Assay and Identification of Clotrimazole contained in a formulation with Metronidazole in Softgels® by HPLCMaría Isabel Morales, Eduardo Rodríguez

© 2010 Banner Pharmacaps Inc. AAPS 2010-15 W4471Research and Development, Gelcaps Exportadora de México S. A. de C.V.

ObjeCtIveSTo develop and validate a method of analysis by HPLC for the identification and assay of Clotrimazole contained in a formulation with Metronidazole in vaginal suppositories (soft-gelatin).

SOft GeLAtIn CAPSuLeS wItH CLOtrIMAzOLe And MetrOnIdAzOLe• The vaginal suppositores contain:

– Clotrimazole .............100 mg

– Metronidazole ...........500 mg • This pharmaceutical dosage form has the characteristic of preserving

and maintaining stability of the active ingredients

– It allows exact dosing of the active

– It is visually pleasant to the consumer• The release of the active ingredient occurs almost immediately after its

administration

AbOut CLOtrIMAzOLe• Chemical name: 1-[(2-Chlorophenyl)-

diphenylmethyl]-1 H-imidazole.

• Chemical formula: C22

H17ClN

2

• Molecular Weight: 344.85

• Clotrimazole is an antifungal drug commonly used in the treatment of fungal infections of both humans and animals such as vaginal yeast infections and ringworm. It is also used to treat athlete’s foot and jock itch.

• Clotrimazole acts against the fungi through inhibition of the synthesis of ergosterol. The inhibition of the synthesis of ergosterol leads to a structural and functional harm of the citoplasmatic membrane.

• The Clotrimazole does not manage the affairs systemically, and following the vaginal application the plasmatic concentrations are minimal. Between 3% and 10% of the dose is absorbed.

MetHOd Of AnALySISPreparation of solutions:• Potassium Phosphate dibasic Solution: Disolved 4.35 g of Potassium

Phosphate dibasic in 700 mL of water. Dilute to 1000 mL and mix.

• Diluent: Mix 250 mL of Potassium Phosphate dibasic Solution and 750 mL of Methanol.

• Standard solution: Weigh approximately 25 mg of standard Clotrimazole in a 25 mL volumetric flask. Add approximately 15 mL of diluent and heat at 50oC / 30 minutes in water bath with constant mechanical shake. Sonicate for 30 minutes. Add 1 mL of Resolution Stock Solution and dilute with diluent and mix. Filter the solution with a 0.45 µm Nylon membrane.

• Resolution Stock Solution: Weigh approximately 5 mg of Clotrimazole Related Compound A in a 10 mL volumetric flask. Add diluent and sonicate for 15 minutes. Dilute with diluent and mix.

• Sample Solution: Weigh a portion of the capsules equivalent to 100 mg of Clotrimazole in a 100 mL volumetric flask. Add approximately 60 mL of diluent and heat at 50°C for 30 minutes in water bath with constant mechanical shake until capsules are dissolved. Sonicate for 30 minutes. Afterwards, shake for 1 minute with mechanical shake. Cool at room temperature and dilute with diluent and mix. Filter the solution with a 0.45 µm Nylon membrane.

Assay and Identification of Clotrimazole contained in a formulation with Metronidazole in Softgels® by HPLCMaría Isabel Morales, Eduardo Rodríguez

© 2010 Banner Pharmacaps Inc. AAPS 2010-15 W4471Research and Development, Gelcaps Exportadora de México S. A. de C.V.

FIGURE 1: Chromatogram of Clotrimazole Standard Solution and Resolution Stock Solution

FIGURE 2: Chromatogram of Clotrimazole Sample Solution

FIGURE 3: Graphic Method Linearity

PrOCedure

CHrOMAtOGrAPHICS COndItIOnS• Detector: WVD or DAD

• Wavelenght: 260 nm

• Column: Spherisorb ODS2, 10 µm, 4.6 x 250 mm

• Column Temperature: 25°C

• Inject volume: 20 µl

• Flow: 1.5 mL/minute

• Mobile phase: Potassium Phosphate dibasic solution / Methanol (25:75 v/v)

Standard solution:Weigh 25 mg of Clotrimazole RS in a 25 mL volumetric flask

Resolution Stock Solution:Weigh 5 mg of Clotrimazole related compound A in a 10 mL volumetric flask

Sample:Weigh a capsule in a 100 mL volumetric flask

Add diluent and heat at 50°C for 30 minutes with constant mechanical shake until the capsule has dissolved

Sonicate 30 minutes and mix for 1 minute with mechanical shake

Cool at room temperature and dilute volume with diluent. Mix and filter

Determine the amount of Clotrimazole with HPLC equipment

Resolution Stock Solution:Add diluent and sonicate 15 minutes. Dilute to volume and mix.

Standard solution:Add diluent, heat at 50°C/30 minutes, sonicate 30 minutes. Cool at room temperature and add 1 ml of Resolution Stock Solution. Dilute to volume and mix. Filter.

Assay and Identification of Clotrimazole contained in a formulation with Metronidazole in Softgels® by HPLCMaría Isabel Morales, Eduardo Rodríguez

© 2010 Banner Pharmacaps Inc. AAPS 2010-15 W4471Research and Development, Gelcaps Exportadora de México S. A. de C.V.

vALIdAtIOn reSuLtS COnCLuSIOn: • The method of analysis is linear, precise, accurate, robust, stable and

specific (selectivity) for the assay and identification of Clotrimazole in soft-gelatin capsules by HPLC.

• The method of analysis is specific to quantify Clotrimazole in soft-gelatin, there is no interference of the excipients with the principal peak.

• The method of analysis is linear between the range of 75% to 125% with a 99.94% recovery and 0.32% RSD.

• The method of analysis fulfills the criteria of precision and accuracy.

• The samples are stable at 5°C and room temperature up until 48 hours.

• The method of analysis is robust to changes of column batch and membrane filter brand.

referenCeS USP 32, pp.1998, The United States Pharmacopeia Convention, 2009.

Validation of Method of Clotrimazole in a formulation with Metronidazole in Soft Gelatin Capsules by HPLC.

Index Merck, p. 423-424, 13th Edition.

Parameters Limits Results

Selectivity

(Placebo analysis)

The response of the method shall only be due to the analyte

There is no interference of the excipients with the analyte

Selectivity

(Sample Degradation)

The degradation substances of the sample must not interfere with the

peak of interest.

Tested samples showed: Acid degradation: 100.00%

Basic degradation: 11.92%

Oxidative degradation: 98.53%

Photolytic degradation: 0.48%

Termic degradation: 2.69%

No degradation product interferes with the peaks of interest

System Linearity

r (correlation coefficient) ≥ 0.99

r2 (determination coefficient) ≥ 0.98

b1 (slope) ≠ 0

CI (confidence interval) of b1 must not include zero value

r = 1.0000

r2 = 0.9999

b1 = 1495.1939

CI (b1) = 1486.5925 to 1503.7954

Method Linearity

r (correlation coefficient) ≥ 0.99

r2 (determination coefficient) ≥ 0.98

CI (confidence interval) of b0 must include zero value

b1 (slope) ≈ 1

CI (confidence interval) of b1 must include the value of 1

% Recovery: CI (confidence interval) (µ) must include the 100% or the average

must be between

98 – 102%

%RSD ≤ 2.0%

r = 0.9999

r2 = 0.9998

CI (b0) = -1.5651 to 0.1384

b1 = 1.0067

CI (b1) = 0.9983 to 1.0151

Average = 99.94%

CI (µ) = 99.76% to 100.11%

RSD = 0.32%

Precision

RepeatabilityRSD ≤ 2.0%

RSD = 1.72%

Average = 104.58%

Precision Reproducibility

RSD ≤ 2.0%

Analysis of Variance (ANOVA):

F cal analyst ≤ F experimental

(0.975 probability)

F cal analyst-day ≤ F experimental

(0.975 probability)

RSD = 1.69%

Average: 103.46 %

ANOVA’s Results:

F cal analyst = 0.82956 < 7.57

F cal analyst-day = 5.46940 < 7.57

Stability of the sample

|di| ≤ 2.0%

(initial percentage – final percentage)

At Room Temperature 24 h 48 h

|di| = 0.85% |di| = 0.80%

At 5°C 24 h 48 h

|di| = 0.79% |di| = 0.31%

Robustness RSD ≤ 2.0%

|di| ≤ 2.0%

Change of batch column (from 0120 to 0123)

RSD =0.44%

|di| = 1.85%

Change of membrane filter brand (form Pall to Agilent Technologies)

RSD = 1.29%

|di| = 0.32%