chapter - 6 simultaneous assay determination of mometasone and
TRANSCRIPT
198
CHAPTER - 6
SIMULTANEOUS ASSAY DETERMINATION OF MOMETASONE AND
TAZAROTENE IN OINTMENT FORMULATION.
6.1 OBJECTIVE
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To develop a simultaneous assay determination method of
Mometasone and Tazarotene in ointment formulation.
6.2 INTRODUCTION
Tazarotene [202] is a new generation of topical receptor selective
retinoid for the treatment of plaque psoriasis. While oral retinoid have
long been used in the treatment of psoriasis, their use has often been
limited to patients with severe psoriasis, due to the potential adverse side
effects associated with their use. This is associated with Mometasone in
ointment formulation. Mometasone [203] is a topical steroid for
dermatologicuse. It is a synthetic steroid with anti-inflammatory activity.
Mometasone reduces or inhibits the action of chemical in the body that
causes inflammation, redness and swelling. Mometasone is used for
inflammation caused by a number of conditions such as allergic
reactions, eczema and psoriasis. Mometasone furoate and tazarotene are
available in the form of ointment of strength 0.1% w/w+0.1% w/w.
Literature survey indicated that HPLC methods for the analysis of
tazarotene and mometasone ointment and pure drugs for individual
determination methods. The present investigation is an attempt to
develop a highly sensitive, simple, precise and rapid analytical method
for the simultaneous estimation of tazarotene and mometasone in
Ointment formulation.
6.3 LITERATURE REVIEW
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Pathare D.B. et al were reported a simple, isocratic, rapid and
accurate reversed phase high performance liquid chromatography
method for the quantitative determination of tazarotene. The
chromatographic separation was done on a Hypersil C18 (250 mm× 118
4.6 mm 5 μm) column using water pH 2.5 with orthophosphoric acid and
acetonitrile in the ration 15:85, v/v as a mobile phase. The
chromatographic resolutions between tazarotene and its potential
impurities A and B were found greater than 3. The limit of detection and
limit of quantification of impurities were found to be 25 and 75 ng /mL.
The developed RPLC method was validated with respect to linearity,
accuracy, precision and robustness [204]
Hecker D. et al were reported Tazarotene in combination with
phototherapy is being used clinically for the treatment of plaque
psoriasis [205].
Saleem S. et al were reported high- performance liquid
chromatographic method for the simultaneous determination of
chlorocresol (CC), mometasone furoate (MF), and fusidic acid (FA) in a
cream formulation. The mobile phase used was 1 .5% w/v of aqueous
ammonium acetate buffer– acetonitrile in the ratio 55:45 v/v and
adjusted pH 3.8. The column used was Symmetry C8, 150× 3.9 mm, 5
µm. The detection and flow rate were 240 nm and 1.0 mL/min. This
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found capable to separate chlorocresol, mometasone furoate, and fusidic
acid in less than 8 min with good resolution and peak shapes [206]
6.4 THEORETICAL ANALYSIS
Solubility and Chemical Information:
Tazarotene is sparingly soluble in acetone, very slightly soluble in
methanol and insoluble in ethanol and water
Figure 6.01
Tazarotene Structural Formula
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Systematic (IUPAC) name is ethyl 6-[2-(4,4-dimethyl-3,4-dihydro-2H-
1-benzothiopyran-6-yl)ethynyl]pyridine-3-carboxylate, formula is
C21H21NO2S and Molecular mass is 351.463 g/mol. Mometasone furoate
is soluble in acetone & in methylene chloride.
Figure 6.02
Mometasone Structural Formula
Chemical name is(9R,10S,11S,13S,14S,16R,17R)-9-chloro-17-(2-
chloroacetyl)-11-hydroxy-10,13,16-trimethyl-3-oxo-6, 7, 8, 9, 10, 11, 12,
13, 14, 15, 16, 17- dodecahydro-3H-cyclopenta[a]phenanthren-17-yl
furan-2-carboxylate, mometasone Furoate is C49H58Cl4O10 C27H30O6Cl2
and Molecular mass is 948.761 g/mol.
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Ointment formulation: Ointment is an emulsion and it will easily
break in tetrahydrofuran, miscible with water and acetonitrile and
methanol.
Scan for UV absorption:
Tazarotene and Mometasone are having UV absorption and the
UVspectras are attached.
6.5 EXPERIMENTAL INVESTIGATIONS
6.5.1 Experiment No.1
The mobile phase was acetonitrile and a solution of 50 mM
ammonium acetate Buffer solution buffer adjusted pH to 3.0 with 10%
solution of phosphoric acid, (60:40; v/v). Mobile phase was filtered
through 0.45 μ membrane filter. Column equipped with instrument was
Inertsil ODS-3V, 250 mm x 4.6 mm, 5 µ and maintained temperature 25
°C. The mobile phase flow rate was maintained at 1.5 ml/min. Standard
tazarotene and mometasone solution was prepared at concentration,
each 20 μg/mL of tazarotene and mometasone in mobile phase. 20 μL
standard solutions were injected two times and average detector
response measured at 254 nm. Chromatograms evaluated with respect to
retention time, resolution and peak shape.
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Both Mometasone and tazarotene peaks were not eluted in 30
minutes, next experiment carried with changing buffer in mobile phase
composition.
6.5.2 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, (80:20; v/v). Mobile phase was filtered through 0.45 μ
membrane filter. Column equipped with instrument was Inertsil ODS-3V,
250 mm x 4.6 mm, 5 µ and maintained temperature 25 °C. The mobile
phase flow rate was maintained at 1.5 ml/min. Standard tazarotene and
mometasone solution was prepared at concentration, each 20 μg/mL of
tazarotene and mometasone in mobile phase. 20 μL standard solutions
were injected two times and average detector response measured at 254
nm. Chromatograms evaluated with respect to retention time, resolution
and peak shape.
Both tazarotene and mometasone peaks were eluted within 40
minutes. Since the run time was more and peak shape found was not
satisfactory, next experiment carried with changing mobile phase
composition.
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6.5.3 Experiment No. 3
The mobile phase was acetonitrile and a solution of 50 mM potassium
dihydrogen phosphate buffer, adjusted pH to 6.8 with 10% solution of
phosphoric acid, (80:20; v/v). Mobile phase was filtered through 0.45 μ
membrane filter. Column equipped with instrument was Inertsil ODS-3V,
250 mm x 4.6 mm, 5 µ and maintained temperature 25 °C. The mobile
phase flow rate was maintained at 1.5 ml/min. Standard Tazarotene and
mometasone solution was prepared at concentration, each 20 μg/mL of
tazarotene and mometasone in mobile phase. 20 μL standard solutions
were injected two times and average detector response measured at 254
nm. Chromatograms evaluated with respect to retention time, resolution
and peak shape.
Both Tazarotene and mometasone peaks were eluted in 30 minutes.
Since the run time was more and peak shape found was not satisfactory,
next experiment carried with changing mobile phase buffer and
composition.
6.5.4 Experiment No. 4
The mobile phase was acetonitrile and a solution of 50 mM potassium
dihydrogen phosphate buffer adjusted pH to 6.8 with 10% solution of
phosphoric acid, tetrahydrofuran (75:20:05; v/v). Mobile phase was
filtered through 0.45 μ membrane filter. Column equipped with
instrument was Inertsil ODS-3V, 250 mm x 4.6 mm, 5 µ and maintained
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tempewrature 25 °C. The mobile phase flow rate was maintained at 1.5
ml/min. Standard tazarotene and mometasone solution was prepared at
concentration, each 20 μg/mL of tazarotene and mometasone in mobile
phase. 20 μL standard solutions were injected two times and average
detector response measured at 254 nm. Chromatograms evaluated with
respect to retention time, resolution and peak shape.
Both Tazarotene and Mometasone peaks were eluted in 30 minutes.
Since the run time was more and peak shape found was not satisfactory,
next experiment carried with changing mobile phase composition.
6.5.5 Experiment No. 5
The mobile phase was acetonitrile and a solution of 50 mM potassium
dihydrogen phosphate buffer adjusted pH to 6.8 with 10% solution of
phosphoric acid, tetrahydrofuran (50:45:5; v/v). Mobile phase was
filtered through 0.45 μ membrane filter. Column equipped with
instrument was Inertsil ODS-3V, 250 mm x 4.6 mm, 5 µ was maintained
temperature 25 °C. The mobile phase flow rate was maintained at 1.5
ml/min. Standard tazarotene and mometasone solution was prepared at
concentration, each 20 μg/mL of tazarotene and mometasone in mobile
phase. 20 μL standard solutions were injected two times and average
detector response measured at 254 nm. Chromatograms evaluated with
respect to retention time, resolution and peak shape.
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Both Tazarotene and mometasone were eluted in 30 minutes. Since
the run time was more and peak shape found was not satisfactory, next
experiment carried with changing mobile phase composition.
6.5.6 Experiment No. 6
The final selected mobile phase was acetonitrile, a solution of 50mM
potassium dihydrogen phosphate buffer adjusted pH to 6.8 with 10%
solution of phosphoric acid, acetonitrile and tetrahydrofuron (30:60:10;
v/v/v). Mobile phase was filtered through 0.45 μ membrane filter. The
mobile phase flow rate was maintained at 1.5 mL/min. Standard
Tazarotene and mometasone solution was prepared at concentration,
each 20 μg/mL of tazarotene and mometasone in mobile phase. 20 μL
standard solutions were injected two times and average detector
response measured at 254 nm. Chromatograms evaluated with respect to
retention time, resolution and peak shape.
6.5.7 Experiment No.7 (Method Validation)
Specificity:
Two types of specificity experiments were performed. In the first one,
specificity assessed by comparing the chromatograms obtained from the
pharmaceutical preparation and the standard solution with those
obtained from excipients which take part in the commercial ointment
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and verifying the absence of interferences. In the second type, forced
degradation performed in order to check the suitability of analytical
conditions for stability study of mometasone and tazarotene. 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 0.2 mg/mL of each mometasone and
tazarotene in acetonitrile was placed in a closed 1 cm quartz cell. The cell
was 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 nm for
15, 30, 60, 120 and 180 minutes. The same procedure was realized for
preparation for LC analysis. Samples, protected in aluminum foil (in
order to perotect from light) were submitted simultaneously to similar
conditions and used as control. After the degradation treatment, the
samples were diluted to 20 μg/ml of each mometasone and tazarotene
with a mixture of acetonitrile, water and tetrahydrofuran (30:60:10,
v/v/v) and immediately analyzed.
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Effect of Oxidation:
Mometasone and tazarotene standards were dissolved in acetonitrile
(0.2 mg/mL of each tazarotene and mometasone), 5 ml of this solution
was transferred to a volumetric flask, where hydrogen peroxide solution
(30%) was added until the final concentration of 10 % and the volume
was completed with acetonitrile. After 20 hours the solution was diluted
until the final concentration of 20 μg/mL of each mometasone and
tazarotene diluted with acetonitrile, water and tetrahydrofuran (30:60:10,
v/v/v), filtered and analysed. Similar procedure was realized for the
ointment, when 25 ml of the initial solution 0.2 mg/mL of each
mometasone and tazarotene, 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 ointment.
Effect of Acid Hydrolysis:
5 ml of 0.2 mg/mL of each mometasone and tazarotene reference
standard solution was transferred to a volumetric flask and HCl was
added until the final concentration of 1M HCl. After 5 hours and 1 and 6
days, one aliquot of the solution was neutralized with NaOH 1M and
diluted with acetonitrile, water and tetrahydrofuran (30:60:10, v/v/v)
until the final concentration of 20 μg/ml of each tazarotene and
mometasone for LC analysis. Similar procedure was realized with the
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ointment, when 25 ml of the initial solution 0.2 mg/mL of mometasone
and tazarotene (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 same circumstances of the ointment.
Effect of Alkaline Hydrolysis:
5 ml of 0.2 mg/mL of mometasone and tazarotene reference standard
solution was transferred to a volumetric flask and NaOH (alkaline
degradation) was added until the final concentration of 1M NaOH. After 5
hours and 1 and 6 days, one aliquot of the solution was neutralized with
HCl 1M and diluted with acetonitrile, water and tetrahydrofuran
(30:60:10, v/v/v) until the final concentration of 20 μg/ml of
mometasone and tazarotene for LC analysis. Similar procedure was
realized with the ointment, when 25 ml of the initial solution 0.2 mg/mL
of mometasone and tazarotene (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 same circumstances of the ointment.
Linearity and Range:
To test linearity, standard plots were construted with six
concentrations in the range 50-150 %. The linearity was evaluated by
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linear regression analysis that was calculated by the least square
regression.
To test linearity, standard plots were constructed with six
concentrations in the range 10-30 μg/mL of each Mometasone and
tazarotene prepared in triplicates. The linearity was evaluated by the
least square regression.
Presission:
The repeatability was verified from six independent sample
preparations in the same day, obtained as described in Sample
preparation for LC analysis. The intermediate precision was tested by
assaying freshly prepared sample solutions at the concentration on two
different days. Precision was reported as %RSD.
Six replicate injections of the standard preparation were madeinto the
HPLC used the methodology given in experimental result.
Six spiked sample preparations and one control sample preparation of
Mometasone and Tazarotene ointment were prepared and injected into
the HPLC using the method as described under experimental result.
Accuracy:
The accuracy was estimated by the recovery of known amounts of
Mometasone and tazarotene standards added to the placebo in the
beginning of the preparative process. The added levels were 80, 100 and
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120% of the nominal drug concentrations. The results were expressed as
the percentage of Mometasone and tazarotene reference standards
recovered from the sample.
The accuracy was estimated by the recovery of known amounts of
Mometasone and Tazarotene to the Placebo in the beginning of the
preparation method. The added levels were 80, 100, and 120% of the
specified limit in triplicate and then proceed with sample preparation as
described under experimental result.
Ruggedness:
Six spiked sample preparations and one control sample preparations
of Mometasone and Tazarotene 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 Mometasone and Tazarotene
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:
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Standard solution, Sample solution were analysed initially and at
different time intervals at room temperature.
System Suitability:
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.
6.6 EXPERIMENTAL RESULTS
On the basis of Mometasone Furoate and Tazarotene analytical method
development experimental trials, RP-HPLC method was suitable for
simultaneous determination of Mometasone Furoate and Tazarotene
assay. Final experiment chromatographic conditions were applied
Preparation of stock solutions: Prepare solution having the concentration
of Mometasone Furoate and Tazarotene 20 ppm, in mobile phase.
Sample preparation: 2 g cream smplewas weighed and transferred
into 100 ml volumetric flask and added 60 ml of acetonitrile. Shaked
mechanically for 5 minutes to disperse the cream and kept the flask in
ultrasonic bath for 10 min with intermediate shaking, made the volume
with acetonitrile and filtered this solution through 0.45 micron
membrane filter.
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Separately injected equal volumes of diluent, standard preparation in
six replicates and sample twice in to equilibrated HPLC system and
record chromatograms and measured the response in terms of peak area.
System suitability parameters occurred during method validation were
Theoretical plates mores than 8000, tailing factor less than 1.5, relative
standard deviation for six replicates of standard solution is less than
2.0%.
6.7 DISCUSSION OF RESULTS
Linearity and range: the correlation coefficients are less than 0.9995
for Mometasone and Tazarotene.
Precision: system precision RSD is less than 2% and method
precision RSD is less than 2% for Mometasone and Tazarotene.
Accuracy: the mean recoveries for Mometasone and Tazarotene acid
are within 98 -102 %.
Specificity: Retention time of Mometasone and Tazarotene peaks in
sample preparation is comparable with respect to retention time of
Mometasone and Tazarotene peaks in standard preparation. Peak purity
passes for Mometasone and Tazarotene peaks in standard and sample
preparations. No intereference was observed at the retention time of
Mometasone and Tazarotene peaks. Peak purity passes for all
degradation conditions.
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Ruggesness: the RSD of twelve results obtained from two different
analysts are within 10 %.
Robustness: Mometasone and Tazarotene acid 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
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 6.01
Peak Purity Data of Tazarotene and mometasone Furoate
Sr. No. Name
Purity Criteria
1 Tazarotene in standard solution Pass
2 Tazarotene in sample solution Pass
3 Mometasone furoate in standard solution
Pass
4 Mometasone furoate in sample solution
Pass
Table 6.02
Recovery Data of Tazarotene by Placebo Spiked Recovery Method
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Level Amount
added(mg) Average
Area
Amount recovered
(mg) Recovery
(%)
Mean
Recovery
(%)
80 Spl-1 0.00787 253346 0.0079 100.5
101.1
80 Spl-2 0.00787 248391 0.0077 98.5
80 Spl-3 0.00787 263101 0.0082 104.4
100 Spl-1 0.00983 317643 0.0099 100.8
100.4
100 Spl-2 0.00983 316889 0.0099 100.6
120 Spl-3 0.00983 314749 0.0098 99.9
120 Spl-1 0.01180 380281 0.0119 100.6
101.2
120 Spl-2 0.01180 384231 0.0120 101.6
120 Spl-3 0.01180 383479 0.0120 101.4
Mean 100.9
S.D 0.43
RSD (%) 0.43
Table 6.03
Recovery Data of Mometasone Furoate by Placebo Spiked Recovery Method
217
Level
Amount added (mg)
Average Area
Amount recovered
(mg) Recovery
(%)
Mean recovery
(%)
80 Spl-1 0.016 584591 0.016 98.8
80 Spl-2 0.016 575011 0.016 97.1 99.3
80 Spl-3 0.016 604438 0.016 102.1
100 Spl-1 0.020 721389 0.020 97.5
100 Spl-2 0.020 732749 0.020 99.0 98.5
100 Spl-3 0.020 733289 0.020 99.1
120 Spl-1 0.024 894231 0.024 100.7
120 Spl-2 0.024 883736 0.024 99.5 100.0
120 Spl-3 0.024 885343 0.024 99.7
Mean 99.29
S.D 0.72
RSD (%) 0.73
Table 6.04
Linearity Data of Tazarotene
Level Conc. (ppm)
Experimental Area (a) Predicted
Area (y)
Residuals (b); b= a-y
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(x) y=mx+c
80% 8.02 253670 254029 -359
90% 9.02 285236 285754 -519
100% 10.02 318943 317479 1464
110% 11.02 349266 349204 61
120% 12.02 380281 380929 -649
Correlation 0.99985
Intercept (c) 229
Slope (m) 31662
Table 6.05
Linearity Data of Mometasone
Linearity Level
Conc.(ppm) Experimental
Area (a)
Predicted Area (y); y=mx+c
Residuals (b); b= a-y (x)
Level-80% 16.04 577741 577756 -14
Level-90% 18.04 649310 650038 -728
Level-100% 20.05 723620 722393 1227
Level-110% 22.05 794394 794604 -209
Level-120% 24.06 866612 866886 -274
Correlation 0.99998
Intercept (c) -506
Slope (m) 36055
Table 6.06
Precision Data of Tazarotene.
Sample Sample wt (mg)
Average Area mg/g
% Assay
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Spl-1 2088.6 334629 0.0501 100.2
Spl-2 2075.1 337115 0.0508 101.6
Spl-3 2079.6 334459 0.0503 100.6
Spl-4 2083.2 334886 0.0503 100.6
Spl-5 2087.9 335541 0.0503 100.5
Spl-6 2081.7 333742 0.0501 100.3
Average 100.65
SD 0.51
RSD 0.50
Table 6.07
Precision Data of Mometasone Furoate
Sample Sample wt (mg)
Average Area Mg/Unit
Assay (%)
Spl-1 2088.6 793774 0.1015 101.5
Spl-2 2075.1 798599 0.1028 102.8
Spl-3 2079.6 785765 0.1009 100.9
Spl-4 2083.2 798912 0.1024 102.4
Spl-5 2087.9 784798 0.1004 100.4
Spl-6 2081.7 793767 0.1018 101.8
Average 100.08
SD 0.53
RSD 0.52
Table 6.08
Intermediate Precision Data of Tazarotene.
Sample Sample wt (mg)
Average Area mg/g
% Assay
220
Spl-1 2108.6 334356 0.0496 99.2
Spl-2 2055.1 333667 0.0508 101.6
Spl-3 2039.6 329915 0.0506 101.2
Spl-4 2043.2 335200 0.0513 102.6
Spl-5 2077.9 337778 0.0508 101.7
Spl-6 2091.7 335538 0.0502 100.4
Average 101.11
SD 1.19
RSD 1.18
Table 6.09
Intermediate Precision Data of Mometasone Furoate
Sample Sample wt (mg)
Average Area Mg/Unit
% Assay
Spl-1 2108.6 794905 0.1007 100.7
Spl-2 2055.1 791391 0.1028 102.8
Spl-3 2039.6 762601 0.0999 99.9
Spl-4 2043.2 784461 0.1025 102.5
Spl-5 2077.9 791391 0.1017 101.7
Spl-6 2091.7 794101 0.1014 101.4
Average 101.51
SD 1.12
RSD 1.11
6.8 SUMMARY, CONCLUSION AND RECOMMENDATIONS
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The proposed method was found to be precise, accurate, simple and
rapid for the determination of Mometasone and Tazarotene from 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 conveniently adopted for routine analysis
of Mometasone and Tazarotene in bulk drugs and the pharmaceutical
dosage forms and also for stability analysis.
222
Figure 6.03
Diluent Chromatograph for Tazarotene and mometasone Furoate Ointment.
Figure 6.04
Standard Chromatograph for Tazarotene and mometasone Furoate Ointment.
223
Figure 6.05
Placebo Chromatograph for Tazarotene and mometasone Furoate Ointment.
Figure 6.06
Sample Chromatograph for Tazarotene and mometasone Furoate Ointment.