CHAPTER - 4
SIMULTANEOUS DETERMINATION OF HALOBETASOL PROPIONATE
AND
SALICYLIC
ACID
RELATED
SUBSTANCES
IN
OINTMENT
FORMULATION AND IDENTIFICATION OF IMPURITIES.
4.1
OBJECTIVE
To develop a simultaneous related substances determination method
for Halobetasol propionate and Salicylic acid in Ointment formulation
and identification of impurities.
4.2
INTRODUCTION
Salicylic acid [182, 183] is a beta hydroxy acid with the formula C6H4
(OH) CO2H, where the OH group is adjacent to the carboxyl group. This
is colourless crystalline organic acid, widely used in organic synthesis
and function as a plant hormone. It is highly soluble in water. Molecular
weight is 138.12, melting point is 1580C, boiling point is 2110C, specific
gravity is 1.44, and solubility in water is slight. Solvent solubility found
to be soluble in alcohol, ether, boiling water, sparingly soluble in
chloroform, and the pH exhibits as 2.4, Stable under normal conditions,
light sensitive.
The literature survey reveals that there are no methods reported for
simultaneous determination of Halobetsol propionate and Salicylic acid
impurities by high performance liquid chromatography. There are very
134
few methods reported for determination of Halobetasol propionate and
Salicylic acid impurities individually and with other components in
human serum by high performance liquid chromatography.
4.3
LITERATURE REVIEW
Covered in Chaper 4.0 and Chapter 7.0
4.4
THEORETICAL ANALYSIS
Halobetasol propionate is soluble in acetone, acetonitrile and
sparingly soluble in methanol and insoluble in water, chemically
halobetasol propionate is 21-chloro-6α, 9-difluoro-11β, 17-dihydroxy16β-methylpregna-1,
4-diene-3-20-dione,
17-propionate,
chemical
formula is C25H31ClF2O5. It has the following structural formula:
Figure 4.01
Halobetasol Propionate Structural Formula
Acid and base functional groups are not present in Halobetasol
propionate structure so buffers are not required for Halobetasol
propionate elution. Based on solubility properties, it required strong
135
organic solvent in mobile phase for elution. Acetonitrile may be preferred
organic modifier in mobile phase.
Salicylic acid is soluble in alcohol, ether, boiling water, sparingly
soluble in chloroform. Molecular formula is C7H6O3, molar mass is
-3
138.12 g mol−1, and Salicylic acid Ka and pKa are 1.06 x 10 and 2.97
Figure 4.02
Salicylic acid Structural Formula
Acid functional group has present in salicylic acid structure, so
buffers are required for salicylic acid elution. Based on solubility
properties it required organic modifier and buffer in mobile phase. Since
proposed method is simultaneous determination method, based on
molecular weight and solubility properties reverse phase chromatography
with ODS column is suitable for both compounds elution. Halobetasol
propionate and salicylic acid are having UV absorption.
Cream formulation: Soluble in tetrahydrofuran, partially soluble in
acetonitrile and water.
136
4.5
EXPERIMENTAL INVESTIGATIONS
4.5.1 Experiment No. 1
Ortho- phosphoric acid (GR grade), acetonitrile HPLC grade, water
(Milli Q) chemicals were used for experiments.
The mobile phase was acetonitrile and a solution of 50 mM potassium
dihydrogen phosphate buffer adjusted pH to 3.0 with 10% solution of
phosphoric acid, (90:10; v/v). Mobile phase was filtered through 0.45 μ
membrane filter. The analytical column, inertsil ODS-3V, 250 mm x 4.6
mm, 5 µ maintained temperature at 25 °C. The mobile phase flow rate
was maintained at 1.5 ml/min. Standard Halobetasol dipropionate and
salicylic acid solution was prepared at concentration, 100 μg/mL of
Halobetasol dipropionate and 6000 μg/mL of salicylic acid in mobile
phase. 20 μL standard solutions were injected two times and average
detector response measured at 210 nm. Chromatograms evaluated with
respect to retention time, resolution and peak shape.
Both the peaks eluted in 10 minutes and salicylic acid eluted in void
volume and next experiment carried with dicresed volume of acetonitrile.
4.5.2 Experiment No. 2
The mobile phase was acetonitrile and a solution of 50 mM potassium
dihydrogen phosphate buffer adjusted pH to 3.0 with 10% solution of
phosphoric acid, (90:10; v/v). Mobile phase was filtered through 0.45 μ
137
membrane filter. The analytical column, an inertsil ODS-3V, 250 mm x
4.6 mm, 5 µ maintained temperature at 25 °C. The mobile phase flow
rate was maintained at 1.5 ml/min. Standard Halobetasol dipropionate
and salicylic acid solution was prepared at concentration, 100 μg/mL of
Halobetasol dipropionate and 6000 μg/mL of salicylic acid in mobile
phase. 20 μL standard solutions were injected two times and average
detector response measured at 210 nm. Chromatograms evaluated with
respect to retention time, resolution and peak shape.
Salicylic acid peaks eluted in 2.5 minutes and halobetasol propionate
at 30 minutes, tailing of salicylic acid peak observed was 1.8 and next
experiment carried with chane in the column Zorbax ODS.
4.5.3 Experiment No. 3
The mobile phase was acetonitrile and a solution of 50 mM potassium
dihydrogen phosphate buffer adjusted pH to 3.0 with 10% solution of
phosphoric acid, (90:10; v/v). Mobile phase was filtered through 0.45 μ
membrane filter. The analytical column, inertsil ODS-3V, 250 mm x 4.6
mm, 5 µ maintained temperature at 25 °C. The mobile phase flow rate
was maintained at 1.5 ml/min. Standard Halobetasol dipropionate and
salicylic acid solution was prepared at concentration, 100 μg/mL of
Halobetasol dipropionate and 6000 μg/mL of salicylic acid in mobile
phase. 20 μL standard solutions were injected two times and average
138
detector response measured at 210 nm. Chromatograms evaluated with
respect to retention time, resolution and peak shape.
Salicylic acid peak eluted in 3.7 minutes and halobetasol propionate
at 9.4 minutes, both salicylic acid and Halobetasol propionate peak
shape was not satisfactory and next experiment carried with inertsil C-8
column.
4.5.4 Experiment No. 4
The mobile phase was acetonitrile and a solution of 50 mM potassium
dihydrogen phosphate buffer adjusted pH to 3.0 with 10% solution of
phosphoric acid, (90:10; v/v). Mobile phase was filtered through 0.45 μ
membrane filter. The analytical column, an inertsil ODS-3V, 250 mm x
4.6 mm, 5 µ maintained temperature at 25 °C. The mobile phase flow
rate was maintained at 1.5 mL/min. Standard Halobetasol dipropionate
and salicylic acid solution was prepared at concentration, 100 μg/mL of
Halobetasol dipropionate and 6000 μg/mL of salicylic acid in mobile
phase. 20 μL standard solutions were injected two times and average
detector response measured at 210 nm. Chromatograms evaluated with
respect to retention time, resolution and peak shape.
Salicylic acid peak eluted in 4.6 minutes and halobetasol propionate
at 11.8 minutes, tailing factor for both the peaks were 1.6 and 1.25
respectively, theoretical plates were found 3352 and 5828 respectively
139
and next experiment carried with chane in the column Zorbax SB
Phenyl, 250 x 4.6 mm, 5 µ.
4.5.5 Experiment No. 5
The mobile phase was acetonitrile and 0.1% orthophosphoric acid.
The analytical column, an inertsil C-8, 250 mm x 4.6 mm, 5 µ
maintained temperature at 40 °C. The mobile phase flow rate was
maintained
at
1.5
mL/min.
in
gradient.
Standard
Halobetasol
dipropionate and salicylic acid solution was prepared at concentration,
100 μg/mL of Halobetasol dipropionate and 6000 μg/mL of salicylic acid
in mobile phase. 20 μL standard solutions were injected two times and
average detector response measured at 210 nm. Chromatograms
evaluated with respect to retention time, resolution and peak shape.
Salicylic acid peak eluted in 4.6 minutes and halobetasol propionate
at 11.8 minutes, and all system suitability parameters are satisfactory
with above analytical conditions.
140
4.5.6 Experiment No. 6
Standard Preparation:
Standard Halobetasol propionate preparation (Stock solution -A):
Weigh accurately about 25mg of Halobetasol propionate, transfer to
100ml volumetric flask. Add about 60 ml of tetrahydrofuran, sonicate to
dissolve, allow cooling at room temperature. Make up volume with the
same. (Concentration of Halobetasol propionate is 250 ppm)
Mix standard preparation:
Weigh accurately about 75mg of Salicylic acid, transfer to 50ml
volumetric flask. Add about 20 ml of tetrahydrofuran, sonicate to
dissolve, add 5.0ml of stock solution A. Make up to volume with
tetrahydrofuran and mix well. 5 ml of above mix stock solution make up
to 50 ml with diluent Filter through 0.45µ Teflon filter, discarding first
few milliliters. (Concentration of Halobetasol propionate and salicylic acid
is 2.5 ppm and 150ppm respectively)
Sample Preparation:
Placed 5 g of sample in a 50 ml volumetric flask, added 25 ml of
diluent and kept on water bath at 80 C for 15-20 min and cooled to room
temperature. Mixed the solution well and chilled the sample in ice bath
and filtered through 0.45 u Teflon filter.
4.5.7 Experiment No. 7 (Method Validation)
141
Specificity:
Diluent, standard solution and sample solution of Halobetasol and
Salicylic acid ointment were prepared and injected into the HPLC as per
methology given in Experimental Results by using a photodiode detector.
A placebo solution of Halobetasol and Salicylic acid ointment was
prepared and injected into the HPLC along with selectivity solution as per
methology given in Experimental results by using a photodiode array
detector.
The accelerated degradation conditions applied were: UV light,
temperature, humidity, oxidant media, acid hydrolysis and alkaline
hydrolysis. Sample were analysed against a freshly prepared control
sample (with no degradation treatment). The peak purity was determined
using the tools of the Waters software. Excipient solutions were
submitted to the same degradation conditions in order to demonstrate no
interference. Specific details of the experiments conditions are described
below:
Effect of UV light:
1 ml of a solution containing 1 mg/mL of Halobetasol and 60 mg/ml
of Salicylic acid in methanol was placed in a closed 1 cm quartz cell. The
cells were exposed to a UV chamber (100 x 18 x 17 cm) with internal
mirrors and UV fluorescent lamp CRS F30W T8 emitting radiation at 254
142
nm for 15, 30, 60, 120 and 180 minutes. The same procedure was
realized for preparation for LC analysis. Protected samples, wrapped in
aluminum foil (in order to perotect from light) were submitted
simultaneously to identical conditions and used as control. After the
degradation treatment, the samples were diluted to 100 μg/ml of
Halobetasol and 6000 μg/ml of Salicylic acid with a mixture of
acetonitrile:methanol:water (6:3:1; v/v/v) and immediately analyzed.
Effect of Temperature (60 °C/24 h):
5 ml of a solution containing 1 mg/mL of Halobetasol and 60 mg/ml
of Salicylic acid in methanol was placed in a 10 ml volumetric flask at 60
°C/24 h for 24 h. After the degradation treatment, the samples were
diluted to 100 μg/ml of Halobetasol and 6000 μg/ml of Salicylic acid
with a mixture of acetonitrile:methanol:water (6:3:1; v/v/v) and analysed
immediately.
Effect of Humidity (25 °C/92% RH for 24 h):
5 ml of a solution containing 1 mg/mL of Halobetasol and 60 mg/ml
of Salicylic acid in methanol was placed in a 10 ml volumetric flask at 25
°C/92% RH for 24 h for 24 h. After the degradation treatment, the
samples were diluted to 100 μg/ml of Halobetasol and 6000 μg/ml of
Salicylic acid with a mixture of acetonitrile:methanol:water (6:3:1; v/v/v)
and analysed immediately.
143
Effect of Oxidation:
Halobetasol and Salicylid standards were dissolved in methanol, this
solution contains 1 mg/mL of Halobetasol and 60 mg/ml of Salicylic acid
(5 ml of this solution were transferred to a volumetric flask, where
hydrogen peroxide solution (30%) was added until the final concentration
of 10 % and the volume was completed with methanol. After 20 hours
the solution was diluted until the final concentration 100 μg/mL, filtered
and analysed. Similar procedure was realized for the commercial
ointment, when 25 mlof the initial solution 6000 μg/ml of Halobetasol
and Salicylid, obtained as described in sample preparation for LC
analysis, were transferred to a volumetric flask and submitted to
degradation. A control solution containing the excipients was prepared
under the same circumstances of the commercial cream.
Effect of Acid and Alkaline Hydrolysis:
5 ml of the Halobetasol and Salicylid reference standard solution
were transferred to a volumetric flask and HCl (acid degradation) or
NaOH (alkaline degradation) was added until the final concentration of
1M in both cases. After 5 hours (basic degradation) and 1 and 6 days
(acid degradation), one aliquot of the solution was neutralized HCl 1M
(alkaline degradation) or with NaOH 1M (acid degradation) and diluted
with acetonitrile, methanol and water (6:3:1, v/v/v) until the final
concentration 100 μg/ml of Halobetasol and 6000 μg/ml of salicylic acid.
144
Similar procedure was realized with the commercial cream, when 25 ml
of the initial solution 100 μg/ml of Halobetasol and 6000 μg/ml of
Salicylic acid (obtained as described in sample preparation for LC
analysis) were transferred to a volumetric flask and submitted to the
degradation. A control solution containing the excipients was prepared
under the samecircumstances of the ointment.
LOD and LOQ:
The qualification and detection limits were obtained based on signalto-noise approach. The background noise was obtained after injection of
the blank, observed over a distance equal to 20 times the width at halfheight of the peak in a chromatogram obtained by the injection 0.5
μg/mL of each reference standards. The signal-to-noise ratio applied was
10:1 for the LOQ and 3:1 for LOD. The results were verified
experimentally.
Based on the determination of prediction linearity and visual
observation of known impurities, six replicate injections were made for
LOD & LOQ.
145
Linearity and Range:
To
test
linearity,
standard
plots
were
construted
with
six
concentrations in the range of 0.03-3.04 μg/mL of Halobetasol, 0.033.02
μg/mL
of
Diflorasone
21
propionate,
0.03-3.02
μg/mL
of
Diflorasone 17 propionate 21 MSC, 0.03-3.02 μg/mL of Halobetasol
propionate in triplicates. The linearity was evaluated by linear regression
analysis that was calculated by the least square regression.
Accuracy:
The accuracy was determined by the recovery of known amounts of
known impurities to the Sample in the beginning of the preparative
process. The added levels were LOQ, 50, 100 and 150% of the specified
limit in triplicate and then proceed with sample preparation as described
under experimental result.
Precision
Six replicate injections of the standard preparation were made into
the HPLC used the methodology given in experimental result.
Six spiked sample preparations and one control sample preparation of
Halobetasol and Salicylic acid ointment were prepared and injected into
the HPLC using the method as described under experimental result.
146
Ruggedness
Six spiked sample preparations and one control sample preparations
of Halobetasol and Salicylic acid ointment were analysed by a different
analyst, using different column, on different day and injected into a
different HPLC using the method as described in experimental result,
along with standard preparation.
Robustness
Standard preparation, diluent, placebo preparation and sample
preparation in triplicate of the sample of Halobetasol and Salicylic acid
ointment were prepared as described in experimental result. The samples
along
with
standard
and
placebo
were
injected
under
different
chromatographic conditions as shown below.
Stability of Analytical Solution
Standard solution, Sample solution were analysed initially and at
different time intervals at room temperature. The system suitability was
verified through the evaluation of the obtained parameters for the
standard elution, such as theoretical plates, peak asymmetry and
retention factor, verified in different days of the method validation.
147
4.6
EXPERIMENTAL RESULTS
Standard Halobetasol propionate preparation (Stock solution -A):
Weigh accurately about 25mg of Halobetasol propionate, transfer to
100ml volumetric flask. Add about 60 ml of tetrahydrofuran, sonicate to
dissolve, allow cooling at room temperature. Make up volume with the
same. (Concentration of Halobetasol propionate is 250 ppm)
Mix standard preparation:
Weigh accurately about 75mg of Salicylic acid, transfer to 50ml
volumetric flask. Add about 20 ml of tetrahydrofuran, sonicate to
dissolve, add 5.0ml of stock solution A. Make up to volume with
tetrahydrofuran and mix well. 5 ml of above mix stock solution make up
to 50 ml with diluent Filter through 0.45µ Teflon filter, discarding first
few milliliters. (Concentration of Halobetasol propionate and salicylic acid
is 2.5 ppm and 150ppm respectively)
On the basis of Halobetasol propionate and Salicylic acid development
experiments, we can conclude that the RP-HPLC methods developed for
simultaneous determination of Halobetasol propionate and Salicylic acid
and their impurities can be used for routine analysis.
Analytical methodology used in related substances:
148
Ortho-phosphoric acid, AR grade, Acetonitrile, HPLC grade, Methanol,
HPLC grade and Water, (Double distilled) Reagents and chemicals were
used for experiment.
Table 4.01
Chromatographic Conditions
Column
Column Oven Temp
Wavelength
Flow Rate
Injection Volume
Run Time
Mobile phase
:
:
:
:
:
:
:
Inertsil, C-8, 250X4.6mm, 5µ.
25°C
231 nm.
1.5 mL / min
50 µL.
40 min.
Buffer : Acetonitrile (50:50) v/v
0.1% ortho phosphoric acid in water was used as buffer and buffer,
acetonitrile and methanol in the ratio of 65:25:10; v/v/v was used as
diluent. Prepared impurity stock standard solution each concentration of
4-hydroxy benzoic acid 600ppm, 4-hydroxy isophthalic acid 300ppm,
Phenol
10ppm,
120ppm,
Salicylic
acid
300ppm,
Diflorasone-21-propionate
Diflorasone-17-propionate-21mesylate
10ppm,
Halobetasol
propionate 10ppm standard solution was prepared by diluting10 ml of
Halobetasol propionate stock solution and 2 ml of 4-hydroxy benzoic
acid, 4-hydroxy isophthalic acid, Phenol , Salicylic acid
related
compounds stock solutions to 100 ml with diluent. (Concentration of 4hydroxy benzoic acid 12ppm, 4-hydroxy isophthalic acid 6ppm, Phenol
2.4ppm,
Salicylic
acid
6ppm,
Diflorasone-
21-propionate
1ppm,
Diflorasone-17 –propionate-21mesylate 1ppm, Halobetasol propionate
149
1ppm) Placebo solution was prepared by dissolving 5 grams placebo in
25 ml diluent at 80 C for 20 min and diluted to 50 ml with diluent.
Chilled this solution and filtered through 0.45 µ Teflon filter. Sample
solution was prepared by dissolving 5 grams sample in 25 ml diluent at
80 C for 20 min and diluted to 50 ml with diluent. Chilled this solution
and filtered through 0.45 µ Teflon filter.
Applied chromatographic conditions given in table and calculated
percent impurities by external standard method. Retention time obtained
for 4-hydroxy benzoic acid was 3.1 min, 4-hydroxy isophthalic acid was
4.6 min, Phenol was 5.0 min, Salicylic acid was 8.3 min, Diflorasone 21
propionate was 20.2 min, Diflorasone 17 propionate 21mesylate was
22.7 min and Halobetasol propionate was 23.9 min. And theoretical
plates obtained are more than 2000, tailing factor less than 2.0 and
percent relative standard deviation less than 2.0 %.
4.7
DISCUSSION OF RESULTS
LOD and LOQ: RSD is less than 33% at LOD level and less than 10%
at LOQ level for Halobetasol, Salicylic acid and known impurities.
Linearity and range: the correlation coefficients are less than 0.9995
for Halobetasol, Salicylic acid and known Impurities.
150
Precision: system precision RSD is less than 5% and method
precision RSD is less than 10% for Halobetasol, Salicylic acid and known
impurities.
Accuracy: the mean recoveries for Halobetasol, Salicylic acid and
known impurities are within 90 -110 %.
Specificity: Retention time of Halobetasol, Salicylic acid peaks and
known peaks in sample preparation is comparable with respect to
retention time of Halobetasol and Salicylic acid and known impurities
peaks in standard preparation. Peak purity passes for Halobetasol,
Salicylic acid and known impurities peaksin standard and sample
preparations. No intereference was observed at the retention time of
Halobetasol, Salicylic acid and known impurities peaks. Peak purity
passes for all degradation conditions.
Ruggesness: the RSD of twelve results obtained from two different
analysts are within 10 %.
Robustness: Halobetasol, Salicylic acid and all known impurities
peaks were resolved with each other and system suitability complies for
all variable conditions, the test method is robust for all variable
conditions.
Stability in analytical solution: Standard and sample solutions are
stable for 12 h at room temperature
151
System suitability: Theoretical plates are less than 2000, tailing factor
is less than 2.0 and relative standard deviation is less than 5.0 for six
standard replicate injections.
Table 4.02
Result table for peak purity
Sr. No.
Name
3
Halobetasol propionate in standard
solution
Halobetasol propionate in sample
solution
Salicylic acid in standard solution
4
Salicylic acid in sample solution
1
2
Purity
Criteria
Pass
Pass
Pass
Pass
Table 4.03
Forced degradation of Salicylic acid in the sample
% Degradation
44-Hydroxy Phenol
Impurity Impurity
Hydroxy
isophthalic
at RRT
at RRT
benzoic
acid
1.4
1.54
acid
Peroxide
0.006
0.002
0.000
0.011
0.003
degradation
Acid
0.000
0.008
0.000
0.013
0.005
degradation
Base
0.007
0.000
0.000
0.000
0.000
degradation
Thermal
0.000
0.000
0.000
0.011
0.003
degradation
Photo
0.000
0.000
0.000
0.011
0.003
degradation
Humidity
0.000
0.000
0.000
0.011
0.003
degradation
152
Table 4.04
Forced degradation of Halobetasol propionate in the sample
Peroxide
degradation
Acid
degradation
Base
degradation
Thermal
degradation
Photo
degradation
Humidity
degradation
% Degradation
Diflorasone 21 Diflorasone 17 Impurity
propionate
propionate 21 at RRT
mesylate
1.02
0.575
0.00
.0.184
0.392
0.00
1.789
22.03
0.00
0.00
0.091
0.00
0.186
0.080
0.00
0.182
0.086
0.00
0.177
Table 4.05
Total all known impurities recovery at LOQ
% RECOVERY
4
4 Hydroxy Phenol
Hydroxy Isophthalic
benzoic acid
acid
Level
Diflorasone Diflorasone
21
17
propionate propionate
21
mesylate
LOQ Spl-1
104.9
97.3
103.5
97.0
104.4
LOQ Spl-2
96.7
96.8
101.2
92.0
95.0
LOQ Spl-3
103.6
95.5
103.9
96.4
101.1
Mean
101.7
96.5
102.9
95.1
100.2
S.D
4.4
0.92
1.45
2.73
4.76
%RSD
4.33
0.96
1.41
2.87
4.76
153
Table 4.06
Total all known impurities recovery at 50, 100 and 150% level
% RECOVERY
4
4 Hydroxy Phenol
Hydroxy Isophthalic
benzoic acid
acid
Level
Diflorasone Diflorasone
21
17
propionate propionate
21
mesylate
50 Spl-1
101.5
105.4
101.4
100.0
93.9
50 Spl-2
102.0
105.7
101.1
100.8
94.8
50 Spl-3
101.8
105.3
101.6
100.0
93.9
100 Spl-1
98.3
99.4
96.0
100.4
97.8
100 Spl-2
98.0
98.8
95.6
99.6
97.0
100 Spl-3
97.8
98.3
95.9
99.2
96.1
150 Spl-1
98.0
98.2
97.6
98.7
98.0
150 Spl-2
97.9
98.2
97.3
98.7
98.0
150 Spl-3
98.1
98.3
97.3
98.9
98.3
Mean
99.3
100.8
98.2
99.6
96.4
S.D
1.88
3.48
2.47
0.76
1.81
%RSD
1.89
3.46
2.52
0.76
1.87
154
Table 4.07
System Precision Data of Halobetasol, salicylic acid and Impurities
4
Hydroxy
benzoic
acid
4 Hydroxy
isophthalic
acid
Phenol Salicylic Halobetasol
acid
propionate
Replicate-1
589795
879152
20656
589104
62117
Replicate-2
589866
880867
20977
589789
62431
Replicate-3
590643
881337
21399
590351
62111
Replicate-4
592426
885240
21436
590325
62819
Replicate-5
589396
873347
19845
588328
62779
Replicate-6
590293
874352
21215
588468
62453
MEAN
590403
879049
20921
589394
62452
S.D
1080.21
4504.53
601.88
896.28
306.78
%RSD
0.18
0.51
2.88
0.15
0.49
155
Table 4.08
Method Pecision Data of Halobetasol Impurities in %
Diflorasone Diflorasone Impurity Total
21
17
at RRT
impurities
propionate propionate 1.02
21
mesylate
Spl. 1
1.002
0.964
0.185
2.151
Spl. 2
1.007
0.944
0.159
2.11
Spl. 3
1.007
0.942
0.174
2.123
Spl. 4
0.991
0.937
0.171
2.099
Spl. 5
0.987
0.939
0.159
2.085
Spl. 6
0.989
0.939
0.173
2.101
Ave.
0.997
0.944
0.17
2.112
S.D
0.009
0.01
0.01
0.023
%RSD
0.924
1.062
5.834
1.093
Table 4.09
156
Intermediate Precision Data of Salicylic Acid, Halobetasol and
Impurities in %
4
Hydroxy
benzoic
acid
4 Hydroxy
isophthalic
acid
Spl. 1
0.197
0.100
0.040
0.012
0.003
0.352
Spl. 2
0.196
0.100
0.040
0.012
0.003
0.351
Spl. 3
0.195
0.100
0.039
0.012
0.003
0.349
Spl. 4
0.194
0.099
0.040
0.012
0.003
0.348
Spl. 5
0.194
0.099
0.039
0.012
0.003
0.347
Spl. 6
0.194
0.099
0.039
0.012
0.003
0.347
Ave.
0.195
0.100
0.04
0.012
0.003
0.349
0.65
0.55
1.39
0
0
0.6
%RSD
Phenol Salicylic Halobetasol Total
acid
propionate
impurities
Table 4.10
Halobetasol propionate and salicylic acid LOD and LOQ Data in
µg/ml.
Name of Compound
LOD
LOQ
4 Hydroxy Benzoic acid
0.036
0.121
4 Hydroxy isophathlic acid
0.036
0.12
Phenol
0.036
0.12
Salicylic acid
0.031
0.104
0.07
0.021
Diflorasone 17 propionate 21
mesylate
0.073
0.022
Halobetasol propionate
0.057
0.017
Diflorasone 21 propionate
157
Table 4.11
Linearity Data of Halobetasol Propionate and Impurities
Diflorasone 21
propionate
Diflorasone 17
propionate 21
mesylate
Halobetasol
propionate
Conc.
(µg/mL)
Area
Conc.
(µg/mL)
Area
Conc.
(µg/mL)
Area
Lev. LOQ
0.069
1385
0.071
1153
0.057
1765
Lev. 50%
0.493
29856
0.491
26915
0.505
32006
Lev. 80%
0.790
47911
0.785
43360
0.807
51200
Lev. 90%
0.888
54006
0.883
48813
0.908
57666
Lev. 100%
0.987
59164
0.982
53424
1.009
63099
Lev. 110%
1.086
65316
1.080
59035
1.110
69477
Lev. 120%
1.184
72435
1.178
65376
1.211
77225
Lev. 150%
1.48
90432
1.472
81048
1.514
96005
Slope
62672
57001
64395
Intercept
-2095
-2009
-1264
Correlation
coefficient
0.9996
0.9996
0.9997
Table 4.12
158
Linearity Data of Salicylic Acid and Impurities
4 Hydroxy
benzoic acid
4 hydroxy
isophthalic acid
Phenol
Salicylic acid
Level
Conc.
(µg/mL)
Area
Conc.
(µg/mL)
Area
Conc.
(µg/mL)
Area
Conc.
(µg/mL)
Area
LOQ
0.118
6415
0.12
9299
0.12
1606
0.103
6822
50%
5.911
311638
3.012
453251
1.195
14206
2.996
302869
80%
9.458
498793
4.819
726880
1.912
21961
4.793
485049
90%
10.64
561784
5.422
817451
2.151
24639
5.392
544673
100%
11.82
613916
6.024
878306
2.39
26889
5.992
596324
110%
13
678178
6.626
983895
2.629
29841
6.591
655896
120%
14.18
752253
7.229
1090428
2.868
32859
7.19
726542
150%
17.73
937076
9.036
1360643
3.586
40512
8.987
906572
Slope
52705
150761
11196
100890
Intercept
-989
-6904
481
-2136
0.9998
0.9996
0.9998
0.9998
Correlation
coefficient
Table 4.13
Solution Stability Data of Impurities
159
4 Hydroxy
benzoic
acid
4 Hydroxy Phenol Diflorasone
Isophthalic
21
acid
propionate
Diflorasone
17
propionate
21 mesylate
Initial
0.203
0.106
0.04
1.052
0.956
8 h.
0.205
0.105
0.04
1.015
0.951
12 h.
0.205
0.105
0.042
1.039
0.941
Mean
0.204
0.105
0.041
1.035
0.949
S.D
0.001
0.001
0.001
0.019
0.008
%RSD
0.565
0.548
2.83
1.81
0.805
Table 4.14
RT, RRT and RRF Data of Halobetasol impurities
Sr.No
Name of the
compound
RT
RRT
RRF
1
Halobetasol
propionate
26.8
1
1
2
Halobetasol
(Base)
9.8
0.37
1.002
3
Diflorasone 21
propionate
10.78
0.4
1.007
4
Diflorasone 17
propionate 21
mesylate
21.42
0.8
1.039
Table 4.15
RT, RRT and RRF Data of Salicylic Acid Impurities
160
Sr.No
Name of the
compound
RT
RRT
RRF
1
Salicylic acid
8.3
1
1
2
4-hydroxy benzoic
acid
3.1
0.37
0.5
3
4-hydroxy
isophthalic acid
4.6
0.55
1.49
4
Phenol
5
0.6
0.088
Figure 4.03
Diluent chromatograph for Halobetasol propionate and salicylic acid
Ointment RS
Figure 4.04
161
Placebo chromatograph for Halobetasol propionate and salicylic acid
Ointment RS
Figure 4.05
Sample chromatograph for Halobetasol propionate and salicylic acid
Ointment RS
Figure 4.06
162
Standard chromatograph for Halobetasol propionate and salicylic
acid Ointment RS
Figure 4.07
Spiked sample chromatograph for Halobetasol propionate and
salicylic acid Ointment RS
Figure 4.08
Sample chromatograph for Halobetasol propionate and salicylic acid
Ointment RS
163
4.8
Summary, Conclusion and Recommendations.
The proposed method was found to be simple, precise, accurate and
rapid for the determination of Halobetasol propionate and Salicylic acid
related substance from pure and its dosage forms, the mobile phase is
simple to prepare and economical. The sample recoveries in all the
formulations were in good agreement with their respective label claim
and their suggestive not interference of formulation excipients in the
estimation.
Hence this method can be easily and conveniently adopted for routine
analysis of Halobetasol propionate and Salicylic acid related substance in
bulk drugs and the pharmaceutical dosage forms and also for stability
analysis.
CHAPTER - 5
SIMULTANEOUS ASSAY DETERMINATION OF MEFENAMIC ACID
164