Assay and Identification of Clotrimazole contained in a formulation with Metronidazole in Softgels by HPLC

Mara Isabel Morales, Eduardo Rodrguez

ObjeCtIveS To 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: C22H17ClN2 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 athletes 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 AnALySIS Preparation 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 m 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 50C 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 m Nylon membrane.

Research and Development, Gelcaps Exportadora de Mxico S. A. de C.V.

2010 Banner Pharmacaps Inc. AAPS 2010-15 W4471

Assay and Identification of Clotrimazole contained in a formulation with Metronidazole in Softgels by HPLC
Mara Isabel Morales, Eduardo Rodrguez

PrOCedure
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 Standard solution: Add diluent, heat at 50C/30 minutes, sonicate 30 minutes. Cool at room temperature and add 1 ml of Resolution Stock Solution. Dilute to volume and mix. Filter. Resolution Stock Solution: Add diluent and sonicate 15 minutes. Dilute to volume and mix.

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

CHrOMAtOGrAPHICS COndItIOnS Detector: WVD or DAD Wavelenght: 260 nm Column: Spherisorb ODS2, 10 m, 4.6 x 250 mm Column Temperature: 25C Inject volume: 20 l Flow: 1.5 mL/minute Mobile phase: Potassium Phosphate dibasic solution / Methanol (25:75 v/v)
FIGURE 1: Chromatogram of Clotrimazole Standard Solution and Resolution Stock Solution

Sonicate 30 minutes and mix for 1 minute with mechanical shake

FIGURE 2: Chromatogram of Clotrimazole Sample Solution

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

FIGURE 3: Graphic Method Linearity

Determine the amount of Clotrimazole with HPLC equipment

Research and Development, Gelcaps Exportadora de Mxico S. A. de C.V.

2010 Banner Pharmacaps Inc. AAPS 2010-15 W4471

Assay and Identification of Clotrimazole contained in a formulation with Metronidazole in Softgels by HPLC
Mara Isabel Morales, Eduardo Rodrguez

vALIdAtIOn reSuLtS
Parameters Selectivity (Placebo analysis) Limits The response of the method shall only be due to the analyte Results There is no interference of the excipients with the analyte Tested samples showed: Acid degradation: 100.00% Basic degradation: 11.92% Selectivity (Sample Degradation) The degradation substances of the sample must not interfere with the peak of interest. Oxidative degradation: 98.53% Photolytic degradation: 0.48% Termic degradation: 2.69% No degradation product interferes with the peaks of interest r (correlation coefficient) 0.99 System Linearity r2 (determination coefficient) 0.98 b1 (slope) 0 CI (confidence interval) of b1 must not include zero value r (correlation coefficient) 0.99 r2 (determination coefficient) 0.98 CI (confidence interval) of b0 must include zero value b1 (slope) 1 Method Linearity CI (confidence interval) of b1 must include the value of 1 % Recovery: CI (confidence interval) () must include the 100% or the average must be between 98 102% %RSD 2.0% Precision Repeatability RSD 2.0% 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 = 1.72% Average = 104.58% RSD = 1.69% Average: 103.46 % ANOVAs Results: F cal analyst = 0.82956 < 7.57 F cal analyst-day = 5.46940 < 7.57 At Room Temperature 24 h 48 h Stability of the sample |di| 2.0% (initial percentage final percentage) 24 h |di| = 0.79% |di| = 0.85% |di| = 0.80% 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 () = 99.76% to 100.11% RSD = 0.32% r = 1.0000 r2 = 0.9999 b1 = 1495.1939 CI (b1) = 1486.5925 to 1503.7954

COnCLuSIOn: The method of analysis is linear, precise, accurate, robust, stable and specific (selectivity) for the assay and identification of Clotrimazole in softgelatin 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 5C 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.

Precision Reproducibility

At 5C 48 h |di| = 0.31%

Change of batch column (from 0120 to 0123) RSD =0.44% Robustness RSD 2.0% |di| 2.0% |di| = 1.85% Change of membrane filter brand (form Pall to Agilent Technologies) RSD = 1.29% |di| = 0.32%

Research and Development, Gelcaps Exportadora de Mxico S. A. de C.V.

2010 Banner Pharmacaps Inc. AAPS 2010-15 W4471