Estimation of Levocetirizine
in Bulk and Formulation by Second Order Derivative Area under Curve
UV-Spectrophotometric Methods
Audumbar
Digambar Mali*, Manojkumar Patil
Department of Pharmaceutics, Sahyadri College of Pharmacy, Methwade,
Sangola-413307, Solapur MS India.
*Corresponding Author E-mail: maliaudu442@gmail.com
ABSTRACT:
Simple, fast and reliable spectrophotometric methods were developed
for determination of Levocetirizine
in bulk and pharmaceutical dosage forms. The solutions of standard and the
sample were prepared in Methanol. The quantitative determination of the drug
was carried out using the second order Derivative Area under Curve method
values measured at 247-255nm. Calibration graphs constructed at their
wavelengths of determination were linear in the concentration range of Levocetirizine
using 5-25μg/ml (r˛=0.999) for second order Derivative Area under Curve
spectrophotometric method. The proposed methods have been extensively validated
as per ICH guidelines. There was no significant difference between the
performance of the proposed methods regarding the mean values and standard
deviations. The developed methods were successfully applied to estimate the
amount of Levocetirizine
in pharmaceutical formulations.
KEY WORDS:
Levocetirizine,
Second order Derivative, Area under Curve (AUC), Precision, Accuracy.
INTRODUCTION:
Chemically Levocetirizine chemically is [2-[4- [(r)-(4-chlorophenyl) phenylmethyl]-1- piperazinyl] ethoxy] acetic acid is a third generation non-sedative
antihistamine, developed from the second generation antihistamine cetirizine. [1, 2] It is the L enantiomer
of the cetirizine racemate.
Levocetirizine works by blocking histamine receptors.
It does not prevent the actual release of histamine from mast cells, but
prevents it binding to its receptors. [3, 4] This in turn prevents the release
of other allergy chemicals and increased blood supply to the area, and provides
relief from the typical symptoms of hay fever.
In our Literature survey reveals that for Levocetirizine
Spectrophotometric [5, 6] methods and HPLC [7, 8, 9]
methods have been reported for its determination in commercial formulation.
To our notice, no UV- spectrophotometric method using Second Order
Derivative Area under Curve has been reported for the determination of Levocetirizine in bulk and tablets. Hence an attempt has
been made to develop new Second Order Derivative Area under Curve
spectrophotometric method for estimation of Levocetirizine
in bulk and pharmaceutical formulations with good accuracy simplicity,
precision and economy.
Fig. 1 Structure
of Levocetirizine
MATERIALS
AND METHODS:
Derivative
Spectrophotometric Methods:-
The second derivative spectrophotometry
was used in the wavelength ranges from 247 and 255 nm.
[d2A/dλ2]= f(λ):
second order
The second derivative spectrum of an absorption
band is characterized by a maximum, a minimum, and a cross-over point at the
λ max of the absorption band. [10, 11]
Area under curve (Area
calculation):
In this study area was integrated between wavelength ranges from
247 and 255 nm.
Area calculation: (α+β) =
Where, α is area of portion bounded by curve data and a
straight line connecting the start and end
point, β is the area
of portion bounded
by a straight
line connecting the
start and end point on curve data and horizontal axis,
λ1 and λ2 are wavelength range
start and end point of curve region. [12, 13]
Apparatus and
instrumentation:
A Shimadzu 1800 UV/VIS double beam spectrophotometer with 1cm matched
quartz cells was used for all spectral measurements. Single Pan Electronic
balance (Contech, CA 223, India) was used for
weighing purpose. Sonication of the solutions was carried out using an
Ultrasonic Cleaning Bath (Spectra lab UCB 40, India). Calibrated volumetric
glassware (Borosil®) was used for the validation
study.
Materials:
Reference standard of Levocetirizine API
was supplied as gift sample by Lupin Laboratory Park,
Aurangabad. Methanol was obtained from Research-Lab Fine HYPERLINK "http://www.indiamart.com/company/3928956/"ChemHYPERLINK
"http://www.indiamart.com/company/3928956/" Industries, HYPERLINK
"http://www.indiamart.com/company/3928956/"Islampur,
Mumbai, and Maharashtra. Capsule sample
with label claim 5 mg per Tablet were purchased from local market Mangalwedha, Solapur,
Maharashtra, India.
Method development:
Preparation of Standard and
Sample Solutions:-
Stock solution of 10μg/ml of Levocetirizine
was prepared in Methanol, for Second Order Derivative Area under Curve
spectrophotometric analysis. The standard solutions were prepared by dilution
of the stock solution with Methanol in a concentration range of 5, 10, 15, 20
and 25μg/ml with Methanol for Second Order
Derivative Area under Curve spectrophotometric methods. Methanol was used as a
blank solution.
Fig.
2 Second order derivative Area under Curve spectrum of
Levocetirizine in Methanol (25µg/ml).
Fig.
3 Second order derivative spectrum of Levocetirizine in Methanol (25µg/ml).
Calibration curve for Levocetirizine:
The dilutions were made from Standard Stock solution to get
concentration of 5, 10, 15, 20, and 25µg/ml respectively. These solutions were
scanned from 400 to 200 nm and First Order Derivative Area under Curve values
was integrated in the range of 224-231 nm. The calibration curve was plotted
between areas under curve values against concentration.
Fig. 4 Linearity of Levocetirizine.
Assay of tablet
formulation:-
Twenty tablets each containing 5 mg of Levocetirizine
were weighed crushed to powder and average weight was calculated. Powder
equivalent to 10 mg of Levocetirizine was transferred
in 100 ml of volumetric flask. A 50 ml of Methanol was added and sonicated for 15 minutes. Then solution was further diluted
up to the mark with Methanol. The solution was filtered using Whatman filter
paper no. 41, first 5 ml of filtrate was discarded.
This solution was further diluted to obtain 10µg/mL
solution with water, subjected for UV analysis using Methanol as blank. This
procedure was repeated three times.
Fig. 5 Second
order derivative Area under Curve spectrum of Levocetirizine
of dosage form in Methanol (25µg/ml).
Fig. 6 Second
order derivative spectrum of Levocetirizine of dosage
form in Methanol (25µg/ml).
Fig. 7 Second
order derivative overlay of Levocetirizine at diff.
Concentration.
Table 1: Assay of tablet
dosage form:-
|
Sr.No.
|
Sample Solution Concentration (µg/ml)
|
Amount found (%)
|
Mean % found*
|
%RSD*
|
|
1
|
25
|
98.59
|
|
|
|
2
|
25
|
99.52
|
99.00
|
0.2961
|
|
3
|
25
|
98.90
|
|
|
*n=3, % RSD = % Relative Standard Deviation.
Method Validation:[14]
The above method
was validated for
various parameters such as
Accuracy, Linearity, Precision,
Limit of detection
(LOD) and Limit of Quantitation (LOQ) according to ICH guideline.
Accuracy:-
The accuracy for
the analytical method
was evaluated at
80%, 100% and
120% levels of 25µg/ml Sample solution. Second Order
Derivative Area under curve (AUC) was measured in wavelength range 247-255 nm
and results were obtained in terms of percent recovery. Three determinations at
each level were performed and % RSD was calculated for each level.
Table
2: Accuracy results for Levocetirizine:-
|
Accuracy level
|
Sample Conc. (µg/ml)
|
Std. Conc.
|
Total amount.
Added (µg/ml)
|
% Recovery
|
Mean % Recovery
|
% RSD
|
|
80
|
25
|
12
|
22
|
100.12
|
|
|
|
100
|
25
|
15
|
25
|
100.29
|
100.49
|
0.2397
|
|
120
|
25
|
18
|
28
|
101.08
|
|
|
Precision:-
The precision of an analytical procedure expresses the closeness
of an agreement (degree of scatter) between a series of measurements obtained
from multiple sampling of the same homogeneous sample under the prescribed
conditions intraday precision was studied by integrating area of standard solution
of 25µg/ml concentration at six independent series in the same day. Interday
precision studies were performed by integrating area of standard solution of
25µg/mlconcentration on three consequent days. The
%RSD Was calculated.
Table
3: Precision Study:-
|
Parameter
|
Intra
day
|
Inter-day
|
|
Sample sol conc.µg/ml
|
25
|
25
|
|
AUC (mean)
|
0.5326
|
0.5237
|
|
%RSD
|
0.8369
|
0.8620
|
Limit of Detection and Limit
of Quantification:-
The Limit of Detection (LOD) is the smallest concentration of the analyte that gives the measurable response. LOD was
calculated using the following formula
LOD = 3.3 σ /S
The Limit of Quantification (LOQ) is the smallest concentration of
the analyte, which gives response that can be
accurately quantified. LOQ was calculated using the following formula
=
10 σ/S
Where, σ is standard deviation of the response and
S is the slope
of the calibration curve.
LOD and LOQ of Levocetirizine was found to be 0.53µg/ml and1.59µg/ml respectively.
Table
4: Summary of validation parameters:-
|
Parameter
|
Result
|
|
λ range
|
247-255
|
|
Regression Equation (y=mx+c)
|
Y=0.023x + 0.003
|
|
Linearity range
|
5-25µg/ml
|
|
Slope
|
0.023
|
|
Intercept
|
0.003
|
|
Correlation coefficient (R2)
|
0.999
|
|
Limit of Detection (LOD) µg/ml
|
0.53
|
|
Limit of Quantitation
(LOQ) µg/ml
|
1.59
|
|
Accuracy (Mean % Recovery)
|
100.49
|
|
Precision (%RSD)
|
0.2397
|
RESULTS
AND DISCUSSION:
The UV visible spectroscopic method for the Levocetirizine
by Second order derivative Area under Curve was found to be simple, accurate,
economical and reproducible. The
drug concentrations were
found to be linear
in the range
of 05-25 µg/ml and the
correlation coefficient value of 0.999 indicates that developed
method was linear.
For Precision the
percent relative standard
deviation (% RSD) was found to be
0.2397 while,
intra-day and inter-day
precision results in
terms of percent relative standard deviation
values were found to be 0.5326 and 0.5237 respectively thus the method
is observed as precise. The accuracy of the method was assessed by recovery
studies at three different levels i.e. 80%, 100%, 120%. The values of standard
deviation were satisfactory and the recovery studies were close to 100%. The %
RSD value is ≤ 2 indicates the accuracy of the method. The
Limit of Detection
and Limit of Quantitation
values were found to
be 0.53µg/ml and 1.59µg/ml
respectively. The result
of the analysis
for pharmaceutical
formulation by the
developed method was
consistent with the
label claim, highly reproducible
and reliable. The method can be
used for routine
quality control analysis
of Levocetirizine
in bulk
and pharmaceutical formulations.
CONCLUSION:
The UV spectroscopic AUC method for the analysis of Levocetirizine by Second order derivative Area under Curvewas found to be simple, precise, and accurate; can be
used for assay of bulk drug and pharmaceutical dosage formulations.
ACKNOWLEDGEMENT:
The authors are highly thankful to the Sahyadri
College of Pharmacy, Methwade, Sangola,
Solapur, Maharashtra, India
for proving all the facilities to carry out the research work.
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