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Development of RP-HPLC method for simultaneous determination of

Brimonidine tartrate and Brinzolamide by QbD approach and its validation

Article · October 2016

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Eurasian J Anal Chem, 2016, 11(2), 63-78

Development of RP-HPLC
Method for Simultaneous
Determination of Brimonidine
Tartrate and Brinzolamide by
QbD Approach and Its
Validation
Varsha P. Agrawal, Sonal S. Desai & Girish K. Jani
Department of Quality Assurance Techniques, S.S.R. College of Pharmacy, D & N. H,
INDIA.

ŸReceived 02 July 2015 ŸRevised 28 September 2015 ŸAccepted 01 October 2015

A simple, rapid, accurate and precise reversed phase high performance liquid
chromatographic method has been developed for the simultaneous determination of
brimonidine tartrate (BRT) and brinzolamide (BRZ). A 32 factorial design was utilized to
aid in method development and optimization. Effective chromatographic separation was
achieved using C18 column (250 × 4.6 mm, 5μm) as a stationary phase and mobile phase
consisted of methanol: 0.01 M ammonium acetate buffer (49.5: 50.5, v/v), pH adjusted to
3.8 with acetic acid at a flow rate of 1.1 mL/min at a detection wavelength of 260 nm.
The injection volume was 20 µL. Quality by design approach was applied to evaluate the
effect of two factors i.e. mobile phase composition and flow rate on the various
chromatographic responses (area, number of theoretical plates, resolution, retention
time and tailing factor). The retention time of BRT and BRZ were found to be 3.96 and
8.34 min; respectively. Calibration curves were found to be linear over the concentration
range of 0.2-1.4 µg/mL for BRT and 1-7 µg/mL for BRZ. The limit of detection and limit
of quantitation for BRT were found to be 0.03 µg/mL and 0.09 µg/mL whereas those for
BRZ were found to be 0.018 µg/mL and 0.051 µg/mL; respectively. The % recovery of
the drugs by developed method was found in the range of 99.04 to 101.67 %. The
proposed method was found to be precise as well as robust. The method was
successfully applied for quantitative determination of BRT and BRZ in in-house dosage
form i.e. suspension.

Keywords: brimonidine tartrate, brinzolamide, RP-HPLC, method validation, quality by

design

INTRODUCTION

Glaucoma is an eye disease, wherein the intraocular pressure within the eye is

Correspondence: Dr. Sonal S. Desai

Department of Quality Assurance Techniques, S.S.R. College of Pharmacy, Sayli,
Silvassa, D & N.H - 396230, INDIA
E-mail: sdesai6381@gmail.com
doi: 10.12973/ejac.2016.122a

Copyright © 2014 by iSER, International Society of Educational Research

ISSN: 1306-3057

V. P. Agrawal, S. S. Desai & G. K. Jani

enough so as to cause damage to the optic nerve [1]. Brimonidine tartrate (BRT),
chemically 5-bromo-6 (2-imidazolidinylideneamino) quinoxaline L- tartrate is a α2-
adrenoreceptor agonistused for the treatment of open-angle glaucoma [2]. The
ocular hypotensive effect of this molecule is because of its ability to decrease
aqueous humor production [3]. Brinzolamide (BRZ), chemically (R) – 4- (ethyl
amino)-3,4-dihydro-2-(3-methoxy propyl)-2H thienol[3,2-e]- 1,2-thiazine-6
sulphonamide 1,1-dioxide, a non-competitive reversible carbonic anhydrase
inhibitor is indicated for the treatment of elevated intraocular pressure in patients
with glaucoma [4]. Simbrinza ophthalmic suspension is available as a fixed dose
combination of BRT (0.2% w/w) and BRZ (1% w/w), which is indicated for the
treatment of glaucoma.
Quality by Design (QbD) is a systematic approach that focuses on understanding
and control of processes to provide continuous improvement in method
development with the desired critical quality attributes [5]. QbD-based analytical
method development helps to recognize and reduce sources of variability that may
lead to poor method performance. It also ensures that the method meets its
proposed performance requirements throughout the product and method life cycle
[6-8]. Quality is built into the development of the method itself, resulting in
improved separations. USFDA also proposed QbD as important criteria for method
development.
Several analytical methods such as UV [9,10], RP-HPLC [11-13], HPTLC [14],
UPLC [15], spectrofluorimetric [16], HILIC [17], GC-MS [18], LC/MS/MS [19] and
capillary electrophoresis methods [20] are reported for the determination of BRT
alone. Few UV [21-24], RP-HPLC [25-28] and HPTLC [29] methods have been
reported for estimation of BRT and Timolol (TM). BRZ is official in IP [30] and
USP[31]. Methods such as UV spectrophotometry [32], HPLC and HPTLC [33] are
reported for simultaneous estimation of BRZ and TM. Few UV derivative
spectrophotometric methods [34, 35] have been reported for the determination of
BRT and BRZ. To the best of our knowledge, till now no reversed phase high
performance liquid chromatographic method has been reported for simultaneous
determination of BRT and BRZ utilizing experimental design. Thus, the aim of the
present study was to develop, optimize and validate a simple and rapid RP-HPLC
method for the simultaneous determination of BRT and BRZ using QbD approach.

Figure 1. Chemical structure of brimonidine tartrate (BRT) and brinzolamide (BRZ).

MATERIALS AND METHODS

Chemicals and reagents

Reference standards of BRT (purity 98 % w/w) and BRZ (purity 98 % w/w)

were obtained from Sun Pharmaceutical Pvt. Ltd, Halol, Gujarat, India. HPLC grade

64 © 2016 iSER, Eurasian J Anal Chem, 11(2), 63-78

 Development of RP-HPLC Method

methanol and water were purchased from Fisher scientific India Pvt. Ltd, Powai,
Mumbai. HPLC grade ammonium acetate was purchased from Rankem (RFCL),
Haryana, India.

Instruments

Analysis was performed on Cyber lab LC 100HPLC system equipped with binary
LC P-100 pump, high pressure gradient mixer (1500 µL) and a UV detector. Data
acquisition and processing was done using WS- Workstation software. Equitron
digital ultrasonic cleaner was used for mixing the solutions. Precisa digital weighing
balance was used for weighing. Equiptronics digital pH meter was used for all pH
measurements.

Selection of wavelength

For both drugs, standard solutions of 10 μg/mL were prepared in methanol

individually and were scanned in the wavelength range of 200-400nm and the
overlain spectrum was obtained. From the overlain spectrum, isoabsorptive point
was found to be at 260 nm (Figure 2). Thus, 260 nm was selected as detection
wavelength for the simultaneous estimation of both the drugs.

Figure 2. Overlain spectrum of BRT and BRZ.

Method optimization

Initially various mobile phases such as methanol: water (80: 20, v/v);
acetonitrile: water (80: 20, v/v); methanol: 0.01 M phosphate buffer (pH adjusted to
3.14 with ortho phosphoric acid) (40: 60, v/v); acetonitrile: methanol: 0.01M
phosphate buffer (pH 3.14) (10: 40: 50, v/v/v); methanol: 0.4 % TEA in water (pH
adjusted to 3.0 with o-phosphoric acid) (25: 75, v/v) etc. were tried at different flow
rates but they didn’t produced satisfactory results. After evaluating all the factors
like resolution, peak symmetry, number of theoretical plates, time required for
analysis; the mobile phase consisting of methanol: 0.01 M ammonium acetate buffer

© 2016 iSER, Eurasian J Anal Chem, 11(2), 63-78 65

V. P. Agrawal, S. S. Desai & G. K. Jani

(pH adjusted to 3.8 with acetic acid) (45: 55, v/v) at a flow rate of 1.0 mL/min was
selected for further optimization by QbD.

Software aided method optimization

A 32 factorial experimental design was separately applied for both drugs to

optimize the chromatographic conditions. A 32 factorial design indicates that there
are three levels and two factors involved in it. The three levels were low (-1),
medium (0) and high (+1) whereas the factors were A (mobile phase ratio) and B
(flow rate). The chromatographic responses involved in the trial were area (Y1, Y6),
number of theoretical plates (Y2, Y7), resolution (Y3, Y8), retention time (Y4, Y9) and
tailing factor (Y4, Y10). This design was specifically selected since it required fewer
runs (13) as compared to the others. It was suitable for exploring response surface
and creating different models with Design Expert ® (Version 9.0.4, Trial version).
The levels selected for both the drugs are described in Table 1. About 13
experimental runs were carried out for both drugs using the different
chromatographic conditions and responses were observed as described in Table 2
and Table 3.

Table 1: Experimental factors and levels used in factorial design.
Factor Level used
Independent variable Low (-1) Medium (0) High (+1)

A= Mobile phase ratio (v/v) 50 : 50 45 : 55 40 : 60

B = Flow rate (mL min-1 ) 0.5 1.0 1.5
Dependent variable
Y1,Y6 = Area of BRT and BRZ; respectively
Y2, Y7 = No. of theoretical plates of BRT and BRZ; respectively
Y3, Y8 = Resolution of BRT and BRZ; respectively
Y4, Y9 = Retention time of BRT and BRZ; respectively
Y5, Y10 = Tailing factor of BRT and BRZ; respectively

Analysis of variance (ANOVA) was applied to the response variables to examine
the significance of the model. Lack of fit test, which indicated insignificant lack of fit
value corresponding to a higher p-value as compared to the model F-value, was also
used to examine the applied model.

Chromatographic conditions

Chromatographic separation was achieved on Sun chrome C-18 column (250 ×

4.6 mm, 5 μm) and the software aided optimized mobile phase was methanol: 0.01
M ammonium acetate buffer (pH adjusted to 3.8 with acetic acid) (49.5: 50.5, v/v).
The flow rate of mobile phase was 1.1 mL min-1. The detection was carried out at
260 nm. The injection volume was 20 µL. The chromatographic run time was 10
min. The mobile phase was filtered before use through a 0.2 μ membrane filter
(Sartorius Stedium Biotech, Germany) and degassed for about 15 min.

Preparation of mobile phase

Accurately weighed 0.3854 g of ammonium acetate was dissolved in 500 mL of

HPLC grade water. The solution was adjusted to pH 3.8 with 1M acetic acid. The
resulting buffer was filtered through a 0.2 µ membrane filter. Required volume of
the mobile phase was prepared by mixing methanol and ammonium acetate buffer
(49.5: 50.5, v/v). Then the mixture was sonicated for about 15 min to ensure proper
mixing and then filtered through a 0.2 µ membrane filter.

66 © 2016 iSER, Eurasian J Anal Chem, 11(2), 63-78

 Development of RP-HPLC Method

Table 2: Observed responses of 13 experimental runs for BRT.

Run Level Factor Response
Mobile phase Flow rate Retention Area Resolution No. of Tailing
(v/v) (mL/min) time (mAU) theoretic factor
(min) al plates
1 1, 1 50:50 1.5 3.188 6418.3 15.30 5673.18 1.68
2 0, 0 45:55 1.0 3.580 6891.2 19.18 5918.94 1.54
3 0, 0 45:55 1.0 3.580 6891.2 19.18 5918.94 1.54
4 -1,+1 40:60 1.5 3.912 7011.9 24.97 6165.57 1.73
5 -1,-1 40:60 0.5 6.612 11911.0 26.97 6848.36 2.15
6 0,0 45:55 1.0 3.580 6891.2 19.18 5918.94 1.54
7 -1,0 40:60 1.0 4.144 7326.9 24.76 6316.25 2.15
8 0,0 45:55 1.0 3.580 6891.2 19.18 5918.94 1.54
9 0,-1 45:55 0.5 5.975 11544.8 21.62 6584.08 1.90
10 +1,0 50:50 1.0 3.440 6853.7 15.22 5736.26 1.58
11 +1,-1 50:50 0.5 5.636 11342.2 17.04 6299.36 1.66
12 0,+1 45:55 1.5 3.373 6484.0 19.47 5922.00 1.66
13 0,0 45:55 1.0 3.580 6891.2 19.18 5918.94 1.54

Table 3: Observed responses of 13 experimental runs for BRZ.
Run Level Factor Response
Mobile Flow rate Retention Area Resolution No. of Tailing
phase (mL/min ) time (mAU) theoretic factor
(v/v) (min) al plates
1 +1,+1 50:50 1.5 6.570 3818.5 15.30 9299.64 1.22
2 0, 0 45:55 1.0 8.802 4452.4 19.18 9651.61 1.18
3 0,-1 45:55 0.5 14.741 7167.5 21.62 13043.5 1.18
4 0,0 45:55 1.0 8.802 4452.4 19.18 9651.61 1.18
5 0,0 45:55 1.0 8.802 4452.4 19.18 9651.61 1.18
6 +1,-1 50:50 0.5 11.629 6833.4 17.04 12292.7 1.22
7 0,0 45:55 1.0 8.802 4452.4 19.18 9651.61 1.18
8 -1,-1 40:60 0.5 19.918 7417.3 26.97 14261.1 1.18
9 -1,0 40:60 1.0 12.425 4455.0 24.76 11647.3 1.16
10 0,0 45:55 1.0 8.802 4452.4 19.18 9651.61 1.18
11 -1,+1 40:60 1.5 11.678 4087.8 24.97 12212.8 1.09
12 +1,0 50:50 1.0 7.108 4316.4 15.22 8951.51 1.06
13 0,+1 45:55 1.5 8.298 3961.5 19.47 10084.2 1.18

Preparation of solutions

Preparation of standard stock solutions

Standard stock solutions of BRT and BRZ were prepared by dissolving 10 mg of

each drug separately in separate 100 mL volumetric flasks using methanol as a
solvent up to 50 mL, then sonicated for 15 minutes and the final volume was made
up to 100 mL with methanol to get the standard stock solutions containing 100
μg/mL of each of BRT and BRZ.

Preparation of working standard solutions

Working standard solution of BRT (10 μg/mL) was prepared by transferring

about 1 mL of stock solution of BRT into 10 mL volumetric flask and the volume was
made up to the mark by using mobile phase as diluent. Working standard solution of
BRZ (10 μg/mL) was prepared by transferring about 5 mL of stock solution of BRZ
into 50 mL volumetric flask and diluting it up to mark with mobile phase.

© 2016 iSER, Eurasian J Anal Chem, 11(2), 63-78 67

V. P. Agrawal, S. S. Desai & G. K. Jani

Preparation of mixed standard solution

Required mixed standard solution containing BRT and BRZ was prepared by
transferring accurate volumes of each of the working solution of BRT as well as BRZ
to a 10 mL volumetric flask and diluting it up to mark with mobile phase.

Preparation of sample solution

Accurately measured 1 mL of in-house suspension containing 0.2 % BRT and 1 %

BRZ was taken and transferred to 100 mL volumetric flask. About 50 mL of
methanol was added into the flask and sonicated for 15 minutes. Then, the final
volume was made up to 100 mL with methanol to produce solution containing 20
μg/mL of BRT and 100 μg/mL of BRZ. From this stock, 1 mL of solution was taken
and diluted up to10 mL with methanol to obtain a solution containing 2 μg/mL of
BRT and 10 μg/mL of BRZ. The resulting solution was further diluted to get a final
solution containing 0.8 μg/mL of BRT and 4 μg/mL of BRZ and then filtered through
0.2 μm filter to get a clear solution.

Method validation

The developed and optimized method was validated as per ICH guidelines [36]
for various parameters such as specificity, system suitability, linearity and range,
LOD, LOQ, accuracy, precision and robustness.

Specificity

The specificity of the method was assessed by comparing chromatograms

obtained from drug standards with that obtained from sample solution.

System suitability

System suitability parameters like number of theoretical plates, resolution and

tailing factor were evaluated by injecting six replicates of working standards
containing 0.8 μg/mL of BRT and 4 μg/mL of BRZ. Then the % RSD was calculated.

Linearity and range

The linearity of the developed method was estimated using standard solutions of
seven different concentrations in the range of 0.2–1.4 μg/mL for BRT and 1–7
μg/mL for BRZ. Each solution was injected in triplicate. A graph of average area vs.
concentration was plotted and regression coefficients (R2) for both the drugs were
calculated. The linearity equations for both the drugs were obtained by linear
regression analysis, using GraphPad Prism software.

Limit of Detection (LOD) and Limit of Quantitation (LOQ)

LOD and LOQ of BRT and BRZ were evaluated using standard deviation method.
Calibration curves were plotted in the range of 0.025-0.2 μg/mL for BRT and 0.1-0.6
μg/mL for BRZ.LOD and LOQ of both the drugs were calculated using formula 3.3
σ/S and 10 σ/S, respectively, where σ is the standard deviation of intercepts and S is
the slope of the calibration curve.

68 © 2016 iSER, Eurasian J Anal Chem, 11(2), 63-78

 Development of RP-HPLC Method

Accuracy

The accuracy of the proposed method was determined by standard addition

method by calculating the percentage recoveries of both the drugs. The study was
carried out at three different concentration levels. Known amount of standard
solution of BRT and BRZ (0.4μg/mL and 2 μg/mL; 0.8 μg/mL and 4 μg/mL; 1.2
μg/mL and 6 μg/mL) were spiked into the prequantified sample solution of BRT and
BRZ (0.1 μg/mL and 0.5 μg/mL); respectively. Area was measured in triplicates,
concentrations of both the drugs were calculated and % recovery was determined at
each level using following formula:
𝑂𝑏𝑠𝑒𝑟𝑣𝑒𝑑𝑣𝑎𝑙𝑢𝑒
% 𝑅𝑒𝑐𝑜𝑣𝑒𝑟𝑦 = ∗ 100%
𝑇𝑟𝑢𝑒𝑣𝑎𝑙𝑢𝑒

Precision

The precision studies were carried out as inter-day and intra-day precision
studies at three different concentration levels of test solution. The concentrations of
BRT and BRZ at 50% level were 0.4 μg/mL and 2 μg/mL, respectively; at 100 % level
were 0.8 μg/mL and 4 μg/mL, respectively and at 150 % level were 1.2 μg/mL and 6
μg/mL, respectively. Intraday precision studies were carried out on the same day at
different time intervals whereas intraday studies were carried out on three different
consecutive days. Area of both the drug at each concentration level was measured in
triplicate and % RSD was calculated.

Robustness

The robustness of the method was evaluated by varying method parameters such
as flow rate (1.0 mL/min, 1.2 mL/min); detection wavelength (259 nm, 261 nm);
mobile phase composition (50:50, 49:51) and pH (3.7, 3.9). It was assessed by
injecting the standard solution (0.8 μg/mL of BRT and 4 μg/mL of BRZ) six times and
test solution (0.8 μg/mL of BRT and 4 μg/mL of BRZ) twice and calculating the
values of % RSD. The data were evaluated using one-way analysis of variance
(ANOVA).

Analysis of in-house suspension

An in-house suspension containing 0.2 % BRT and 1% BRZ was prepared,

suitable dilution was done and then analyzed. The % content of each drug was
determined using the following formulas:
𝐴 9: 𝑊;: 1 0.8 10 100 10
% 𝑜𝑓𝐵𝑅𝑇 = × × × × × × ×𝑃:
𝐴;: 100 10 10 𝑊9: 1 4

𝐴 9C 𝑊;C 1 4 10 100 10
% 𝑜𝑓𝐵𝑅𝑍 = × × × × × × ×𝑃C
𝐴;C 100 10 10 𝑊9C 1 4

Where, AT1 andAT2are the average area of test solutions of BRT and BRZ,
respectively; AS1 and AS2 are the average area of standard solutions of BRT and BRZ,
respectively; WT1 and WT2 are the weights of BRT and BRZ, respectively in the
sample; WS1 and WS2 are the weight of standards of BRT and BRZ, respectively; P1
and P2 are the purity of standards of BRT and BRZ, respectively.

© 2016 iSER, Eurasian J Anal Chem, 11(2), 63-78 69

V. P. Agrawal, S. S. Desai & G. K. Jani

RESULTS AND DISCUSSION

Design of experiment

A 32 full factorial design was performed using 13 experimental runs for BRT as
well as for BRZ. The dependent as well as independent variables of all runs are
shown in Table 1. The proposed regression equations for various chromatographic
responses of both the drugs are given in the Table 4.
It was observed that the best fitted model for BRT was the quadratic model. In
case of BRZ for all the responses quadratic model was found to be the best fitted
model except for tailing factor where in the best fitted model was linear (Table 5).A
positive value represents an effect that favors the optimization, while a negative
value indicates an inverse relationship between the factor and the response. In case
of BRT, it is clear from the equations that the factor A (mobile phase composition)
and factor B (flow rate) had negative effect on all the chromatographic responses. In
case of BRZ, the factor A had negative effect on area, number of theoretical plates,
resolution and retention time and it had a positive effect on tailing factor whereas
the factor B had negative effect on all the chromatographic responses. Interaction of
A and B had a negative effect on Y1and Y4 and had a positive effect on Y2, Y3 and Y5
with reference to BRT. For BRZ, the square of the factor A2 was having a positive
impact while B2 was having a negative impact on response area. The source sum of
squares (Source SS) in ANOVA indicates that the contribution of factor A (mobile
phase) (SS=993.31) is higher than factor B (flow rate) (SS =34.56) for optimizing the
response term resolution.

Table 4: Regression equations for various chromatographic responses.
Drug Regression equation
BRT Y1= 6897.08 – 272.60*A – 2480.63 *B – 6.20 * AB + 178.51 *A2 + 2102.61 * B2
Y2 = 5934.63 – 270.23 *A – 328.51 *B + 14.15 *A*B + 52.41 *A2 + 279.20 *B2
Y3 = 19.22 – 4.86 *A – 0.98 *B + 0.06 *A*B + 0.67 *A2 + 1.22 *B2
Y4 = 3.59 – 0.41 *A – 1.29 *B – 0.06 *A*B + 0.19 *A2 + 1.07 *B2
Y5 = 1.58 – 0.185 *A – 0.11 *B + 0.11 *A*B + 0.18*A2 + 0.09 *B2
BRZ Y6 = 4446.68 – 165.30 *A – 1591.73 * B + 78.65 *A*B – 46.69 *A2 + 1132.11 *B2
Y7 = 9678.51– 1262.89 *A – 1333.43 *B – 236.20 *A*B + 553.60 *A2 + 1818.07
*B2
Y8= 19.22 – 4.86 *A – 0.98*B + 0.06 *A*B + 0.67*A2 + 1.23*B2
Y9 = 8.81 – 3.12 *A – 3.29 *B + 0.79 *A*B + 0.95 *A2 + 2.70 *B2
Y10 = 1.17 + 0.01 *A – 0.02 *B

The values of R2for Y1, Y2, Y3, Y4 and Y5 for full model in case of BRT were 0.9998,
0.9927, 0.9993, 0.9994, 0.8433; respectively whereas in BRZ were 0.9985, 0.9968,
0.9988, 0.9930 and – 0.0933; respectively (Table 5). For BRT, all model terms were
found to be significant whereas in case of BRZ all model terms except tailing factor
were found to be significant. In case of BRT, the calculated F values for full models of
area, number of theoretical plates, resolution, retention time and tailing factor were
5865.76, 191.49, 2082.03, 2374.31 and 7.53; respectively whereas that of BRZ were
930.09, 432.40, 2082.03, 340.98 and 0.49; respectively.
3D response surface plots presented as Figure 3a–e for BRT and as Figure 4a–e
for BRZ which were used to determine the relationship between the response and
the factors. In case of BRT, the plot (Figure 3a) indicates that both the mobile phase
(A) and flow rate (B) had a negative effect on area. With the decrease in flow rate,
the area increases. It is evident from Fig. 3b, that an increase in mobile phase
composition or flow rate decreases the number of theoretical plates. A response
surface plot (Figure 3c, 4c) indicates the negative effect of both the factors on
resolution. The retention time and tailing factor decreases with the increase in flow

70 © 2016 iSER, Eurasian J Anal Chem, 11(2), 63-78

 Development of RP-HPLC Method

rate (Figure 3d-e). In case of BRZ, both mobile phase (A) and flow rate (B) had a
negative effect on area as well as on number of theoretical plates as shown in Figure
4a-b. It is obvious from Figure 4d that a decrease in flow rate causes an increase in
retention time. When considering the response term tailing factor, the response
surface plot (Figure 4e) indicates the positive effect of mobile phase composition on
the response term.

Table 5: Regression analysis summary for the finally suggested models.
Drug Respons Model R2 Adjusted Predicted SD % CV Adequate
e R 2 R 2 precision
BRT Y1 Quadratic 0.9998 0.9996 0.9977 42.38 0.53 191.25
Y2 Quadratic 0.9927 0.9876 0.9477 38.01 0.62 46.37
Y3 Quadratic 0.9993 0.9988 0.9944 0.12 0.61 144.28
Y4 Quadratic 0.9994 0.9990 0.9944 0.036 0.86 138.77
Y5 Quadratic 0.8433 0.7314 -0.2419 0.12 6.73 8.81
BRZ Y6 Quadratic 0.9985 0.9974 0.9849 64.60 1.31 80.06
Y7 Quadratic 0.9968 0.9945 0.9697 126.52 1.17 63.30
Y8 Quadratic 0.9993 0.9988 0.9944 0.12 0.61 144.28
Y9 Quadratic 0.9959 0.9930 0.9584 0.31 2.91 62.85
Y10 Linear 0.0889 - 0.0933 -0.8719 0.047 4.03 2.35

Method optimization

The final mobile phase ratio optimized for the simultaneous determination of
BRT and BRZ was done using Design Expert ® (Version 9.0.4, Trial version) after
interpreting the various response surface plots. In the optimization step, the effect of
two factors i.e. mobile phase composition and flow rate on the various
chromatographic responses were evaluated. The desirability plot for both the drugs
was generated by the software. In case of BRT, the desirability factors of mobile
phase and flow rate were found to be 0.906 and 0.240; respectively (Figure 3f)
whereas incase of BRZ, they were found to be 0.976 and 0.846; respectively (Figure
4f). As per desirability factors, different combinations of methanol and acetate
buffer at suggested flow rate were tried and responses for both the drugs were
evaluated. The optimized mobile phase selected was methanol: 0.01M ammonium
acetate buffer (pH 3.8) (49.5: 50.5, v/v) at flow rate of 1.1 mL/min, which resulted in
desired resolution and peak symmetry and require low solvent consumption.

Figure 3. 3D surface plots of BRT for various chromatographic responses (a) area; (b) number of
theoretical plates

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V. P. Agrawal, S. S. Desai & G. K. Jani

Figure 3. 3D surface plots of BRT for various chromatographic responses (c)
resolution; (d) retention time; (e) desirability.

Figure 4. 3D surface plots of BRZ for various chromatographic responses (a) area;
(b) number of theoretical plates; (c) resolution

72 © 2016 iSER, Eurasian J Anal Chem, 11(2), 63-78

 Development of RP-HPLC Method

Figure 4. 3D surface plots of BRZ for various chromatographic responses (e) desirability.

Specificity

The proposed HPLC method was found to be specific as there was no interference
found from the solvent, mobile phase or excipients present in the suspension
(Figure 5 and 6)

Figure 5. Representative chromatogram of standard BRT and BRZ using optimized mobile phase.

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V. P. Agrawal, S. S. Desai & G. K. Jani

Figure 6. Representative chromatogram of BRT and BRZ in sample.

System suitability

The column efficiency as determined from the number of theoretical plates for
both the drugs was found to be more than 4000; resolution was more than 14 and
tailing for the same peak was found to be less than 2. Also the % RSD for all these
parameters was found to be less than 2 %. System suitability analysis of both the
drugs is represented in Table 6.

Table 6: System suitability parameters.
Parameters BRT BRZ
Mean± S.Da %RSDa Mean± S.Da %RSDa
Number of theoretical plates 4906.63±69.84 1.42 8461.43±113.18 1.34
Resolution 14.57±0.17 1.15 14.57±0.17 1.15
Tailing factor 1.32±0.02 1.79 1.12±0.02 1.82
a = average of six determinations

Linearity and Range

The proposed method showed linearity over concentration range of 0.2 -1.4
μg/mL for BRT and 1 -7 μg/mL for BRZ with regression coefficients 0.9998 and
0.9993; respectively (Table 7). Statistically calculated F value for linearity regression
was found to be Fcal 92271for BRT and Fcal 28594 for BRZ as compared to F Crit
4.3807(DFn, DFd = 1.0, 19.0) indicating the statistical significance of method
linearity.

Table 7. Regression analysis data for the proposed method
Parameters BRTb BRZb
Wavelength (nm) 260 260
Linearity (μg mL-1 ) 0.2 -1.4 1 -7
Regression equation Y= 5465x+ 82.97 Y= 4031x- 13.50
Slope 5465 4031
Intercept 82.97 13.50
Correlation coefficient ( R2) 0.9998 0.9993
LOD(μg mL-1 ) 0.0171 0.0296
LOQ(μg mL-1 ) 0.0518 0.0898
b = three determinations, LOD=Limit of detection, LOQ= Limit of quantification

74 © 2016 iSER, Eurasian J Anal Chem, 11(2), 63-78

 Development of RP-HPLC Method

Accuracy
The percentage recoveries of both the drugs were found to range between 99.04
– 101.67 % which are within the acceptance limit as shown in the Table 8.

Table 8: Accuracy of the proposed HPLC method.
% Level Amount Presentc Amount recoveredc % Recovery ± S.Dc
(µg mL-1 ) (µg mL-1 )
BRT BRZ BRT BRZ BRT BRZ
50 0.41 1.98 0.41 1.96 100.80 ± 1.07 99.04± 0.30
100 0.80 4.00 0.80 3.99 100.67 ± 0.88 99.80± 0.28
150 1.21 5.96 1.23 5.95 101.67±0.19 100.02± 1.02
c = three determinations.

Precision
Intraday as well as intraday precision studies were carried out for evaluating the
precision of the proposed method and the % RSD was found to be less than 2 at each
level as represented in Table 9. Thus, the developed method was found to be precise.

Table 9: Intra-day and inter-day precision of the proposed HPLC method.
Concentration Intraday Precision (% RSD)d Interday Precision (% RSD)d
Drugs
(µg/mL) Day 1 Day 1 Day 2 Day 3
BRT 0.4 0.76 1.26 1.46 1.28
0.8 0.38 0.28 0.65 0.70
1.2 0.16 0.40 0.51 0.25
BRZ 2 0.12 0.39 0.65 0.42
4 0.27 0.60 0.48 0.14
6 0.83 0.88 0.48 0.19
d = three determinations

Robustness
In robustness study, % RSD was found to be less than 2 % in case of area of
standard solutions and % content was found to be between 98-102 % (Table 10).
Although the calculated F-value was higher than the critical F-value but the values of
% RSD obtained for area, retention time and % w/w of drug were found to be less
than 2.0. Hence, the developed method was robust.

Table 10: Evaluation of robustness for determination of BRT and BRZ.
Parameter Areae Average (% RSD) % Content (w/w)
Retention timee
BRT BRZ BRT BRZ BRT BRZ
Flow rate (mL/min)
1.0 4536.62(0.57) 16188.13(0.41) 4.07(0.65) 8.45(0.70) 100.83(0.25) 98.18(0.68)
1.1 4469.52(0.52) 15943.13(0.32) 4.00(0.26) 8.34(0.41) 99.15(0.52) 98.65(0.32)
1.2 4263.45(0.75) 15332.25(0.14) 3.73(0.62) 7.73(0.54) 101.22(0.14) 99.70(0.22)
Fcal/Fcri 44.67 127.58 117.45 114.63 2.29 8.96
Wavelength (nm)
259 4543.83(0.81) 15861.75(0.31) 3.87(0.54) 8.13(0.49) 98.17(0.81) 98.50(0.46)
260 4469.52(0.52) 15943.13(0.32) 4.00(0.26) 8.34(0.41) 99.15(0.52) 98.65(0.32)
261 4357.82(1.40) 15884.48(0.55) 3.91(1.22) 8.39(1.62) 98.50(1.37) 98.15(0.78)
Fcal/Fcri 7.65 0.69 8.01 4.20 0.45 0.64
Mobile phase ratio (Methanol: Buffer, v/v)
50:50 4466.98(0.94) 16044.40(0.35) 3.91(0.63) 8.14(0.36) 100.28(0.94) 98.99(0.14)
49.5:50.5 4469.52(0.52) 15943.13(0.32) 4.00(0.26) 8.34(0.41) 99.15(0.52) 98.65(0.32)
49:51 4456.77(0.69) 15935.43(0.17) 3.96(0.45) 8.87(0.46) 99.76(0.69) 99.23(0.31)
Fcal/Fcri 0.07 2.79 10.18 186.41 0.96 2.44
pH
3.7 4431.27(0.98) 15908.47(0.31) 3.93(0.86) 8.11(0.86) 99.95(0.98) 98.88(0.36)
3.8 4469.52(0.52) 15943.13(0.32) 4.00(0.26) 8.34(0.41) 99.15(0.52) 98.65(0.32)
3.9 4455.45(0.75) 15737.18(0.23) 3.92(0.98) 8.81(0.69) 99.21(0.75) 98.95(0.11)
Fcal/Fcri 0.52 1.26 3.42 65.23 0.54 0.30
e = six determinations

© 2016 iSER, Eurasian J Anal Chem, 11(2), 63-78 75

V. P. Agrawal, S. S. Desai & G. K. Jani

Assay

The prepared in-house suspension was analyzed using the developed method.
The content of BRT was found to be 99.17 % and that for BRZ was found to be 98.33
% (Table 11). Thus, the above developed method can be applied for the routine
analysis of formulations containing BRT and BRZ.

Table 11: Results for analysis of in-house suspension.
Amount taken (µg mL-1 ) Amount found (µg mL-1 ) g ± S.D % w/w g± S.D
BRT BRZ BRT BRZ BRT BRZ
0.8 4 0.81 ± 0.06 4.00 ± 0.01 99.17 ± 0.18 98.33 ± 0.34
g = Average of three determinations

CONCLUSION

A simple, rapid, sensitive, specific, accurate and precise RP-HPLC method has
been developed for the first time and optimized utilizing QbD for the simultaneous
determination of BRT and BRZ. The method is rapid as the run time is relatively
short (10 min) within which the two drugs are well resolved. The main aim of
implementing analytical QbD in method optimization was to identify the failures and
the critical quality attributes so as to establish a design space such that there is no
requirement of revalidation in case of any changes in method parameters. The QbD
was applied in HPLC method development so as to verify robustness of the method.
The developed HPLC method was suitable for routine quality control analysis.

Acknowledgment

Authors are thankful to Sun Pharmaceuticals Pvt. Limited, Halol, Gujarat, Indiafor
providing gift samples of brimonidine tartrate and brinzolamide. Authors would also
like to thank Mr. Virag A. Shah for his help in implementing principles of QbD.

References

1. Sharma HL & Sharma KK (2011) Principles of Pharmacology, 2nd Edition, Paras

Publication: Delhi, p. 135-138.
2. Maryadele JO, Patricia EH & Kristin JR (2006) The Merck Index: An Encyclopedia of
Chemicals, Drug and Biological, 14th Edition, Merck & Co. Inc.: New Jershey, p.1375-
1376.
3. Laurence LB, John SL & Keith LP (2006) Goodman and Gilman’s: The Pharmacological
Basis of Therapeutics, 11th Edition, Mc Graw Hill Publication: New York, pp. 1723.
4. Tripathi KD (2008) Essentials of Medical Pharmacology, 2nd Edition, Jaypee Brother’s
Medical Publisher’s Ltd: New Delhi, p. 144-148.
5. Ayre A, Mane P, Ghude K, Nemade M & Gide P (2014) Implementing Quality by Design-A
methodical approach in the RP-HPLC method development process. Int J Adv Pharm
Anal, 4 (1): 1-6.
6. Bajaj M & Nanda S (2015) Analytical Quality by Design (AQBD): New paradigm for
analytical method development. Int J Dev Res, 5 (2): 3589-3599.
7. Bhutani H, Kurmi M, Singh S, Beg S & Singh B (2014) Quality by Design (QbD) in
Analytical Sciences: An Overview. Pharma Times, 46 (8): 71-75.
8. Narwade NN, Jadhav JB & Patil RD (2014) Implementation of QBD to analytical method
development processes. World J Pharm Res, 4 (1): 1520-1535.
9. Jain PS, Khatal RN, Jivani HN & Surana SJ (2011) 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, 1 (3): 323-329.
10. Bhagav P, Deshpande P, Pandey S & Chandran S (2010) Development and validation of
stability indicating UV spectrophotometric method for the estimation of brimonidine

76 © 2016 iSER, Eurasian J Anal Chem, 11(2), 63-78

 Development of RP-HPLC Method

tartrate in pure form, formulations and preformulation studies. Scholars Res Lib, 2 (3):
106-122.
11. Angirekhula N & Annapurna MM (2012) Liquid chromatographic method for the
analysis of brimonidine in ophthalmic formulations. E- J of Chemistry, 9 (3): 1327-1331.
12. Karamanos NK, Lamari F, Katsimpris J & Gartaganis S (1999) Development of an HPLC
method for determining the alpha 2-adrenergic receptor agonist brimonidine in blood
serum and aqueous humor of the eye. Biomed Chromatogra, 13 (1): 86-88.
13. Shirke RR & Pai N (2002) RP-HPLC determination of brimonidine tartrate in
Brimonidine tartrate eye drops. Indian drugs, 39 (9): 484-486.
14. Anand M, Fonseca A, Santosh GV & Padmanabh DB (2010) Development and validation
of high performance thin layer chromatographic method for estimation of brimonidine
tartrate as bulk drug and in ophthalmic solutions. Int J Chem Tech Res, 2 (3): 1376-
1379.
15. Sonanis MC & Rajput AP (2011) Development and validation of a new stability
indicating analytical method for the determination of related components of
brimonidine tartrate in drug substances and drug product using UPLC. Int J Pharm
Pharm Sci, 3 (1): 145-150.
16. Sunitha G, Bhagirath R, Alapati VR, Ramakrishna K, Subramanayam CVS & Anumolu PD
(2013) Fluorimetric quantification of brimonidine tartrate in eye drops. Indian J Pharm
Sci, 75 (6): 730–732.
17. Ali MS, Khatri AR, Munir IM & Ghori M (2009) A stability-indicating assay of
brimonidine tartrate ophthalmic solution and stress testing using HILIC.
Chromatographia, 70 (3): 539-544.
18. Acheampong A, Diane DS & Tang L (1995) Measurement of brimonidine concentrations
in human plasma by a highly sensitive gas chromatography/mass spectrometric assay. J
Pharm Biomed Anal, 13 (8): 995-1002.
19. Jiang S, Chappa AK & Proksch JW (2009) A rapid and sensitive LC/MS/MS assay for the
quantitation of brimonidine in ocular fluids and tissues. J Chromatogr B, 877 (3): 107–
114.
20. Tzovolou DN, Lamari F, Mela EK, Gartaganis SP & Karamanos NK (2000) Capillary
electrophoretic analysis of brimonidine in aqueous humor of the eye and blood sera
and relation of its levels with intraocular pressure. Biomedic Chromatogr, 14 (2): 301-
305.
21. Desai HH & Captain AD (2014) Three simple validated UV spectrophotometric methods
for the simultaneous estimation of timolol maleate and brimonidine tartrate and their
comparison using ANOVA. Int J Pharm Res Anal; 4 (3): 168-177.
22. Mohamed SR, Merey HA, Tawakkol SM & Sweilam MN (2014) Simultaneous
determination of timolol maleate and brimonidine tartrate in their pharmaceutical
dosage form. Taylor and Francis, 4 (2):132–145.
23. Mohamed SR, Merey HA, Tawakkol SM & Sweilam MN (2014) Applicability of Bivariate
calibration algorithm and Vierordt method for simultaneous determination of timolol
maleate and brimonidine tartrate in their binary mixture and pharmaceutical dosage
form. Int J Pharm Sci Res, 5 (2): 2631-2641.
24. Popaniya HS & Patel HM (2014) Simultaneous determination of brimonidine tartrate
and timolol maleate in combined pharmacetical dosage form using two different green
spectrophotometric methods. World J Pharm and Pharm Sci, 3 (3): 1330-1340.
25. Elshanawane AA, Hafez HM, Abdelaziz LM & Mohram MS (2014) Development and
validation of HPLC method for simultaneous estimation of brimonidine tartrate and
timolol maleate in bulk and pharmaceutical dosage form. J Chromat Separation Techniq,
5 (3): 1-5.
26. Hafez HM, Elshanawane AA, Abdelaziz LM & Mohram MS (2014) Development and
validation of HPLC method for simultaneous estimation of brimonidine tartrate and
timolol maleate in bulk and pharmaceutical dosage form. J App Pharm, 6 (14): 398-407.
27. Nagaraju P, Kumar KS & Chaudhary P (2014) Development and validation of RP-HPLC
method for the simultaneous estimation of brimonidine tartrate and timolol maleate in
combined dosage form. Asian J Chem Pharm Res, 2 (1): 58-64.
28. Phogat A, Kumar MS & Mahadevan N (2011) Simultaneous estimation of brimonidine
tartrate and timolol maleate in nanoparticles formulation by RP-HPLC. Int J Rec Adv
Pharm Res, 3 (2): 31-36.

© 2016 iSER, Eurasian J Anal Chem, 11(2), 63-78 77

V. P. Agrawal, S. S. Desai & G. K. Jani

29. Jain PS, Khatal RN, Jivani HN & Surana SJ (2011) Development and validation of TLC-
densitometry method for simultaneous estimation of brimonidine tartrate and timolol
maleate in bulk and pharmaceutical dosage form. J Chromatograph Separat Techniq, 2
(3): 1-5.
30. Indian Pharmacopeia (2014) Ministry of Health and Family Welfare, Govt. of India. 7th
ed., p. 1201-1202.
31. United States Pharmacopeia National Formulary (USP 34 NF 29) (2011) USP 34th ed.
and NF 29th ed., p. 2074-2075.
32. Shah PA, Kadikar AS, Katira RM, Patel KG & Gandhi TR (2014) Simultaneous
determination of brinzolamide and Timolol maleate using three different
Spectrophotometric methods. World J Pharm and Pharm Sci, 3 (2): 1955-1967.
33. Shah PA, Kadikar AS, Gevariya NR and Patel KG (2014) Simultaneous estimation of
brinzolamide and timolol maleate using chromatographic methods. Res J Pharm Biol
Chem Sci, 5 (5): 1010- 1017.
34. Vijay P, Patel D, Desai S & Meshram D (2014) Development and validation of derivative
spectrophotometric method for simultaneous estimation of brimonidine tartrate and
brinzolamide in combined dosage form. Indo American J Pharm Res, 4 (3): 1472-1478.
35. Mashru R & Senta B (2014) Development and validation of spectrophotometric method
for simultaneous estimation of brinzolamide and brimonidine tartrate. Asian J Pharm
Life Sci, 4 (2): 16- 20.
36. International Conference on Harmonization (2005) Q2 (R1), Validation of analytical
procedures: text and methodology. Geneva.


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