Ibandronate Tablets

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Jineetkumar B G et al. World Journal of Pharmacy and Pharmaceutical Sciences

DEVELOPMENT AND VALIDATION OF REVERSE PHASE HPLC

DISSOLUTION METHOD FOR DETERMINATION OF

IBANDRONATE SODIUM IN TABLET DOSAGE FORM

Jineetkumar B. Gawad*1

, Pritam S. Jain2, Atul A. Shirkhedkar

2, Sanjay J. Surana

2

1St. John Institute of Pharmacy and Research, Palghar (E), M.S India.

2R. C. Patel Institute of Pharmaceutical Education and Research, Shirpur (M.S.) India.

ABSTRACT

A simple, accurate and sensitive liquid chromatographic method has

been developed for the determination of ibandronate sodium drug

substance in tablet dosage form. The separation was achieved on

Hypersil BDS C18 (250mm X 4.6mm), 5µm column. The mobile

phase consisted of Buffer: ACN (95:05) v/v; flow rate 1.0 ml min−1

at

ambient temperature. The analytes were monitored by PDA detector.

The method was validated for specificity, precision, linearity, solution

stability and accuracy. The average recoveries for ibandronate were in

the range of 99.0–102.0% and the method can be successfully applied

for the routine analysis of ibandronate sodium drug substance.

KEY-WORDS: Reverse Phase HPLC, Ibandronate sodium, Stability indicating, Validation.

INTRODUCTION

Ibandronate sodium [(1-hydroxy-3-(methyl pentyl amino) propylidene bisphosphonic acid

monosodium monohydrate)] is the sodium salt of ibandronic acid, a synthetic nitrogen-

containing bisphosphonate drug.[1,2,3]

This new third generation bisphosphonate is used to

treat patients with bone disease like Paget’s disease, malignant hypercalcemia and

postmenopausal osteoporosis.[4,5,6]

For assay of ibandronate sodium, few analytical methods

have been reported. Indirect fluorescence detection was used in a high performance ion

exchange chromatographic method based on the formation of the non-fluorescent Al3+

-

ibandronate complex after post-column addition of the fluorescent Al3+

-morin reagent.[7]

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Article Received on

01 January 2013,

Revised on 20 January 2013,

Accepted on 28 January 2013

*Correspondence for

Author:

* Jineetkumar B. Gawad

St. John Institute of Pharmacy

and Research, Palghar (E), M.S

India.

[email protected]

mailto:[email protected]

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ibandronate was determined by high performance ion exchange chromatography with UV

detection at 240nm after complex formation with Cu2+

ion. Ibandronate sodium was

determined by capillary zone electrophoretic method within direct detection at 254nm, The

limit of detection (LOD) values reported for ibandronate was 352–1760µgml−1

. [8-13]

The aim of this study was to develop a simple, sensitive, precise liquid chromatographic

method with PDA detection for the determination of ibandronate sodium.

Fig. 1: Structure of ibandronate sodium

MATERIALS AND METHODS

Chemicals and reagents

The standard sample of ibandronate sodium drug substance was procured from Aarti Drugs

Ltd, Boisar-Thane. Analytical reagent (AR grade) Disodium Edetate, Sodium acetate

(trihydrate)Thomas Baker, Pentanesulfonic acid sodium salt HPLC Grade, Merck,

Triethylamine HPLC Grade, Rankem, Orthophosphoric acid (OPA) HPLC Grade, Potassium

dihydrogen phosphate, Rankem, Water HPLC grade procured from Milli-Q system.

High Performance Liquid Chromatography

Agilent HPLC 1200 series chromatograph equipped with binary pump, 2695 Photodiode

Array Detector with data processing capacity was used. A Hypersil BDS column C18 (250

mm x 4.6 mm, 5 mm) was used. The pH measurement was performed by using LAB INDIA-

PICO controlled pH analyzer equipped with pH electrode. Mobile phase filtration was

performed by vacuum pump using 0.45 μm filter paper. As a degasser, PCI Analytics Pathak

ultrasonicator was used. Typical operating conditions include flow rate, 1 ml/min; injection

volume, 20 μl; wavelength, 200nm; column compartment temperature, 350C; and operating

P

O

OH

O-

PO

OHOH

OH

N CH3

CH3Na

+

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condition, room temperature. The retention times of the ibandronate sodium peak is at about

26.79 min. Relative standard deviation for the peak areas of the six replicate injections for

ibandronate peak is not more than 1.0%.

Preparation of stock and standard solutions

Weigh accurately and transfer about 67 mg of ibandronate sodium to 50 mL of volumetric

flask. Add 30 mL of dissolution medium and sonicate to dissolve. Allow to equilibrate to

room temperature and dilute to volume with dissolution medium, mix. Dilute 2 mL with 25

mL with dissolution medium, mix.

Figure.2: HPLC chromatogram of standard (water)

Preparation of Sample

Pour 500 mL of dissolution medium in each vessel. Allow sufficient time for dissolution

medium to equilibrate at 370

C± 0.50

C. Adjust stirring element speed to 50 rpm with USP

type II apparatus . Place 1 tablet in each vessel taking care to exclude air bubbles from the

surface of dosage form unit. Start apparatus. At the end of specific time, withdraw 20 mL

aliquot from the zone midway between surface of dissolution medium and top of the rotating

paddle and filter through GFC filter, discarding first 5 mL of filtrate. Dilute 5 mL of solution

to 20 mL with dissolution medium.

Filter Compatibility

Sample solution was prepared by accurately weighing 100 mg dissolved in 100 ml diluent,

this solution was sonicated for 10mins in ice cold condition, volume made up to mark with

diluent. The following variations were carried out at the sample filtration stage: one aliquot of

the sample solution was centrifuged; other aliquots of the sample solution were filtered

through Whatman GF/C filter (Glass fiber), 0.45 μm PVDF (Polyvinylidene difluoride) and

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0.45 μm nylon filter. The obtained filtered solution was analysed. The absolute difference

between the results obtained with centrifuged solution and filtered solution were calculated.

Results are shown in Table No.5a,b

Filter Saturation

The saturation of 0.45 μm nylon filter was optimized by filtering and discarding 2.0 ml and

4.0 ml sample solution using three separate filters, followed by filtration of further 10 ml

aliquots and collection of the filtrates in three separate test tubes. Each sample was analyzed

and the results calculated. The absolute difference in the results obtained between two

consecutively filtered aliquots was calculated and the minimum volume of solution required

to saturate the filter was determined. Results are shown in Table No.6.

Figure.3: Overlay Chromatogram of Sample (water)

Figure.4: Overlay Chromatogram of Standard (0.1N HCl)

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Figure.5: Overlay Chromatogram of Sample (0.1N HCl)

Figure.6: Overlay Chromatogram of Standard (Acetate buffer pH 4.5)

Figure.7: Overlay Chromatogram of Sample (Acetate buffer pH 4.5)

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Figure.8: Overlay Chromatogram of Standard (Phosphate buffer pH 6.8)

Figure.9: Overlay Chromatogram of Sample (Phosphate buffer pH 6.8)

METHOD VALIDATION

Linearity

In order to prepare stock solution, 128mg Ibandronate sodium was accurately weighed,

dissolved in diluent with sonication and diluted to 100 ml with the diluent. The mobile phase

was filtered through 0.45-μm membrane filter and delivered at 1ml/min for column

equilibration; the baseline was monitored continuously during this process. The detection

wavelength was 200 nm. The prepared dilutions were injected in series, peak area was

calculated for each dilution, and concentration was plotted against peak area.

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Figure.10: Linearity of Ibandronate Sodium

Accuracy

The accuracy of an analytical procedure expresses the closeness of agreement between the

value which is accepted either as a conventional true value or an accepted reference value and

the value found. This is also termed as trueness. It was done by recovery study. Sample

solutions were prepared with 100% in triplicate.

System Precision (Repeatability)

Repeatability expresses the precision under the same operating conditions over a short

interval of time. Repeatability is also termed intra-assay precision. Solutions of Ibandronate

Sodium were prepared as per test method and injected for 6 times. The mean SD and RSD

were checked for precision.

Range

The range of an analytical procedure is the interval between the upper and lower

concentration (amounts) of analyte in the sample (including these concentrations) for which it

has been demonstrated that the analytical procedure has a suitable level of precision, accuracy

and linearity. Range to be inferred from the data of linearity, recovery and precision

experiments.

Analytical Solution Stability

The stability of the drug in solution during analysis was determined by repeated analysis of

standard and sample. The standard and sample were prepared and injected into HPLC at

initial and different time intervals up to 24 hrs.

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RESULTS AND DISCUSSION

Method Development and Optimization

As there is no chromophore present in ibandronate sodium, there was no possibility for UV or

fluorescence detection and no suitable groups are present for derivatization. Ibandronate

sodium; for this reason water was chosen as diluents. Preliminary experiments were carried

out Using Hypersil BDS C18 Column with Buffer: ACN (70:30) v/v (adjusted to pH 2 using

OPA) Ibandronate was lost peak shape while on Inertsil ODS Column, with Buffer: ACN

(40:60) v/v peak was tailed with asymmetry 2.41.Ibandronate was determine on Hypersil

BDS C18 column, peak was separated using phosphate buffer with pH 7.0 (1.75gm

pentanesulfonic acid sodium salt+100mg EDTA in 900ml of water +6ml TEA, dilute upto

1000ml, adjust pH with OPA); Buffer: ACN (95:05) v/v. Better resolution obtained using

acetonitrile as organic modifier. Satisfactory separation and good peak shapes were achieved

within a reasonable time using a mobile phase of 95:5% (v/v) mixture of buffer and ACN

with a flow rate of 1.0 ml min−1

. The effect of column temperature on separation was studied

at different temperatures ranging from 350C to 65

0C. Ambient temperature was found to be

optimal from the point of view of both resolution and peak shape.

Table 1: Result of Dissolution Study in Water

Time

in

min

Sample

area

%

Release

%

Relative/Withdraw

vol.

%

Cumulative

Mean

%

Min

%

Max

%

%

RSD

30 95756 102 2 102 104 105 102 1.9

10022 107 2 107

93130 100 2 100

10075 108 2 108

45 10624 114 2 110 100 102 100 1.0

10528 113 2 109

10447 112 2 100

10691 114 2 104

∞ 11023 118 2 110 102 100 101 2.0

11067 118 2 114

10321 110 2 105

10138 108 2 112

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Table 2: Result of Dissolution Study in 0.1N HCl

Time

in min

Sample

area

%

Release

% Relative/

Withdraw

vol.

%

Cumulative

Mean

%

Min

%

Max

%

%

RSD

30 10878 100 2 93 100 115 107 1.0

10195 101 2 101

10296 99 2 99

10215 101 2 98

45 10807 101 2 112 101 110 111 1.8

10146 102 2 103

10768 100 2 110

10570 102 2 105

∞ 10507 101 2 101 101 120 105 2.0

11152 100 2 103

10685 101 2 113

10764 102 2 114

Table 3: Result of Dissolution Study in Acetate Buffer pH 4.5

Time

in

min

Sample

area

%

Release

% Relative/

Withdraw vol.

%

Cumulative

Mean

%

Min

%

Max

%

%

RSD

30 10853 100 2 105 103 105 112 2.0

11474 105 2 111

11373 104 2 109

11484 106 2 104

45 11148 100 2 106 103 109 113 1.9

11234 102 2 100

11378 100 2 108

12004 105 2 104

∞ 11381 106 2 106 103 114 109 2.0

11225 102 2 103

11426 103 2 109

12354 104 2 101

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Table 4: Result of Dissolution Study in Phosphate Buffer pH 6.8

Time

in

min

Sample

area

%

Release

%

Relative/Withdraw

vol.

%

Cumulative

Mean

%

Min

%

Max

%

%

RSD

30 69173 91 2 92 98 95 100 2.0

70333 93 2 95

69902 94 2 102

71439 100 2 98

45 72024 99 2 97 100 92 105 3.0

64158 92 2 92

71125 102 2 99

70426 95 2 97

∞ 70162 91 2 93 103 100 108 2.0

74662 96 2 95

68912 101 2 94

70042 100 2 96

Table 5a: Filter Compatibility Study

Condition Sample Preparation

Centrifuged 10 mL of stock centrifuged at 4000 RPM for 5 min, 5 mL to 25 mL with

diluent

GFC 10 mL of stock filtered through GFC- filtrate is so hazy.

0.45µm

Nylon

10 mL of stock filtered through 0.45 µm nylon, 5 mL to 25 mL with

diluent.

Whatman 41 10 mL of stock filtered through whatman 41- filtrate is so hazy.

Whatman 42 10 mL of stock filtered through whatman 42- filtrate is so hazy.

Table 5b: Result of Filter Compatibility Study

Condition Area % Assay

Centrifuged 44655 97.5

0.45 µm Nylon 45689 99.2

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Table 6: Filter Saturation Study

Sr.

No.

Condition Lable

Claim

(mg)

Average

wt

(mg)

Sample

wt

mg

Area %

Assay

mg/

Tab

1 0.2µm Nylon 1mL

Discard

150 527.6 527.6 21554 99.9 149.9

2 0.2µm Nylon 2mL

Discard

150 527.6 527.6 21553 99.9 150.5

3 0.2µm Nylon 4mL

Discard

150 527.6 527.6 21651 100.4 147.2

4 0.45µm Nylon 1mL

Discard

150 527.6 527.6 21658 98.4 146.5

5 0.45µm Nylon 2mL

Discard

150 527.6 527.6 21218 99.2 149.3

6 0.45µm Nylon 4mL

Discard

150 527.6 527.6 21416 99.8 148.7

7 0.45µm PVDF 1mL

Discard

150 527.6 527.6 21533 99.8 150.9

8 0.45µm PVDF 2mL

Discard

150 527.6 527.6 21536 99.6 146.8

9 0.45µm PVDF 4mL

Discard

150 527.6 527.6 21529 98.9 149.0

10 Centrifuged 150 527.6 527.6 21433 99.4 149.6

Method validation

The proposed method was validated as per ICH guidelines. The drug solutions were prepared

as per the earlier adopted procedure given in the experiment.

Linearity

Stock solution: 128mg ibandronate sodium in 100 mL of water (1200 ppm)

The linearity of photodiode array detector response of ibandronate sodium at different

concentrations was studied in the range 150-450 µgml−1

for ibandronate sodium. The data was

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subjected to statistical analysis using a linear-regression model. The regression equations for

ibandronate sodium is y = 139.8x - 735.9. The statistical parameters slope and correlation

coefficient values were calculated and shown in Table No.7.

Table 7: Linearity Study

Sample Name PPM R.T Area I Area II Area III Mean SD RSD

50 % Level 150 3.93 20308 20468 20314 20363 906 0.45

80 % Level 240 3.94 33014 32537 32726 32759 2404 0.73

90 % Level 270 3.92 40872 36859 37077 36962 1092 0.30

100 % Level 300 3.95 45776 41011 41202 41028 1656 0.40

110 % Level 330 3.93 45776 45755 45784 45772 151 0.03

120 % Level 360 3.96 49511 49126 49542 49393 2315 0.47

150 % Level 450 3.93 62676 62442 61784 62301 4624 0.74

Accuracy

Accuracy of method was determined by recovery experiments using standard addition

technique. Recoveries were determined by adding the ibandronate sodium in triplicate i.e.

100%. Average recovery values are given in Table No.8.

Table 8: Results for Accuracy of Ibandronate Sodium

Sr. No Spiked

Sample

(mg)

Area I Area II Area III Mean Recovery % Recovery

100 % Level 574.20 366299 366299 366299 366299 574.36 100.0

100 % Level 574.30 364985 364985 364985 364985 572.30 99.7

100 % Level 574.10 365375 365375 365375 365375 572.92 99.8

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System Precision (Repeatability)

Solutions of Ibandronate sodium were prepared as per test method and injected for 6 times.

The mean SD and RSD were checked for precision. Results are shown in Table No.9.

Table 9: System Precision

Sr.No. Area % Released

1 43519 99.82

2 43133 98.93

3 43472 99.71

4 43500 99.77

5 43396 100.16

6 43667 99.65

7 Mean 99.65

8 SD 0.41

9 % RSD 0.41

Range

Range to be inferred from the data of linearity, recovery and precision experiments it was

found to be 50-450 ppm.

Analytical Solution Stability

The stability of the drug in solution during analysis was determined by repeated analysis of

standard and sample. The standard and sample were prepared and injected into HPLC at

initial and different time intervals up to 24 hrs. Results are shown in Table No.10

Table 10: Analytical Solution Stability

Standard Ibandronate Standard Ibandronate Sample

Time Area % Diff w.r.t.

initial

Area % Diff w.r.t.

initial

Ini4ial 40680 43519

1 hr 40886 -0.5 43982 -1.1

4 40747 -0.2 43119 0.9

8 40563 0.3 43148 0.9

12 40881 -0.5 42987 1.2

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16 41273 -1.5 43328 0.4

20 40435 0.6 43398 0.3

24 40224 1.1 43684 -0.4

RESULTS AND DISCUSSION

The linear regression analysis data for the calibration plots showed good linear relationship

with r2 = 0.998. The method was validated for linearity, accuracy, system precision and

solution stability. Statistical analysis proves that the method is repeatable, selective and

accurate for the determination of investigated drug.

CONCLUSION

The proposed stability indicating HPLC method provides simple, accurate and reproducible

quantitative analysis for determination of Ibandronate sodium. The developed stability

indicating HPLC method has advantages over other analytical methods due to selectivity and

better sensitivity.

ACKNOWLEDGEMENTS:

The authors are mostly thankful to Aarti Drugs Ltd, Boisar (M.S) India for providing valuable

drug sample.

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Ibandronate Tablets