INTRODUCTION:
Validation is defined as
establishing Documentevidences which provides a high degree assurance that a
specific process will consistently produce a product meeting its pre-
determined specification and quality characteristics. This medication is used
to treat certain bladder/urinary tract symptoms. Flavoxate is a smooth-muscle
relaxant. It works by relaxing the muscles in the bladder. Flavoxate helps to
reduce leaking of urine, feelings of needing to urinate right away, frequent
trips to the bathroom, and bladder pain. This medication does not treat the
cause of your bladder symptoms (e.g., urinary tract/bladder/prostate infection
or inflammation). flavoxate hydrochloride (FLX), 3-methylfl
avone-8-carboxylic acid β-piperidinoethyl esterhydrochloride is strong
smooth muscle relaxant, with selective action on the pelvic. Itis used for the
symptomatic relief of pain, urinary frequency, and incontinence associated with
inflammatory disorders of the urinary tract1-5.
MATERIAL AND METHOD:
Flavoxate was procured from yarrow Chem, Mumbai;
Distilled water was obtained from local market. Methanol, Hydrochloric Acid,
Sodium Hydroxide and acetonitrile was obtained from S.D Fine Chem, Pvt. Ltd,
Mumbai. All the other chemicals and reagents used were of analytical grade and
used as obtained.
Experimental Work:
The experimental work of stability indicating UV-
Spectrophotometry for the estimation of Flavoxate is given below.
Selection of solvent system:
The selection of solvent was made after checking the
solubility of drug in different solvents like water, methanol, Acetonitrile,
0.1 N NaOH, 0.1 N HCl etc. Flavoxate
was found to be completely
soluble in Methanol, Acetonitrile. Thereforeon the basis of solubility and
economical point of view Methanol and water (60:40) is selected as a solvent
system6-8.
Preparation of standard stock solution (100”g/ml):
Accurately weighed 10mg of Flavoxate and transferred
into 100ml volumetric flask. Add sufficient amount of water and sonicated it
for 15 min. After Sonication, volume was made upto 100ml with water to get
final concentration 100”g/ml9-12.
Preparation of working standard solution (10”g/ml):
From the above stock solution 1 ml of Flavoxate was
taken and transferred into 10ml volumetric flasks and then volume was made up
to 10ml with water for make the 10”g/ml as final concentration13-16.
RESULTS AND DISCUSSION:
Selection of analytical wavelength:
Working standard solution 10”g/ml of Flavoxate was scanned
in the UV-region i.e. 400 to 200nm. In UV- Spectrophotometer the λmax was
obtain 242nm.
Fig. 1: UV-spectrum ofFlavoxate HCl
Selection of analytical concentration range and
linearity study:
The standard stock solution of Flavoxate were diluted
with water to get series of concentration of 2, 4, 6, 8, 10, 12.μg/ml.
Absorbances of these solutions were measured at 242nm for Flavoxate in 1cm cell
using solvent blank. The linearity of proposed method was found to be in
between 2-12”g/ml for Flavoxate. Limit of detection were found to be
0.513094μg/ml and Limit of quantitation were found to be
1.552849μg/ml for DRUG. Plot of absorbance Vs concentration were
found to be linear and is depicted in Fig.
Fig. 2: Calibration curve of Flavoxate
Analysis of marketed formulation:
Accurately weighed 10 tablets of marketed
formulation Flavoxate HCL 200 and average weight were found to be 335mg. Then
these tablets were crushed to fine powder and from this 167mg of powder weighed
containing equivalent weight of 100mg of DRUG. It has been transferred into
100ml of volumetric flask and volume was made up to the mark with waterand this
mixture is sonicated for 15 min. After Sonication, it was filtered through
Whatmann filter paper no. 41. The solution was further diluted with waterto get
a final concentration of 8μg/ml of DRUG. The solution was scanned in
UV-range (i.e., 200-400nm). The analysis procedure was repeated six times with
tablet formulation.
Method validation:
Validation of an analytical method is the
process by which it is established by laboratory studies, that the performance
characteristics of the method meet the requirements for the intended analytical
application. Method validation is performed to ensure that an analytical
methodology is accurate, specific, reproducible and rugged over the specific
range that an analyte will be analyzed.
Linearity:
The linearity study of Flavoxate was performed by
preparing standard solution of 2, 4, 6, 8, 10, 12”g/ml. The calibration graph
of concentration versus absorbance is plotted for Flavoxate.
Precision:
The precision of an analytical procedure express the
closeness of agreement between a series of measurements obtained from the
multiple sampling of the same homogeneous sample under the prescribed
condition. The precision of the method was evaluated by intraday and inter-day
variation studies. In intraday studies, working solutions of standard and
sample were analyzed thrice in a day and the percentage of relative standard
deviation (% RSD) was calculated. In case of inter-day variation studies, the
working solution of standard and sample were analyzed onthree consecutive days
and the percentage of relative standard deviation (% RSD) was calculated. To
check the degree of repeatability of the methods, suitable statistical
evaluation was carried out. Six samples of the tablet formulation were analyzed
for the repeatability study.
Accuracy:
Recovery studies were carried out at three different
levels (80%, 100% and 120%) by standard addition as per ICH guidelines to
ascertain the accuracy of the proposed method. As per the label claim, tablet
contains 200mg of Flavoxate.
To perform recovery study at 80%, tablet powder is
weighed about 167mg containing equivalent to 100mg of Flavoxate and to this
adds standard 80mg of Flavoxate. The mixture is triturated well and from this
powder is taken and added into 100ml of volumetric flask and add into solvent
methanoland water (60:40) and prepare 8μg/ml concentration solution of
Flavoxate.
To perform recovery study at 100%, tablet powder is
weighed about 167mg containing equivalent to 100mg of Flavoxate and to this
adds standard 100mg of Flavoxate. The mixture is triturated well and from this
powder is taken and added into 100ml of volumetric flask and make 8μg/ml
concentration solution of Flavoxate.
To perform recovery study at 120% tablet powder is
weighed about 167mg containing equivalent to 100mg of Flavoxate and to this
standard 120mg of Flavoxate is added. The mixture is triturated well and from
this powder is taken and added into 100ml of volumetric flask and by serial
dilution technique required concentration solution is prepared containing
8μg/ml of Flavoxate.
Limit of Detection:
The detection limit is of an individual
analytical procedure is the lowest amount of analyte in a sample which can be
detected but not necessarily quantitated as an exact value. The limit of
detection are calculated by using the formula DL = 3.3*σ/S where, σ
is SD of the response and S is the slope of the calibration curve.
Limit of Quantitation:
The quantitation limit of an individual analytical
procedure is the lowest amount of analyte in a sample which can be
quantitatively determined with suitable precision and accuracy. The
quantitation limit may be expressed as QL = 10*σ/S, where σ is the SD
of the response and S is the slope of the calibration curve.
Robustness:
The robustness of an analytical procedure is a measure
of its capacity to remain unaffected by small but deliberate changes in method
parameter an indication of itsreliability during normal usage. It is the
ability to provide accurate and precise results under a variety of conditions.
Forced degradation study:
The stress conditions applied for degradation study
involved acid hydrolysis, base hydrolysis, oxidative degradation, thermal
degradation (60șC), UV photolysis (211nm). After the fixed time period the
treated drug solutions were diluted with solvent. For every stress condition
three solutions were prepared 8”g/ml of Flavoxate. The specific stress conditions
are described as follows.
Acid hydrolysis:
Accurately weighed 10mg of Flavoxate and transferred
it to 100ml volumetric flasks, added 90ml of methanol and water (60:40) and
10ml 0.1N HCl. These flasks were placed at room temp for 24 hr. After 24 hr solutions
were neutralized with 0.1N NaOH. Finally make the solution of 8”g/ml
concentration of Flavoxate and absorbance was measured at 242nm.
Fig. 3: Acid degradation of Flavoxate
Alkali hydrolysis:
Accurately weighed 10mg of Flavoxate and transferred
into 100mL volumetric flasks, added 90ml methanol and water (60:40) and 10ml
0.1N NaOH. These flasks were placed at room temp for 24 hr. After 24 hr
solutions were neutralized with 0.1N Hcl. Finally prepare solutions of 8”g/ml
concentration of Flavoxate and absorbance was measured at 242nm. Finally
absorbance of sample was compared with standard absorbance and percent
degradation was calculated.
Fig. 4: Alkali degradation of Flavoxate
Oxidative degradation:
Accurately weighed10mg of Flavoxate and transferred
into100mL volumetric flasks, added 90mlmethanol and water (60:40) and 10ml H2O2
Solution (3%). These flasks were placed at room temp for 24 hr. After 24 hr
from the above solution make the 8 ”g/ml solution and absorbance of sample was
taken at 242nm. And compared with standard absorbance and percent degradation
was calculated.
Fig. 5: Oxidative degradation of Flavoxate
Neutral degradation:
Pure drug were add in to water for 1hrs at 60°C. The
samples were diluted with methanol and water (60:40) to get 8mg/ml concentration solution of Flavoxate
and absorbance was measured at 242nm. Finally absorbance of sample was compared
with standard absorbance and percent degradation was calculated.
Fig. 6: Photolytic degradation of Flavoxate
Thermal degradation:
Thermal degradation was carried out by exposing pure
drugs to dry heat at 60oC for 1hrs.The samples after exposure to
heat were diluted with water to get 8mg/ml
concentration solution of Flavoxate and absorbance was measured at 242nm.
Finally absorbance of sample was compared with standard absorbance and percent
degradation was calculated.
UV light degradation:
UV light degradation was carried out by exposing pure
drugs in sunlight for 1hrs.The samples after exposure to UV light were diluted
with water to get 8mg/ml concentration
solution of Flavoxate and absorbance was measured at242nm. Finally absorbance
of sample was compared with standard absorbance and percent degradation was
calculated.
Fig. 7: Thermal degradation of Flavoxate
Fig. 8: UV light degradation of Flavoxate
RESULTS AND DISCUSSION:
Analysis of marketed formulation:
The results of marketed formulation
aregiven below, the percent amount found of the Flavoxate in tablet formulation
was found to be 98.81 ± 0.39 is within the limit.
Table 1: Analysis of Marketed formulation
|
Sr. no.
|
Formulation
|
Lable claim
(mg)
|
Actual conc.
of DRUG (”g/ml)
|
Amount found
(”g/ml)
|
% Amount found
|
|
1
|
Flavoxate 200 mg
Tablets
|
200
|
8
|
8.04
|
100.5
|
|
2
|
8
|
8.08
|
101.9
|
|
3
|
8
|
7.95
|
99.4
|
|
4
|
8
|
7.87
|
98.45
|
|
5
|
8
|
7.99
|
99.93
|
|
6
|
8
|
8.04
|
100.6
|
Table 2: Statistical validation: Analysis of Marketed
formulation
|
Name of the
drug
|
Mean*
|
SD*
|
%RSD*
|
|
Flavoxate
|
99.72
|
1.2000
|
0.0120
|
*Average of six determinants
Evaluation of analytical method validation:
Linearity:
The calibration curves were found to be linear over
concentration range of 2, 4, 6, 8, 10, 12 ”g/ml. The results of linearity study
are given intable.
Table 3: Linear regression data for calibration curve
of Flavoxate
|
Drug
|
Linearity
(”g/ml)
|
r2
|
Slope
|
Intercept
|
|
Flavoxate
|
2-12 ”g/ml
|
0.999
|
0.111
|
0.0092
|
Precision:
For the precision study 8 ”g/ml concentration solution
was used to Intra- day and Interday study. The results are given below:
i. Inter-day Precision of Drug:
Table 4: Inter day Precision data of Flavoxate
|
Sr. No.
|
Interval of
Time
|
Absorbance
|
Mean
Absorbance
|
Amount found
(”g/ml)
|
% Amount found
|
|
Day I
|
|
0.563
|
0.561
|
0.558
|
0.560
|
8.023
|
100.6
|
|
Day II
|
Inter-day
|
0.533
|
0.553
|
0.547
|
0.544
|
7.963
|
99.66
|
|
Day III
|
|
0.544
|
0.539
|
0.550
|
0.544
|
7.906
|
98.90
|
Table 5: Statistical validation of Inter-day precision
data
|
Name of the
drug
|
Mean*
|
SD*
|
% RSD*
|
|
Flavoxate
|
99.72
|
1.20004
|
0.01203
|
*Average
of three determinants
ii. Intra-day precision of LMS
Table 6: Intra-day Precision data of
Flavoxate
|
Sr. No.
|
Interval of
Time
|
Absorbance
|
Mean Abs.
|
Amount found (”g/ml)
|
% Amount found
|
|
11.30 am
|
|
0.336
|
0.335
|
0.337
|
0.336
|
2.99
|
99.70
|
|
2.30 pm
|
Intra-day
|
0.338
|
0.337
|
0.336
|
0.337
|
3.0
|
100
|
|
5.30 pm
|
|
0.340
|
0.337
|
0.341
|
0.339
|
3.01
|
100.59
|
iii. Repeatability data (Intra-assay
precision)
Table 8: Repeatability data
|
Sr. No.
|
Concentration
(”g/ml)
|
Absorbance
|
Amount found
(”g/ml)
|
Amount found
(%)
|
|
1
|
8
|
0.336
|
2.991
|
99.70
|
|
2
|
8
|
0.340
|
3.026
|
100.89
|
|
3
|
8
|
0.334
|
2.973
|
99.10
|
|
4
|
8
|
0.338
|
3.008
|
100.29
|
|
5
|
8
|
0.337
|
3.000
|
100
|
|
6
|
8
|
0.339
|
3.017
|
100.59
|
Table 9: Statistical validation of repeatability data
|
Name of the
drug
|
Mean*
|
SD*
|
%RSD*
|
|
Flavoxate
|
100.09
|
0.641
|
0.64
|
*Average of six determinants
Accuracy (Recovery study):
To determine the accuracy of the proposed methods,
recovery studies were carried at three different levels (80%, 100% and 120%)
its shows in table.
Table 10: Recovery study data of Flavoxate
|
Level of
Recovery
|
Amount present
(mg)
|
Added
concentration (mg)
|
Amount
recovered (mg)
|
% Recovery
|
|
|
8
|
80
|
14.60
|
101.4
|
|
80%
|
8
|
80
|
14.58
|
101.3
|
|
|
8
|
80
|
14.25
|
99.01
|
|
|
8
|
100
|
16.24
|
101.5
|
|
100%
|
8
|
100
|
16.25
|
101.6
|
|
|
8
|
100
|
16.14
|
101.9
|
|
|
8
|
120
|
17.82
|
101.3
|
|
120%
|
8
|
120
|
17.91
|
101.8
|
|
|
8
|
120
|
17.6
|
100
|
Table 11: Statistical validation of recovery study data
of Flavoxate
|
Level of
Recovery
|
% Mean
Recovery*
|
SD*
|
% RSD
|
|
80%
|
100.57
|
1.351
|
1.34
|
|
100%
|
101.33
|
0.378
|
0.373
|
|
120%
|
101.03
|
0.929
|
0.919
|
*Average of three determinants
Robustness:
Table 12: Robustness data of Flavoxate
|
Sr. no.
|
Concentration (”g/ml)
|
241 nm
|
242 nm
|
243 nm
|
|
1
|
8.0 ”g/ml
|
0.335
|
0.336
|
0.334
|
|
2
|
0.337
|
0.338
|
0.339
|
|
3
|
0.335
|
0.336
|
0.337
|
|
4
|
0.336
|
0.336
|
0.335
|
|
5
|
0.337
|
0.337
|
0.335
|
|
6
|
0.336
|
0.337
|
0.338
|
|
Mean
|
0.336
|
0.336
|
0.336
|
|
SD
|
0.0008
|
0.0008
|
0.001
|
|
% RSD
|
0.26
|
0.24
|
0.58
|
Force degradation studies:
Table 13: Force degradation study data
|
Sr. No.
|
Conditions
|
% Degradation
|
% Assay
|
|
1.
|
Acid hydrolysis
(0.1N HCl, room
temp, 24 hrs)
|
79.06
|
21.04
|
|
2.
|
Base hydrolysis
(0.1N NaOH, room
temp, 24 hrs)
|
51.88
|
48.36
|
|
3.
|
Oxidative
degradation
(3% H2O2,
room temp, 24 hr)
|
56.49
|
43.80
|
|
4.
|
Photolytic
degradation
(UV-radiation,
room temp,6hrs)
|
4.86
|
95.62
|
|
5.
|
Thermal
degradation
(60șC,room temp,
24 hrs)
|
10.92
|
89.53
|
|
6.
|
Sunlight
degradation
( keep under sunlight,6
hr )
|
2.83
|
98.12
|
Table 14: Summary of validation data of proposed UV
method
|
Parameters
|
Flavoxate
|
|
Wavelength(λmax)
|
211 nm
|
|
Linearity range
|
1-9 ”g/ml
|
|
Regression
equation
|
y = 0.111x+0.0092
|
|
Regression
coefficient (r2)
|
0.9991
|
|
Precision
|
Repeatability
|
100.09 ± 0.64
|
|
Intra-day
|
100.09 ± 0.75
|
|
Inter-day
|
98.91 ± 0.75
|
|
Accuracy
(% recovery)
|
80% ± %RSD
|
100.20± 0.26
|
|
100% ± %RSD
|
100.13 ± 0.16
|
|
120% ± %RSD
|
100.11 ± 0.21
|
|
Limit of
Detection (”g/ml)
|
0.719
|
CONCLUSION:
The UV-Spectrophotometric stability indicating assay
method was developed and validated as per ICH guidelines and suitable for estimation of Flavoxate in bulk and tablet dosage form. The developed method wassimple,
sensitive, accurate, precise and also economic in time as well as compared to
chromatographic method. Recovery
studies were showed that there is no interference of excipients. Forced degradation studies were carried for
Flavoxate which can prove the
extent of degradation in chosen experimental conditions. Stability study showed
that need to prevent drug from acidic, basis and oxidative conditions for the
safety of the drug. Hence,
these methods can be used in quality control and routine analysis of the
finished product.
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DOI: 10.52711/2231-5675.2022.00002