Development and validation of a clomifene citrate quantification method for the stability study of an investigational medical product

Agnaoui Samy [1] , Marilly Elisa [1], Marchand Chloé [1], Salmon Damien [1][2], Dhelens Carole [1], Filali Samira [1][2], Merienne Camille [1], Pirot Fabrice [1][2], Pivot Christine [1]
[1] Pharmacie, Hôpital Edouard-Herriot, Lyon, France.
[2] Laboratoire de Pharmacie Galénique Industrielle, ISPB, Lyon, France.
[*] Contact details: name.surname@chu-lyon.fr except for damien.salmon01@chu-lyon.fr

Context
Capsules of clomifene citrate (CC) 50 mg and their placebo were requested to our hospital pharmacy for a clinical trial.

Objectif
The main goal of this study is to develop and to validate a stability indicating quantification method for CC using High-Performance Liquid Chromatography and Ultra-Violet detection (HPLC-UV). This method is meant for stability assessment and post-production controls of the capsules.

Materials and methods
The calibration standard range is situated between 20 and 100 µg/mL. The validation standard range is situated between 30 and 90 µg/mL The HPLC system consists in a C18 stationary phase (150 x 4.6 mm, 5 µm) set at 40°C and a methanol / phosphate buffer pH 8.0 (88:12, v/v) mobile phase. The detection wavelength used is 290 nm. The accuracy profile, used as a validation tool following SFSTP recommendations enables to estimate (i) linearity, (ii) specificity and selectivity, (iii) accuracy, (iv)quantification limits and (v) measurement bias based on the validation standards concentrations. A forced degradation study (FDS) [1] is performed on the capsules content using (i) alkaline (2 M NaOH, 5h), (ii) acidic (2 M HCl, 5h), (iii) oxidative (H2O2 3%, 9:1, 24h), (iv) heat (80°C, 3h) and (v) photo-oxidative (UV 265 nm, 30 min) condition in order to identify potential degradation products.

Results
No interferences are observed between the signals of the active pharmaceutical ingredient, the excipients and the degradation products (chromatographic resolution ≥ 1.5). CC retention time is 2.3 min. All the validation parameters are compliant with the following specifications: specificity and selectivity, linearity (R² > 0.99), precision (intra and inter day Coefficient of Variation (CV) < 5% and 8% respectively) and accuracy (CV < 10%). The tolerance range limit is within the acceptability range limit (10%). The FDS in heat condition reveals degradation products with retention times at 0.73, 0.81 and 1.94 min. The 1.94 min compound is also encountered with the photo-oxidative condition. No other degradation products can be identified during the other FDS conditions.

Discussion / Conclusion
This CC stability indicating quantification method based on the use of HPLC-UV is validated, allowing to perform the stability study of the CC capsules in the context of a clinical trial.

References
[1] Coord. V. Sautou. 1ère éd. SFPC/GERPAC, 2013. Guide méthodologique des études de stabilité des préparations.

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