Acta Scientific Pharmaceutical Sciences (ASPS)(ISSN: 2581-5423)

Research Article Volume 5 Issue 4

Chemo-metric Assisted UV-Spectrophotometric Methods for the Simultaneous Estimation of Brimonidine Tartrate and Timolol Maleate

Mukthinuthalapati Mathrusri Annapurna* and Yenda Manishankar

Department of Pharmaceutical Analysis and Quality Assurance, GITAM Institute of Pharmacy, GITAM (Deemed to be University), Visakhapatnam, India

*Corresponding Author: Mukthinuthalapati Mathrusri Annapurna, Department of Pharmaceutical Analysis and Quality Assurance, GITAM Institute of Pharmacy, GITAM (Deemed to be University), Visakhapatnam, India.

Received: March 29, 2021; Published: April 30, 2021

Abstract

  Two Chemo-metric assisted UV-spectrophotometric methods have been developed for the simultaneous determination of Timolol maleate and Brimonidine tartrate in ophthalmic dosage forms. Timolol is a beta blocker used for the patients suffering from ocular hypertension and glaucoma and Brimonidine tartrate is a an alpha-2 adrenergic receptor agonist. Simultaneous equation method and absorbance ratio method were proposed for the simultaneous determination of Timolol maleate and Brimonidine tartrate in ophthalmic preparations. The two methods were validated and were found to be linear over 1 - 60 μg/mL for Timolol maleate and 1 - 40 μg/mL for Brimonidine tartrate. The two proposed methods find application in the quality control of pharmaceuticals.

Keywords: Timolol Maleate; Brimonidine Tartrate; Spectrophotometry; Simultaneous Equation Method; Q-Analysis; Validation

Introduction

  Timolol [1] is chemically (S)-1-(tert-butylamino)-3-[(4-morpholin-4-yl-1, 2, 5-thiadiazol-3-yl) oxy] propan-2-ol. Timolol (Figure 1) is used as an anti-hypertensive and has molecular formula, C13H24N4O3S with molecular weight 316.42 g/mol (pKa 9.21). Timolol (TML) is a beta-adrenergic antagonist and levo isomer is more active. Brimonidine tartrate [2] is chemically known as 5-bromo-N-(4,5-dihydro-1H-imidazol-2-yl) quinoxalin-6-amine with molecular formula, C11H10BrN5 and molecular weight 292.14 g/mol. Brimonidine tartrate (BRM) is used to treat open-angle glaucoma or ocular hypertension. Brimonidine tartrate (Figure 2) is used for treating ocular hypertension. Brimonidine tartrate is an α2 adrenergic agonist that acts by the activation of G protein-coupled receptor [3].

Figure 1: Structure of timolol maleate.

Figure 2: Structure of brimonidine tartrate.

  The combination of Timolol maleate and Brimonidine tartrate has a very good combined effect. Analytical techniques such as HPTLC [4], HPLC [5,6] and spectrophotometric [7-12] methods were developed for the simultaneous determination of Timolol maleate and Brimonidine tartrate in pharmaceutical dosage forms. Two UV spectrophotometric methods have been developed and validated [13] as per ICH guidelines for the simultaneous determination of Timolol maleate and Brimonidine tartrate in pharmaceutical formulations.

Materials and Methods

  A double beam UV-VIS spectrophotometer (Shimadzu Model No. UV-1800) with a pair of 10 mm path length matched quartz cells was used for the study. All the drug sample solutions were scanned 200-400 nm with medium scanning speed.

Preparation of phosphate buffer (pH 2.0) solution

  0.136 grams of potassium di hydrogen phosphate was weighed accurately and dissolved in about 800 ml of water in a 1000 ml volumetric flask. The pH of the solution was adjusted to 2.0 with the help of hydrochloric acid and sufficient water was added to make up to volume after sonication.

Preparation of stock solutions

  Stock solutions were prepared in two different volumetric flasks by dissolving 25 mg of each of Timolol maleate and Brimonidine tartrate in methanol and there by diluting the stock solutions as per requirement with phosphate buffer pH 2.0.

Procedure

  Two methods were proposed for the simultaneous determination of Timolol maleate and Brimonidine tartrate i.e. simultaneous equation method (Method A) and Q-analysis or Absorbance ratio method (Method B). A 10 µg/ml solution of both Timolol maleate and Brimonidine tartrate were prepared initially from the stock solutions and the absorption maxima (λmax) was noted. The absorption spectra of Timolol maleate and Brimonidine tartrate were overlaid and shown in figure 3.

Figure 3: Absorption spectrum of timolol maleate (TML) (5 µg/ml), Brimonidine tartrate (BRM) (2 µg/ml) and formulation (Eye drops) (TML: BRM 5: 2) in phosphate buffer pH 2.0.

Simultaneous equation method (Method A)

  In simultaneous equation method the absorption maxima of both Timolol maleate and Brimonidine tartrate were selected. Brimonidine tartrate has shown absorption maxima (λmax) at 246 nm and Timolol maleate has shown absorption maxima (λmax) at 295 nm respectively. The absorptivity values (Ɛ) were calculated from the absorbance values for both the drugs Timolol maleate and Brimonidine tartrate at 246 nm and 295 nm for all the solutions prepared for the linearity study from their individual spectra and substitute in the simultaneous equations.

Q-analysis or Absorbance ratio method (Method B)

  In Q-analysis or absorbance ratio method the wavelength of the absorption maxima (λmax) of one of the drugs and the wavelength of the isosbestic point were selected for the calculation purpose. Two isosbestic (iso-absorptive) points were observed at 268.23 and 319.82 nm from the overlay absorption spectrum of Timolol maleate and Brimonidine tartrate. For Q-Analysis method, the absorption maxima of Brimonidine tartrate (λmax = at 246 nm) and the isosbestic point 268.23 nm were selected for the calculation purpose.

Method validation [13] Linearity

  1-60 µg/ml Timolol maleate and 1-40 µg/ml Brimonidine tartrate solutions were prepared separately from their individual stock solutions and the solutions were scanned against the reagent blank i.e. phosphate buffer pH 2.0. The absorbance values and thereby the absorptivity values were calculated at the selected wavelengths for both Method A and Method B. A calibration curve was drawn by taking the concentration of each of the drug solutions individually on the x- axis and the corresponding absorbance values on the y-axis at the selected wavelengths.

Precision and accuracy

  The intra-day and inter-day precision studies were performed at three different concentration levels (10, 20 and 40 µg/mL) on the same day and on different days and the percentage relative standard deviation (% RSD) was calculated. Accuracy studies were carried out by standard addition method. A fixed concentration of formulation solution was spiked with 80%, 100% and 120% of pure drug solutions and the % recovery was calculated.

Assay of brimonidine tartrate and timolol

  The combination of both Timolol maleate and Brimonidine tartrate is available with brand names Brimocom Eye Drop (Cipla Ltd, India), Brimopress T Eye Drops (Centaur Pharma, India), Combigan eye drops (Allergan plc, India) and Iotim B Drops (FDC Ltd, India) with label claim: Brimonidine tartrate 0.2% w/v and Timolol maleate 0.5% w/v (Brimonidine tartrate: 2 mg/ml and Timolol maleate: 5 mg/ml). Two different brands of these eye drops (Total volume 5 ml) were procured from the medical store and the active constituents were extracted using methanol in to a volumetric flask and sonicated. The extracted solution was filtered and the filtrate was diluted with phosphate buffer pH 2.0 as per the requirement and assay was performed with the two proposed methods. The absorption spectra of Timolol maleate and Brimonidine tartrate were overlaid and shown in figure 3.

Results and Discussion

  Two new spectrophotometric methods, simultaneous equation method (Method A) and Absorbance ratio method (Method B) were proposed for the simultaneous determination of Timolol maleate and Brimonidine tartrate in phosphate buffer pH 2.0. The review of literature of the previously published analytical methods were compared with the present proposed methods and summarized in table 1.

Reagents/Mobile phase (v/v)

Linearity (µg/mL)

λmax (nm)

Observation

Reference

Liquid chromatographic methods

Chloroform: Methanol: Ammonia (30%) (9:1:0.1)

500-1500 ng/spot (TML)

200-600 ng/spot (BRM)

268

HPTLC

[4]

Phosphate buffer: Acetonitrile (65:35)

10-20

295

HPLC

[5]

Ammonium acetate : Methanol (40:60)

10-60

254

HPLC

[6]

Spectrophotometric methods

Phosphate buffer (pH 7.0)

1-120 (TML)

1-60 (BRM)

295

247

Simultaneous equation method

First Derivative method

Multi-component mode method

[7]

Distilled water

1-50 (TML)

4-20 (BRM)

 

255

295

271

295

Simultaneous equation method

 

Q absorbance equation method

[8]

Borate buffer (pH 9.0)

1-60 (TML)

1-40 (BRM)

257

251.5

First Derivative method

Ratio Derivative Spectroscopy

Multi-component mode method

[9]

Distilled water

2-14 (TML)

5-35 (BRM)

247

295

Simultaneous equation method

Q absorbance ratio method

[10]

Borate buffer (pH 9.0)

1-60 (TML)

1-40 (BRM)

295

257

Simultaneous equation method

Q-analysis

[11]

Distilled water

2-50 (TML)

2-14 (BRM)

290

244

Simultaneous equation method

First Derivative method

Ratio first derivative method

[12]

Phosphate buffer (pH 2.0)

1-60 (TML)

1-40 (BRM)

295

246

Simultaneous equation method

Absorbance ratio method

Present work

Table 1: Review of published analytical methods of timolol maleate and brimonidine tartrate.

Method validation Linearity

  Timolol maleate and Brimonidine tartrate obeys Beer-Lambert’s law and have shown linear response over the concentration range 1-60 µg/ml and 1-40 µg/ml respectively (Table 2) for both the methods with linear regression equations y = 0.0223x + 0.0068 (R² = 0.9997) (Figure 4) and y = 0.0676x + 0.0007 (R² = 0.9993) (Figure 5) for Timolol maleate and Brimonidine tartrate respectively.

Conc. (μg/ml)

Timolol maleate

Brimonidine tartrate

1

0.0308

0.0794

2

-

0.1726

5

0.1154

0.3196

10

0.2418

0.6451

20

0.4474

1.3607

30

0.6611

1.9915

40

0.8792

2.7352

50

1.1193

-

60

1.3302

-

Table 2: Linearity of timolol maleate and brimonidine tartrate.
*: Mean of three replicates.

Figure 4: Calibration curve of timolol maleate.

Figure 5: Calibration curves of brimonidine tartrate.

Simultaneous equation method (Method A)

  From the individual absorption spectra of Brimonidine tartrate and Timolol maleate the absorptivity values were calculated from the linearity table and substituted in the simultaneous equation. The specific absorptivity value of any drug is the absorbance of 1%, i.e. g/100ml solution. A1 and A2 represents the absorbance of the formulation solution at 246 nm and 295 nm respectively.

CBRM and CTML are the concentrations of Brimonidine tartrate and Timolol maleate (g/100 ml):

At 246 nm, A1 = 680.2 CBRM+ 49.26 CTML

At 295 nm, A2 = 98.3 CBRM+ 221.8 CTML

ax1 = Absorptivity of Brimonidine tartrate at 246nm = 680.2

ax2 = Absorptivity of Brimonidine tartrate at 295nm = 98.3

ay1 = Absorptivity of Timolol maleate at 246nm = 49.26

ay2 = Absorptivity of Timolol maleate at 295nm = 221.8

Absorbance ratio method (Q Analysis) (Method B)

  Two isosbestic (iso-absorptive) points were observed at 268.23 and 319.82 nm from the overlay absorption spectrum of Timolol maleate and Brimonidine tartrate. For Q-Analysis method, the absorption maxima of Brimonidine tartrate (λmax = at 246 nm) and the isosbestic point 268.23 nm were selected for the calculation purpose. The absorptivity values obtained at the selected wavelengths were substituted in the given equation:

Cx = Qm-Qy/Qx-Qy×A1/ax1

Cy = Qm-Qx/Qy-Qx×A2/ay1

Cx = Concentration of Brimonidine tartrate

Cy = Concentration of Timolol maleate

A1= Absorbance at iso-absorptive wavelength 268.23 nm.

A2 = Absorbance at wavelength 246 nm.

ax1 = Mean absorptivity of Brimonidine tartrate at 268.23 nm. = 115.3

ay1 = Mean absorptivity of Timolol maleate at 246 nm. = 49.26

Qm = Ratio of absorbance of formulation solution at 268.23 and 246 nm.

Qx = Ratio of absorptivity of Brimonidine tartrate at 268.23 and 246 nm. = 0.849

Qy = Ratio of absorptivity of Timolol maleate at 268.23 and 246 nm. = 4.502

Cx = Qm-4.502/0.849-4.502×A1/115.3

Cy = Qm-0.1851/4.502-0.849×A2/49.26.

Precision and accuracy

  The precision studies were performed on the same day (Intraday) and on three different days (Interday) (Intra-day) at three different concentration levels (10, 20 and 40 µg/mL). The % RSD was found to be 0.18-0.81 (Intra-day) and 0.65-1.20 (Interday) for Method A and 0.23-0.78 (Intra-day) and 0.76-0.91 (Interday) for Method B for Timolol maleate. The % RSD was found to be 0.51-0.67 (Intra-day) and 0.81-1.10 (Interday) for Method A and 0.42-0.73 (Intra-day) and 0.61-0.92 (Interday) for Method B for Brimonidine tartrate. The % RSD in precision studies is found to be less than 2 (Table 3) indicating that the methods are precise.

Drugs

Conc (μg/ml)

Intra-day precision

Inter-day precision

*Conc. obtained (µg/ml) ± SD (RSD)

*% Recovery

*Conc. obtained (µg/ml)

± SD (RSD)

*% Recovery

 

 

Method A

Method B

Method A

Method B

Method A

Method B

Method A

Method B

TML

10

9.93 ± 0.02 (0.18)

9.95 ± 0.023 (0.23)

99.3

99.5

9.94 ± 0.06 (0.65)

9.93 ± 0.076 (0.76)

99.4

99.3

20

19.86 ± 0.3 (0.54)

19.84 ± 0.129 (0.65)

99.3

99.2

19.82 ± 0.19 (0.98)

19.88 ± 0.169 (0.85)

99.2

99.4

40

38.9 ± 0.31 (0.81)

39.93 ± 0.312 (0.78)

97.25

99.83

39.5 ± 0.47 (1.20)

39.58 ± 0.36 (0.91)

98.75

98.95

BRM

10

9.98 ± 0.05 (0.51)

9.97 ± 0.042 (0.42)

99.8

99.7

9.93 ± 0.09 (0.98)

9.96 ± 0.079 (0.79)

99.3

99.6

20

19.84 ± 0.10 (0.52)

19.91 ± 0.137 (0.69)

99.2

99.55

19.78 ± 0.21 (1.10)

19.96 ± 0.122 (0.61)

98.9

99.8

40

39.4 ± 0.26 (0.67)

39.63 ± 0.289 (0.73)

98.5

99.08

39.2 ± 0.31 (0.81)

39.73 ± 0.366 (0.92)

98.0

99.33

Table 3: Precision studies of timolol maleate and brimonidine tartrate.
*: Mean of three replicates.

  The % RSD in accuracy was found to be 0.91-1.07 (Method A) and 0.84-1.01 (Method B) for Timolol maleate with a % recovery 98.54 -99.66 and 98.72-99.21 for Method A and Method B respectively. The % RSD in accuracy was found to be 0.88-1.16 (Method A) and 0.97-1.25 (Method B) for Brimonidine tartrate with a % recovery 98.61 -99.42 and 98.10-99.59 for Method A and Method B respectively. The % RSD in accuracy studies is found to be less than 2 (Table 4) indicating that the methods are accurate.

Drugs

Spiked

Conc (μg/ml)

Total

Conc. (μg/ml)

Method A

Method B

Conc. found (μg/ml)

%*Recovery

%RSD

Conc. found (μg/ml)

%RSD

%*Recovery

 

TML

8(80%)

18

17.94

99.66

0.92

17.77

0.92

98.72

10 (100%)

20

19.84

99.19

1.07

19.80

1.01

99.01

12 120%)

22

21.68

98.54

0.91

21.83

0.84

99.21

 

BRM

8 (80%)

18

17.80

98.89

1.16

17.86

1.04

99.20

10 (100%)

20

19.72

98.61

0.98

19.92

1.25

99.59

12 (120%)

22

21.87

99.42

0.88

21.58

0.97

98.10

Table 4: Accuracy studies of timolol maleate and brimonidine tartrate.
*: Mean of three replicates.

Assay of brimonidine tartrate and timolol

  Two different brands of eye drops (Total volume 5 ml) containing the combination of both Timolol maleate and Brimonidine tartrate were extracted using methanol and diluted with phosphate buffer pH 2.0 and the assay was performed with the above two proposed methods. The absorption spectra of the formulation (Eye drops) containing Timolol maleate and Brimonidine tartrate (5: 2) was shown in figure 3. The % recovery (Table 5) was calculated from the absorptivity values and found to be 98.6 - 99.2 for Method A and 98.4 - 99.0 for Method B for Timolol maleate and that of Brimonidine tartrate were found to be 98.5 - 99.0 for Method A and 96.5 - 98.0 for Method B respectively.

Brand

Drug

Label

claim (mg)

*Amount found (mg)

*% Recovery

Method A

Method B

Method A

Method B

Brand I

Timolol

Brimonidine tartrate

5

2

4.93

1.98

4.95

1.96

98.6

99.0

99.0

98.0

Brand II

Timolol

Brimonidine tartrate

5

2

4.96

1.97

4.92

1.93

99.2

98.5

98.4

96.5

Table 5: Assay of timolol maleate and brimonidine tartrate.
*: Mean of three replicates.

Conclusion

  The authors have developed two spectrophotometric methods for the simultaneous determination of Timolol maleate and Brimonidine tartrate in pharmaceutical formulations and the methods are validated. These two methods are simple, precise and accurate and can be successfully applied for the routine quality control of pharmaceuticals in industries.

References

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Citation

Citation: Mukthinuthalapati Mathrusri Annapurna and Yenda Manishankar. “Chemo-metric Assisted UV-Spectrophotometric Methods for the Simultaneous Estimation of Brimonidine Tartrate and Timolol Maleate". Acta Scientific Pharmaceutical Sciences 5.5 (2021): 60-66.

Copyright

Copyright: © 2021 Mukthinuthalapati Mathrusri Annapurna and Yenda Manishankar. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.




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