Development and Validation of Advance
Simultaneous Equation
Methods for Lidocaine Hydrochloride and Clotrimazole in Ear Drop
Bhavisha G. Patel*, Mr. Nirav Gheewala, Hasumati A. Raj
Shree Dhanvantary College of Pharmacy, Kim, Surat, Gujarat, India
ABSTRACT:
A simple, accurate and precise
spectroscopic method was developed for simultaneous estimation of Lidocaine Hydrochloride and Clotrimazole
in Ear Drop by Advance simultaneous equation method. A zero order spectrum was recorded and convert in ratio second order derivative.
The Lidocaine Hydrochloride shows max absorbance at
243.20 nm and Clotrimazole show max absorbance at 225
nm. The method was found to be linear (r2>0.999) in the concentration range of 20-100μg/ml
for Lidocaine Hydrochloride
and 10-50μg/ml for Clotrimazole. The limit of determination was 0.2498μg/ml and
0.0837μg/ml for Lidocaine Hydrochloride and Clotrimazole, respectively. The limit of quantification was
0.7570μg/ml and 0.2537μg/ml for Lidocaine
Hydrochloride and Clotrimazole, respectively. The
accuracy of this method was evaluated by recovery studies and good recovery
result was obtained greater than 99%. The method was successfully applied for
simultaneous determination of Lidocaine Hydrochloride
and Clotrimazole in Ear Drop.
KEY WORDS:
Lidocaine Hydrochloride , Clotrimazole
and Advance simultaneous equation method.
INTRODUCTION:
Lidocaine
Hydrochloride (trade name Xylocaine) is a local
anesthetic and cardiac depressant used
as an antiarrhythmia agent. It is also used following
central spinal, epidural or peripheral administration, for topical management
of major pain. Clotrimazole is an imidazole
derivative with a broad spectrum of antimycotic
activity used as an antifungal. Clotrimazole used in
the treatment of fungal infections (of both humans and other animals) such as vaginal yeast infections, oral thrush, and ringworm. It is also used
to treat athlete's foot
and jock itch.
Fig. 1
Chemical Structure of Lidocaine Hydrochloride(6)
Fig. 2 Chemical Structure of
Clotrimazole(6)
·
A double beam UV/Visible spectrophotometer (Shimadzu model 2450, Japan)
with spectral width of 2 nm, 1 cm quartz cells
was used to measure absorbance of
all the solutions.
·
Spectra were automatically obtained by UV-Probe system software.
·
An analytical balance (Sartorius CD2250,
Gottingen, Germany) was used for
weighing the
samples.
·
Sonicator (D120/2h,
Trans-O-Sonic)
·
All instruments and glass wares were calibrated.
·
Lidocaine hydrochloride
and Clotrimazole raw materials was received as gift
sample from Apex
Healthcare Ltd, Ankleshwar and Luxica Pharma Inc, Panoli, respectively.
·
Methanol AR Grade (Rankem),
Distilled Water, Hcl(Astron), NaOH AR grade
(Rankem) were used for development purpose.
·
Methanol was used for further validation.
Standard
solution of Clotrimazole
(CLO):
Accurately weighed quantity of CLO 10 mg was transferred to
100ml volumetric flask, dissolved, and diluted up to mark with methanol to give
a stock solution having strength 100µg/ml.
Standard solution of Lidocaine Hydrochloride (LIDO):
Accurately weighed quantity of LIDO 10 mg was transferred
into 100 ml volumetric flask, dissolved and diluted up to mark with methanol to
give a stock solution having strength 100µg/ml.
Preparation of standard
mixture (CLO + LIDO):
1ml of standard stock solution of CLO (100μg/ml) and
1ml of standard stock solution of LIDO (100μg/ml) were
pipette out into two 10ml volumetric flasks and volume was adjusted to the mark
with methanol to get 10μg/ml of CLO and 10μg/ml of LIDO.
DEVELOPMENT AND VALIDATION OF METHOD (7-11)
Method:-
Advance Simultaneous Equations:
To determine wavelength for
measurement, standard spectra of Lidocaine
hydrochloride and clotrimazole was scanned between
200-400nm in Methanol. The method was based on the measurement of absorbance of
Lidocaine hydrochloride at 243.20nm and clotrimazole at 225 and s nm in both wavelength.
Equation:
Cx = (A2*ay1 – A1*ay2
/ ax2*ay1 – ax1*ay2)
Cy = (A1*ax2 – A2*ax1 / ay1*ax2
– ay2*ax1)
Where,
Cx = Concentration of Clotrimazole
Cy = Concentration of Lidocaine hydrochloride
A 1 =
Absorbance of test at λ1 (λmax of CLO)
A2 = Absorbance of test at λ2 (λmax of LIDO)
ax1 =
Absorptivity of x drug(CLO) at λ1
ax2 =
Absorptivity of x drug(CLO) at λ2
ay1 =
Absorptivity of y drug(LIDO) at λ1
ay2 =
Absorptivity of y drug(LIDO) at λ2
Selection of
Wavelength of Determination:
The solutions of CLO and LIDO (in the
ratio of 1:2) were scanned separately between 200-400nm.
FIG. 3 Overlain Zero
Order Spectra of CLO
and LIDO (1:2) Ratios, Respectively
For CLO the linearity range 10-50 µg/ml was divided by the
LIDO linearity rang 20-100 µg/ml. 80 µg/ml of LIDO is linear divisor for CLO.
The divided spectra was converted to second
derivative. Measurement of absorbance of Clotrimazole
at 225.00 nm.
For LIDO the linearity range 20-100 µg/ml was divided by
the CLO linearity rang 10-50 µg/ml. 30 µg/ml of CLO is linear divisor for LIDO.
The divided spectra was converted to second
derivative. Measurement of absorbance of Clotrimazole
at 243.20 nm.
Fig. 3
Overlain Ratio Spectra of CLO
and LIDO (1:2) Ratios, Respectively
Fig.4 Zoom View Overlain Ratio of Spectra
of CLO and LIDO (1:2) Ratio, respectively
Fig.5 Overlain Ratio
Second Order Derivative Spectra of CLO and LIDO (1:2) Ratios Respectively
Fig.6
Zoom View Overlain Ratio
Second Order Derivative Spectra of CLO and LIDO (1:2) Ratios, respectively
1. Linearity and
Range:
The ratio second order derivative spectra showed linear absorbance at
225.00 nm for CLO (10-50µg/ml) and
243.20 nm for LIDO (20-100µg/ml).This
method obeyed beer’s law in the concentration range 10-50µg/ml for CLO
and 20-100µg/ml for
LIDO. Correlation coefficient (r2) form calibration curve of CLO and LIDO was found
to be 0.999and 0.999, respectively. The
regression line equation for
CLO and LIDO are as
following,
y = -0.016x+0.122 for CLO _____________ (1)
y = -0.048x -0.019 for LIDO ______________ (2)
FIG.7
Calibration curve for CLO at 225.00
nm
FIG.8 Calibration curve for LIDO at 243.20 nm
2. Precision:
i. Intraday
precision:
The data for intraday precision for combined standard solution of CLO and
LIDO is presented in Table 2.The
% R.S.D was found to
be 0.10-0.28% for CLO and 0.11-0.24% for LIDO. These %RSD value was found to be less than ± 2.0 indicated that the method is precise.
ii.. Interday Precision:
The data for interday precision for combined standard solution of CLO and
LIDO is presented in Table 3.The % R.S.D
was found to be 0.27-0.37% for CLO
and
0.23-0.35% for LIDO.
These %RSD value was found to be less than ± 2.0 indicated that the method
is precise.
TABLE.1 Calibration
data for mixture of CLO and LIDO at 225.00 nm and 243.20
nm, respectively
*(n=6)
|
CLOTRIMAZOLE |
ABSORBANCE
(225.0 nm) Avg ± SD |
LIDOCAINE
HYDROCHLORIDE |
ABSORBANCE
(243.20 nm) Avg ± SD |
|
10 |
-0.048± 0.0005 |
20 |
-0.959±0.0006 |
|
20 |
-0.201± 0.0006 |
40 |
-2.025±0.0006 |
|
30 |
-0.365± 0.0006 |
60 |
-2.945± 0.0010 |
|
40 |
-0.545± 0.0004 |
80 |
-3.844± 0.0007 |
|
50 |
-0.702±0.0006 |
100 |
-4.912± 0.0006 |
TABLE. 2 Intraday precision data for estimation of CLO
and
LIDO *(n=3)
|
Conc. (μg/ml) |
Abs.*
(CLO) Avg. ± SD (225.0nm) |
% RSD |
Abs.*
(LIDO) Avg.± SD (243.20nm) |
% RSD |
|
|
CLO |
LIDO |
||||
|
20 |
40 |
-0.201±0.0005 |
0.28 |
-2.026±0.0023 0.002309 0.002309 0.002309 |
0.11 |
|
30 |
60 |
-0.364±0.0005 |
0.15 |
-2.945±0.0040 |
0.13 |
|
40 |
80 |
-0.545±0.0005 |
0.10 |
-3.848±0.0094 |
0.24 |
TABLE.3 Interday precision data for estimation of CLO
and
LIDO *(n=3)
|
Conc. (μg/ml) |
Abs.*
(CLO) Avg. ± SD (225.0nm) |
% RSD |
Abs.*
(LIDO) Avg. ±SD (243.20nm) |
% RSD |
|
||
|
CLO |
LIDO |
|
|||||
|
20 |
40 |
-0.203 ± 0.0007 |
0.37 |
-2.026±0.0049 |
0.24 |
|
|
|
30 |
60 |
-0.367 ± 0.001 |
0.27 |
-2.948 ± 0.007 |
0.23 |
||
|
40 |
80 |
-0.547 ± 0.002 |
0.36 |
-3.846 ±0.0136 |
0.35 |
||
TABLE.4 Recovery
data of CLO *(n=3)
|
Conc. of CLO from
formulation
(µg/ml) |
Amount of Std. CLO added (µg/ml) |
Total amount
of CLO (µg/ml) |
Total amount
of CLO found (µg/ml)* Mean ± SD |
% Recovery
(n=3) |
% RSD CLO |
|
10 |
8 |
18 |
18.12 ± 0.001 |
101.33 |
0.01 |
|
10 |
10 |
20 |
20.01 ± 0.005 |
100.09 |
0.02 |
|
10 |
12 |
22 |
22.01 ± 0.005 |
100.07 |
0.02 |
TABLE.5 Recovery
data of LIDO *(n=3)
|
Conc. of LIDO from
formulation
(µg/ml) |
Amount of Std. LIDO added (µg/ml) |
Total amount
of LIDO(µg/ml) |
Total amount
of LIDO found (µg/ml)* Mean ± SD |
% Recovery
(n=3) |
% RSD LIDO |
|
20 |
16 |
36 |
36.28 ± 0.011 |
101.61 |
0.03 |
|
20 |
20 |
40 |
40.04 ±0.005 |
100.18 |
0.01 |
|
20 |
24 |
44 |
44.07±0.005 |
100.26 |
0.01 |
3. Accuracy:
Accuracy of the method was determined by recovery study from formulation
at three levels (80%, 100%, and 120%) of standard
addition. Percentage recovery for CLO and LIDOS by this method was found in the range of 100.07-101.33
%and 100.18-101.61%, respectively. The value of %RSD within the limit indicated that the method is accurate and
percentage recovery shows
that there is no interference from
the
excipients.
4. Limit
of Detection
and
Quantification:
The LOD for CLO and LIDO was conformed to be 0.0837µg/ml and 0.2498µg/ml respectively.
The LOQ for CLO and LIDO was conformed to be 0.2537
µg/
ml and 0.7570 µg/ml
respectively. The
obtained LOD and LOQ results are presented in Table 6
5. ROBUSTNESS AND RUGGEDNESS:
The obtained Ruggedness
and Robustness results are presented
in table 7. The % R.S.D
was found to be 0.10- 0.57% for CLO and 0.12 – 0.83% for LIDO. These %RSD value was found to be less than ± 2.0 indicated that the method
is precise. No significant
changes
in the
spectrums were
observed,
proving that the developed method
is rugged and
robust.
TABLE.6 LOD and LOQ data of CLO and LIDO *
(n=10)
|
Conc. (μg/ml) |
Avg. ± SD(225nm)*
CLO |
% RSD |
Avg. ± (243.20
nm)*
LIDO |
% RSD |
|
|
CLO |
LIDO |
||||
|
10 |
20 |
-0.048 ± 0.0004 |
0.83 |
-2.047 ± 0.003 |
0.17 |
|
LOD (μg/ml) |
0.0837 |
0.2498 |
|||
|
LOQ (μg/ml) |
0.2537 |
0.7570 |
|||
TABLE.7 Robustness and Ruggedness data of
CLO and LIDO*(n=3)
|
Condition |
Concentration (µg/ml) |
Change in Wavelength 225.00±0.2nm and
243.20±0.2nm |
Change in Instrument |
||
|
225.2nm |
243.4nm |
UV 1800 |
UV 2450 |
||
|
CLO Mean (n=3) ± % RSD |
20 |
-0.203 ±0.28 |
-0.206 ±
0.48 |
-0.201 ±0.28 |
-0.202 ± 0.37 |
|
30 |
-0.366 ±0.31 |
-0.368 ± 0.27 |
-0.363 ±0.57 |
-0.366± 0.41 |
|
|
40 |
-0.547 ±0.38 |
-0.548 ± 0.18 |
-0.545± 0.10 |
-0.542± 0.28 |
|
|
LIDO Mean
(n=3) ± % RSD |
40 |
-2.034 ± 0.69 |
-2.036 ± 0.35 |
-2.029 ±0.41 |
-2.027 ± 0.51 |
|
60 |
-2.951 ±0.25 |
-2.941 ± 0.34 |
-2.952 ±0.37 |
-2.944 ± 0.12 |
|
|
80 |
-3.850±0.19 |
-3.866 ±0.29 |
-3.859 ±0.68 |
-3.862 ± 0.83 |
|
Application of the Proposed Method for
Analysis of CLO and LIDO In
Formulation (Assay):
CANDID ear drop was used which containing Clotrimazole (1%w/v) and Lidocaine
Hydrochloride (2%w/v) (content 15 ml).From which 10ml transferred in 100 ml
volumetric flask and made up to the mark with the Methanol. Final solution contained
100μg/mL CLO and 200μg/mL
LIDO and From that pipettout
within the linearity. A ratio derivative spectrum of the sample solution containing 20µg/ml of CLO and 40µg/ml of LIDO was recorded and the absorbance at 225.0nm and
243.20nm were noted for estimation of CLO and LIDO, respectively. The concentration of CLO and LIDO
in mixture was determined using
the corresponding calibration
graph. The
results from the analysis of formulation containing Clotrimazole (20mg) and
Lidocaine hydrochloride (40mg) in combination
are
presented in Table 8.The percent assay shows that there is no interference from excipients and the proposed method can successfully applied to analysis of commercial
formulation containing CLO and LIDO.
TABLE.8 Analysis data
of commercial
formulation *(n=3)
|
Sr. No. |
Formulation |
Absorbance* (225.0nm)
CLO |
% Assay CLO ± SD |
Absorbance (243.20.0nm) LIDO |
% Assay LIDO±SD |
|
|
|
CLO |
LIDO |
||||
|
1 |
20 |
40 |
-0.204 |
100.60 ± 0.05 |
-2.042 |
100.52 ± 0.02 |
|
2 |
-0.203 |
-2.014 |
||||
|
3 |
-0.205 |
-2.031 |
||||
TABLE.9 SUMMARY OF VALIDATION
PARAMETER
|
Sr No. |
Parameters |
CLO |
LIDO |
|
1. |
Concentration Range (µg/ml) |
10-50µg/ml |
20-100µg/ml |
|
2. |
Regression Equation |
y = -0.016x+0.122 |
y = -0.048x -0.019 |
|
3. |
Correlation
Coefficient (r2) |
0.999 |
0.999 |
|
4. |
Accuracy
(%Recovery) |
100.49% |
100.68% |
|
5. |
Intraday Precision
(%RSD) |
0.10-0.28% |
0.11-0.24% |
|
6. |
Interday Precision
(%RSD) |
0.27-0.37% |
0.23-0.35% |
|
7. |
Robustness(%RSD) |
0.10- 0.57% |
0.12 – 0.83% |
|
8. |
LOD (µg/ml) |
0.0837µg/ml |
0.2498µg/ml |
|
9. |
LOQ (µg/ml) |
0.2537µg/ ml |
0.7570 µg/ml |
|
10. |
Assay |
100.60 % |
100.52 % |
ACKNOWLEDGEMENT:
We are sincerely thankful to Shree Dhanvantary Pharmacy College, Kim, Surat,
for providing us infrastructure facilities and moral support to carry out this
research work. We are also thankful to SDPARC for giving us their special time
and guidance for this research work. We also thank our colleagues for their
helping hand.
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Received on 06.04.2015 Accepted on 22.04.2015
© Asian Pharma
Press All Right Reserved
Asian J. Pharm.
Res. 5(2): April-June 2015;
Page 108-113
DOI: 10.5958/2231-5691.2015.00016.7