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: lovesoni@hotmail.com

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

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

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

2.1 Standard and working stock solutions of 2.2 Linearity:

EBS and PHE
A stock solution of 100 µg/ml of the ebastine and
phenylephrine hydrochloride prepared by dissolving
10 mg each of the two drugs in 100 ml of methanol.
Working standard solutions of 20 µg/ml of each of the
drugs were prepared and scanned over the range of
400-200nm to obtain spectrum of both drugs (fig.1 and
2). Two wavelengths 252 nm and 216 nm were
selected which are the λmax of two drugs for the
development of the simultaneous equations.The
absorbances of EBS and PHE were measured and
absorptivity values were determined at these two
wavelengths.The concentration of two drugs can be
calculated using the following equations, CEBS =
For linearity study, from the standard stock solutions
of 100µg/ml of both EBS and PHE, different dilutions
were prepared for each drug having concentration as
shown in Table.1 and 2 with methanol. Then these
solutions were scanned over the range of 400-200nm
and absorbances were measured at the respective
analytical wavelengths 252 nm and 216 nm for EBS
and PHE respectively for all the five replicates. The
calibration curves were plotted between the mean
value of the observed absorbance and respective
concentration. From the calibration curve given in
Fig.3 and 4, it was found that both the drug follows
Beer’s – Lamberts law within the range of 5-35 µg/ml
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
concentration ratio of 5:35 ,10:30 , 15:25, 20:20,
25:15, 30:10, 35:5 (µg/ml) were prepared and
absorbance of these mixed standards were analyzed at
the respective analytical wavelengths i.e. 252 nm and
216 nm respectively and percentage of drug content
were calculated. The results were reported in Table 3.
2.4 Analysis of Ebast-Dc:
For analysis of tablet, 20 tablets were weighed
individually and their average weight was determined,
they were then crushed to fine powder, powder
equivalent to 10 mg EBS and 10 mg PHE were taken
in 50ml volumetric flask and dissolved in 30ml of
methanol with vigorous shaking for 5-10minutes. The
volumetric flask though a whatman No. #41 filter
paper. Then volume was made up to mark with
methanol. The above solution is further diluted with
methanol to get a sample solution containing 5µg/ml
of EBS and 5 µg/ml of PHE. The absorbances were
used to calculate the amount of drugs present in the
sample by applying these values in Eqns. The results
of analysis of sample are present in Table 4 and its
statistical validation result is reported in Table 5.
2.5 Accuracy:
To check the accuracy of the developed methods,
analytical recovery experiments were carried out by
the standard addition method (Table 6 and 7).

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
inter-day precision study. Intraday and interday
precision study was calculated by assay of the sample
solution on the same day and on different days at
different time intervals respectively. The standard
deviation (SD) and coefficient of variance (COV) were
calculated (Table 8 and 9).
For limit of detection and quantitation, lower
concentration range solutions of 0.2, 0.3, 0.4, 0.5 and
0.6 µg/ml were prepared and the absorbances were
determined and LOD and LOQ, were calculated by use
of the equation LOD = 3.3 Q/ S and LOQ = 10 Q/ S,
where Q is the standard deviation and S is the slope of
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
The % recovery of EBS and PHE was found to be
close to 100 %. % Coefficient of variance (% COV
<2) and standard deviation (S.D < 1.0) indicating that
developed methods were accurate and precise for
quantitative estimation of both EBS and PHE in tablet
dosage form.
The sample recovery was in good agreement with
respective label claim, which suggested non
interference of formulation additives in estimation.
The developed method is simple, rapid, inexpensive,
accurate, precise and convenient for the routine
analysis.
4. Acknowledgment
The authors are thankful to the Head, School of
Pharmacy, Devi Ahilya University, Indore and Vice-
Chancellor, Devi Ahilya University, Indore for
providing facilities to carry out the research work. One
of the authors (CS) is thankful for AICTE for
providing Junior Research Fellowship. Authors are
grateful to Tonira Pharma Limited (Gujarat) and
Love Kumar Soni et al /Int.J. ChemTech Res.2011,3(4)
Torrent Pharma Ltd. (Gujarat) for providing gift
sample of EBS and PHE respectively.
References
1. Peyri J, Vidal J, Marrón J et al.Ebastine in chronic
urticaria: a double-blind placebo Controlled study
J Dermatol Treat 1991; 2:51-3.
2. Gehanno P, Bremard-Oury C, Zeisser P.
Comparison of ebastine to cetirizine in Seasonal
allergic rhinitis in adults. Annals of Allergy,
Asthma and Immunol 1996; 76 (6): 507-12.
3. Housquet J, Gaudaño EM, Palama Carlos AG et al.
A 12-week, placebo-controlled study of the
efficacy and safety opf ebastine, 10 and 20 mg
once daily, in the treatment of perennial allergic
rhinitis. Allergy 1999; S4: S62-S68.
4. Tagawa M, Kano M, Okamura N, et
al.Neuroimaging of histamine H1-receptor
occupancy in human brain by positron emission
tomography (PET): a comparative study of
ebastine, a second-generation antihistamine, and
(+)-clorphrniramine, a classical antihistamine. Br J
Clin Pharmacl 2001; 52:501-509.
5. Van Cauwenberge P, de Belder T, Sys L. A review
of the second-generation antihistamine ebastine for
the treatment of allergic disorders. Exp Rew
Pharmacother 2004;5(8):1807-13.
6. Salvà M, Carreño B, Pintos M et al. Phase I, single
dose, open label, randomized, Crossover
bioequivalence studies of Ebastine 10 mg and
20 mg regular tablets vs. 10 and 20 mg fast-
dissolving tablets in healthy male volunteers. J
Invest Allergol Clin Immunol 2004; 14(4): S5.
7. J Sastre. Ebastine in allergic rhinitis and chronic
idiopathic urticaria. Allergy 2008; 63:1-2.
8. Tripathi K.D; Essential of Medicinal
Pharmacology, 5th Edn. jaypee Brothers, Medical
Publishers (P) ltd. 2003, 113-114.
9. Rang H.P., Dale M.M., Ritter J.M., and Moore
P.K., Pharmacology, 5th Edn. Churchill
Livingstone, 2003, pages 186,456, 788.
10. Prabhu S.L; Dinesh Kumar C; Shirwaikar A;
Shirwaikar Annie. Determination of ebastine in
pharmaceutical formulation by RP-HPLC. Indian J
Pharm Sci. 2008; 70(3): 406–407.
*****
1925
11. Matsuda Michiaki; Mizuki Yasuyuki; Terauchi
Yoshiaki; Simultaneous determination of the
histamine H1 receptor antagonist Ebastine and it’s
two metabolites, Carebastine and Hydroxyebastine
, in human plasma using HPLC. J. Chromatog. B:
2001, 765 (2), 173-179.
12. Gergov M ; Robson J; Ojanperä I; Vuori J;
Simultaneous screening and quantitation of 18
antihistamine drugs in blood by liquid
chromatography ionspray tandem mass
spectrometry. Forensic Science International.
2001, 121 (1-2), 108-115.
13. Kang W; Liu K H; Ryu J Y; Shin J G;
Simultaneous determination of Ebastine and its
three metabolites in plasma using liquid
Chromatography-tandem mass spectrophometry. J
Chrom. B, 2004, 813 (1-2), 75-80.
14. Ashok P ; Meyyanathan S N; Pandilla Bharani ;
Suresh B ; Analysis of ebastine in pharmaceutical
preparations by high performance thin layer
chromatography. J. Planar Chrom., 2003, 16 (2),
167-169.
15. Sharma S.A; Spectrophotometric determination of
phenylephrine hydrochloride and orphenadrine
citrate in pure and in dosage forms. J Pharm
Biomed Anal. 2002; 30 (4):1385-92.
16. Muszalska I ; Zajac M ; Wróbel G; Nogowska
M ; UV/ VIS spectrophotometric methods for
determination of caffeine and phenylephrine
hydrochloride in complex pharmaceutical
preparations and Validation of the methods. Cta
Pol Pharm; 57(4): 247-52.
17. Erk N; Quantitative analysis of chlorpheniramine
maleate and phenylephrine hydrochloride in nasal
drops by differential – derivative
spectrophotometric , zero crossing first derivative
UV spectrophotometric and absorbance ratio
methods. J Pharm Biomed Anal. 2000; 23
(6):1023-31.
18. Erk Nevin; Kartal Murat; Simultaneous HPLC and
derivative ratio spectra spectrophotometry
determination of chlorpheniramine maleate and
phenylephrine hydrochloride. Il Farmaco 1998, 53
(8-9), 617-622.
19. Korany MA ; Wahbi AM ; Mandour S; Elsayed
MA; Determination of certain drugs in
multicomponent formulations by first derivative
ultraviolet spectrophotometry. Analytical
Letters,1985, 18 (1), 21-34.