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STABILITY INDICATING ANALYTICAL METHOD DEVELOPMENT … · The HPLC system was controlled with...
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STABILITY INDICATING ANALYTICAL METHOD DEVELOPMENT
AND VALIDATION FOR THE SIMULTANEOUS ESTIMATION OF
BRIMONIDINE TARTRATE AND TIMOLOL MALEATE USING RP-
HPLC METHOD IN BOTH BULK AND PHARMACEUTICAL DOSAGE
FORM
Jomol Joseph1*, N. J. R. Hepsebah
2 and K. Deepthi
1
1Department of Pharmaceutical Analysis and Quality Assurance, St. Pauls College of
Pharmacy, Turkayamzal, Hayathnagar (M), Rangareddy (Dt), Hyderabad.
2Department of Pharmaceutical Analysis and Quality Assurance, Vijaya College of Pharmacy, Munaganur
(Village), Hayathnagar (Mandal), Hyderabad.
ABSTRACT
A simple and effective RP-HPLC method had been developed for the
estimation of Brimonidine Tartrate and Timolol Maleate in Ophthalmic
dosage form, using Inertsil ODS C18 (250 x 4.6 mm, 5.), mobile
phase Phosphate Buffer and Acetonitrile (68:32), detection wavelength
at 320 nm, at flow rate of 0.8 ml/min at retention time 2.85 min for
Brimonidine Tartrate, 3.68 min for Timolol Maleate. Linearity was
obtained in the range of 25 µg/ml to 150 µg/ml for Brimonidine
Tartrate and 62.5 µg/ml to 375 µg/ml for Timolol Maleate,
respectively. The correlation coefficient was found to be 0.999 for both
the drugs. The Recovery studies were performed for Brimonidine
Tartrate and Timolol in the range of 50% - 150 %. The % Assay for
Brimonidine Tartrate is 99.85 % and Timolol Maleate is 99.77 %.
Forced Degradation studies were conducted according to the ICH guidelines and the Drug
Product was found to be stable in all conditions. Hence, the method could be successfully
applied for routine analysis of Brimonidine Tartrate and Timolol Maleate in combined
ophthalmic dosage form.
KEY WORDS: Brimonidine Tartrate and Timolol Maleate, RP-HPLC, ICH.
WORLD JOURNAL OF PHARMACY AND PHARMACEUTICAL SCIENCES
SJIF Impact Factor 6.041
Volume 5, Issue 5, 650-662 Research Article ISSN 2278 – 4357
*Corresponding Author
Jomol Joseph
Department of
Pharmaceutical Analysis and
Quality Assurance, St. Pauls
College of Pharmacy,
Turkayamzal, Hayathnagar
(M), Rangareddy(Dt),
Hyderabad.
Article Received on
01 March 2016,
Revised on 21 March 2016,
Accepted on 12 April 2016
DOI: 10.20959/wjpps20165-6177
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INTRODUCTION
Glaucoma[1]
is a slowly progressive optic neuropathy disease in this there is loss of retinal
ganglion cells, excavation of the optic nerve head with varying degree of visual impairment
or blindness. chemically Brimonidine tartrate (BRT)[2]
(5-bromo-6 (2-
imidazolidinylideneamino) quinoxaline L-Tartrate )is a selective alpha-2 adrenergic agonist.
It is usually given in eyedrop form. Its ocular hypotensive effect is due to its ability to
decrease aqueous humor production and increase uveoscleral outflow. Its selectivity towards
alpha-2 adrenergic receptors and its neuroprotective activity on retinal ganglionic cells makes
it as an important therapeutic agent for the treatment of open angle glaucoma. Its superiority
in treating glaucoma in cardiopulmonary patients makes this drug as a better alternative to
timolol. Timolol maleate (TM)[3]
((S)-1-[1, 1-dimethylethyl) amino]-3-[[4-(4-morpholinyl)-1,
2, 5- thiadiazol-3-yl] oxy]-2-propanol, (Z)-2-butenedioate (1:1) salt.) is a nonspecific β-
adrenergic blocker. It was the first β-blocker to be used as an antiglaucoma agent. None of the
newer β-blockers were found to be more effective than timolol. In its oral form it is used to
treat high blood pressure and prevent heart attacks, and occasionally to prevent migraine
headaches. In its ophthalmic form it is used to treat open-angle and occasionally secondary
glaucoma.
Fig 1: structure of Brimonidine Tartrate Fig 2: Structure of Timolol maleate
The combination of Brimonidine tartrate and Timolol maleate is very useful in the treatment
of glaucoma.On literature survey, it was found that only ratio difference spectroscopic
method has been reported for the simultaneous estimation of Brimonidine tartrate and
Timolol maleate in combined dosage forms and no method is available in the
pharmacopoeias. In the view of the need for a suitable method for routine analysis in
combined formulations, attempts are being made to develop simple, precise and accurate
analytical methods for simultaneous estimation of titled ingredients and extend it for their
determination in formulation.[4-9]
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MATERIALS AND METHODS
Chemicals and reagents
Analytically pure sample of Brimonidine tartrate and Timolol maleate with purities greater
than 99% were obtained as gift samples from Chandra labs (Hyderabad, India) and tablet
formulation COMBIGEN was procured from Apollo pharmacy, Hyderabad, India with
labelled amount 500mg each of troxerutin and calcium dobesilate. Acetonitrile (HPLC grade)
was obtained from Sigma Aldrich (Hyderabad, India), Water (HPLC grade), Potassium
dihydrogen orthophosphate (AR grade) and phosphoric acid (AR Grade) were obtained from
SD Fine chemicals (Hyderabad, India). 0.45μm Nylon membrane filters were obtained from
Spincotech private limited, Hyderabad, India.
Instrument
HPLC analysis was performed on Shimadzu LC-20AD Prominence Liquid Chromatograph
comprising a LC-20AD pump, Shimadzu SPD-20A Prominence UV-Vis detector and Enable
C18G reverse phase C18 column (250X4.6 mm, 5 micron particle size). A manually
operating Rheodyne injector with 20 μL sample loop was equipped with the HPLC system.
The HPLC system was controlled with “Lab solutions lite” software. In addition, an
electronic analytical weighing balance (0.1mg sensitivity, Shimadzu AY 220), digital pH
meter (DELUX model 101), a sonicator (sonica, model 2200 MH) and UV-Visible
Spectrophotometer (Shimadzu UV-1800 series, software-UV probe version 2.42) were used
in this study.
Selection of wavelength: Initially method development work was started by taking UV-
visible spectra from 400-200 nm of Brimonidine and Timolol standard solutions. By
observing the spectra of standard solutions λmax 320 nm was chosen as the desired
wavelength. The spectrum was shown in Fig No: 3.
295.8
247.2
319.6
AU
0.00
0.05
0.10
0.15
0.20
0.25
0.30
0.35
0.40
0.45
nm
220.00 240.00 260.00 280.00 300.00 320.00 340.00 360.00 380.00
Fig 3: Overlay Spectra of Brimonidine tartrate and Timolol maleate
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Chromatographic conditions
The separation of the drugs was achieved on a C18 column reverse phase (4.6 mm X 250
mm, 5 micron particle size). The mobile phase consists of a mixture of Potassium Phosphate
Buffer: Acetonitrile as the mobile phase in the ratio 68:32 at a flow rate of 0.8 ml/minute and
the volume injected was 20 μl for every injection .the detection wavelength was set at 320
nm.
Preparation of Buffer: Accurately weighed 2.72 gm of Potassium dihydrogen ortho
phosphate in a 1000 ml of volumetric flask add about 900 ml of milli-Q water, sonicate and
finally made up the volume with water and pH adjusted to 3.8 with dil. Orthophosphoric acid
solution.
Mobile Phase: Prepared a degassed and filtered mixture of Buffer and Acetonitrile were
taken in the ratio 68:32 v/v. Peaks of Brimonidine tartrate and Timolol were well resolved
with the solvent system of Buffer and Acetonitrile in the ratio of 68:32 v/v.
Sample Preparation: From the Above formulation 5 ml was taken and diluted to 10 ml with
diluents. Further 1ml was taken from the sample stock solution in to a 10 ml volumetric flask
and made up with diluents.
RESULTS AND DISCUSSION
Method Development
A Reverse phase HPLC method was developed keeping in mind the system suitability
parameters i.e. resolution factor (Rf) between peaks, tailing factor (T), number of theoretical
plates (N), runtime and the cost effectiveness. The optimized method developed resulted in
the elution of Brimonidine at 2.85 min and Timolol at3.66 min. Figure 4 represent
chromatograms of mixture of standard solutions. The total run time is 8 minutes with all
system suitability parameters as ideal for the mixture of standard solutions.
System‐suitability tests are an integral part of method development and are used to ensure
adequate performance of the chromatographic system. Retention time (Rt), number of
theoretical plates (N), peak resolution (Rs) and peak Tailing factor (T) were evaluated for six
replicate injections of the standards at working concentration. The results given in Table 1
were within acceptable limits.
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Table 1: System suitability studies results.
Parameters* Required Limits Brimonidine Timolol
Retention time (min) % RSD < 1% 2.86 3.86
Number Of Theoretical plates (N) Not less Than 2000 5482 8345
Tailing factor (T) Not More Than 2 1.32 1.36
In order to test the applicability of this method developed to a commercial formulation,
„COMBIGAN„ was chromatographed at working concentration equivalent to standard
working and it is shown in Figure 5. The sample peaks were identified by comparing the
relative retention times with the standard drugs mixture, Fig: 4.
Fig. 4: Typical chromatogram of the mixture of Standard solution
Method validation
Validation of the analytical method is the process that establishes by laboratory studies in
which the performance characteristics of the method meet the requirements for the intended
analytical application. The RP-HPLC method developed was validated according to
International Conference on Harmonization[10]
guidelines for validation of analytical
procedures. The method was validated for the parameters in terms of system suitability,
selectivity, linearity, accuracy, precision, ruggedness, robustness, limit of detection(LOD) and
limit of quantitiation(LOQ).
Linearity: For the linearity 100% each of working standard solution of Brimonidine and
Timolol were injected at Concentration 25-150 μg/ml and 62.5-375 μg/ml respectively and
the results obtained are tabulated as follows in the table 2,3 and calibration curves are shown
in figure 5,6 The results show an excellent correlation exists between mean peak area and
concentrations level of drugs within the concentration range. The correlation coefficient and
% curve fitting for Brimonidine and Timolol were found to be 0.999, 99.9% and 0.999,
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99.9% respectively which is well within the acceptance criteria limits. the results are
tabulated in table 4
Table 2: Linearity of Brimonidine
S. No Concentration (μg/ml) Area
1 25 266769
2 50 497616
3 75 765644
4 100 1022668
5 125 1280202
6 150 1531526
Table 3 Linearity of Timolol Maleate
S. No Concentration (μg/ml) Area
1 62.5 470827
2 125 934610
3 187.5 1408163
4 250 1902898
5 312.5 2367398
6 375 2804850
Figure 5 Linearity curve for Brimonidine
Figure 6Linearity curve for Timolol Maleate
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Table 4 Linearity Report
Parameters Results (n=6)
Brimonidine Timolol
Linearity range 25-150 g/ml 62.5-375 g/ml
Slope 10209 7529
Intercept 650.4 965.8
Correlation
coefficient 0.999 0.999
Precision: The precision of the analytical method is determined by assaying sufficient
number of sample and relative standard deviation is calculated.
Intraday Precision (Repeatability)
This Study was carried out by injecting six dilutions of the standard concentrations of
Brimonidine and Timolol on the same day by the same analyst.the results of whioch are
shown in table 5. %RSD was determined for peak areas of Brimonidine and Timolol. The
acceptance limit should be not more than 2% and the results obtained were found to be within
the acceptance limits. The RSD values of intraday precision for replicate injections of
Brimonidine and Timolol were found to be between 0.2 and 0.2 respectively which are well
within the acceptance criteria limit (RSD ≤ 2).
Table 5 Intraday Precision of Brimonidine and Timolol
Injections (n) Brimonidine Timolol
Peak area Retention time Tailing factor Peak area Retention
time
Tailing
factor
1 1038712 2.848 1.32 1966404 3.651 1.35
2 1040436 2.848 1.31 1969136 3.652 1.35
3 1041110 2.849 1.33 1965525 3.653 1.35
4 1044061 2.849 1.33 1975011 3.653 1.35
5 1037862 2.85 1.34 1964311 3.655 1.32
6 1037031 2.851 1.3 1961916 3.655 1.3
Mean 1039869
1967051
SD 2563.4 4567.9
% RSD 0.2 0.2
Accuracy: Accuracy was determined at three different level 50 %, 100 % and 150 % of the
target concentration in triplicate At each level, three determinations were performed and
results obtained. The accuracy was calculated from the test results as the percentage of the
analyte recovered by the assay. The amounts recovered, values of percent mean recovery
were calculated as shown in Tables 6 and 7 The accepted limits of mean recovery are 98%-
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102% and all observed data are within the required range that indicates good recovery values
and hence the accuracy of the method developed.
Table 6: Results of Accuracy studies for bromidine
Concentration level (%) Amount added *Amount recovered *% Mean recovery
(µg/ml) (µg/ml)
50 50 50.21 100.42
100 100 100.24 100.24
150 150 148.35 98.9
*Mean of three replicates
Table 7: Results of Accuracy studies for Timolol
Concentration level (%) Amount added *Amount recovered *% Mean recovery
(µg/ml) (µg/ml)
50 125 50.69 101.38
100 250 250.20 100.08
150 375 378.75 101
Robustness
The robustness of an analytical method is a measure of its capacity to remain unaffected by
small but deliberate variations in method parameters and provides an indication of its
reliability during normal usage. It is concluded that the method is robust as it is found that the
% RSD is less than 2 concerning % assay despite deliberate variations done concerning flow
rate (± 0.2), pH (± 0.2) and % organic phase (± 5%).
Sensitivity
The sensitivity of measurement of Brominidine and Timolol by use of the proposed method
was estimated in terms of the limit of quantitation(LOQ) and the limit of detection
(LOD).The limit of detection (LOD) was obtained as 0.21mcg/ml for Brominidine and 0.42
µg/ml for Timolol. The limit of quantitation (LOQ) was obtained as 0.64mcg/ml for
Brominidine and 1.28 µg/ml for Timolol
FORCED DEGRADATION[11]
Procedure
Brimonidine tartrate and Timolol Maleate Opthalmic drops was stressed with following
mentioned conditions and solutions were prepared with respective stressed samples and each
sample solution was injected into the Chromatographic system as per methodology.
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a. Acid degradation
b. Base degradation
c. Peroxide degradation
d. Photolytic degradation
e. Thermal degradation
f. Neutral degradation
Oxidation: To 1 ml of stock solutionof Brimonidine and Timolol, 1 ml of 20% hydrogen
peroxide (H2O2) was added separately. The solutions were kept for 30 min at 600c. For
HPLC study, the resultant solution was diluted to obtain 100 µg/ml & 250 µg/ml
solution and 10 µl were injected into the system and the chromatograms were recorded to
assess the stability of sample
Acid Degradation Studies: To 1 ml of stock solution Brimonidine and Timolol, 1 ml of
2N Hydrochloric acid was added and refluxed for 30mins at 600c .The resultant
solution was diluted to obtain 100 µg/ml & 250 µg/ml solution and 10 µl solutions were
injected into the system and the chromatograms were recorded
Alkali Degradation Studies: To 1 ml of stock solution Brimonidine and Temolol, 1 ml of
2N sodium hydroxide was added and refluxed for 30mins at 600c. The resultant solution
was diluted to obtain 100 µg/ml & 250 µg/ml solution and 10 µl were injected into the
system and the chromatograms were recorded
Dry Heat Degradation Studies: The standard drug solution was placed in oven at 600c
for 6 h to study dry heat degradation. For HPLC study, the resultant solution was diluted to
100 µg/ml & 250 µg/ml solution and10µl were injected into the system and the
chromatograms were recorded
Photo stability studies: The photochemical stability of the drug was also studied by
exposing the 300 µg/ml & 10 µg/ml & 25 µg/ml solution to UV Light by keeping the beaker
in UV Chamber for 24 hrs or 200 Watt hours/m2 in photo stability chamber.For HPLC study, the
resultant solution was diluted to obtain 100 µg/ml & 250 µg/ml solutions and 10 µl were
injected into the system.The chromatograms were recorded
Neutral Degradation Studies: Stress testing under neutral conditions was studied by
refluxing the drug in water for 24 h r s at a temperature of 60º. For HPLC study, the
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resultant solution was diluted to 100 µg/ml & 250 µg/ml solution and 10 µl were injected
into the system and the chromatograms were recorded and are shown from figures 7-11. The
results of these are recorded in table 8 and 9
Figure 7 Chromatogram of Acid Sample (2 N Hcl)
Figure 8 Chromatogram of Base Sample (2 N NaoH)
Figure 9 Chromatogram of Peroxide Sample (20% H2O2)
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Figure 10 Chromatogram of Thermal Sample (600C for 6 h)
Figure 11 Chromatogarm of Neutral Sample (Reflux for 24 hrs at 60ºC)
Table 8: forced degradation studies of Timolol
Degradation
mechanism / condition Component % Assay % Degradation
Purity
Angle
Purity
Threshold
Remarks
Undegraded sample Timolol
Maleate 99.85 -- 0.151 0.727 Passed
Thermal at - 105°C for
48 Hr
Timolol
Maleate 95.33 4.51 0.96 0.418 Passed
Photolytic at 254nm -
168 Hrs
Timolol
Maleate 94.38 5.47 0.089 0.430 Passed
Acid degradation Timolol
Maleate 92.50 7.35 0.151 0.727 Passed
Base degradation Timolol
Maleate 93.30 6.55 0.078 0.281 Passed
Peroxide degradation Timolol
Maleate 94.38 5.47 0.067 0.278 Passed
Neutral Degradation. Timolol
Maleate 99.66 0.19 0.155 0.361 Passed
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Table 9: forced degradation studies of Brimonidine
Degradation mechanism
/ condition Component % Assay % Degradation
Purity
Angle
Purity
Threshold
Remarks
Undegraded sample Brimonidine
Tartrate 99.77 -- 0.151 0.727 Passed
Thermal at - 105°C for 48
Hr
Brimonidine
Tartrate 95.10 0.8 0.96 0.418 Passed
Photolytic at 254 nm -
168 Hrs
Brimonidine
Tartrate 98.8 0.8 0.089 0.430 Passed
Acid degradation Brimonidine
Tartrate 92.39 -- 0.151 0.727 Passed
Base degradation Brimonidine
Tartrate 93.34 11.84 0.078 0.281 Passed
Peroxide degradation Brimonidine
Tartrate 94.64 15.65 0.067 0.278 Passed
Neutral Degradation Brimonidine
Tartrate 99.09 8.87 0.155 0.361 Passed
CONCLUSION
Simultaneous determination of Brimonidine and Timolol maleate in their pharmaceutical
formulation using HPLC has been successfully achieved. The method is accurate and precise
for reliable quality control evaluation of drugs with good accuracy and precision. From these
values it is concluded that the new HPLC method is suitable for the simultaneous
determination of these two components in their pharmaceutical formulations.
ACKNOWLEDGEMENT
Authors are very much thankful to Chandra Labs for providing the gift samples of
Bromonidine and Timolol Maleate, respectively and also wish our sincere thanks to St.Pauls
College Of Pharmacy, Hyderabad, for giving permission to carry out our research work.
REFERENCES
1 Jaypee, Tripathi.K.D. Essentials of Medical Pharmacology, 6th
edition Pg.no.144.
2 Laurenc L. Brunton, john S. Lazo, Keith L. Parker; „Goodman and Gilman‟s the
pharmacological basis of Therapeutics: McGraw Hill, Newyork, U.S.A; 2001; 10: 256,
278, and 1721.
3 John H.B, John M.B; Wilson and Gisvold‟s textbook of Organic Medicinal
&Pharmaceutical Chemistry, Lippincott Williams & Wilkins, WoltersKluner Company.
2004; 11: 534.
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Joseph et al. World Journal of Pharmacy and Pharmaceutical Sciences
4 John H.B, John M.B; Wilson and Gisvold‟s textbook of Organic Medicinal
&Pharmaceutical Chemistry, Lippincott Williams & Wilkins, WoltersKluner Company.
2004; 11: 534.
5 www.pharmaresearchlibrary.com/development-and-validation-of-rp-hplc-method-for-the-
simultaneous-estimation-of-brimonidinetartrate-and-timolol-maleate-in-combined-
dosage-form/
6 Arun Phogat, Murugesan Senthil kumar, Nanjaian Mahadevan. Simultaneous Estimation
of Brimonidine Tartrate and Timolol Maleate in Nanoparticles Formulation by RP-HPLC.
International Journal of Recent Advances in Pharma Res. Jul 2011; 3: 31-6.
7 Madhav.A, Naidu A, Subbarao D.V, Srinivasu.P. Developed and validated the new LC
method for the analysis of brimonidine Tartrate and related compounds.
8 Shirke RR, Pai N (2002) Rp-hplc determination of brimonidine tartrate in brimonidine
tartrate eye drops Ind. Drugs 2002; 39: 484-486. Indian drugs ISSN 0019-462X Coden
Indrba.
9 Mahajan Ananad, Athensla Fonseca, Gandhi Santosh V, Deshpande padmanabh
B,development and validation of high performance thin layer chromatograph method for
the estimation of brimonidine tartrate as Bulk drug in ophthalmic solutions,
IJPTR(International Journal of Physical Therapy and Rehabilitation), 2(3): 1376-1379,
July 2010; 2(3): 1376-9.
10 Pritam.S.Jain et.al, Development and validation of first order derivative UV-
spectrophotometric method for determination of Brimonidine tartrate in bulk and in
formulation. Asian J Pharm Biol Res. Jul-Sep 2011; 1(3): 323-9.
11 International Conference on Harmonization of Technical Requirements for Registration of
Pharmaceuticals for Human use. Validation of Analytical Procedures: Text and
Methodology ICH Q2 (R1). 2005.
12 Stabilitystudies.wordpress.com/2011/11/25/forced-degradation-studies.