chapter 7 simultaneous assay estimation of...

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CHAPTER – 7

SIMULTANEOUS ASSAY ESTIMATION OF BUTENAFINE

HYDROCHLORIDE AND BETAMETHASONE FROM CREAM

FORMULATION.

7.1 OBJECTIVE

To develop a simultaneous assay eatimation method for Butenafine

hydrochloride and Betamethasone from cream formulation.

7.2 INTRODUCTION

Butenafine hydrochloride [207,208] (n-4-ter-butyl-benzyl-N-methyl-1-

naphthaline methylamine hydrochloride) is a derivative of benzylamine

having same mode of action and chemical structure to allylamine

antifungal agent. It has the molecular weight of 353.93 and an empirical

formula C23H27N.HCl. It leads to accumulation of squalene and converts

squalene to lanosterol. It is primarily fungicidal against dermatophytes

and inhibits squalene epoxidase an enzyme.

The azoles have been used as antifungal to treat dermatophytosis

since the 1970s. The most used topical agents are miconazole and

clotrimazole. The cytochrome P450-dependent enzymes inhibit lanosterol

14-demethylase and these act as fungi-static agents, a component of

fungal cell membrane, this is important for the biosynthesis of ergosterol,

Butenafine hydrochloride is slightly soluble in water, freely soluble in

ethanol, methanol and chloroform.

225

Betamethasone dipropionate [209, 210] is chemically 9-fluoro-11b-

hydroxy-16b-methyl-3, 20-dioxopregna-1, 4-diene-17, 21-diyl

dipropionate. It has molecular weight of 504.6 and the empirical formula

C28H37FO7. Betamethasone dipropionate is almost white to white

crystalline powder, practically insoluble in water, sparingly soluble in

alcohol, freely soluble in acetone and in methylene chloride.

The cream formulation contains 0.05% betamethasone as

betamethasone dipropionate, a synthetic corticosteroid is member of the

class of steroids and 1% butenafine hydrochloride, a synthetic antifungal

agent.

7.3 LITERATURE REVIEW

Yan-Yan L.B. et al were reported High performance liquid

chromatography determination of the sample, after extraction with

methanol under ultrasonic bath, was separated on HP Hypersil column

C18 (150 mm x 4.6 mm 5u ), with a mobile phase, consisted of methanol

and 0.05 mol/L ammonium acetate, a flow rate of 0.8 ml/min, a

detecting wavelength at 223 nm, the column temperature at 40 °C,

within the range of 0.09-0.45 μg, the calibration curve was

linear(r=0.9997), the mean recovery rate was 9.89 mg/g, accorded label

content with the 10 mg/g [211].

Chunying W. et al were reported HPLC determination of butenafine

hydrochloride ointment content. Kromasil-C18 column with acetonitrile -

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methanol-0.0 5mol/L ammonium acetate buffer pH 6.8, (66:19:15) for

the mobile phase, detection wavelength of 282 nm. The sample size in

the range of 4.1 ~ 20.5 μg with a good linear relationship, the average

recovery was 98.17% and the relative standard deviation (RSD) was

0.13% [212].

Rui-Guo C, Liu YC et al were reported the objective HPLC

determination of butenafine hydrochloride in gel formulation. Waters

Symmetry C18 (3.9 mm × 150mm, 5μm) column with methanol and

sodium acetate buffer in the ratio 73:27, as mobile phase at a flow rate

1.0 mL/min, detection wavelength of 282nm, column temperature of 35

°C. Butenafine hydrochloride was linear in the range of 5.194 ~ 519.4

μg/mL having good linear relationship, the minimum detection limit of

0.39 ng. The average recovery was 99.72% and RSD was 0.52% (n = 6)

[213].

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

method using UV detection for simultaneous determination of Fusidic

acid and betamethasone dipropionate in a cream formulation. A

supelcosil C18 column was used for chromatographic separation. The

mobile phase used was mixture of acetonitrile and 0. 01M sodium

hydrogen orthophosphate in the ratio of 70:30, %v/v and pH was

adjusted to 6.0 with glacial acetic acid. Chromatographic system was

operarted at a flow rate of 1.0 ml/ min with the UV detector set at 235

227

nm. The method was found satisfactory with respect to all validation

parameters [214].

Kedor-Hackmann E.R. et al were reported 27the simultaneous

determination of betamethasone dipropionate and salicylic acid in both

ointment and topical solution using HPLC. The method was optimized

using a Licrosphere 100 RP -18 (125 x 4 mm, 5 microns) column,

tetrahydrofuran- acetonitrile- acetic acid 1% (20:25:55 v/v), pH 3.3, as

mobile phase, and UV detection was carried at 254 nm. The method was

found satisfactory with respect to linearity and range, recory and

precission [215]

Dyderski S. et al were reported were reported A reverded- phase HPLC

method was developed for the determination of betamethasone

dipropionate in lipophylic bases. Analysis was performed using a PLRP

column with a mobile phase of water- methanol- acetonitrile and

ultraviolet detection at 254 nm. The calibration curve was constructed

for concentration (0-50 μg/mL the method is simple, accurate and

precise [216].

Ankam R. et al were reported RP-HPLC for the simultaneous

determination of betamethasone and butenafine hydrochloride in cream

formulation. The determination was carried out on lichrocart

lichrosphere RP-select B (250Χ4.6 mm, 5 μ) column in isocratic mode,

the mobile phase used was mixture of 50 mM ammonium acetate buffer

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and acetonitrile in the ratio of 60:40, adjusted to pH 4.5 ± 0.1 with

glacial acetic acid. The flow rate was maintained at 2.0 ml/min and

elution was monitored at 254 nm [217].

Minshan S et al were reported RP-HPLC method was developed for

simultaneous determination of betamethasone, dipropionate salicylic

acid and their related compounds in Diprosalic Lotion. A YMC Jsphere

ODS (150 mm x 4.6 mm id.) column at 35°C and UV detection at 240 nm

was used. A gradient elution was employed using acetonitrile and 0.05%

v/v methanesulfonic acid solution as mobile phases. All compounds were

evaluated in 38 min. All impurities and degradation products were well

resolved, this indicating capacity of this method [218].

7.4 THEORETICAL ANALYSIS

Butenafine hydrochloride chemical structure and mode of action is

similar to allylamine antifungals. It has the empirical formula

C23H27N.HCl and a molecular weight of 353.93.

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Figure 7.01

Butenafine hydrochloride

Butenafine Hydrochloride is Structural formula reveals that the molecule

is ionic and somewhat non polar. Systematic name is [(4-tert-

butylphenyl) methyl](methyl)(naphthalen-1-ylmethyl)amine. It require

buffer to elute smoothly, hence 0.1% ortho-phosphoric acid can be

selected as buffer as the acidic pH is more robust than the neutral.

Figure 7.02

Betamethasone Structural formula

Betamethasone dipropionate is [(8S,9R,10S,11S,13S,14S,16S,17R)-9-

fluoro-11-hydroxy-10,13,16-trimethyl-3-oxo-17-(2-propanoyloxyacetyl)-6,

7, 8, 11, 12, 14, 15, 16-octahydrocyclopenta[a]phenanthren-17-yl]

propanoate, chemical formula is C28H37FO7 and molecular mass is

504.59 g/mol. Structural formula reveals that the molecule have no

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

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

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



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acidic or basic functional groups, hence it will not demand for buffer, as

it is nonpolar hence it require solvent strength for its elution. Hence

methanol can be selected as initial solvent because of its low cost even if

is more viscous with aqueous mobile phase than acetonitrile. As the

analyte is combination of BH and BP, initial trials are selected with 0.1%

OPA and methanol. Simple C-18 column was selected for initial trials as

it is stable and rugged. Both the molecules having UV absorption hence

simple UV detector selected, Betamethasone has lowest amount in the

formulation and hence priority given to betamethasone, selectively 254

nm wavelength was selected for monitoring the both analytes. Initial

trials were done on 25°C and flow rate at 1.5ml/minutes to get faster

elution. Diluent selected based on the solubility of both analytes. Water

and methanol in equal volume were used for initial trials.

7.5 EXPERIMENTAL INVESTIGATIONS

The reference standard of Butenafine hydrochloride was procured

from Hetero Drugs Limited, (Hyderabad, India) betamethasone

dipropionate and cream sample were prepared in In-house facility.

Creams are claimed to contain 1% w/w of Butenafine hydrochloride,

0.05% w/w synthetic corticosteroid betamethasone as betamethasone

dipropionate and the following inactive ingredients: benzyl alcohol, ethyl

alcohol, cetyl alcohol, and sodium benzoate, emulsifying wax, disodium

edentate, polyethylene glycol 40 stearate, liquid petrolatum, polysorbate

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60, propylene glycol, simethicone, white petrolatum, sodium hydroxide

and water. All reagents were analytical or HPLC grade ammonium

acetate, acetonitrile, and glacial acetic acid were procured from Merck

India Limited (Mumbai, India. Purified water was obtained by a Millipore®

Dire-Q 3 UV with pump (Molsheim, France)

The HPLC system (Jasco, Tokyo, Japan) consisted of an AS-2057

intelligent sampler, PU-2080 quaternary pump, UV-2075 intelligent

UV/Vis detector, and CO-2065 intelligent column oven and detector

seted at 254nm. The analytical column, a lichrocart lichrosphere RP-

select B (250 mm x 4.6 mm i.d., 5 μmparticle size) (Merck, Germany) was

operated in ambient temperature (25 °C).

7.5.1 Experiment No.1

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

acid buffer pH 3.0 adjusted with 10% solution of phosphoric acid, (30:70;

v/v). Mobile phase was filtered through 0.45 μ Teflon membrane filter.

The mobile phase flow rate was maintained at 1.0 ml/min. Standard

solutions of butenafine hydrochloride 200 μg/mL and betamethasone 10

μg/mL were prepared in mobile phase. These standard solutions were

injected two times and detector response measured at 254 nm.

Both the peaks were not eluted until 30 minutes, therefore next

experiment carriedout with increased organic modifier in mobile phase.

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7.5.2 Experiment No. 2.

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, (40:60; v/v). Mobile phase was filtered through 0.45 μ

Teflon membrane filter. The mobile phase flow rate was maintained at

1.0 ml/min. Standard solutions of butenafine hydrochloride 200 μg/mL

and betamethasone 10 μg/mL were prepared in mobile phase. These

standard solutions were injected two times and average detector

response measured at 254 nm.

Both butenafine and betamethasone 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.


The final selected mobile phase was acetonitrile and a solution of

50mM ammonium acetate buffer adjusted pH to 4.5 with 10% solution of

acetic acid (80:20; v/v). Mobile phase was filtered through 0.45 μ

membrane filter. The mobile phase flow rate was maintained at 1.0

ml/min. Standard solutions of butenafine hydrochloride 200 μg/mL and

betamethasone 10 μg/mL were prepared in mobile phase. These

standard solutions were injected two tomes and detector response

measured at 254 nm.

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All the actives and impurities were not separated in dilute standard

and sample spiked impurities. Therefore experiment carried with

different mobile phase composition.



50mM ammonium acetate buffer adjusted pH to 4.5with 10% solution of





standard solutions were injected two times and average detector

response measured at 254 nm.

All the actives and impurities were separated in dilute standard while

in sample spiked with impurities in 50 min. since run time is more, next

experiment carried with fine tuning of mobile phase.


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50mM ammonium acetate buffer adjusted pH to 4.5with ten percent

solution of acetic acid (60:40; v/v). Mobile phase was filtered through

0.45 μ membrane filter. The mobile phase flow rate was maintained at

1.0 ml/min. Standard solutions of butenafine hydrochloride 200 μg/mL

and betamethasone 10 μg/mL were prepared in mobile phase. These

standard solutions were injected two times and detector response

measured at 254 nm.

All the actives and impurities were separated in dilute standard as

well as sample spiked with impurities in 30 min. Since the run time was

more, next experiment carried with increased flow rate.



50mM ammonium acetate buffer adjusted pH to 4.5with 10% solution of





standard solutions were injected two times and detector response

measured at 254 nm.

Both Butenafine hydrochloride and Betamethasone peaks were eluted

in 15 minutes. Since the run time peak shapes found were satisfactory.

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In-house developed R&D samples (Lot-1, 2 and 3) each two tubes

about 5 g sample was taken in 100 ml beaker and mixed properly. Cream

approximately 20 mg of butenafine hydrochloride and 1 mg of

betamethasone equivalent sample was taken in 100 ml volumetric flask

and dissolved in mobile phase and made volume up to the mark. Above

sample was transferred to 200 ml volumetric flask and chilled in ice bath

for 10 min. supernatant solution was filtered through Whatman filter

paper No. 40. The further aqliquot solution was further diluted to get

final concentration. The solution was evaluated at 254 nm. The

chromatographic conditions utilized were proper for proper resolution of

peaks.

7.5.7 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 cream 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 Butenafine and Betamethasone. The

accelerated degradation conditions applied were: light (UVC), acid, basic

basic and oxidant media. Samples were analysed and compared with

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control sample solution (with no degradation treatment) and under light

protection. The peak purity was evaluated using the tools of the Waters

software. Excipient solutions were submitted to the same degradation

conditions in order to explain 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

butenafine and 0.01 mg/mL of betamethasone 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 were submitted simultaneously to

identical conditions and used as control. After the degradation treatment,

the samples were diluted to 200 μg/ml with a mixture of water and

acetonitrile, (60:40; v/v) and immediately analyzed.

Effect of Oxidation: Butenafine and betamethasone standards were

dissolved in acetonitrile (0.2 mg/mL of butenafine and 0.01 mg/mL of

betamethasone), 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 with a mixture of water and

acetonitrile, (60:40; v/v) to final concentration of 200 μg/mL of

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butenafine hydrochloride and 10 μg/mL of betamethasone, filtered and

analysed. Similar procedure was realized for the commercial cream,

when 25 ml of the initial solution 0.2 mg/mL of butenafine and 0.01

mg/mL of betamethasone, 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 0.2 mg/mL of butenafine and 0.01 mg/mL of betamethasone

reference standard solution was transferred to a volumetric flask and

HCl (acid degradation) 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 and water

(40:60, v/v) until the final concentration of 200 μg/ml of butenafine

hydrochloride and 10 μg/ml of betamethasone for LC analysis. Similar

procedure was realized with the cream, when 25 ml of the initial solution

0.2 mg/mL of butenafine and 0.01 mg/mL of betamethasone (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 cream.

Effect of Alkaline Hydrolysis:

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5 ml of 0.2 mg/mL of butenafine and 0.01 mg/mL of betamethasone

reference standard solution was transferred to a volumetricflask 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 and water

(40:60, v/v) until the final concentration of 200 μg/ml of butenafine

hydrochloride and 10 μg/ml of betamethasone for LC analysis. Similar

procedure was realized with the cream, when 25 ml of the initial solution

0.2 mg/mL of butenafine and 0.01 mg/mL of betamethasone (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 cream.

Precission:

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.

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Six spiked sample preparations and one control sample preparation of

Butenafine hydrochloride and betamethasone cream were prepared and

injected into the HPLC using the method as described under

experimental result.

Accuracy:

The accuracy was determined by the recovery of known amounts of

Butenafine and betamethasone standards added to the placebo in the

beginning of the preparative process. The added levels were 80, 100 and

120% of the nominal drug concentrations. The results were expressed as

the percentage of Butenafine hydrochloride and Betamethasone reference

standards recovered from the sample

Ruggedness:

Six spiked sample preparations and one control sample preparations

of Butenafine hydrochloride and Betamethasone 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 Butenafine hydrochloride and

Betamethasone Cream were prepared as described in experimental

240

result. The samples along with standard and placebo were injected under

different chromatographic conditions as shown below.

Linearity and Range

To test linearity, standard plots were constructed with six

concentrations in the range of 100 to 300 μg/mL of Butenafine

hydrochloride and 5 to 15 μg/mL Betamethasone prepared in triplicates.

The linearity was evaluated by the least square regression

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.

The objective of validation of an analytical procedure is to

demonstrate that the method suitable for its intended purpose. The

method was validated for linearity, precision (repeatability and

intermediate precision), accuracy, specificity, robustness and system

suitability.

7.6 EXPERIMENTAL RESULTS

241

On the basis of butenafine Hydrochloride and betamethasone

analytical method development experimental trials, RP-HPLC method

was suitable for simultaneous determination of butenafine hydrochloride

and betamethasone assay. Final experiment chromatographic conditions

were applied

Preparation of stock solutions: Prepare solution having the

concentration of Butenafine hydrochloride 200 ppm and betamethasone

10 ppm in mobile phase.

Sample preparation: 5 g cream smple was weighed and transferred

into 100 ml beaker and mixed properly. An accurately weighed quantity

of cream equivalent to 20 mg of butenafine hydrochloride and 1 mg of

betamethasone was taken in 100 ml volumetric flask and dissolved in

mobile phase. Whole solution was transferred in 250 ml volumetric flask

and chilled it in ice bath for 10 min. The solution was filtered through

Whatman filterpaper No.40. The aliquot portion of the filtrate was further

diluted to get final concentration.

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 5000, tailing factor less than 1.5, relative

242

standard deviation for six replicates of standard solution is less than

2.0%.

7.7 DISCUSSION OF RESULTS

Linearity and range: the correlation coefficients are less than 0.9995

for Butenafine hydrochloride and Betamethasone.

Precision: system precision RSD is less than 2% and method

precision RSD is less than 2% for Butenafine hydrochloride and

Betamethasone.

Accuracy: the mean recoveries for Butenafine hydrochloride and

Betamethasone are within 98 -102 %.

Specificity: Retention time of Butenafine hydrochloride and

Betamethasone peaks in sample preparation is comparable with respect

to retention time of Butenafine hydrochloride and Betamethasone peaks

in standard preparation. Peak purity passes for Butenafine hydrochloride

and Betamethasone peaks in standard and sample preparations. No

intereference was observed at the retention time of Butenafine

hydrochloride and Betamethasone peaks. Peak purity passes for all

degradation conditions.

Ruggesness: the RSD of twelve results obtained from two different

analysts are within 10 %.

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Robustness: Butenafine hydrochloride and Betamethasone 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 7.01

Peak Purity Data of Butenafine Hydrochloride and Betamethasone

Sr. No. Name

Purity Criteria

1 Butenafine hydrochloride in standard solution

Pass

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2 Butenafine hydrochloride sample

solution Pass

3 Betamethasone in standard solution Pass

4 Betamethasone in sample solution Pass

Table 7.02

Recovery Data of Butenafine hydrochloride

Levels (%)

Added

conc. (μg/mL)

Average Area

Recovered

conc. (μg/mL)

Recovery (%)

Mean

recovery (%)

80 Spl. 1 00787 8874137 0.0079 100.5 100.6

80 Spl. 2 00787 8551706 0.0076 96.8

80 Spl. 3 00787 9215850 0.0082 104.4

100 Spl. 1 00983 11126339 0.0099 100.8 100.4

100 Spl. 2 00983 11099909 0.0099 100.6

100 Spl. 3 00983 11024949 0.0098 99.9

120 Spl. 1 01180 13320382 0.0119 100.6 101.2

120 Spl. 2 01180 13458769 0.0120 101.6

120 Spl. 3 01180 13432412 0.0120 101.4

Mean (%) 100.7

SD 1.96

RSD (%) 1.94

Table 7.03

Recovery Data of Betamethasone.

Added levels (%)

Added

conc. (μg/mL)

Average Area

Recovered

conc. (μg/mL)

Recovery (%)

Mean

recovery (%)

245

80 Spl. 1 15.74 1483116 15.78 98.8

99.6

80 Spl. 2 15.93 1452469 15.64 96.7

80 Spl. 3 15.82 1550707 15.70 103.3

100 Spl. 1 19.87 1818759 20.08 96.9

98.2

100 Spl. 2 19.95 1858994 20.02 99.0

100 Spl. 3 20.27 1850217 20.65 98.6

120 Spl. 1 24.22 2268677 23.99 100.7

100.0

120 Spl. 2 23.82 2242050 24.09 99.5

120 Spl. 3 23.75 2246127 23.52 99.7

Mean (%) 99.2

SD 1.98

RSD (%) 1.99

Table 7.04

Linearity Data of Butenafine HCl

Levels (%) x a (μg/mL) y=mx+c b=a-y

50 101.87 6118269 6094915.2 23353.8

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x=concentration, a=experimental area, y=predicted area and b=residuals.

Table 7.05

Linearity Data of Betamethasone

Table 7.06

Method Precision Data of Butenafine HCl

Sample

Sample wt

(mg) Average Area Mg/Unit % Assay

Spl-1 2015.5 11711740 202922 103.7

75 152.80 9141247 9064783.1 76463.9

100 203.74 11855406 12034651.0 -179245

125 254.67 15040202 15004518.9 35683.1

150 305.60 18018131 17974386.8 43744.2

Correlation 0.99980

Intercept (c) 155179.40

Slope (m) 58308.31

Levels (%) x a (μg/mL) y=mx+c b=a-y

50 5.01 929154 936553 -7399

75 7.52 1415671 1396485.3 19185.7

100 10.02 1836002 1856417.6 -20415.6

125 12.53 2329220 2316349.9 12870.1

150 15.04 2772041 2776282.2 -4241.2

Correlation 0.9998

Intercept (c) 16688.40

Slope (m) 183532.44

247

Spl-2 2055.1 11687605 203922 101.5

Spl-3 2039.6 11556181 203231 101.1

Spl-4 2043.2 11741303 202345 102.6

Spl-5 2077.9 11831622 201654 101.6

Spl-6 2091.7 11753160 203154 100.3

Average 101.8

SD 1.19

RSD 1.17

Table 7.07

Method Precision Data of Betamethasone

Sample Sample wt


Spl-1 2015.5 2007806 2954606 104.8

Spl-2 2055.1 1977300 2985641 101.2

Spl-3 2039.6 1930924 2945618 99.6

Spl-4 2043.2 1990189 2948612 102.5

Spl-5 2077.9 2007770 2924332 101.6

Spl-6 2091.7 2014646 2944233 101.3

Average 101.8

SD 1.73

RSD 1.69

Table 7.08

Intermediate Precision Data of Butenafine HCl.

Sample

Sample wt


Spl-1 2088.6 11808871 0.0495 101.0

248

Spl-2 2090.1 11615746 0.0509 99.3

Spl-3 2079.6 11750392 0.0496 100.9

Spl-4 2083.2 11730322 0.0499 100.6

Spl-5 2087.9 11753248 0.0496 100.5

Spl-6 2081.7 11560647 0.0508 99.2

Average 100.2

SD 0.81

RSD 0.81

Table 7.09

Intermediate Precision Data of Betametasone.

Sample Sample wt


Spl-1 2088.6 1968150 2.0199 99.2

Spl-2 2090.1 2000687 2.0387 100.8

Spl-3 2079.6 1983349 2.0021 100.4

Spl-4 2083.2 2001989 2.0256 101.2

Spl-5 2087.9 1991043 1.9770 100.4

Spl-6 2081.7 1988428 2.0071 100.6

Average 100.4

SD 0.66

RSD 0.66

Figure 7.03

Sample chromatograph of Betamethasone and Butenafine HCl

0

250

500

750

1000

mV 4.703

7.767

249

Typical chromatogram of butenafine hydrochloride (200 µg/ml, tR =4.703 min) and betamethasone (10

µg/ml, tR=7.767 min) sample under optimized conditions.

7.8 SUMMARY, CONCLUSION AND RECOMMENDATIONS

The reversed phase LC method proposed was found to be simple, fast,

accurate, precise, linear, robust and specific and it is powerful tool to

investigate chemical stability of butenafine hydrochloride and

betamethasone in cream formulation. The robustness of the method was

verified with small variation on pH, concentration of organic phase,

detector wavelength, column manufacturer and analysis temperature. All

the parameters meet the criteria of the ICH guidelines for method

validation. Its chromatographic retention time 4.7 of Butenafine

hydrochloride and 7.76 min of betamethasone allows the analysis of a

large number of samples in an adequate period of time. Therefore the

method could be recommended for routine quality control analysis

commercial cream, as well as for routine quality control analysis of raw

material and of cream. The method applicability to stability studies was

proved through the evaluation of the main factors that affect the drug

content in solution and through the butenafine and betamethasone

chapter 7 simultaneous assay estimation of...

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