www.wjpps.com
287
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]
WWOORRLLDD JJOOUURRNNAALL OOFF PPHHAARRMMAACCYY AANNDD PPHHAARRMMAACCEEUUTTIICCAALL SSCCIIEENNCCEESS
VVoolluummee 22,, IIssssuuee 11,, 228877--330011.. RReesseeaarrcchh AArrttiiccllee IISSSSNN 2278 – 4357
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.
www.wjpps.com
288
Jineetkumar B G et al. World Journal of Pharmacy and Pharmaceutical Sciences
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
+
www.wjpps.com
289
Jineetkumar B G et al. World Journal of Pharmacy and Pharmaceutical Sciences
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
www.wjpps.com
290
Jineetkumar B G et al. World Journal of Pharmacy and Pharmaceutical Sciences
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)
www.wjpps.com
291
Jineetkumar B G et al. World Journal of Pharmacy and Pharmaceutical Sciences
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)
www.wjpps.com
292
Jineetkumar B G et al. World Journal of Pharmacy and Pharmaceutical Sciences
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.
www.wjpps.com
293
Jineetkumar B G et al. World Journal of Pharmacy and Pharmaceutical Sciences
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.
www.wjpps.com
294
Jineetkumar B G et al. World Journal of Pharmacy and Pharmaceutical Sciences
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
www.wjpps.com
295
Jineetkumar B G et al. World Journal of Pharmacy and Pharmaceutical Sciences
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
www.wjpps.com
296
Jineetkumar B G et al. World Journal of Pharmacy and Pharmaceutical Sciences
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
www.wjpps.com
297
Jineetkumar B G et al. World Journal of Pharmacy and Pharmaceutical Sciences
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
www.wjpps.com
298
Jineetkumar B G et al. World Journal of Pharmacy and Pharmaceutical Sciences
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
www.wjpps.com
299
Jineetkumar B G et al. World Journal of Pharmacy and Pharmaceutical Sciences
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
www.wjpps.com
300
Jineetkumar B G et al. World Journal of Pharmacy and Pharmaceutical Sciences
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.
REFERENCES
1. Bauss F, Lalla S, Endele R, Hothorn LA. Effects of treatment with ibandronate on
bone mass architecture biomechanical propertites and bone concentration of
ibandronate in ovariectomized aged rats. J. Rheumatol, 2002; 29: 2200–2208.
2. Bauss F, Graham RR. Ibandronate in osteoporosis preclinical data and rationale for
intermittent dosing. Osteoporos Int, 2004; 15: 423–433.
3. Lovdahl MJ, Pietrzyk DJ. Anion-exchange separation and determination of
bisphosphonates and related analytes by post-column indirect fluorescence detection.
J. Chromatogr. A, 2000; 868: 141–142.
4. My H, Xq Z, Bc W. Determination of ibandronate by high performance ion exchange
chromatography. Se Pu, 2000; 18: 254–255.
www.wjpps.com
301
Jineetkumar B G et al. World Journal of Pharmacy and Pharmaceutical Sciences
5. Rodriguez JAB, Desimone MF, Iglesias SL, Giorgieri SL, Diaz LE. Validation of a
capillary electrophoresis method for the analysis of ibandronate related impurities. J.
Pharm. Biomed. Anal, 2007; 44: 305–308.
6. Jiang Y, Xie Z. Determination of Ibandronate and its degradation products by ion- pair
RP LC with evaporative light-scattering detection. Chromatog, 2005; 62: 257–261.
7. Zacharis CK, Tzanavaras PD. Determination of bisphosphonate active pharmaceutical
ingredients in pharmaceuticals and biological material a review of analytical methods.
J. Pharm. Biomed. Anal, 2008; 48: 483–496.
8. ICH, Q2A, harmonized tripartite guideline, text on validation of analytical procedures,
IFPMA, in proceeding of international conference on harmonization, Geneva; March
1994.
9. ICH, Q2B, harmonized tripartite guideline, text on validation of analytical procedures,
IFPMA, in proceeding of international conference on harmonization, Geneva; March
1996.
10. Skoog D. Leqary J. Principle of Instrumental Analysis. 54th
ed., Singapore; Thomson
Asia Pvt Lt: 2004, 3-8.
11. Willard HH, Metritt LL, Dean JA, Settal FA. Instrumental methods of analysis. 7th
ed.,
New Delhi; CBS Publisher and Distributors: 1986, 118.
12. Munson JW. Pharmaceutical Analysis, Modern Methods: Part-B: Marcel Dekker.,
New York; 1992, 155-176.
13. US FDA Technical Review Guide: Validation of Chromatographic Methods. Center
for Drug Evaluation and Research (CDER). Rockville, MD 1993.