.39
4	110	991.94482	987.63763	989.12244	989.5683	110.308	100.28
5	120	1072.18787	1085.57324	1083.63306	1080.4647	120.440	100.37

Remark:

· Average Recovery of Ibandronate Sodium is found within the acceptable imit.

% RSD of recovery is within acceptable limit

Repeatability (Precision): Table 4

Repeatability expresses the precision under the same operating conditions over a short interval of time.

Repeatability or Precision study is carried on 5 different samples of Cadronate 150mg tablets, and results are as follows:

Observations
Standard Weight :	25.2mg
Standard Purity :	99.24%
Standard Area :	805.29915

STD IBANDRONATE SODIUM		PEAK AREA OBTAINED
1		806.20221
2		806.31476
3		803.38049
AVG		805.29915
Std Dev		1.66
%RSD		0.21

Observations :
Sr.No.	Avg Spl Weight (gm)	Spl Weight taken (gm)	Area of Ibandronate Sodium	Mg of Ibandronate Sodium	% Of L.C.
1	0.6025	0.4042	873.30481	151.319	100.88
2	0.6041	0.409	879.8009	151.055	100.70
3	0.6068	0.4042	876.09424	152.885	101.92
4	0.6019	0.4079	875.15784	150.115	100.08
5	0.6056	0.4083	874.73059	150.816	100.54
				% MEAN :	100.83
				% SD :	0.68
				%RSD:	0.68

Remark :

· Values are found well within acceptable limits

· Hence method found to be precise.

Analysis of tablets

To determine the content of Ibandronic acid in tablets (label claim: 150 mg); twenty tablets were weighed; their average weight determined and were finely powdered in mortar and pestle . The correct amount of powder equivalent to 25mg of Ibandronate sodium was weighed in 25ml Volumetric flask and further dissolved in water and sonicated for 10 min., the final volume was made up with water and mix⁵.

The excipients were separated by filtration. After filtration, Further dilutions are made with mobile phase to get working standard solution and Sample solution containing 1.0 mg/mL of Ibandronic acid.

Validation:

The methods were validated with respect to Specificity, System Suitability, linearity, accuracy, precision (Repeatability and ruggedness) ^4,5.

The specificity test was performed by injecting sequentially the Product placebo, Standard Ibandronic Solution and Sample Solution and observed no interference of blank (Water) and Placebo at the retention time of Ibandronate Sodium. Refer AMV Report (1.Selectivity).

On the standard solution System suitability testing is performed to ensure system performance before and during analysis, which demonstrates that system is operating properly and is ready to deliver results with acceptable precision ⁹. Refer AMV Report ( 2. System Suitability).

The Linearity of Measurement was evaluated by analyzing different concentration of the standard Ibandronate Sodium i.e. Concentration range was 0.5, 0.75, 1.0, 1.25 and 1.5mg/ml and the linear regression was calculated found Correlation coefficient 1.0. Refer AMV Report (3. Linearity).

To ascertain the accuracy of the proposed method experiments were carried out by standard addition method at 80, 90, 100, 110 and 120 % level by spiking in the product placebo. From the total amount, drug recovery found was 100.28 to 100.40%, the percentage recovery RSD % was calculated and found 0.05%. Refer AMV Report ( 6. Accuracy).

The repeatability of this sample solution was determined by injecting five times and recorded the chromatograms the results obtained were in the range from 100.08 to 101.92 and RSD % observed 0.68%. Also it was tested for Intermediate precision on different day, analyst and Instrument and using same lot of sample, the %RSD observed was 0.24%. Refer AMV Report (7.Precision (Repeatability) and 8.Precision (Intermediate precision).

CONCLUSION:

After the review of Analytical Method Validation report and observing the results obtained, it is summarized that the methods discussed in the present work provide a convenient and accurate way for analysis of Ibandronic Sodium in its pharmaceutical tablet dosage form ⁹.

REFERENCES:

Journal Articles:

1. Snyder L. Kirkland J., and Glajch J., in “Practical HPLC Method Development”, 2^nd Edition -Published by John Wiley and Sons, Inc. (New York), 1997.

2. Skoog D.A., West D.M., Holler J.F in “Fundamentals of Analytical Chemistry”, 7^th edition -Published by Saunders College, U.S.A., 1996.

3. Sanjay Kumar R., Karthikeyan C., Moorthy N. S. H. N., Trivedi P., Indian Journal of Pharmaceutical Sciences, 2006, Vol. 68.

Book Chapter:

4. Analyst, July-1997, Vol. 122, (83R- 88R).

5. Kubala T, Gambhir B., Borst S. I., Drug development and industrial pharmacy , 1993, Vol. 19, 749-757.

6. Gurdeep. R. Chatwal and Sham K. Anand. Instrumental Method of Chemical Analysis, 5^th edition

Books:

7. Published by John Wiley and sons, Chromatographic Separations by Peter A. Sewell and Brian Clarke, , 1987

8. Published by CBS publishers and Distributors, Instrumental Method of Analysis, 6^th edition–Author Willard Merritt and Dean Settle.

Web Abstracts:

9. ICH, Guidelines for Validation of Analytical Methods, CDER, Rockville, URL : http://www.chromatographyonline.com/ lcgc/data/articlestandard/lcgc/492001/3266/article.pdf

10. Pecherstorfer Renal safety of intravenous ibandronate 6mg infused over 15 or 60 minutes in patients with breast cancer and bone metastases: A randomized, open-label study. Meeting: 2007 ASCO Annual Meeting, No:1114 First Author: R. von Moos Category: Breast Cancer - Metastatic Breast Cancer - Metastatic Breast Cancer URL:http://www.ncbi.nlm.nih.gov/pubmed /12783289

Received on 28.08.2013 Modified on 15.09.2013

Asian J. Research Chem. 6(11): November 2013; Page 1003-1006