Balakrishna et al Journal of Drug Delivery & Therapeutics.

2020; 10(1):92-96

Available online on 15.01.2020 at http://jddtonline.info

Journal of Drug Delivery and Therapeutics

Open Access to Pharmaceutical and Medical Research
© 2011-18, publisher and licensee JDDT, This is an Open Access article which permits
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Open Access Research Article

Analytical method development and validation for the determination of
Brinzolamide by RP-HPLC
Tiwari Balakrishna 1*, Shirsat Mrunal K. 2, Kulkarni Amol 3
1 Research Scholar, Pacific University, Udaipur, India
2 Dean/Principal, PAHER University, Udaipur, India
3 Director, Institute of Pharmaceutical Science and Research (for girls), Swami Chincholi, Daund, Maharashtra, India

ABSTRACT
Brinzolamide is inhibitor of carbonic anhydride and is highly specific and non-competitive. The aim of the present study is to develop a simple,
precise, accurate, sensitive RP-HPLC method for the determination of bulk drug. The objective of the method validation is to demonstrate
whether the method was suited for the intended purpose. The method was validated as per the ICH guidelines. The method was validated for
linearity, precision (repeatability, intermediate precision), accuracy, specificity, robustness, ruggedness, limit of detection and limit of
quantification. Cosmosil (4.6X250mm, 5 μ) column was used for separation. The selected wavelength for Brinzolamide was 254 nm. The mobile
phase consists of Acetonitrile: Potassium dihydrogen phosphate buffer (40:60). Flow rate was delivered at 1.0 mL/min. Appropriate dilutions of
standard stock solutions were prepared to get desired concentrations in the range of 100-500 mcg/ml. The equation od standard curve was y =
441.8x + 1132 and R2 = 0.998. The RT obtained was 6.6167 minutes.
Keywords: Brinzolamide, UV spectroscopy, RP-HPLC, ICH

Article Info: Received 03 Nov 2019; Review Completed 21 Dec 2019; Accepted 29 Dec 2019; Available online 15 Jan 2020
Cite this article as:
Tiwari B, Shirsat MK, Kulkarni A, Analytical method development and validation for the determination of Brinzolamide by
RP-HPLC, Journal of Drug Delivery and Therapeutics. 2020; 10(1):92-96 http://dx.doi.org/10.22270/jddt.v10i1.3845

*Address for Correspondence:

Tiwari Balakrishna, Research Scholar, Pacific University, Udaipur, India

INTRODUCTION: After, doing in depth study in the present research work, it

was found that the present research work is having various
Brinzolamide is inhibitor of carbonic anhydride and is highly advantages over the previous work. The advantages include
specific and non-competitive. Chemically Brinzolamide is less retention times of the component, with good resolution.
(4R)-4-(ethylamino)-2-(3-methoxypropyl)-1,1-dioxo- The % RSD of robustness was found to be less. The results
2H,3H,4H-1λ⁶-thieno[3,2-e] [1,2]thiazine-6-sulfonamide. obtained from the validation suggest that the method was
Carbonic anhydrase is a type of enzyme that can be found in found to be precise, accurate, linear and robust enough and
several body tissues including eye. It is responsible for the method was also found to be economical.
catalysation of the reversible reaction indulged in carbon
dioxide hydration and carbonic acid dehydration. In human
beings, carbonic anhydrase is present in form of various
isoenzymes among which the most active one is carbonic
anhydrase II. During the ciliary processes of eye when
carbonic anhydrase is inhibited it results in reduction of
secretion of aqueous humour, most probably by decreasing
the creation of bicarbonate ions with further decrease in
transportation of sodium and fluid. This result in decrease of
intraocular pressure.
Literature review suggest few HPLC ,HPTLC, spectroscopic ,
Stability indicating HPLC determinations were performed.1-13 Figure 1: Sturucture of Brinzolamide
The aim of the present study is to develop a simple, precise,
accurate, sensitive HPLC method for the determination.
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Balakrishna et al Journal of Drug Delivery & Therapeutics. 2020; 10(1):92-96

EXPERIMENTAL WORK: Validation procedure:

Chemicals and reagents: HPLC grade acetonitrile, The objective of the method validation is to demonstrate
methanol, from Spectro Chemie. Ammonium acetate, acetic whether the method was suited for the intended purpose.
acid of analytical grade was used. Millipore grade water was The method was validated as per the ICH guidelines. The
used. The reference standard samples of Brinzolamide was method was validated for linearity, precision (repeatability,
provided was provided by Ajanta Pharma. intermediate precision), accuracy, specificity, robustness,
ruggedness, limit of detection, limit of quantification. A
Instrumentation and analytical conditions: calibration graph was constructed by taking six different
The analysis was carried out by using Younglin (S.K) concentrations, ranging from 50 – 150 μg/mL. The peak area
Gradient System UV Detector, 4.6X250mm cosmosil column, was calculated, and calibration curve was constructed by
20ml loop size HPLC (With UV/Vis Detector. Other taking peak area and concentration on both the axis. The
instrumentation includes Double beam UV- Visible linearity was evaluated by linear regression analysis. The
spectrophotometer Simadzu UV-1800, digital balance precision studies were demonstrated by two parameters
(metler tolado), vacuum pump (Gelman science), pH meter inter day and intraday precision. Intraday precision was
(poloman). performed by injecting six replicated injections to the
chromatographic system on the same day and calculated the
Chromatographic conditions: %RSD. The inter day precision was performed by injecting
Cosmosil (4.6X250mm, 5 μ) column was used for separation. six replicated injections at two consecutive days. From the
peak area of the chromatograms, the %RSD was calculated.
Preparation of Standard stock solution: The accuracy was determined by adding a known amount of
the standard to the sample, and the percentage recovery was
100 mg of Brinzolamide was weighed in to 100 ml
estimated. The robustness was determined by incorporating
volumetric flasks. The drug was dissolved in 40 ml of solvent,
deliberate changes into the method conditions like the
and shaken manually for 10 min. The volume was made up to
change in flow rate, pH, and gradient. Ruggedness was
the mark with solvent and the final strength obtained was
performed by carrying out the proposed method with two
1000 µg/ml.
different analysts.
Preparation of sample solution:
RESULTS AND DISCUSSION:
1, 2, 3, 4, 5ml of the standard stock solution of Brinzolamide
were transferred separately into five 10ml volumetric flasks Selection of wavelength:
and volume was made up to mark to get the final UV spectrum was obtained by preparing a solution by taking
concentrations of 10, 20, 30, 40, 50µg/ml of Brinzolamide. diluents and scanned between 200 to 400 nm. Brinzolamide
The chromatograms were recorded and a standard curve shows λmax at 254 nm. So, it is selected as a detection
was plotted by taking concentrations on x-axis and area wavelength.
under curve on y-axis. The equation of standard curve was
generated to determine the concentration of drug in pure
and degradation products.

Figure 2: Spectrum of Brinzolamide

Development and optimization of the HPLC method: delivered at 1.0 mL/min with detection wavelength at 254
nm. A 20 μL was injected to the chromatographic system
For getting an optimized chromatographic condition, A
with ambient temperature. Acetonitrile: Potassium
Cosmosil (4.6X250mm, 5 μ) column was used for separation.
dihydrogen phosphate buffer (40:60) was the optimized
The mobile phase consists of Acetonitrile: Potassium
mobile phase selected for experimentation. The RT obtained
dihydrogen phosphate buffer (40:60). Flow rate was was 6.6167 minutes.

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Balakrishna et al Journal of Drug Delivery & Therapeutics. 2020; 10(1):92-96

Figure 3: RT of Brinzolamide

Method validation: 500 mcg/ml. Then the concentrations were plotted against
the area under curves to get the equation of standard curve,
Linearity:
as presented in Table 1.
Appropriate dilutions of standard stock solutions were
prepared to get desired concentrations in the range of 100-

Table 1: Dilutions for linearity study

Stock solution Diluted (ml) Final
(µl) concentration
100 10 10
200 10 20
300 10 30
400 10 40
500 10 50

Figure 4: Standard curve of Brinzolamide

Table 2: Linearity Data
S. N. Conc. in (µg/ml) Mean AUC (n = 5) % RSD
1 10 5236 0.453
2 20 10113 0.243
3 30 14705 0.428
4 40 18999 0.651
5 50 22884 0.766

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Balakrishna et al Journal of Drug Delivery & Therapeutics. 2020; 10(1):92-96

Precision:
Precision is determined at two levels: a) Repeatbility b) Intraday and Interday Precision.
Table 3: Precision Data
Parameter Amount taken (µg/ml) Amount found* (µg/ml) %RSD
System Precision 60 59.94 0.475
Method Precision 60 60.15 0.683
*Mean of Six observations
Table 4: Intraday and Interday Precision Data
Concentration of Brinzolamide Intraday* %RSD Interday * %RSD

20µg/ml 20.07 0.083 19.92 0.138

40µg/ml 39.99 0.091 39.94 0.129

60 µg/ml 60.09 0.074 59.91 0.144

*Mean of three observations

Accuracy:
To check the accuracy of the developed methods, analytical recovery experiment was carried out by standard addition method.
Recovery study was performed by adding 80, 100 and 120 % of the test concentration as per ICH guidelines.
Table 5: Accuracy
Concentration of Drug Concentration of Drug
Sr. No. % Recovery± SD
Added (µg/ml) Added (µg/ml)
1 64 10.4 99.99 ± 0.006
2 80 13 99.96 ± 0.018
3 96 15.6 96.95 ±0.027

Limit of Quantification (LOQ) and Limit of Detection Robustness was studied by observing the change in
(LOD): following parameters, and then observation of each
parameter change was done to access their effect on system
The limit of quantification (LOQ) is defined as the lower
suitability and assay. Change in mobile phase composition
concentration of an analyte in a sample that can be
was done by ± 5.0 ml of organic solvent and the change in
determined with acceptable precision and accuracy under the detection wavelength ± 10 nm was done.
the stated operational conditions of the method. The limit of
detection (LOD) is defined as the lowest concentration of an Change in mobile phase composition: The sample solution
analyte in a sample that can be detected, not quantified. The at test concentration (60μg/ml of drug was injected thrice
LOD and LOQ were estimated from the set of 5 calibration with the mobile phase composition changed by ± 5.0ml of
curves used to determine method linearity. The LOD and organic solvent from the developed method.
LOQ were calculated using following formula
Change in detection wavelength: The sample solution at
LOD = 3.3× σ/S test concentration (60μg/ml of drug was injected thrice with
the change in detection wavelength by ± 10 nm from the
LOQ= 10× σ/S
developed method. The % assay of drug after changes in
Where, σ = Standard deviation of the Y- intercepts of the 5 method parameters were observed.
calibration curves, S = Mean slope of the 5 calibration curves.
CONCLUSION:
The LOQ and LOD parameters of Brinzolamide are provided
A precise RP – HPLC method was developed for the
in Table 5.
determination of Brinzolamide. The shorter run time elutes
Table 6: LOD and LOQ parameters Erlotinib hydrochloride with good resolution, and
symmetry. The method was validated as per the ICH
Parameter Values guidelines and the method was found to be simple, precise,
S.D. of Intercept* 0.531037 linear, accurate, rugged and robust enough.

Mean Slope of Calibration Curve* 441.8

LOD (μg/ml) 0.00396
LOQ (μg/ml) 0.01202
Robustness:

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Balakrishna et al Journal of Drug Delivery & Therapeutics. 2020; 10(1):92-96

Table 7: Summary of Validation parameters (5) Devi, C. M.; Kalita, P.; Dutta, R. S. Development and validation
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Intra-day (n = 3) 0.074 - 0.091 Simultaneous Estimation of Brinzolamide and Timolol
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Inter-day (n = 3) 0.129 - 0.144 2016, 7 (3), 1290–1298.
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