STABILITY INDICATING ANALYTICAL METHOD DEVELOPMENT … · The HPLC system was controlled with “Lab solutions lite” software. In addition, an electronic analytical weighing balance

www.wjpps.com Vol 5, Issue 5, 2016.

650

Joseph et al. World Journal of Pharmacy and Pharmaceutical Sciences

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


651


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,

http://www.drugs.com/combigan.html


655


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%-


657


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.


658


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


659


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.

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STABILITY INDICATING ANALYTICAL METHOD DEVELOPMENT … · The HPLC system was controlled with “Lab solutions lite” software. In addition, an electronic analytical weighing balance