chapter 3 simultaneous determination of...

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CHAPTER – 3

SIMULTANEOUS DETERMINATION OF HALOBETASOL

PROPIONATE AND FUSIDIC ACID RELATED SUBSTANCES IN

CREAM FORMULATION AND IDENTIFICATION OF IMPURITIES.

3.1 OBJECTIVE

To develop a simultaneous related substances determination method

for Halobetasol propionate and Fusidic acid in cream formulation and

identification of Impurities.

3.2 INTRODUCTION

Halobetasol propionate is one of the potent corticosteroid indicated in

the relief of the inflammatory and pruritic manifestations of

corticosteroid-responsive dermatitis [164,165]. Important treatment goals

include providing moisture to the skin, preventing dryness and avoiding

prurities, as a result the occurrence of inflammation should

concomitantly decline.

Fusidic acid is bacteriostatic antibiotic used in the treat of primary

and secondary skin infections caused by sensitive strains of S.aureus,

Strepto cocci species and C. Minutissimum [167] Halobetasol [169-172]

propionate is a synthetic corticosteroid. It is having anti-inflammatory,

antipruritic and vasoconstrictor activities. Fusidic acid is the steroidal

antibiotic is used to treat the gram positive infections. It acts by

preventing translocation of peptidyl tRNA. Resistant mutants are easily

106

selected, even during therapy and therefore fusidic acid is usually

administered in combination with another antibiotic. This helps reduce

the risk of selecting resistant mutants. To survive, the fusidic acid

resistant mutants become resistant to the antibiotic given in combination

[173, 174].

3.3 LITERATURE REVIEW:

Giancarlo C. et al were reported degradation of halobetasol propionate

in the presence of bases. The cyclization product was separated by

isolation technique and fully characterized by MS, NMR and X-ray

crystallography [175].

Hikal A.H. et al were reported a new HPLC method for the assay of

sodium fusidate or fusidic acid in formulations and compared to a

microbiological assay method. It was perfomred using test organism and

agar diffusion technique with Staphylococcus aureus. With five test

levels of the standard, potencies were interpolated from standard curve

using a log transformation straight-line method with least-squares fitting

(r = 0.99+). Both methods were applied to fusidic acid in tablets, a

suspension, and an ointment. Excellent agrement was observed between

results of the two methods [176].

Krzek J. et al were reported a thin-layer chromatography

densitometric method for the identification and quantification of fusidic

acid RF=0.53, methyl hydroxybenzoate RF=0.64, propyl hydroxybenzoate

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RF=0.72, and butylated hydroxyanisol RF=0.77, which are the

components of a dermatological ointment. Silica gel 60 F254 TLC plates

were used as the stationary phase and mobile phase used was n-

hexane-ethyl acetate-glacial acetic acid in the ratio of 6:3:1, v/v/v. Spots

on chromatograms were detected by densitometric measurements at

three different wavelengths of 240 nm for fusidic acid, 260 nm for methyl

hydroxybenzoate, propyl hydroxybenzoate, and 290 nm for butylated

hydroxyanisol, used for decreased interferences and increased selectivity

of the peaks. Methdo found to be pricise, accuracte, and shown high

sensitivity [177].

Shaikh S. et al were reported a simple, specific, and precise HPLC

method for the simultaneous determination of chlorocresol, mometasone

furoate, and fusidic acid in a cream. The mobile phase used was 1.5%

w/v aqueous ammonium acetate buffer and acetonitrile, 55:45 v/v and

adjusted pH 3.8, operated at the flow rate of 1.0mL/min. The column

used was C8, 150 x 3.9 mm, 5 microm. The detection was done at 240

nm. The method resolves chlorocresol, mometasone furoate, and fusidic

acid in less than 8 min with good resolution [178].

Kok-Khiang P. et al were reported a simple and selective HPLC with

UV detection for simultaneous determination of fusidic acid and

betamethasone dipropionate in a cream formulation. Chromatographic

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separation was carried out by using C18 column and mobile phase

consisting of acetonitrile and 0.01 M disodium hydrogen orthophosphate

in the ratio 70:30, % v/v and adjusted to pH 6 with glacial acetic acid.

Analysis was done at a flow rate of 1.0 mL/min; detection was carried at

235 nm [179].

3.4 THEORETICAL ANALYSIS

Sample information:

Halobetasol propionate [180] is a molecule having chemical name as

21-chloro-6 alpha, 9-difluoro-11ß, 17-dihydroxy-16 ß-methylpregna-1,

4-diene-3-20-dione, 17-propionate, molecular formulae of C25H31ClF2O5

and molecular weight reported was 485 g/mole. Halobetasol propionate

is soluble in Acetone, Acetonitrile and sparingly Soluble in Methanol, pKa

of 7.2 at 25°C and pH in the range of about 6.5-7.9.

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Fig. 3.01

Halobetasol propionate Structural formula

Fusidic acid[181] is a molecule having chemical name as 2-(16-

acetyloxy-3, 11 –dihydroxy-4,8,10,14-tetramethyl-2,3,4, 5,6,7, 9,11,12,

13,15,16-dodecahydro- 1H- cyclopenta [a] phenanthren-17-yelidene) -6-

methyl-hept-5-enoic acid. Molecular weight reported was 516.709.

Fusidic acid is practically insoluble in water, freely soluble in ethanol

(96%) and acetonitrile. Molecular formula is C31H48O6 and it is a weak

acid with a pKa of 5.7. Fusidic acid is mostly ionised in tissue and

plasma at the physiological pH of 7.4.

http://en.wikipedia.org/wiki/Carbon

http://en.wikipedia.org/wiki/Hydrogen

http://en.wikipedia.org/wiki/Oxygen

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Fig. 3.02

Fusidic acid Structural formula

Sample selected was in solid state and complex mixture of Halobetasol

propionate, Fusidic acid and other excipients. Both active ingredients are

in cream formulation and which are soluble in acetonitrile and other

excipients are not soluble in acetonitrile, so acetonitrile and water

combination as a solvent can be selected for extraction of these drugs

from cream formulation. Halobetasol propionate and Fusidic acid are

having UV absorption.

In summary, reverse phase chromatographic separation is suitable for

method development. Columns suitable for sibutramine hydrochloride

and orlistst compounds in reversed phase chromatography are C18, C8,

phenyl, and cyano.

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Table 3.01

Initial HPLC Method Development Conditions for Halobetasol

propionate and Fusidic Acid.

Separation Variable Preferred Initial choice

Column

Dimensions(length,ID) 15x0.46 cm

Particle size 5 μma

Stationary phase C8 or C18

Mobile Phase

Solvents A and B Buffer-acetonitrile

% B 80-100%b

Buffer(compound, pH,

concentration)

25 mM potassium

phosphate, 2.0<pH<3.0c

Additives(e.g.,amine modifiers,

ion-pair reagents)

Not recommended initially

Flow rate 1.0-2.0 ml/min

Temperature 25 °C

Sample size 20 μL

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3.5 EXPERIMENTAL INVESTIGATIONS:

Ortho-Phosphoric Acid, AR grade, Triethylamine, AR grade, Acetonitrile,

HPLC grade, Methanol, HPLC grade and Milli Q water chemicals were

used for experiments.

Transferred 1 mL of ortho phosphoric acid and in 1000 mL of water and

adjusted pH to 3.0(±0.05) with Ortho phosphoric Acid. This solution was

used as buffer in mobile phase preparation. Water and methanol were

mixed in the ratio of 10: 90 v/v and this was used as diluent for

standard and sample preparation

3.5.1 Experiment No. 1

The mobile phase was acetonitrile and a solution of 0.1% phosphoric

acid 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 was

maintained temperature at 25 °C. The mobile phase flow rate was

maintained at 1.5 ml/min. Standard Halobetasol dipropionate and

Fusidic acid solution was prepared at concentration, 100 μg/mL of

Halobetasol dipropionate and 4000 μg/mL of Fusidic acid in mobile

phase. 20 μL standard solutions were injected two times and average

detector response measured at 240 nm. Chromatograms evaluated with

respect to retention time, resolution and peak shape.

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Both the peaks eluted within 40 minutes, run time is more and forced

degradation solutions are injected and found co-elution. Next experiment

carried with decreased non polarity of column.

3.5.2 Experiment No.2




The analytical column, inertsil ODS-3V, 250 mm x 4.6 mm, 5 µ


maintained at 1.5 ml/min. Standard Halobetasol dipropionate and

Fusidic acid solution was prepared at concentration, 100 μg/mL of

Halobetasol dipropionate and 4000 μg/mL of Fusidic acid in mobile

phase. 20 μL standard solutions were injected two times and average



Both the peaks eluted in 40 minutes, run time is more and forced

degradation solutions are injected and found co-elution. Next experiment

carried with dicresed non polarity of column.

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The analytical column, a Zorbax SB Phenyl, 250 x 4.6mm, 5 µ and


maintained at 1.5 mL/min. Standard Halobetasol propionate and Fusidic

acid solution was prepared at concentration, 100 μg/mL of Halobetasol

propionate and 4000 μg/mL of Fusidic acid in mobile phase. 20 μL

standard solutions were injected two times and average detector

response measured at 240 nm. Chromatograms evaluated with respect to

retention time, resolution and peak shape.

Both the peaks eluted in 40 minutes, run time is more and the forced

degradation solutions are injected and found to co-elution and next

experiment carried with addition of amine modifers


The mobile phase was acetonitrile and a solution of 0.1%

triethylamine buffer adjusted pH to 2.0 with 10% solution of phosphoric

acid, (70:30; v/v). Mobile phase was filtered through 0.45 μ membrane

filter. The analytical column, Zorbax SB Phenyl, 250 x 4.6mm, 5 µ was


maintained at 1.5 ml/min. Halobetasol propionate and Fusidic acid

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solution was prepared at concentration, 100 μg/mL of Halobetasol

dipropionate and 4000 μg/mL of Fusidic acid in mobile phase. 50 μL

standard solutions were injected two times and average detector

response measured at 240 nm. Chromatograms evaluated with respect to

retention time, resolution and peak shape.

Both the peaks eluted in 40 minutes Halobetasol 9.2 minutes and

Fusidic acid in 29.0 minutes, the forced degradation solutions are

injected and found to co-elution and next experiment carried with solvent

mixtures methnaol and acetonitrile


The mobile phase was acetonitrile, methanol and a solution of 0.1%

phosphoric acid buffer adjusted pH to 2.0 with 10% solution of

phosphoric acid, (35:13:52 v/v/v). Mobile phase was filtered through

0.45 μ membrane filter. The analytical column, a Zorbax SB Phenyl, 250

x 4.6mm, 5 µ was maintained temperature at 25 °C. The flow rate of

mobile phase was maintained at 1.5 ml/min. Standard Halobetasol

propionate and Fusidic acid solution was prepared at concentration, 100

μg/mL of Halobetasol dipropionate and 4000 μg/mL of Fusidic acid in

mobile phase. 20 μL standard solutions were injected in duplicate and

average detector response measured at 240 nm. Chromatograms

evaluated with respect to retention time, resolution and peak shape.

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All the actives and impurities are separated in dilute standard and in

sample spiked impurities are not well separated and next experiment

carried with fine tuning of acetonitrile and methanol.


The mobile phase was acetonitrile, methanol and a solution of 0.1%

triethylamine buffer adjusted pH to 2.0 with 10% solution of phosphoric

acid, (35:20:45; v/v/v). Mobile phase was filtered through 0.45 μ

membrane filter. The analytical column, a Zorbax SB Phenyl, 250 x

4.6mm, 5 µ was maintained temperature at 25 °C. The flow rate of


dipropionate and Fusidic acid solution was prepared at concentration,

100 μg/mL of Halobetasol dipropionate and 4000 μg/mL of Fusidic acid

in mobile phase. 20 μL standard solutions were injected in duplicate and

average detector response measured at 240 nm. Chromatograms

evaluated with respect to retention time, resolution and peak shape.



carried with fine tuning of acetonitrile and methanol, wavelength was

found to be suitable at 240 nm for all substances.

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The Final mobile phase was acetonitrile, methanol and a solution of

0.1% triethylamine buffer adjusted pH to 2.0 with 10% solution of

phosphoric acid, (35:25:40; v/v/v). Mobile phase was filtered through

0.45 μ membrane filter. The analytical column, Zorbax SB Phenyl, 250 x

4.6mm, 5 µ was maintained temperature at 25 °C. The flow rate of


dipropionate and Fusidic acid was prepared at concentration, 100 μg/mL

of Halobetasol dipropionate and 4000 μg/mL of Fusidic acid in mobile

phase. 20 μL standard solutions were injected in duplicate and average





carried with fine tuning of acetonitrile and methanol, wavelength was

found to be suitable at 240 nm for all substances.

3.5.8 Experiment No.8

Standard Preparation:

Stock solutions 150 ppm of Halobetasol propionate, 100 ppm of

diflorasone 21 propionate,100 ppm of diflorasone 17 propionate 21

mesylate, 400 ppm of Fusidic acid, 800 ppm of 3 ketofusidic acid, 800

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ppm of 11 keto fusidic acid 800 ppm, 16 disacetyl fusidic acid 800 ppm

were Prepared in diluent.

Standard solution was prepared by mixing and diluting 1 ml each

Halobetasol and its impurities solutions and 10 ml Fusidic acid, 1 ml

each fusicisic acid solutions to 100 ml with diluent and obtained

concentration of Halobetasol propionate 1.5 ppm, diflorasone 21

propionate1 ppm, diflorasone 17 propionate 21 mesylate 1 ppm, Fusidic

acid 40 ppm, 3 ketofusidic acid 8 ppm, 11 keto Fusidic acid 8 ppm, 16

disacetyl Fusidic acid 8 ppm.

Sample Preparation:

Placed 5 g of sample in a 50 ml volumetric flask, added 20 ml of

acetonitrile and kept on water bath at 80 C for 15-20 min and then

cooled to room temperature. Added 5 ml of water to the above solution

and mixed well, chilled the sample solution in ice bath and finally filtered

through 0.45 u Teflon filter.

3.5.9 Experiment No.9 (Method Validation)

Specificity:

Diluent, standard solution and sample solution of Halobetasol and

Fusidic acid cream were prepared and injected into the HPLC as per

methology given in Experimental Results by using a photodiode detector.

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A placebo solution of Halobetasol and Fusidic cream 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. The peak purity was detected by using the tools of the Waters

software. Excipient solutions were submitted to the same degradation

conditions in order to explain no interference. Evident details of the

experiments conditions are described below:

Effect of UV light:

1 ml of a solution containing 1 mg/mL of Halobetasol and 40 mg/mL

of Fusidic 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

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 process, the

samples were diluted to 100 μg /ml of Halobetasol and 4000 μg/ml of

Fusidic acid with a mixture of acetonitrile:methanol:water (6:3:1; v/v/v)

and immediately analyzed.

120

Effect of Temperature (60 °C/24 h):


of Fusidic 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 4000 μg/ml of Fusidic 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):


of Fusidic acid in methanol was placed in a 10 ml volumetric flask at 25

°C/92% RH for 24 h. After the degradation treatment, the samples were

diluted to 100 μg/ml of Halobetasol and 4000 μg/ml of Fusidic acid with

a mixture of acetonitrile:methanol:water (6:3:1; v/v/v) and analysed

immediately.

Effect of Oxidation:

Halobetasol and Fusidic acid standards were dissolved in methanol (1

mg/ml of Halobetasol, 40 mg/mL of Fusidic acid and 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 of Halobetasol and 4000

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μg/mL of Fusidic acid, filtered and analysed. Similar procedure was

realized for the commercial cream, when 25 ml of the initial solution 100

μg/ml of Halobetasol and 4000 μg/ml of Fusidic acid, 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 Hydrolysis:

5 ml of the Halobetasol and Fusidic acid reference standard solution

were transferred to a volumetric flask and HCl was added until the final

concentration of 1M in both cases. After 5 hours and 1 and 6 days, one

aliquot of the solution was neutralized with NaOH and diluted with

acetonitrile, methanol and water (6:3:1, v/v/v) until the final

concentration 100 μg/ml of Halobetasol and 4000 μg/ml of Fusidic acid

for LC analysis. Similar procedure was realized with the commercial

cream, when 25 ml of the initial solution 100 μg/ml of Halobetasol and

4000 μg/ml of Fusidic 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 same circumstances of the commercial cream.

Effect of alkaline hydrolysis:

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5 ml of the Halobetasol and Fusidic acid reference standard solution

were transferred to a volumetric flask and NaOH was added until the

final concentration of 1M in both cases. After 5 hours and 1 and 6 days,

one aliquot of the solution was neutralized with 1M HCl and diluted

with acetonitrile, methanol and water (6:3:1, v/v/v) until the final

concentration 100 μg/ml of Halobetasol and 4000 μg/ml of Fusidic acid

for LC analysis. Similar procedure was realized with the commercial

cream, when 25 ml of the initial solution 100 μg/ml of Halobetasol and

4000 μg/ml of Fusidic acid 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

commercial cream.

LOD and LOQ:

The qualification and detection limits were obtained based on signal-

to-noise approach. The background noise was obtained after injection of

the blank, observed over a distance equal to 20 times the width at half-

height 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.

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Based on the determination of prediction linearity and visual

observation of known impurities, six replicate injections were made for

LOD &LOQ.

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, 0.03-

3.02 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

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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 Fusidic acid cream were prepared and injected into the

HPLC using the method as described under experimental result.

Ruggedness

Six spiked sample preparations and one control sample preparations

of Halobetasol and Fusidic acid cream 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 Fusidic acid

cream were prepared as described in experimental result. The samples

along with standard and placebo were injected under different

chromatographic conditions.

Stability of Analytical Solution

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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.

2.6 EXPERIMENTAL RESULTS

On the basis of Halobetasol and Fusidic acid Analytical method

development experimental trials, RP-HPLC method was suitable for

simultaneous determination of Halobetasol, Fusidic acid and their

impurities.

Final experiment chromatographic conditions were applied for

Identification as well as quantification of related substance. Impurities

were identified by spiking known impurities in to sample preparation.

Preparation of stock solutions: Prepare solution having the concentration

of Halobetasol 100 ppm and Fusidic acid 4000 ppm, in acetonitrile.

Sample preparation: Five gram cream sample quivalent to 2.5 mg of

Halobetasol and 40 mg Fusidic acid was transferred to 50 ml volumetric

flask and added 20 ml of acetonitrile and kept on water bath at 80 °C for

15-20 min, then cooled to room temperatiure. Added 5 ml of water to this

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solution and mixed well, chilled the sample in ice-bath and finally filtered

through 0.45 μ Teflon filter.

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 not less than 8000, tailing factor not more than 1.5,

relative standard deviation for six replicates of standard solution is not

more than 2.0%.

2.7 DISCUSSION OF RESULTS:

LOD and LOQ: RSD is less than 33% at LOD level and less than 10%

at LOQ level for Halobetasol and Fusidic acid and known impurities.

Linearity and range: the correlation coefficients are less than 0.999

for Halobetasol, Fusidic acid and known Impurities.

Precision: system precision RSD is less than 5% and method

precision RSD is less than 10% for Halobetasol, Fusidic acid and known

Impurities.

Accuracy: the mean recoveries for Halobetasol, Fusidic acid and known

impurities are within 90 -110 %.

Specificity: Retention time of Halobetasol and Fusidic acid and known

peaks in sample preparation is comparable with respect to retention time

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of Halobetasol and Fusidic acid and known impurities peaks in standard

preparation. Peak purity passes for Halobetasol and Fusidic acid and

known impurities peaksin standard and sample preparations. No

intereference was observed at the retention time of Halobetasol and

Fusidic 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 and Fusidic 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

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 3.02

Peak Purity Data for Halobetasol Propionate and Fusidic Acid

Sr. No. Name

Purity

Criteria

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1 Halobetasol propionate in standard

solution Pass

2 Halobetasol propionate in sample

solution Pass

3 Fusidic acid in standard solution Pass

4 Fusidic acid in sample solution Pass

Table 3.03

RT, RRT, LOD and LOQ Data of Halobetasol Propionate Impurities

HBI D21PI D17P21MI HP

RT 5.7 6.3 10.3 11.4

RRT 0.500 0.553 0.904 1.00

LOD 0.012 0.013 0.011 0.018

LOQ 0.03 0.03 0.03 0.03

Table 3.04

RT, RRT, LOD and LOQ Data of Fusidic Acid Impurities

11 KFA 3 KFA FA 16DAFA

RT (min) 14.9 16.0 18.2 26.6

RRT 0.819 0.879 1.000 1.462

LOD (μg/mL) 0.03 0.02 0.02 0.03

LOQ 0.09 0.08 0.08 0.09

Table 3.05

Recovery Data of Halobetasol Propionate Impurities at LOQ Level

Level HBI (%) D21PI (%) D17P21MI (%) HP (%)

LOQ 96.60 99.60 96.34 103.21

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LOQ 98.20 96.20 98.60 102.42

LOQ 99.34 98.34 99.39 100.12

Mean 98.05 98.05 98.11 101.92

SD 1.38 1.72 1.58 1.61

%RSD 1.40 1.75 1.61 1.58

Table 3.06 Recovery Data of Halobetasol Propionate Impurities at 50, 100 and

150 % Level

Level HBI (%) D21PI (%) D17P21MI (%) HP (%)

Lev. 50% 101.26 99.21 100.23 101.23

Lev. 50% 102.33 102.26 101.11 102.25

Lev. 50% 99.15 101.43 98.26 100.43

Lev. 100% 98.34 100.23 102.26 102.26

Lev. 100% 98.23 102.26 98.60 101.23

Lev. 100% 100.23 100.23 99.39 98.60

Lev. 150% 102.26 101.23 102.26 99.39

Lev. 150% 97.47 102.42 100.23 100.23

Lev. 150% 102.11 100.76 100.23 100.23

Avearge 100.15 101.11 100.29 100.65

SD 1.92 1.1 1.42 1.23

%RSD 1.92 1.09 1.42 1.22

Table 3.07

Recovery Data of Fusidic Acid Impurities at LOQ Level

LEVEL % Recovery

3 keto

fusidic acid

11 keto

fusidic acid

16

Disacetyl fusidic acid

Fusidic

acid

LOQ 96.60 99.60 103.21 103.21

LOQ 103.21 103.21 102.42 102.42

LOQ 102.42 102.42 99.39 100.12

Mean 100.74 101.74 101.67 101.92

SD 3.61 1.9 2.02 1.61

%RSD 3.58 1.87 1.98 1.58

Table 3.08

Recovery Data of Fusidic Acid Impurities at 50, 100 and 150% level

LEVEL % Recovery

130

3 keto

fusidic acid

11 keto

fusidic acid

16

Disacetyl fusidic acid

Fusidic

acid

Lev. 50% 101.26 99.21 100.23 101.23

Lev. 50% 102.33 98.20 103.11 102.25

Lev. 50% 99.15 99.34 98.26 98.20

Lev. 100% 98.34 98.92 102.26 99.34

Lev. 100% 98.20 99.39 98.60 99.93

Lev. 100% 99.34 99.22 99.39 98.60

Lev. 150% 98.63 101.23 102.26 99.39

Lev. 150% 97.47 102.42 100.55 100.23

Lev. 150% 102.11 101.76 101.23 102.26

Avearge 99.65 99.96 100.65 100.16

SD 1.8 1.45 1.71 1.48

%RSD 1.8 1.45 1.69 1.48

Table 3.09

Linearity Data of Halobetasol Propionate and Impurities

Halobetasol

Diflorasone 21

propionate

Diflorasone 17 propionate 21

MSC

Halobetasol

propionate

Level

Conc.

(μg/mL)

Peak

area

Conc.

(μg/mL)

Peak

area

Conc.

(μg/mL)

Peak

area

Conc.

(μg/mL)

Peak

area

LOQ 0.03 1949 0.03 1963 0.03 1965 0.03 1863

Lev. 50% 0.76 47079 0.76 46433 0.76 46481 0.78 45929

Lev. 75% 1.14 74740 1.13 74290 1.13 74367 1.16 70505

Lev. 100% 1.52 97432 1.51 98125 1.51 98226 1.55 93128

Lev.125% 1.90 122979 1.89 123853 1.89 123981 1.94 115680

Lev. 150% 2.28 147707 2.27 146795 2.27 146946 2.33 139315

Lev. 200% 3.04 195936 3.02 196348 3.02 196550 3.10 189137

Corr. coff. 0.9999 0.9998 0.99984 0.99986

R. square 0.9998 0.9997 0.99968 0.99971

131

Table 3.10

Linearity Data of Fusidic acid and Impurities

Fusidic acid 3 Keto fusidic

acid 11 Keto fusidic

acid

16 Disacetylfusidic

acid

Level Conc.

(μg/mL) Peak area

Conc. (μg/mL)

Peak area

Conc. (μg/mL)

Peak area

Conc. (μg/mL)

Peak area

LOQ 0.08 1275 0.08 1234 0.09 1394 0.09 1399

50% 20.06 314436 21.10 322878 22.10 364747 21.55 358892

75% 30.09 489059 31.65 473394 33.15 534781 32.33 522463

100% 40.12 637543 42.20 617122 44.20 697146 43.10 699321

125% 50.15 804707 52.75 778931 55.25 879938 53.88 882683

150% 60.18 979266 63.30 935557 66.30 1056873 64.65 1067164

200% 80.24 1282099 84.40 1247204 88.40 1408932 86.20 1420321

Corr. Coff. 0.9998 0.9999 0.99992 0.99992

R. square 0.9997 0.9998 0.99984 0.99984

132

Fig. 3.03

Halobetasol + Fusidic acid and Impurities Standard

Figure 3.04

Halobetasol + Fusidic acid Cream Impurities Spiked Sample chromatograph

133

3.8 Summary, Conclusion and Recommenddations.

A simple fast, accurate, precise, and cost effective isocratic stability

indicating reverse phase high performance liquid chromatographic

method was developed for simultaneous determination of Halobetasol

propionate and fusidic acid as well as its impurities in the newly

developed cream formulation. The chromatography equipped with Zorbax

SB phenyl (250X4.6mm) column using a mobile phase of buffer 0.1%

triethylamine buffer pH adjusted to 2.0 with Orthophosphoric acid

acetonitrile, methanol in the ratio of 40:35:25 v/v with a flow rate of 1.5

ml/min, with UV configured at 240nm. These impurities were identified

by spiking known impurities in to sample preparation.

Hence this method can be conveniently adopted for routine analysis

of sibutramine hydrochloride and orlistat in bulk drugs and the

pharmaceutical dosage forms and also for stability analysis.

chapter 3 simultaneous determination of...

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