Development and validation of a method for the quantitative determination of baclofen and its degradation products for the stability study of experimental medicine

Z. Mokdadi, E. Vissac, M. Venet, C. Marchand, C. Dhelens, C. Merienne, F. Pirot
Hôpital Edouard Herriot, Lyon, France

Context
The hospital pharmacy was involved into the development two formulations of baclofen capsules (B) 10 mg, 20 mg and theirs placebos for a clinical trial. The main objective of our study was to develop and validate a method for the dosing of B and its impurity A, a stability indicator, by HPLC-DAD-SQ for the control of these capsules and the stability assay.

Materials and methods
The calibration range was performed with 3 calibration standards: 18, 25 and 32 µg/mL, the validation standards were 20, 25 and 30 µg/mL. The HPLC consisted of an octadecylsilylated stationary phase thermostated at 20°C (150 mm x 2.1 mm, 2.6 m). The mobile phase, composed of 5 mM ammonium formate buffer (AFB) (pH = 3) and methanol + 2% AFB 5 mM (pH = 3) was injected in gradient mode (0-3 min: 70% AFB, 3-4 min: 30% AFB, 4-6 min: 70% AFB). The injection volume was 5 µL, the pump flow rate was 0.4 mL/min and the wavelength used was 330 nm. The simple quad parameters were as followed, the gas temperature was 350°C, the flow rate was 12 L/min with a pressure of 35 psig and a voltage of 3000 V. Detection was at 214 m/z and 196 m/z respectively for baclofen and its impurity A. The accuracy profile carried out in accordance with GERPAC and SFSTP recommendations evaluated linearity, precision, accuracy, limits of quantification and measurement inaccuracy as a function of concentration. Different forced degradation (FD) was performed on pharmaceutical B powder: acid (2 M HCl, 3 h), alkaline (2 M NaOH, 3 h), photo-oxidant (265 nm, 6 h), thermal (60 °C, 15 days), and oxidizer (H2O2 3%, 9 days) treatment to identify degradation products.

Results
The retention time for B was 1.64 min and 3.87 min for impurity A. The validation parameters were all within specification: linearity R2 = 0.998 (R2 > 0.99), repeatability at 1.48% and reproducibility at 2.7% (CV<5% and CV<8% by ANOVA) and accuracy at 1.1% (CV<10%).
The limits of the tolerance interval were all within the predefined acceptability range (10%). FDs under acidic, alkaline, and photo-oxidising conditions did not allow degradation of B, and no impurities and/or degradation products appeared.
Under thermal conditions, DFs slightly degraded B, but showed impurity A. B was degraded under oxidative conditions but did not reveal the impurity and/or degradation products.

Discussion / Conclusion:
A quantitative stability-indicating assay for B-capsules by HPLC-DAD-SQ has been validated. This specific, simple, and rapid technique allows us to carry out the stability study of the capsules in this clinical trial.

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