International Journal of ChemTech Research

CODEN( USA): IJCRGG ISSN : 0974-4290

Vol. 3, No.4, pp 1918-1925, Oct-Dec 2011

Development and validation of UV-

Spectrophotometric assay protocol for
simultaneous estimation of Ebastine and
Phenylephrine Hydrochloride in tablet dosage
form using simultaneous equation method
Love Kumar Soni*, Tamanna Narsinghani, Charu Saxena

School of Pharmacy, Devi Ahilya University, Takshashila Campus, Ring Road,

Indore -452017, India.

*Corres.author: [email protected]

Abstract: The present work deals with the simultaneous estimation of ebastine and phenylephrine hydrochloride
in tablet dosage form, without prior separation by simultaneous equation method. The absorption maxima at 252
nm and 216 nm were used for the estimation of ebastine and phenylephrine hydrochloride, respectively. Both the
drugs obey Beer-Lamberts law within the range of 5-35 µg/ml for ebastine and 5-35 µg/ml for phenylephrine
hydrochloride, respectively. The method was validated according to ICH guidelines with respect to linearity,
accuracy, limit of detection/quantitation and precision. The result of analysis has been validated statistically.
Recovery study confirmed the accuracy of the proposed method. The proposed procedure is simple, inexpensive,
accurate and precise, require no separation steps and can be used for the routine analysis of both drugs.
Key words: Simultaneous equation method, Ebastine, Phenylephrine hydrochloride.

1. Introduction: ,systolic and diastolic pressure.It is indicated for nasal

congestion, minor eye irritations and open angle
Ebastine (EBS) is second generation H1 – receptor glaucoma [8,9].The estimation of PHE using UV-
antagonist and antihistaminic agent which binds spectroscopy has been reported in combination with
preferentially to peripheral H1 receptor without central other drugs[15-19]. The objective of the work is to
side effects. It is indicated for the sympatomatic develop new simultaneous equation method for
treatment of seasonal and perennial allergic and estimation of EBS and PHE in tablet dosage form with
chronic urticaria [1-7]. EBS has been determined by RP- good accuracy, simplicity, precision and economy.
HPLC method [10,11], liquid chromatography ionspray
tandem mass spectrometry [12,13], high performance thin 2. Materials and Methods
layer chromatography [14] in single and combination
The instrument used for the analysis was UV double
with other drugs . Phenylephrine hydrochloride (PHE)
beam spectrophotometer (Shimadzu Model 1700).Gift
is α1-adrenoceptor agonist which stimulates
sample of ebastine was procured from Tonira Pharma
postsynaptic alpha receptor cause vasoconstriction
Love Kumar Soni et al /Int.J. ChemTech Res.2011,3(4) 1919

Ltd. Gujarat and phenylephrine hydrochloride was A2ay1-A1ay2 / ax2ay1-ax1ay2…..(1) , CPHE = A1ax2-
procured from Torrent Pharma Ltd. Gujarat. Combined A2ax1 / ax2ay1-ax1ay2 ….(2) , where CEBS and CPHE
ebastine and phenylephrine hydrochloride tablets were the concentrations of EBS and PHE respectively
(Ebast-Dc containing ebastine-10 mg and in the diluted sample, A1 and A2 are the absorbances of
phenylephrine hydrochloride -10 mg and manufactured the sample at 216 and 252 nm , respectively , ax1 and
by micro labs ltd.Pondicherry) were purchased from ax2 were absorptivities of EBS at 252 and 216 nm, ay1
local market. Analytical grade methanol was used as and ay2 were absorptivities of PHE at 252 and 216 nm,
the common solvent. respectively.

2.1 Standard and working stock solutions of 2.2 Linearity:

EBS and PHE For linearity study, from the standard stock solutions
A stock solution of 100 µg/ml of the ebastine and of 100µg/ml of both EBS and PHE, different dilutions
phenylephrine hydrochloride prepared by dissolving were prepared for each drug having concentration as
10 mg each of the two drugs in 100 ml of methanol. shown in Table.1 and 2 with methanol. Then these
Working standard solutions of 20 µg/ml of each of the solutions were scanned over the range of 400-200nm
drugs were prepared and scanned over the range of and absorbances were measured at the respective
400-200nm to obtain spectrum of both drugs (fig.1 and analytical wavelengths 252 nm and 216 nm for EBS
2). Two wavelengths 252 nm and 216 nm were and PHE respectively for all the five replicates. The
selected which are the λmax of two drugs for the calibration curves were plotted between the mean
development of the simultaneous equations.The value of the observed absorbance and respective
absorbances of EBS and PHE were measured and concentration. From the calibration curve given in
absorptivity values were determined at these two Fig.3 and 4, it was found that both the drug follows
wavelengths.The concentration of two drugs can be Beer’s – Lamberts law within the range of 5-35 µg/ml
calculated using the following equations, CEBS = for both drugs respectively (Table 1 and 2).

Table 1. Linearity of EBS at 252nm

Absorbance of EBS at concentration (µg/ml)
Replicate no.
5 10 15 20 25 30 35
Replicate 1 0.294 0.585 0.875 1.165 1.446 1.745 2.035
Replicate 2 0.290 0.588 0.878 1.166 1.459 1.746 2.038
Replicate 3 0.291 0.585 0.879 1.168 1.458 1.748 2.033
Replicate 4 0.288 0.592 0.881 1.162 1.465 1.751 2.032
Replicate 5 0.291 0.586 0.882 1.163 1.462 1.752 2.036
Mean 0.290 0.588 0.878 1.165 1.459 1.749 2.035
Standard Deviation (S.D.) 0.0015 0.0052 0.004 0.0064 0.0072 0.0022 0.0026
Standard Error (S.E) 0.0006 0.0023 0.0018 0.0028 0.0032 0.0010 0.0116

Table 2. Linearity of PHE at 216 nm

Absorbance of PHE at concentration (µg/ml)
Replicate no.
5 10 15 20 25 30 35
Replicate 1 0.235 0.481 0.699 0.946 1.167 1.399 1.634
Replicate 2 0.238 0.478 0.698 0.941 1.168 1.397 1.638
Replicate 3 0.239 0.475 0.696 0.943 1.171 1.398 1.636
Replicate 4 0.232 0.477 0.698 0.945 1.170 1.396 1.638
Replicate 5 0.232 0.482 0.697 0.942 1.169 1.399 1.637
Mean 0.235 0.478 0.698 0.942 1.169 1.398 1.635
Standard Deviation (S.D.) 0.025 0.0028 0.0022 0.002 0.0028 0.0018 0.0030
Standard Error (S.E.) 0.011 0.0012 0.0092 0.0089 0.0012 0.0083 0.0013
Love Kumar Soni et al /Int.J. ChemTech Res.2011,3(4) 1920

Figure 1. Spectrum of EBS Figure 2. Spectrum of PHE

Calibration Curve of EBS

2.5

A
2
b
s 1.5
o
r
b 1
a
n
c 0.5
e

0
0 5 10 15 20 25 30 35
Concentration (µg/ml)

Figure 3. Linearity curve of EBS

Love Kumar Soni et al /Int.J. ChemTech Res.2011,3(4) 1921

Calibration Curve of PHE

2
A
b
s 1.5
o
r
b 1
a
n
c 0.5
e
0
0 5 10 15 20 25 30 35
Concentration (µg/ml)

Figure 4. Linearity curve of PHE

2.3 Analysis of mixed standards: supernatant liquid was transferred to 50 ml of

Seven mixed standards of EBS and PHE in the volumetric flask though a whatman No. #41 filter
concentration ratio of 5:35 ,10:30 , 15:25, 20:20, paper. Then volume was made up to mark with
25:15, 30:10, 35:5 (µg/ml) were prepared and methanol. The above solution is further diluted with
absorbance of these mixed standards were analyzed at methanol to get a sample solution containing 5µg/ml
the respective analytical wavelengths i.e. 252 nm and of EBS and 5 µg/ml of PHE. The absorbances were
216 nm respectively and percentage of drug content used to calculate the amount of drugs present in the
were calculated. The results were reported in Table 3. sample by applying these values in Eqns. The results
of analysis of sample are present in Table 4 and its
2.4 Analysis of Ebast-Dc: statistical validation result is reported in Table 5.
For analysis of tablet, 20 tablets were weighed
individually and their average weight was determined, 2.5 Accuracy:
they were then crushed to fine powder, powder To check the accuracy of the developed methods,
equivalent to 10 mg EBS and 10 mg PHE were taken analytical recovery experiments were carried out by
in 50ml volumetric flask and dissolved in 30ml of the standard addition method (Table 6 and 7).
methanol with vigorous shaking for 5-10minutes. The

Table 3 . Analysis of mixed standard

Amount Present Amount Found
S.
(µg/ml) (µg/ml) %
No.
EBS PHE EBS PHE EBS PHE
1 5 40 4.95 39.97 99.01 99.90
2 10 35 10.24 34.52 102.40 98.60
3 15 30 14.85 29.67 99.00 98.90
4 20 25 19.83 24.95 99.15 99.80
5 25 20 24.75 19.70 99.08 98.50
6 30 15 29.55 14.69 98.50 98.00
7 35 10 34.69 9.85 99.11 98.50
Love Kumar Soni et al /Int.J. ChemTech Res.2011,3(4) 1922

Table 4. Result of analysis of EBAST –DC

Lable Claim Conc. Found
S. % Found
(mg/Tab) (mg/Tab)
No.
EBS PHE EBS PHE EBS PHE
1 10 10 9.90 9.99 99.01 99.9
2 10 10 9.98 10.016 99.8 100.16
3 10 10 9.85 10.020 98.5 100.2
4 10 10 9.82 10.010 98.2 100.1
5 10 10 10.01 10 100.1 99.9

Table 5 . Result of statistical validation of EBAST-DC

% coefficient of
S. Mean
Drug S.D. variance S.E.
No. %
(COV)
1 EBS 99.12 0.2825 0.2824 0.12
2 PHE 100.07 0.4925 0.4905 0.21

Table 6. Result of recovery study of EBAST-DC

Amount taken Amount added
Replicate % Recovery
(µg/ml) (µg/ml)
no.
EBS PHE % EBS PHE EBS PHE
Replicate-1 10 10 7 6.5 98.45 99.75
Replicate-2 10 10 7 6.5 98.67 99.75
80
Replicate-3 10 10 7 6.5 98.52 100.14
Replicate-1 10 10 10 10 98.30 101.86
Replicate-2 10 10 100 10 10 98.23 101.84
Replicate-3 10 10 10 10 98.23 101.82
Replicate-1 10 10 13 11.5 98.81 101.34
Replicate-2 10 10 13 11.5 98.81 101.34
120
Replicate-3 10 10 13 11.5 98.93 101.55

Table 7. Result of statistical validation of recovery study

S. No. Drug Mean (%) S.D. % COV
EBS 98.55 0.134 0.137
80%
PHE 99.88 0.200 0.200
EBS 98.25 0.102 0.103
100%
PHE 101.84 0.010 0.009
EBS 98.85 0.089 0.089
120%
PHE 101.41 0.121 0.119

2.6 Precision: 2.7 Detection and quantitation limits:

Precision study was carried out by doing intra and For limit of detection and quantitation, lower
inter-day precision study. Intraday and interday concentration range solutions of 0.2, 0.3, 0.4, 0.5 and
precision study was calculated by assay of the sample 0.6 µg/ml were prepared and the absorbances were
solution on the same day and on different days at determined and LOD and LOQ, were calculated by use
different time intervals respectively. The standard of the equation LOD = 3.3 Q/ S and LOQ = 10 Q/ S,
deviation (SD) and coefficient of variance (COV) were where Q is the standard deviation and S is the slope of
calculated (Table 8 and 9). the calibration curve (Table 10 and 11).
Love Kumar Soni et al /Int.J. ChemTech Res.2011,3(4) 1923

Table 8. Intra and inter day precision study of EBS

% Obtained % Obtained
Replicate no. st
1 hr 2nd hr 3rd hr Day 1 Day 2 Day 3
Replicate - 1 99.85 99.72 99.65 99.92 98.35 98.15
Replicate - 2 99.78 99.65 99.52 99.78 98.28 98.08
Replicate - 3 99.25 99.15 98.84 99.85 98.15 97.94
Replicate - 4 99.62 99.55 99.41 99.68 98.42 98.25
Replicate - 5 99.92 99.67 99.35 99.35 98.18 97.85
Mean 99.68 99.54 99.35 99.71 98.23 98.10
S.D. 0.266 0.230 0.309 0.233 0.080 0.169
% COV 0.267 0.231 0.311 0.223 0.081 0.173

Table 9. Intra and inter day precision study of PHE

% Obtained % Obtained
Replicate no.
1st hr 2nd hr 3rd hr Day 1 Day 2 Day 3
Replicate - 1 100.21 100.15 100.11 100.12 99.32 98.85
Replicate - 2 100.28 100.22 100.16 100.18 99.41 98.68
Replicate - 3 100.58 100.43 100.32 99.93 99.28 98.52
Replicate - 4 101.08 100.97 100.85 99.85 98.92 98.44
Replicate - 5 99.85 99.72 99.54 99.78 98.35 98.44
Mean 100.4 100.29 100.14 99.97 99.15 98.57
S.D. 0.460 0.455 0.469 0.172 0.253 0.188
% COV 0.458 0.454 0.468 0.172 0.255 0.191

Table 10. LOD and LOQ of EBS

Absorbance of EBS at concentration (µg/ml)
Replicate no.
0.2 0.3 0.4 0.5 0.6
Replicate - 1 0.049 0.062 0.075 0.085 0.095
Replicate -2 0.048 0.061 0.076 0.084 0.092
Replicate -3 0.049 0.060 0.078 0.085 0.094
Replicate -4 0.051 0.063 0.074 0.086 0.093
Replicate -5 0.050 0.062 0.075 0.083 0.095
Mean 0.049 0.062 0.076 0.085 0.094
S.D. 0.00114 0.00114 0.00151 0.00114 0.0013

Table 11. LOD and LOQ of PHE

Absorbance of PHE at concentration (µg/ml)
Replicate no.
0.2 0.3 0.4 0.5 0.6
Replicate - 1 0.018 0.028 0.036 0.047 0.055
Replicate -2 0.017 0.029 0.035 0.048 0.055
Replicate -3 0.018 0.030 0.034 0.045 0.056
Replicate -4 0.019 0.026 0.036 0.046 0.055
Replicate -5 0.021 0.028 0.033 0.048 0.052
S.D. 0.00114 0.00148 0.0013 0.0013 0.00187
Love Kumar Soni et al /Int.J. ChemTech Res.2011,3(4) 1924

LOD and LOQ of EBS

0.1
A
b
0.09
s 0.08
o
r 0.07
p 0.06
t
i 0.05
v 0.04
i
t 0.03
y
0.02
0.01
0
0.2 0.3 0.4 0.5 0.6

Concentration (µg/ml)

Figure 5. Calibration curve of LOD and LOQ for EBS

LOD and LOQ of PHE

A 0.06
B
S 0.05
O
R 0.04
P
T 0.03
I
V 0.02
I
T
0.01
Y
0
0.2 0.3 0.4 0.5 0.6

CONCENTRATION (µg/ml)

Figure 6. Calibration curve of LOD and LOQ for PHE

3 Results and Discussion accurate, precise and convenient for the routine
analysis.
The % recovery of EBS and PHE was found to be
close to 100 %. % Coefficient of variance (% COV 4. Acknowledgment
<2) and standard deviation (S.D < 1.0) indicating that The authors are thankful to the Head, School of
developed methods were accurate and precise for Pharmacy, Devi Ahilya University, Indore and Vice-
quantitative estimation of both EBS and PHE in tablet Chancellor, Devi Ahilya University, Indore for
dosage form. providing facilities to carry out the research work. One
The sample recovery was in good agreement with of the authors (CS) is thankful for AICTE for
respective label claim, which suggested non providing Junior Research Fellowship. Authors are
interference of formulation additives in estimation. grateful to Tonira Pharma Limited (Gujarat) and
The developed method is simple, rapid, inexpensive,
Love Kumar Soni et al /Int.J. ChemTech Res.2011,3(4) 1925

Torrent Pharma Ltd. (Gujarat) for providing gift 11. Matsuda Michiaki; Mizuki Yasuyuki; Terauchi
sample of EBS and PHE respectively. Yoshiaki; Simultaneous determination of the
histamine H1 receptor antagonist Ebastine and it’s
two metabolites, Carebastine and Hydroxyebastine
References , in human plasma using HPLC. J. Chromatog. B:
2001, 765 (2), 173-179.
1. Peyri J, Vidal J, Marrón J et al.Ebastine in chronic 12. Gergov M ; Robson J; Ojanperä I; Vuori J;
urticaria: a double-blind placebo Controlled study Simultaneous screening and quantitation of 18
J Dermatol Treat 1991; 2:51-3. antihistamine drugs in blood by liquid
2. Gehanno P, Bremard-Oury C, Zeisser P. chromatography ionspray tandem mass
Comparison of ebastine to cetirizine in Seasonal spectrometry. Forensic Science International.
allergic rhinitis in adults. Annals of Allergy, 2001, 121 (1-2), 108-115.
Asthma and Immunol 1996; 76 (6): 507-12. 13. Kang W; Liu K H; Ryu J Y; Shin J G;
3. Housquet J, Gaudaño EM, Palama Carlos AG et al. Simultaneous determination of Ebastine and its
A 12-week, placebo-controlled study of the three metabolites in plasma using liquid
efficacy and safety opf ebastine, 10 and 20 mg Chromatography-tandem mass spectrophometry. J
once daily, in the treatment of perennial allergic Chrom. B, 2004, 813 (1-2), 75-80.
rhinitis. Allergy 1999; S4: S62-S68. 14. Ashok P ; Meyyanathan S N; Pandilla Bharani ;
4. Tagawa M, Kano M, Okamura N, et Suresh B ; Analysis of ebastine in pharmaceutical
al.Neuroimaging of histamine H1-receptor preparations by high performance thin layer
occupancy in human brain by positron emission chromatography. J. Planar Chrom., 2003, 16 (2),
tomography (PET): a comparative study of 167-169.
ebastine, a second-generation antihistamine, and 15. Sharma S.A; Spectrophotometric determination of
(+)-clorphrniramine, a classical antihistamine. Br J phenylephrine hydrochloride and orphenadrine
Clin Pharmacl 2001; 52:501-509. citrate in pure and in dosage forms. J Pharm
5. Van Cauwenberge P, de Belder T, Sys L. A review Biomed Anal. 2002; 30 (4):1385-92.
of the second-generation antihistamine ebastine for 16. Muszalska I ; Zajac M ; Wróbel G; Nogowska
the treatment of allergic disorders. Exp Rew M ; UV/ VIS spectrophotometric methods for
Pharmacother 2004;5(8):1807-13. determination of caffeine and phenylephrine
6. Salvà M, Carreño B, Pintos M et al. Phase I, single hydrochloride in complex pharmaceutical
dose, open label, randomized, Crossover preparations and Validation of the methods. Cta
bioequivalence studies of Ebastine 10 mg and Pol Pharm; 57(4): 247-52.
20 mg regular tablets vs. 10 and 20 mg fast- 17. Erk N; Quantitative analysis of chlorpheniramine
dissolving tablets in healthy male volunteers. J maleate and phenylephrine hydrochloride in nasal
Invest Allergol Clin Immunol 2004; 14(4): S5. drops by differential – derivative
7. J Sastre. Ebastine in allergic rhinitis and chronic spectrophotometric , zero crossing first derivative
idiopathic urticaria. Allergy 2008; 63:1-2. UV spectrophotometric and absorbance ratio
8. Tripathi K.D; Essential of Medicinal methods. J Pharm Biomed Anal. 2000; 23
Pharmacology, 5th Edn. jaypee Brothers, Medical (6):1023-31.
Publishers (P) ltd. 2003, 113-114. 18. Erk Nevin; Kartal Murat; Simultaneous HPLC and
9. Rang H.P., Dale M.M., Ritter J.M., and Moore derivative ratio spectra spectrophotometry
P.K., Pharmacology, 5th Edn. Churchill determination of chlorpheniramine maleate and
Livingstone, 2003, pages 186,456, 788. phenylephrine hydrochloride. Il Farmaco 1998, 53
(8-9), 617-622.
10. Prabhu S.L; Dinesh Kumar C; Shirwaikar A;
Shirwaikar Annie. Determination of ebastine in 19. Korany MA ; Wahbi AM ; Mandour S; Elsayed
pharmaceutical formulation by RP-HPLC. Indian J MA; Determination of certain drugs in
Pharm Sci. 2008; 70(3): 406–407. multicomponent formulations by first derivative
ultraviolet spectrophotometry. Analytical
Letters,1985, 18 (1), 21-34.

*****