Development and Validation of Spectrophotometric Method for
Simultaneous estimation of Levocetrizine Dihydrochloride and
Phenylephrine Hydrochloride in their Tablet Dosage Form
Urwashi Tiwari 1*, S. P. Wate1, R. S. Jumle
Sharad Pawar College of Pharmacy, Wanadongari, Nagpur
Corresponding author : tiwariurwashi@yahoo.in
ABSTRACT:
Simple and sensitive spectrophotometric method has been developed for the estimation
of Levocetirizine dihydrochloride (LEC) and Phenylephrine hydrochloride (PHE) in their
tablet dosage forms. Method involved solving simultaneous equations based on
measurement of absorbances at two wavelengths 230.5 nm and 275.5 nm, theλ max of
Levocetrizine dihydrochloride and Phenylephrine hydrochloride, respectively. Beer’s
law was obeyed in the concentration range of 2.5 -12.5µg/ml for LEC and 5-25 µg/ml
for PHE with line of regression 0.0673x + 0.2386 with correlation coefficient of 0.9983
and 0.3459x – 2.522and correlation coefficient 0.9830 respectively..The mobile selected
was based upon the absorption and λmax. Methanol:distilled water (90:10) was used
as a solvent for PHE and Levo. Overlain spectra of PHE and LEVO suggested that both
the drugs show same absorbance at 250.5 nm i.e. at isobestic point .Commercially
available tablets were analyzed and the results are statistically compared with those
obtained by the reference method and validated by recovery studies. The results are found
satisfactory and reproducible and can be used for routine analysis of both drugs in quality
control laboratories. These methods offer the advantages of rapidity, simplicity and
sensitivity and normal cost and can be easily applied to resource –poor settings without
the need for expensive instrumentation and reagents.
KEYWORDS:
Levocetirizine
dihydrocloride,
Phenylephrine
hydrochloride,
simultaneous equation method, methnol:water(90:10), validation of analytical method.
INTRODUCTION:
1,2
Levocetirizine
dihydrochloride
(LEVO),
is
R2(2(4(4Chlorophenyl)phenylmethyl)1piperazinyl)ethoxy)acetic acid hydrochloride. It
is indiacted for the relief of symptoms associated with seasonal allergic rhinitis (SAR)
and perennial allergic rhinitis (PAR), and for the treatment of the uncomplicated skin
manifestations
of chronic idiopathic urtcaria(CIU).It is official in Indian
Pharmacopoeia.3Similarly, Phenylephrine hydrochloride (PHE), is R-2Methylaminol(3hydroxyphenyl)ethanol hydrochloride. It is an alpha –adrenergic receptor, producing
pronounced vasoconstriction.4-9 Since no spectrophtometric method had been reported
for the simultaneous estimation of Levocetirizine dihydrochloride and Phenylephrine
hydrochloride, a successful attempt has been made to estimate both these drugs
simultaneously by simple UV spectrophotometric method in their tablet dosage form.
Suitable statistical tests were performed on validation data. Few HPLC methods and
10-12
very few UV spectrophotopmetric methods have been reported for simultaneous
estimation of Levocetirizine dihyrochloride and Phenylephrine hydrochloride in tablet
dosage form in combination with other two drugs in the literature in pharmaceutical
formulation.
13
Levocetirizine was officially assayed by Liquid chromatography using stainless steel
column 25mm x4.Phenylephrine was also assayed by potntiometrically.Mobile phase
was used as 0.05 m potassium dihydrogen phosphate and acetonitrile( 60:40v/v withpH
adjusted with and 10% w/v sodium hydroxide, packed with octa desyl silane bonded
with silica.14 Phenylephrine hydrochloride was assayed (I.P) by potntiometrically using
0.1 M HCl and 50 ml ethanol (95%) and titrate with 0.1 M ethanolic NaOH. Many
researchers have dealt with the development of methods that quantify LEVO in pure
and in tablets.15,16 Methods that include validation of UV Spectrophotometric methods
for simultaneous estimation of Levocetrizine dihydrochloride in bulk and
pharmaceutical formulation when present alone or in combination with Ambroxol
hydrochloride.
MATERIAS AND METHOD:
Levocetirizine dihyddrrochloride and Phenylephrine hydrochloride were obtained from
Zim laboratory, Kalmeshwar, Nagpur as a gift sample and were used as working
standards. Methanol of analytical grade and double distilled water were used
throughout the analysis. The pharmaceutical formulation of LEVO and PHE that is
Levocet D +(Heteo Health care,Mumbai) the commercial formulation of Levocetirizine
and Phenylephrine are available in ratio of 1:2 as tablet.
Instrument:
Shimadzu UV/Vis double beam spectrophotometer, model 1700 Pharmaspec with 1 cm
quartz cells was used for all spectral measurements.
.2HCl
Fig-1: Chemical structure of Levocetirizine dihydrochloride
.HCl
Fig-2:Chemical structure of Phenylephrine hydrochloride
Preparation of Standard and sample drug stock solution :
Standard stock solution of phenylephrine hydrochloride and levocetrizine dihydrochloride was
prepared by dissolving 100mg of PHE and 50 mg of LEVO in 100ml volumetric flask using
methanol:water (90:10) as solvent and volume was made up to 100 ml to produce
1000μg/ml of PHE and 500 µg of LEVO. From these stock solutions, working standard
solutions of concentration were prepared by appropriate dilutions. Working standard solutions
were scanned in the entire UV range to determine the λmax.The λmax of PHE and LEVO were
found to be 275.5 nm and 230. 5 nm respectively.
Calibration curves:
Five standard dilutions of each drug were prepared having concentration of 2.5 – 25 µg/ml, the
absorbances of these solutions were measured at 275.5 nm and 230.5 nm and calibration
curves were plotted. The absorptiviy coefficients of the two drugs were determined using
calibration curves(graph 1and 2).
Concentration( µg/ml)
Fig-3: Calibration Plot of Phenylephrine
Concentration (µg/ml )
Fig-4: Calibration Plot of Levocetirizine
Preparation of sample solution:
Sample solution containing both the drugs was prepared by dissolving 20 mg of PHE and 10
mg of LEVO in 100ml volumetric flask using 90 ml of methanol and 10ml of double distilled
water and made 200 µg/ml and 100µg/ml of PHE and LEVO stock solution
respectively.,working standard of 10 µg/ml concentration was prepared by appropriate
dilution. Five standard solution of PHE of concentration 5,10,15.20,25 and five standard
solution of LEVO of concentration 2.5, 5,7.5,10.5,12.5 were prepared from wokking standard
solution.The absorbance of this sample solution was measured at 275.5 nm and 230.5 nm and
their concentrations were determined using proposed analytical methods.
Simultaneous equation method:
Two wavelengths selected for the method are 275.5 nm and 230.5 nm that are absorption
maxima of PHE and LEVO respectively in methanol:water (90:10).The absorbasences were
measured at the selected wavelengths and absorptivities (A 1%, 1 cm) for both the drugs were
determined as the mean of the five independent determinations. Concentrations in the sample
were obtained by using following equationsCx = (A2ay1‐A1ay2)/ (ax2ay1‐ax1ay2)
…………….1)
Cy = (A1ax2‐A2ax1)/ (ax2ay1‐ax1ay2)
……………..2)
Where,
A1 and A2 are absorbances of mixture at 275.5 nm and 230.5nmrespectively.
ax1 and ax2 are absorptivities of PHE at λ1 and λ2 respectively.
ay1 and ay2 are absorptivities of LEVO at λ1 and λ2 respectively.
Cx and Cy are concentrations of PHE and LEVO respectively.
Fig-5: UV Spectrum of PEN
Fig-6:UV Spectrum of LEVO
Fig-7:UV overlain spectra of PHE and LEVO
Estimation in the marketed formulation :
Twenty tablets were accurately weighed. Average weight of tablet was calculated. The
tablets were crushed to fine powder and mixed thoroughly. A quantity of tablet powder
equivalent to 10 mg of PEN and 5 mg of LEVO was transferred to 100 ml volumetric
flask and dissolved in distilled water with vigorous shaking and volume was made to
100 ml with same solvent. The solution was filtered through Whatman filter paper no.
41. The aliquot portion of filtrate was further diluted with solvent to get final
concentration of about 10 µg/ml and 5 µg/ml of PEN and LEVO respectively. The
absorbance of resulting solutions was measured at 230.5 nm and 275.5nm in 1 cm cell
against blank.The content of PEN and LEVO in tablet was calculated using the formula
as follows.
Where,
Cx or Cy = Concentration of PEN and LEVO in g/100 ml
W
= Average weight of tablet
Wm = Weight of sample
L
= Label claim of sample taken
Thr result of analysis of the formulation is shown in table 1.
Method validation:
The method validation parameters Like accuracy, precision,rudgeness, linearity and
range were checked as per ICH guidelines.The accuracy of the method was determined
by the recovery studies. The recovery studies were performed by the standard addition
method at 80%,100% and 120% and the percentage recoveries were calculated and are
shown in Table 1 The Precision of the method was evaluated by interday and intraday
variation studies.In intraday studies ,working solution of standard and sample were
analysed thrice in a day and percentage relatve standard deviation (%RSD) was
calaculaed,The data is shown in table2. The linearity for the PHE and LEVO were
determined at five concentration levels, ranging from 80-120 % of the test
concentration and absorbance were recorded at 230.5 nm and 275.5 nm. PEN and
LEVO were found to be linear in 80-120% of the test concentration. Results of
linearity and range studies are shown in Table – 2,3.
RESULTS AND DISCUSSION :
In the present work, new method , namely (Vierordt’s method ) was used for the
simultaneous spectroscopic estimation of Phenylephrine HCl and Levocetirizine
dihyrochloride in commercially available tablet dosage form. The concentrations in the
range of 2.5 – 25 µg/ml of working standard and two sampling wavelengths of 275.5
nm (λ max of PHE HCl), and 230.5 nm (λ max of LEVO dihydrochloride) gave
optimum accuracy, precision, economy and sensitivity for this method. The proposed
procedure was successfully applied to the determination of PHE and LEVO l in the
commercially available tablet dosage form.
Recovery studies were carried out at different concentrations by spiking a known
concentration of standard drug to the preanalysed sample and contents were reanalyzed
by proposed methods.The results of marketed formulation analysis and recovery studies
are depicted in table 1.The method was validated statistically for range , linearity,
precision,accuracy and results are depicted in table in 2. Accuracy was ascertained on
the basis of recovery studies.Precision was calculated as inter ahd intraday variation for
boththe drugs. Th recovery experiment indicated the absence of interference from the
commonly encountered pharmaceutical excipients present in formulations.The
proposed method is found to be simple, sensitive and can be used for routine quality
control analysis of PHE and LEVO in bulk and tablet dosage form.
Table-1:Result of analysis of recovery study of marketed formulation.
Dosage
Form
Labelled
claim
%
Estimated
%
Recovery
S.D
COV(%)
S.E
Tablet
10
100.70
101.18
0.3159
0.3122
0.4066
5
100.8
100.54
0.2146
0.2134
0.2793
Table-2: Validation parameters for Phenylephrine HCl
Parameters
Results
1.
λmax
275.5 nm
2.
Beer’s law limit(ug/ml)
5-25 ug/ml
3.
Regression equation
a.Slope
b.Intercept
0.3459
-2.522
4.
Correlation coefficient (r2 )
-2.522
5.
Molar absorptivity
35958.61
6.
Precision
100.17,99.10
7.
Specificity
A 10 µg/ml solution of drug in
solvent(methanol and distilled
water
in
ratio
of
90:10Respectively) at UV detection
of 275.5nm will show an
absorbance value of 0.1903
Table-3: Validation parameters for Levocetirizine dihydrochloride
Parameters
Results
1. λmax
230.5 nm
2. Beer’s law limit (µg/ml)
2.5-12.5 µg/ml
3.
Regression equation
a)Slope
b)Intercept
0.0673
4.
Correlation coefficient(r2)
0.9983
5.
Molar absorptivity
35958.61
6.
Precision
99.83, 98.87
7.
Specificity
A 10 µg/ml solution of candidate drug
in solvent(methanol and distilled
water in ratio of 90:10 respectively) at
UV detection of 230.5nm will show an
absorbances value of 0.4583
0.2386
4: CONCLUSION:
The Vierodt’s method permits simple, rapid and direct determination of Phenylephrine
hydrochloride and Levocetirine dihydrochloride is commercially available tablet
dosage form without previous separation.The results of analysis of two drugs from
tablet formulation using method was found close to 100%, standard deviation was
satisfactorily low indicating which showed that there is no interference of excipients.
5. ACKNOWLEDGMENT:
The authors wish to thank Zim Laboratories, Nagpur, Maharastra for providing the gift
samples of Phenylephrine HCl and Levocetirizine dihydrochloride.
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