Development and validation of a method for the quantitative determination of baclofen and its degradation products for the stability study of experimental medicine.
 : Pharmacie, Hôpital Edouard Herriot, Lyon France
 : Laboratoire de Pharmacie Galénique Industrielle, ISPB, Lyon, France
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 µm). 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.
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.